Encyclopedia of Dietary Supplements (Print and Online) 0824755049, 9780824755041, 9780429171178

Table of contents :
Encyclopedia of Dietary Supplements (Online)
Edited By:
Paul M. Coates, M. Coates Paul, Marc Blackman, Marc R. Blackman, Gordon M. Cragg, Mark Levine, Jeffrey D. White, Joel Moss
Edition 1st Edition
First Published 2004
eBook Published 22 March 2013
Pub. LocationBoca Raton
Imprint CRC Press
DOI https://doi.org/10.1201/b13959
Pages 840
eBook ISBN 9780429171178
Subjects Bioscience, Medicine, Dentistry, Nursing & Allied Health

Encyclopedia of Dietary Supplements (Online)
Edited ByPaul M. Coates, M. Coates Paul, Marc Blackman, Marc R. Blackman, Gordon M. Cragg, Mark Levine, Jeffrey D. White, Joel Moss
Edition1st Edition
First Published2004
eBook Published22 March 2013
Pub. LocationBoca Raton
ImprintCRC Press
DOIhttps://doi.org/10.1201/b13959
Pages840
eBook ISBN9780429171178
SubjectsBioscience, Medicine, Dentistry, Nursing & Allied Health

Citation preview

Encyclopedia of

Dietary

Supplements edited by Paul M. Coates Marc R. Blackman

Gordon M. Cragg Mark Levine Joel Moss

Jeffrey D. White

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Brief Contents

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or ere er 151

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159 167 177 189 197

Feverfew (Tanacetum parthenium) .......... 211 DR Se aS are eee aa ee > eee Caste (AUT) SOTVUM) wn ee ee aesee 229 iouveer (Zineiber officinale)... 1... i we ee es 241 ROO

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Ginseng, American (Panax quinquefolium) ..... Ginseng, Asian (Panax ginseng) ............ Ee Rhee Gg Gos ek pao 6 a Se Ree AN eee git dc Ss oy ba 8 or a Goldenseal (Hydrastis canadensis) .......... Reeds ee ER Gs cee ee es ioteen Toa Polyphenols 2.266... eee ees

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Reviewers

The Editors wish to thank the outside reviewers, who lent their time, shared their expertise, and volunteered their editorial insights. Please note that some of the following read more than one article and two reviewers requested they remain anonymous.

Salvatore Alesci, M.D., Ph.D.

Marilyn Barrett, Ph.D.

/ National Institutes of Health, Bethesda, Maryland, U.S.A. / Pharmacognosy Consulting Services, San Carlos, California, U.S.A.

Melinda A. Beck, Ph.D.

/ University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

Joseph M. Betz, Ph.D.

/ National Institutes of Health, Bethesda, Maryland,

John Beutler, Ph.D. Mark Blumenthal

U.S.A.

/ National Cancer Institute, Frederick, Maryland, U.S.A. / American

Richard A. Bone, Ph.D.

Botanical Council and HerbalGram,

Austin, Texas, U.S.A.

/ Florida International University, Miami, Florida, U.S.A.

Linda S. Brady, Ph.D.

/ National Institutes of Health, Bethesda, Maryland, U.S.A.

Alan L. Buchman, M.D., M.S.P.H Illinois, U.S.A.

/ Feinberg School of Medicine at Northwestern University, Chicago,

John H. Cardellina, If, Ph.D. / National Cancer Institute, Frederick, Maryland, U.S.A. Lucas R. Chadwick, Ph.D. / UJC/NIH Center for Botanical Dietary Supplements Research in Women’s Health, Chicago, Illinois, U.S.A.

Yung-Chi Cheng, Ph.D.

/ Yale School of Medicine, New Haven, Connecticut, U.S.A.

George P. Chrousos, M.D.

/ National Institutes of Health, Bethesda, Maryland, U.S.A.

G. H. Constantine, Ph.D. Steven Dentali, Ph.D.

/ Oregon State University College of Pharmacy,

Corvallis, Oregon, U.S.A.

/ American Herbal Products Association, Silver Spring, Maryland, U.S.A.

Edzard Ernst, M.D., Ph.D., F.R.C.P. Plymouth, Exeter, Devon, U.K. Norman R. Farnsworth, Ph.D. Guylaine Ferland, Ph.D.

/

/ University of Illinois at Chicago, Chicago, Illinois, U.S.A. Université de Montréal, Montreal,

Lorraine A. Fitzpatrick, M.D. Sherwood L. Gorbach, M.D.

/ Peninsula Medical School of the Universities of Exeter &

Canada

/ Women’s Health Fellowship Mayo Clinic, Rochester, Minnesota, U.S.A. / Tufts University School of Medicine, Boston, Massachusetts,

U.S.A.

Tory M. Hagen, Ph.D.

/ Oregon State University, Corvallis, Oregon, U.S.A.

Mary L. Hardy, M.D.

/ David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A.

Jane Higdon, Ph.D.

/ Oregon State University, Corvallis, Oregon, U.S.A.

Richard B. Kreider, Ph.D.

/ Baylor University, Waco, Texas, U.S.A.

Norman I. Krinsky, Ph.D. / School of Medicine and Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, U.S.A.

Oran Kwon, Ph.D.

/ Korea Food and Drug Administration, Seoul, South Korea Benjamin H.S. Lau, M.D., Ph.D. / Loma Linda University, Loma Linda, California, U.S.A. Gian Paolo Littarru, M.D. / Polytechnic University of Marche, Ancona, Italy Yuan Chun Ma, Ph.D. / Canadian Phytopharmaceuticals Corp., Richmond, British Columbia, Canada Craig J. McClain, M.D. / University of Louisville, Louisville, Kentucky, U.S.A. Donald B. McCormick, Ph.D. / Emory University School of Medicine, Atlanta, Georgia, U.S.A. Joshua W. Miller, Ph.D. / University of California School of Medicine, Davis, California, U.S.A. / National Institutes of Health, Bethesda, Maryland, U.S.A. Richard L. Nahin, Ph.D., M.P.H. Jac B. Park, Ph.D. / United States Department of Agriculture, Beltsville, Maryland, U.S.A. Vii

Vili Greg Pennyroyal

/ Natural Product Solutions LLC, Temecula,

J. David Phillipson, D.Sc., Ph.D.

California,

U.K. William F. Popin, M.S. / Young Living Essential Oils, Lehi, Utah, U.S.A. A. Catharine Ross, Ph.D. / The Pennsylvania State University, University Park, Pennsylvania, U.S.A. Filippo Rossi-Fanelli

/ University of London, London,

/ Universita degli Studia di Roma, Rome, Italy

Norman Salem, Jr., Ph.D.

/ National Institutes of Health, Rockville, Maryland,

Manickam Sugumaran, M.Sc., Ph.D.

/ University

U.S.A.

of Massachusetts, Boston, Masschusetts,

U.S.A.

Ronald S. Swerdloff, M.D. / Harbor-UCLA Medical Center and the David Geffin School of Medicine, Torrance, California, U.S.A. Barbara N. Timmermann, Ph.D. / University of Arizona College of Pharmacy, Tucson, Arizona, U.S.A. Roy Upton, Herbalist

/ American Herbal Pharmacopoeia®, Scotts Valley, California, U.S.A.

Hildebert Wagner, Ph.D. W. Allan Walker, M.D.

/ University of Munich, Munchen, Germany / Harvard Medical School, Boston, Massachusetts,

U.S.A.

Contributors

Steve F. Abcouwer

/ University of New Mexico School of Medicine, Albuquerque, New Mexico, U.S.A.

Gianluca Aimaretti

/ University of Turin, Turin, Italy

Salvatore Alesci / Clinical Neuroendocrinology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A. Lindsay H. Allen / United States Department of Agriculture—Western Human Nutrition Research Center, University of California, Davis, California, U.S.A. John J.B. Anderson / Schools of Public Health and Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A. Decio Armanini

/ University of Padua, Padua, Italy

Emanuela Arvat

/ University of Turin, Turin, Italy

Dennis V.C. Awang Pamela Bagley

Matteo Baldi Rudolf Bauer John Beard

/ MediPlant Consulting Inc., White Rock, British Columbia, Canada

/ Biomedical Libraries, Dartmouth College, Hanover, New Hampshire,

U.S.A.

/ University of Turin, Turin, Italy / Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz, Graz, Austria / The Pennsylvania State University, University Park, Pennsylvania,

Gary R. Beecher

/ Lothian, Maryland, U.S.A.

Joseph M. Betz

/ National Institutes of Health, Bethesda, Maryland, U.S.A.

Jens Bielenberg

/ Division of Endocrinology,

Marc R. Blackman

Nancy L. Booth

University of Padua, Padua, Italy

/ Endocrine Section, Laboratory of Clinical Investigation, National Center for

Complementary and Alternative Medicine,

Maryland,

U.S.A.

National Institutes of Health, Bethesda,

U.S.A.

/ UIC/NIH Center for Botanical Dietary Supplements Research,

Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Christelle Bourgeois Francois G. Brackman

/ Institute of Medical Biochemistry, / Fournier Pharma,

Garches,

Medical University of Vienna, Vienna, Austria

France

Raymond F. Burk / Clinical Nutrition Research Unit, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A. Werner R. Busse

Shenglin'Chen

/ Dr. Willmar Schwabe

GmbH

& Co. KG, Karlsruhe,

Germany

/ Molecular and Clinical Nutrition Section, Digestive Diseases Branch,

National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Emily Y. Chew / Division of Epidemiology and Clinical Research, National Eye Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Carolyn S. Chung / Children’s Hospital Oakland Research Institute, Oakland, California, U.S.A.

Daniel O. Clegg / George E. Wahlen Department of Veterans Affairs Medical Center and University of Utah School of Medicine, Salt Lake City, Utah, U.S.A. Dallas L. Clouatre / Glykon Technologies Group, L.L.C., Santa Monica, California, U.S.A. Jerry M. Cott

/ Food and Drug Administration,

Rockville, Maryland,

U.S.A.

Edward M. Croom, Jr. / School of Pharmacy, University of Mississippi, Oxford, Mississippi, U.S.A. Gustav Dallner / Stockholm University, Stockholm, Sweden

x

logy Branch, Pedro Del Corral / Clinical Neuroendocrinology Unit, Pediatric Reproductive Endocrino Bethesda, Health, of Institutes National nt, National Institutes of Child Health and Human Developme Maryland, U.S.A. of Pharmacy, Brigit Dietz / UIC/NIH Center for Botanical Dietary Supplements Research, College University of Illinois at Chicago, Chicago, Illinois, U.S.A. York, Linda C. Duffy / Infectious Diseases Division, University of Buf falo—State University of New Women and Children’s Health Research Foundation,

Women and Children’s Hospital/Kaleida Health,

Buffalo, New York, U.S.A. of Peter Eck / Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute U.S.A. Maryland, Bethesda, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Alan Edgar

/ Fournier Pharma,

Memory P.F. Elvin-Lewis Jan Engle

Garches,

France

/ Washington University, St. Louis, Missouri, U.S.A.

/ UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy,

University of Illinois at Chicago,

Chicago, Illinois, U.S.A.

Daniel S. Fabricant / UIC/NIH Center for Botanical Dietary Supplements Research for Women’s Health, Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy (M/C-877), University of Illinois at Chicago, Chicago, Illinois, U.S.A. Norman R. Farnsworth / UIC/NIH Center for Botanical Dietary Supplements Research for Women’s Health, Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy (M/C-877), University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Cristina Fiore / University of Padua, Padua, Italy Sanford C. Garner / Constella Group, Inc., Durham, North Carolina, U.S.A. Ezio Ghigo / University of Turin, Turin, Italy Roberta Giordano / University of Turin, Turin, Italy Elizabeth Griffiths / Infectious Diseases Division, University of Buf falo—State University of New York, Women and Children’s Health Research Foundation, Women and Children’s Hospital/Kaleidaz Health, Buffalo, New York, U.S.A. Peter Hadley

/ Delft University of Technology, Delft, FGN, The Netherlands

William S. Harris

/ Lipid and Diabetes Research Center, Mid America Heart Institute, Saint Luke’s

Hospital, Kansas City, Missouri, U.S.A.

Robert P. Heaney Chi-Tang Ho

/ Creighton University, Omaha, Nebraska,

U.S.A.

/ Cook College, Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A.

Curtiss D. Hunt / United States Department of Agriculture, Agriculture Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota, U.S.A. Christopher G. Jackson / University of Utah School of Medicine and George E. Wahlen Department of Veterans Af fairs Medical Center, Salt Lake City, Utah, U.S.A. C. Jakobs / VU University Medical Center, Clinical Chemistry, The Netherlands

Metabolic

Unit, Amsterdam,

Elizabeth J. Johnson / Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, U.S.A. Katharine M. Jones / United States Department of Agriculture—Western Human Nutrition Research Center, University of California, Davis, California, U.S.A.

Wiltrud Juretzek

/ Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany

Chithan Kandaswami / State University of New York at Buffalo, Buffalo, New York, U.S.A. Arie Katz / Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

Kara M. Kelly / Division of Pediatric Oncology, Integrative Therapies Program for Children with Cancer, College of Physicians and Surgeons, Columbia University Medical Center, New York, New York, U.S.A.

Ikhlas A. Khan

/ National Center for Natural Products Research, Research Institute of Pharmaceutical

Sciences, School of Pharmacy,

Janet C. King

University of Mississippi, University, Mississippi, U.S.A. / Children’s Hospital Oakland Research Institute, Oakland, California, U.S.A.

Marguerite A. Klein

/ Division of Extramural Research and Training, National Center for

Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland,

U.S.A.

xi

Leslie M. Klevay

/ Grand Forks Human Nutrition Research Center, Agricultural Research Service,

Grand Forks, North Dakota,

Egon Koch

U.S.A.

/ Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany

David J. Kroll / Natural Products Laboratory, Research Triangle Institute (RTI International), Research Triangle Park, North Carolina, U.S.A. Oran Kwon / Korea Food and Drug Administration, Seoul, Korea Elena Ladas / Division of Pediatric Oncology, Integrative Therapies Program for Children with Cancer, College of Physicians and Surgeons, Columbia University Medical Center, New York, New York, U.S.A.

Joshua D. Lambert / Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A.

Fabio Lanfranco / University of Turin, Turin, Italy Benjamin Z. Leder / Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A. Jee-Hyuk Lee / Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

James E. Leklem

/ Oregon State University, Corvallis, Oregon, U.S.A.

Albert Y. Leung

/

Mark Levine

Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of

/

Phyto-Technologies,

Inc., Woodbine,

Iowa, U.S.A.

Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

Walter H. Lewis / Washington University, St. Louis, Missouri, U.S.A. Thomas S.C. Li / Agriculture and Agri-Food Canada, Pacific Agri-Food Research Center, Summerland, British Columbia, Canada Tieraona Low Dog / University of Arizona Health Sciences Center, Tucson, Arizona, U.S.A. Shelly C. Lu) / USC Research Center for Liver Diseases, USC-UCLA Alcoholic Liver and Pancreatic Disease Center, The Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

José M. Mato / CIC-Biogune, Metabolomics Unit, Technological Park of Bizkaia, Derio, Bizkaia, Spain Mauro Maccario / University of Turin, Turin, Italy Gail B. Mahady / UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Irini Manoli_ / ‘Endocrine Section, Laboratory of Clinical Investigation, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland, U.S.A.

Lisa Marafetti / University of Turin, Turin, Italy Valentino Martina / University of Turin, Turin, Italy Donald B. McCormick / School of Medicine, Emory University, Atlanta, Georgia, U.S.A. Dennis J. McKenna / Center for Spirituality and Healing, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, U.S.A. Mark Messina / School of Public Health, Loma Linda University, Loma Linda, California, U.S.A. Joanna Michel / UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. J.A. Milner / National Institutes of Health, Bethesda, Maryland, U.S.A. Homan Miraliakbari / Memorial University of Newfoundland, St. John’s, Newfoundland, Canada

Donald M. Mock / University of Arkansas for Medical Sciences, Little Rock, Arkansas, U.S.A. Joel Moss / National Institutes of Health, NHLBI, Pulmonary-Critical Care Medicine Branch, Bethesda, Maryland, U.S.A. / National Center for Natural Products Research, Research Institute of Ilias Muhammad Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi, U.S.A. Steven M. Musser / Office of Scientific Analysis and Support, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, Maryland, U.S.A. Koji Nakanishi

/

Columbia

University, New York, New York, U.S.A.

xii

/ Clinical Nutrition Research Unit, Vanderbilt University School of Medicine,

Brooke K. Norsworthy Nashville,

Tennessee,

U.S.A.

York, / Infectious Diseases Division, University of Buf falo—State University of New

Pearay Ogra

Women and Children’s Health Research Foundation,

Women and Children’s Hospital/Kaleida Health,

Buffalo, New York, U.S.A. Adewole L. Okunade

/

Washington

University, St. Louis, Missouri,

U.S.A.

Karel Pacak / Clinical Neuroendocrinology Unit, Pediatric Reproductive Endocrinology Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. Sebastian J. Padayatty / Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Jae B. Park / Phytonutrients Laboratory, BHNRC, ARS, United States Department of Agriculture, Beltsville, Maryland, U.S.A. Cesare Patrini

/ University of Pavia, Pavia, Italy Colleen E. Piersen / UIC/NIH Center for Botanical Dietary Supplements Research, Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Gregory A. Plotnikoff / Center for Spirituality and Healing, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, U.S.A. Haiping Qiao / Infectious Diseases Division, University of Buffalo—State University of New York, Women and Children’s Health Research Foundation,

Women and Children’s Hospital/Kaleida Health,

Buffalo, New York, U.S.A.

Eugenio Ragazzi / University of Padua, Padua, Italy Charles J. Rebouche / Carver College of Medicine, University of Iowa, Iowa City, Iowa, U.S.A.

Gianguido Rindi

/ University of Pavia, Pavia, Italy

Richard S. Rivlin / Clinical Nutrition Research New York, U.S.A.

Unit, Institute for Cancer Prevention,

New York,

P.J. Rohdewald / Institute of Pharmaceutical Chemistry, Westfdlische Wilhelms-Universitat Munster, Munster, Germany

A. Catharine Ross Robert K. Rude

/ The Pennsylvania State University, University Park, Pennysylvania,

U.S.A.

/ University of Southern California, Los Angeles, California, U.S.A.

Robert M. Russell / Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, U.S.A. Rosalie Sagraves / UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. G.S. Salomons / VU University Medical Center, Clinical Chemistry, Metabolic Unit, Amsterdam, The Netherlands Shengmin Sang / Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A. John Paul SanGiovanni / Division of Epidemiology and Clinical Research, National Eye Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

Steven J. Schwartz Mariangela Seardo Fereidoon Shahidi Barry Shane

/ The Ohio State University, Columbus, Ohio, U.S.A. / University of Turin, Turin, Italy / Memorial University of Newfoundland, St. John’s, Newfoundland,

Canada

/ University of California, Berkeley, California, U.S.A.

William L. Smith / University of Michigan Medical School, Ann Arbor, Michigan, U.S.A. Fabio Soldati / Pharmaton SA, Head of Research and Development, Bioggio, Switzerland

Jiannan Song

/ School of Public Health and School of Medicine, University of North Carolina,

Chapel Hill, North Carolina,

Stephen Sporn

U.S.A.

/ Springfield, Missouri, U.S.A.

Roland Stocker / Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia

xiii

Kristian Strgmgaard

/ The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark

J.W. Suttie / College of Agricultural and Life Sciences, University of Wisconsin-Madison, Wisconsin, U.S.A.

Lawrence Sweetman / Mass Spectrometry Laboratory, Institute Medical Center, Dallas, Texas, U.S.A. Anne L. Thurn

of Metabolic Disease, Baylor LR)

/ Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland, U.S.A.

Maret G. Traber Roy Upton

Madison,

/ Linus Pauling Institute, Oregon State University, Corvallis, Oregon, U.S.A.

/ American Herbal Pharmacopoeia®, Scotts Valley, California, U.S.A.

Stine B. Vogensen

/ The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark

Yaohui Wang / Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Solomon P. Wasser

Karin Woelkart

Richard J. Wurtman M. Wyss

/ Institute of Evolution, University of Haifa, Mount Carmel, Haifa, Israel

/ Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz, Graz, Austria / Massachusetts Institute of Technology, Cambridge, Massachusetts,

U.S.A.

/ DSM Nutritional Products Ltd., Basel, Switzerland

Chung S. Yang / Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A.

Steven H. Zeisel / School of Public Health and School of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A. Jianping Zhao / National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi, U.S.A.

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ee ee Sg Ws ee ale ee 6.05 0 bh Iron’ / John Beard 2 ee ee ee ae 0h 3 Isoflavones 9° Mark: Messing 00.040. ... ere.-es eee e eee 2.0 c... Kava (Piper methysticum) / Steven M. Musser’.. Lactobacilli and Bifidobacteria / Linda C. Duffy, Stephen Sporn, Elizabeth Griffiths,

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401 409

en ee ae eg = ee 2 Ne © cay see / Peter Hadley and’ Steven Jo SCHWGTIZ, Maca (Lepidium meyenii) / Ilias Muhammad, Jianping Zhao, and Ikhlas A. Khan ..........-.

421

Lycopene

445

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457

/ Elena Ladas, David J. Kroll, and Kara M. Kelly..........

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S37 545 555 569 577

Red Clover (Trifolium pratense)

587

| Nancy L. Booth and Colleen E. Piersen.................

Reishi or Ling Zhi (Ganoderma lucidum)

Ribollayin.

| Richard 'S. Rivlin

Saw Palmetto (Serenoa repens) Selenium

0.

/ Solomon P. Wasser...............0.0...20-.

0.2

x.

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qeauet VE

000

/ Raymond F. Burk and Brooke K. Norsworthy

Shiitake (Lentinus edodes)

Seal

/| Edward M. Croom, Jr........

_/ Solomon P. Wasserve:

St. John’s Wort (Hypericum perforatum)

// “Gianguido,Rindi andsCesare,Patrinichads

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701 715

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733

Vitamin MAO Yohimbe Zine j Index

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745

fey) 771 783 791

Preface

Welcome to the Encyclopedia of Dietary Supplements, reflecting the combined efforts of more than 100 authors on more than 75 different topics. We expect this work to become a valuable reference for students and researchers of physiology and chemistry, for healthcare providers, and for consumers who are interested in understanding the kind of science that is—or is not—behind the claims that are made for dietary supplements that are sold throughout the world, where standards of government regulation differ from country to country.

In the United States, sales of products in the dietary supplement market approached $20 billion in 2003. Their form and their labeling are regulated by the Food and Drug Administration (FDA) as a result of legislation passed in 1994 called the Dietary Supplement Health and Education Act (DSHEA). The dietary supplement category in the United States includes vitamins, minerals, and other ingredients that are found in foods, as well as ingredients not ordinarily found in foods—such as extracts of herbs and other natural products—that are used by consumers for their potential health-promoting, diseasepreventing, performance-enhancing or healing properties. Many of these are represented in the chapters of this book. The Encyclopedia is not just for consumers in the U.S. market, although we acknowledge that the term “dietary supplements’’ is an American expression. We are not aware of any other single term that describes all of the substances that we wish to include in this encyclopedia, even though some may not consider it appropriate to certain products not marketed in the United States. Consumers in all parts of the world ingest the substances that we have covered in this reference. Sometimes the claims for benefit of specific products are borne out by well-documented scientific studies. In other cases, they are not,

and enthusiasm for their use is based on popular legend or on longstanding patterns of use in traditional healing systems. In this encyclopedia, we hope that readers will be able to examine the types of evidence that have been used to support claims of benefit. The goal of the Encyclopedia of Dietary Supplements is to provide readers with comprehensive, yet accessible, information on the current state of science for individual

supplement ingredients or extracts. To this end, each entry reviews the basic information available about the ingredient, including where applicable its chemistry and functions, before detailing the pre-clinical and clinical literature. Articles outline the regulatory status of each substance, and then conclude with references to the relevant literature.

Dietary supplements included for this first edition of this Encyclopedia were selected in large part because of their popularity in the marketplace. It is clear that the level of scientific information available differs markedly among the various entries. For many ingredients, the chemistry and physiology, pre-clinical and clinical information, and mechanism of action are well known. For others, by contrast, some or many pieces of these data are missing. The preparation of some commercial products is of high quality and follows good agricultural, laboratory, and manufacturing practices. Again, by contrast, the preparations for others have not been reliable, making them subject to high variability in content and contamination. As dietary supplement use becomes more widespread, there are growing concerns about the safety of some ingredients, including possible harmful interactions between supplements and prescribed drugs. These issues should form the basis

for future: research. The field of dietary supplements is a rich one, and the science related to this large class of ingredients is expanding all the time. Thus, an important feature of this encyclopedia is XVii

XViii

that, after this first edition appears in print and online at www.dekker.com, future updates will be made online and on a regular basis. Topics that have not been covered in this edition can be included in future online versions. The first online update, for example, will include an article on regulation of these products around the world. Likewise, information

that requires, it can be updated promptly via the online updates, without having to wait for a revised printed edition. Two of the topics in this edition of the Encyclopedia—Ephedra and Androstenedione—were commissioned before their status as dietary supplements in the U.S. market was changed. In February 2004, the FDA announced a ban on ephedra-containing products from the dietary supplement market in the United States (http: //www.cfsan. fda. gou/~lrd/fpephed6. html). In March 2004, the FDA issued warning letters to companies that market products containing androstenedione (http. //www.cfsan. fda.gov/~dms/ andltr.html). The regulatory status of these products as dietary supplements is therefore in question. Nevertheless, until recently, both ephedra and androstenedione were widely consumed in the United States. We felt, therefore, that discussion of the science of these

ingredients was important. We express our thanks to the authors of the individual articles. This is a challenging and somewhat controversial field, but we believe that our authors have provided a balanced and current view of the literature. We also acknowledge with gratitude the hard work and guidance of Marcel Dekker’s editorial staff, particularly Jinnie Kim, Sapna Maloor, and Oona Schmid. Finally, we wish to emphasize that the inclusion of articles on particular dietary supplements in this Encyclopedia does not imply that we endorse them. Paul M. Coates Marc R. Blackman

Gordon M. Cragg Mark Levine Joel Moss

Jeffrey D. White

Encyclopedia of

Dietary

Supplements

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S-Adenosylmethionine José M. Mato CIC-Biogune, Metabolomics Unit, Technological Park of Bizkaia, Derio, Bizkaia, Spain

Shelly C. Lu USC Research Center for Liver Diseases, USC-UCLA Alcoholic Liver and Pancreatic Disease Center, The Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California,

Los Angeles, California, U.S.A.

INTRODUCTION S-Adenosyl-L-methionine (SAMe) has been shown to regulate key cell functions. Abnormalities in SAMe content have been linked to the development of liver disease and to depression. This article reviews the biochemistry and functions of SAMe, its deficiency in liver

of liver disease and in patients with liver disease. Both serum and cerebrospinal fluid (CSF) levels of this methionine metabolite have been reported to be low in depressed patients; the possibility of SAMe therapy has therefore been considered in this condition. The effect of SAMe in the treatment of other diseases, such as osteoarthritis, has also been investigated.

disease and depression, and SAMe treatment in liver disease, depression, and osteoarthritis.

BIOCHEMISTRY AND FUNCTIONS

COMMON

AND SCIENTIFIC NAME

S-Adenosyl-L-methionine—also known as 5’-[(3-amino3-carboxypropyl)-methylsulfonio]-5'-deoxyadenosine and S-(5'-desoxyadenosin-5-yl)-methionine—has the chemical formula [C);H23N6O;S]". It is abbreviated in the scientific literature as AdoMet, SAM, or SAMe.

In the early literature, before the identification of its structure, SAMe was known as “‘active methionine.”’

GENERAL

DESCRIPTION

SAMe was discovered by Giulio Cantoni in 1953 and since then has been shown to regulate key cellular functions such as differentiation, growth, and apoptosis. Abnormal SAMe content has been linked to the development of experimental and human liver disease, and this has led to the examination of the effect of SAMe supplementation in a variety of animal models

José M. Mato, Ph.D., is Professor and Director at CIC-Biogune, Metabolomics Unit, Technological Park of Bizkaia, Derio, Bizkaia,

Spain. Shelly C. Lu, M.D., is Professor at USC Liver Diseases, USC-UCLA

Research Center for Alcoholic Liver and Pancreatic Disease

Center, The Division of Gastrointestinal and Liver Diseases, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

Encyclopedia of Dietary Supplements DOT: 10.1081 /E-EDS-120022079 Copyright © 2005 by Marcel Dekker. All rights reserved.

Discovery Though SAMe was discovered 50 years ago, its story begins in 1890 with Wilhelm His. When he fed pyridine to dogs, he was able to isolate N-methylpyridine from the urine—His emphasized the need to demonstrate both the origin of the methyl group as well as the mechanism of its addition to the pyridine (reviewed

in Ref!!), Both questions were addressed by Vincent du Vigneaud, who, during the late 1930s, demonstrated that the sulfur atom of methionine was transferred to cysteine through the “trans-sulfuration’’ pathway, and discovered the “‘transmethylation’’ pathway, that is, the exchange of methyl groups between methionine, choline,

betaine,» and

demonstrated

creatine.

In

that a liver homogenate

1951,

Cantoni

supplemented

with ATP and methionine converted nicotinamide to N-methylnicotinamide. Two years later, he established S-Adenosylmethionine AdoMet, SAM, SAMe

0 ~ N= N

S Ahi ts

N

NAN

O

' vs 7

CH, O

O

Fig. 1 Structure of SAMe.

N

S-Adenosylmethionine

4

that methionine and ATP reacted to form a product, which he originally called “active methionine,”’ capable of transferring its methyl group to nicotinamide or guanidoacetic acid to form N-methylnicotinamide or creatine in the absence of ATP. After determination

of its structure,

he called it AdoMet

(Fig. 1). Subsequently, Cantoni and his colleagues discovered methionine adenosyltransferase (MAT)— the enzyme that synthesizes SAMe, S-adenosylhomocysteine (SAH)—the product of the transmethylation reactions, and SAH-hydrolase—the enzyme that converts SAH to adenosine and homocysteine (Hcy). At about the same time, Peter Bennett discovered that folate and vitamin B,, could replace choline as a

source of methyl groups in rats maintained on diets containing Hcy in place of methionine, a finding that led to the discovery of methionine synthase (MS). In 1961, John Tabor demonstrated that the propylamino moiety of SAMe is converted via a series of enzymatic steps to spermidine and spermine. In the biosynthesis of polyamines, _5/-deoxy-5’-methylthioadenosine (MTA) was identified as an end product. Thus, by the beginning of the 1960s, Laster’s group could finally provide an integrated view, similar to that depicted in Fig. 2, combining the transmethylation and transsulfuration pathways with polyamine synthesis. Since then, SAMe has been shown to donate: 1) its methyl group to a large variety of acceptor molecules, including DNA, RNA, phospholipids, and proteins; 2) its sulfur atom, via a series of reactions, to cysteine and glutathione (GSH), a major cellular antioxidant; 3) its propylamino group to polyamines, which are required for cell growth; and 4) its MTA moiety, via a complex set of enzymatic reactions known as the “methionine salvage pathway,’’ for the resynthesis of this amino acid. These reactions can affect a wide spectrum of biological processes ranging from metal detoxication and catecholamine metabolism to membrane fluidity, gene expression, cell growth, differentiation, and apoptosis (reviewed in Ref.”!), to establish what Cantoni called the “AdoMet empire.”’ :

Synthesis In mammals, there synthesize SAMe: MATI and MATIII while MATII is the

are three distinct enzymes that MATI, MATII, and MATIII. are the gene products of MATIA, gene product of MAT2A (reviewed

in Ref.) In adults, MATIA

is expressed exclusively in

the liver and pancreas, whereas MAT72A is expressed in all tissues, including the liver. In fetal rat liver, MATIA

expression increases progressively from gestation, increases 10-fold immediately and reaches a peak at 10days of age, slightly by adulthood. Conversely, MAT2A

day 20 of after birth, decreasing expression

MTA Patrescnesit & Spermidine

> MTA Spermine

MAT Glyci

Serine

Met

"Z THF

5,10-MTHF MS MT

Pe

N,N-Dimethyl-Gly

BHMT

SAMe

ms ) GNMT

Betaine

Ot

= X-CH,

Hey padtegti oy CBS

Cystathionine o-Ketobutyrate

Cys ——>

——

GSH

Fig. 2 Hepatic metabolism of SAMe. Methionine (Met) is converted to homocysteine (Hcy) via S-adenosylmethionine (SAMe) and S-adenosylhomocysteine (SAH). The conversion of Met to SAMe is catalyzed by methionine adenosyltransferase (MAT). After decarboxylation, SAMe can donate the remaining propylamino moiety attached to its sulfonium ion to putrescine to form spermidine and methylthioadenosine (MTA) and to spermidine to form spermine and a second molecule of MTA. SAMe donates its methyl group in a large variety of reactions catalyzed by dozens of methyltransferase (MTs), the most abundant in the liver being glycineN-methyltransferase (GNMT). The SAH thus generated is hydrolyzed to form Hcy and adenosine through a reversible reaction catalyzed by SAH hydrolase. Hcy can be remethylated to form methionine by two enzymes: methionine synthase (MS) and betaine methyltransferase (BHMT). In the liver, Hcy can also go through the trans-sulfuration pathway to form cysteine via a two-step enzymatic process. In the presence of serine, Hcy is converted to cystathionine in a reaction catalyzed by cystathionine f-synthetase (CBS). Cystathionine is then hydrolyzed by cystathionase to form cysteine, a precursor for the synthesis of glutathione (GSH). In tissues other than the liver, kidney, and pancreas, cystathionine is not converted to GSH due to the lack of expression of one or more enzymes of the trans-sulfuration pathway. The expression of BHMT is also limited to the liver. All mammalian tissues convert Met to Hcy, via SAMe and SAH, and remethylate Hcy to Met via the MS pathway. Other abbreviations in this figure: THF, tetrahydrofolate; 5,10-MTHF, methylenetetrahydrofolate; 5-MTHF, methyltetrahydrofolate; Ser, serine; Gly, glycine; X, methyl acceptor molecule; X-CH3, methylated molecule.

decreases after birth, increases threefold in the newborn, and decreases further in postnatal life, reaching

a minimum in the adult liver (about 5% that of MATIA). Due to differences in the regulatory and kinetic properties of the various MATs, MATII cannot maintain the same high levels of SAMe compared to the combination of MATI and MATIII (reviewed in Ref.)), Consequently, in MATIA knockout mice, despite a significant increase in MAT2A expression,

S-Adenosylmethionine

3

the liver content of SAMe is reduced about threefold from birth, when the switch from MAT2A to MATIA

takes place.)

(caused by both inactivation of the enzyme and reduced expression of MATIJA due to the spontaneous methylation of the gene promoter) and are predisposed to develop HCC.”:"°

DEFICIENCY

In Depression

In Liver Disease

Mice lacking MATIA have hepatic hyperplasia and spontaneously develop nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC).°-4 It is also well known that when rats and mice are fed a diet deficient in methyl groups (choline, methionine, folate, and vitamin Bj), the liver develops steatosis

within a few days (reviewed in Refs.'-*!). If the diet continues, NASH,

fibrosis of the liver, and cirrhosis

result, with some animals developing HCC. Numerous nutritional studies have shown that dietary methyl deficiency causes a decrease in the hepatic content of SAMe, an increase in the concentration of SAH, and

an elevation of plasma Hcy levels. It has been demonstrated,

for

example,

that

disruption

of the

gene

encoding for 5,10-methylenetetrahydrofolate reductase (MTHFR), which synthesizes 5-methyltetrahydrofolate, required by methionine synthase to remethylate Hcy to methionine (see Fig. 2), results in elevated plasma Hcy levels, and reduced content of hepatic betaine, glycerophosphocholine, and phosphocholine, the intracellular

storage forms of choline, as well as increased content

of SAH and reduced SAMe.!”! Plasma Hcy decreased and hepatic phosphocholine increased in MTHFR knockout mice fed a diet supplemented with betaine; while knockout mice fed a control diet developed severe steatosis, those on a diet supplemented with betaine had only moderate or mild steatosis.!7! The observation that MAT/A knockout mice have hepatic hyperplasia, are more susceptible to develop liver injury in response to a choline-deficient ‘diet, and spontaneously develop NASH and Hcc®4) strongly suggests that shortage of SAMe may be a key component of the mechanism by which a deficiency in methyl groups causes hepatic lesions. Microarray and proteomic experiments using liver

from MATIA

knockout mice! indicate that SAMe

regulates the expression of a large and diverse set of genes, including many metabolic genes that are affected in 3-mo-old knockout mice long before the appearance of any sign of histological lesion. This surprising result suggests that abnormal SAMe levels may cause liver injury and cancer through perturbation of multiple metabolic pathways in the cell. The medical implications of these observations are obvious,

since cirrhotic patients, independent

of the

etiology of their disease, have impaired metabolism of methionine, reduced hepatic synthesis of SAMe

Major depression has been associated with a deficiency in methyl groups (folate, vitamin B,2, and SAMe)

(reviewed in Ref."!), Thus, depressed patients often have low plasma folate and vitamin B,, and reduced

SAMe content in the CSF. Moreover, patients with low plasma folate appear to respond less well to antidepressants. The mechanism by which low SAMe concentrations may contribute to the appearance and evolution of depression is, however, not well known. SAMe-dependent methylation reactions are involved in the synthesis and inactivation of neurotransmitters, such as noradrenaline, adrenaline, dopamine, serotonin, and histamine, and the administration of drugs

that stimulate dopamine synthesis, such as L-dihydroxyphenylalanine, causes a marked decrease in SAMe concentration in rat brain, and in plasma and CSF in

humans. Moreover, various drugs that interfere with monoaminergic neurotransmission, such as imipramine

and

desipramine,

reduce

brain

SAMe

content

in mice (reviewed in Ref.!'!). As in the liver, these results suggest that abnormally low SAMe levels may cause depression through perturbation of multiple metabolic pathways in the brain.

INDICATIONS AND USAGE Treatment in Animal Models of Liver Disease

The importance of the metabolism of methyl groups in general, and SAMe in particular, to normal hepatic physiology, coupled with the convincing body of evidence linking abnormal SAMe content with experimental and human liver disease, led to the study of the effect of SAMe supplementation in a variety of animal models of liver disease. SAMe administration to alcohol-fed rats and baboons reduced GSH depletion and liver damage (reviewed in Ref"). It improved survival in animal models of galactosamine-,

acetaminophen-,

and

thioacetamide-induced

hepatotoxicity, and in ischemia—reperfusion-induced liver injury (reviewed in Ref.!'?}), SAMe treatment also lowered liver fibrosis in rats treated with carbon tetrachloride (reviewed in Ref.!'3}), and reduced neoplastic hepatic nodules in animal models of HCC (reviewed in Ref.!'*)),

S-Adenosylmethionine

4

Treatment of Human

randomized clinical trials.’’"*! The AHRQ reached a similar conclusion: ‘‘For liver conditions other than

Diseases

SAMe has been used in humans for the past 20 years for the treatment of osteoarthritis, depression, and liver disease. In 2002, the Agency for Healthcare Research and Quality (AHRQ) reviewed 101 indivi-

dual clinical trials of SAMe.!'*! Of these, 47 focused

on depression, 14 on osteoarthritis, and 40 on liver disease. Of the 41 studies on liver disease, 9 were for

cholestasis

of pregnancy,

cholestasis,

7 for cirrhosis,

12 for

other

causes

8 for chronic

of

hepatitis,

and 4 for various other chronic liver diseases. Pharmacokinetics Orally

administered

SAMe

has

low

bioavailability,

presumably due to a significant first-pass effect (degradation in the gastrointestinal tract) and rapid hepatic metabolism. Plasma concentrations obtained with an enteric-coated tablet formulation are dose related, with peak levels of 0.5-Img/L achieved 3—Shr after single doses ranging from 400 to 1000mg."'*! The levels decline to baseline within 24hr. One study showed a significant gender difference in bioavailability, with women showing three- to sixfold greater peak plasma values than men.''*! Plasma-protein binding of SAMe is no more than 5%. SAMe crosses the blood-brain barrier, with slow accumulation in the CSF. Unmeta-

bolized SAMe is excreted in urine and feces. Parenterally administered SAMe has much higher bioavailability.

However,

this form

is currently

not

approved for use in the United States. Liver disease

Of the 40 studies on liver disease analyzed by the AHRQ, 8 were included in a meta-analysis of the efficacy of SAMe in relieving pruritus and decreasing elevated serum bilirubin levels associated with cholestasis of pregnancy.''*! Compared to placebo, treatment with SAMe was associated with a significant decrease in pruritus and serum bilirubin levels. Similar results were obtained when 6 studies were included in a meta-analysis of the efficacy of SAMe in relieving pruritus and decrease bilirubin levels associated with cholestasis caused by a variety of liver diseases. In 2001, the Cochrane Hepato-Biliary Group analyzed 8 clinical trials of SAMe treatment of alcoholic liver disease involving 330 patients.“ This meta-analysis found that SAMe decreased total mortality [odds ratio (OR) = 0.53, 95% confidence interval (CI) = 0.22-1.29] and liver-related mortality (OR = 0.63, 95% CI = 0.25-1.58). However, since many of the studies were small and their quality varied greatly,

the

Cochrane

Group

concluded,

“SAMe

should not be used for alcoholic liver disease outside

cholestasis,

additional

smaller

trials should

be con-

ducted to ascertain which patient populations would benefit more from SAMe, and what interventions (dose and route of administration) are most effective.) The Cochrane Hepato-Biliary Group also concluded that only 1 trial involving 123 patients with alcoholic cirrhosis used adequate methodology and reported clearly on mortality and liver transplantation. In this study,"'7] mortality decreased from 30% in the placebo group to 16% in the SAMe group (p = 0.077). When patients with more advanced cirrhosis (Child score C) were excluded from the analysis (a total of 8 patients), the mortality was significantly less in the SAMe group (12%) compared to the placebo group (25%, p = 0.025). In this study, 1200mg/day was administered orally.

Depression Of the 40 studies on depression analyzed by the AHRQ, 28 were included in a meta-analysis of the efficacy of SAMe in decreasing symptoms of depression.''>! Compared to placebo, treatment with SAMe was associated with an improvement of approximately 6 points in the score of the Hamilton Rating Scale for Depression measured at 3 weeks (95% CI = 2.2-9.0). This degree of improvement was statistically as well as Clinically significant. However, compared to treatment with conventional antidepressant pharmacology, treatment with SAMe was not associated with a statistically significant difference in outcomes. With respect to depression, the AHRQ report concluded: ‘“Good dose-escalation studies have not been performed using the oral formulation of SAMe for depression.’’"*! The AHRQ report also concluded that “‘additional smaller clinical trials of an exploratory nature should be conducted to investigate uses of SAMe to decrease the latency of effectiveness of conventional antidepressants and to treat postpartum depression.’’!'*!

Osteoarthritis

Of the 13 studies on osteoarthritis analyzed by the AHRQ, 10 were included in a meta-analysis of the efficacy of SAMe in decreasing pain of osteoarthritis." Compared to placebo, one large randomized clinical trial showed a decrease in the pain of osteoarthritis with SAMe treatment. Compared to treatment with nonsteroidal anti-inflammatory medications, treatment with oral SAMe was associated with fewer adverse effects while being comparable in reducing pain and improving functional limitation.

S-Adenosylmethionine

Adverse effects

ACKNOWLEDGMENTS

The risks associated with SAMe are minimal. It has been used in Europe for 20 years and is available under prescription in Italy, Spain, the United Kingdom, and Canada, and over the counter as a dietary supplement in the United States. The most common side effects of SAMe are nausea and gastrointestinal disturbance, which occur in less than 15% of treated subjects.

This work was supported by NIH grants DK51719 (to S.C. Lu), AA12677, AA13847, and AT-1576 (to S.C. Lu and J.M. Mato), and Plan Nacional de I + D 2002-00168 (to J.M. Mato).

Interactions with herbs, supplements, and drugs Theoretically, SAMe might increase the effects and adverse effects of products that increase serotonin levels, which include herbs and supplements such as Hawaiian baby woodrose, St. John’s wort, and L-tryptophan, as well as drugs that have serotonergic effects. These drugs include tramadol (Ultram®), pentazocine (Talwin®), clomipramine (Anafranil®), fluoxetine (Prozac”), paroxetine (Paxil®), sertraline (Zoloft®), amitriptyline (Elavil®), and many others. It is also recommended that SAMe be avoided in patients taking monoamine oxidase inhibitors or within 2 weeks of discontinuing such medication.

REFERENCES 1.

Finkelstein, J.D. Homocysteine: a history progress. Nutr. Rev. 2000, 58 (7), 193-204.

2.

Mato, J.M.; Corrales, F.J.; Lu, S.C.; Avila, M.A.

a

regulates liver function. 15-26.

FASEB

control

switch

that

J. 2002, /6 (1),

Sela She! Alvate7, |awnuane 7./ 60Net ele n An, W.; Corrales, F.J.; Avila, M.A.; Kanel, G.; Mato, J.M. Methionine adenosyltransferase 1A

4.

knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (10), 5560-5565. Martinez-Chantar, M.L.; Corrales, F.J.; MartinezCruz, A.; Garcia-Trevijano, E.R.; Huang, Z.Z.; Chen, L.X.; Kanel, G.; Avila, M.A.; Mato, J.M.; Lu, S.C. Spontaneous oxidative stress and liver

CONCLUSIONS Although evidence linking abnormal SAMe content with the development of experimental and human liver disease is very convincing, the results of clinical trials of SAMe treatment of liver disease are not conclusive. Consequently, SAMe

S-Adenosylmethionine:

in

tumors in mice transferase 1A. 1292-1294. 5.

L.A.

and

Tissue

levels of

S-adenosylhomo-

cysteine in rats fed methyl-deficient, amino acid-

defined diets for one to five weeks. Carcinogenesis 1983, 4 (8), 1051-1057. 6.

Koteish, A.; Diehl, A.M. Animal models of steato-

hepatitis. Best Pract. Res. Clin. Gastroenterol. 2002, 16 (5), 679-690. i

Schwahn-maB.Carm Chen, . Z.:.. Laryeass MDE Wendel, U.; Lussier-Cacan, S.; Genest, J., Jr.; Mar, M.H.; Zeisel, S.H.; Castro, C.; Garrow, T.; Rozen, R. Homocysteine—-betaine interactions

in a murine model of 5,10-methylenetetrahydrofolate

reductase

deficiency.

FASEB

J.

2003,

17 (3), 512-514. 8.

Santamaria, E.; Avila, M.A.; Latasa, M.U.; Rubio, As -Martin-Duce;~As. Luj: $.@gs:Mato;. IM; Corrales, F.J. Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine. Proc. Natl.

Acad. Sci. U.S.A. 2003, /00 (6), 3065-3070.

With respect to osteoarthritis, as of now, there is no

evidence associating a deficiency in SAMe with the appearance of the disease. Moreover, the efficacy of SAMe in the treatment of osteoarthritis is also not convincing at present.

N.; Poirier,

S-adenosylmethionine

should not be used outside clin-

ical trials for the treatment of liver conditions other than cholestasis. A new clinical study enrolling a larger number of patients should be carried out to confirm that SAMe decreases mortality in alcoholic liver cirrhosis. This is important because if SAMe improves survival, it will become the only available treatment for patients with alcoholic liver cirrhosis. Although depression has been associated with a deficiency in SAMe, it is not yet clear whether this is a consequence or the cause. To clarify this point, more basic research and the development of new experimental models are needed. Clinical trials indicate that SAMe treatment is associated with an improvement of depression. Dose studies using oral SAMe should be performed to determine the best dose to be used. New studies should also be carried out in which the efficacy of SAMe is compared with that of conventional antidepressants.

Shivapurkar,

lacking methionine adenosylFASEB J. 2002, J/6 (10),

9.

Duce,

A.M.;

Ortiz,

P.;

Cabrero,

C.;

Mato,

J.M. S-Adenosyl-t-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis. Hepatology 1988, 8 (1), 65-68.

S-Adenosylmethionine

10.

11:

2.

Avila, M.A.; Berasain, C.; Torres, L.; MartinDuce, A.; Corrales, F.J.; Yang, H.P.; Prieto, J.; Lu, S.C.; Caballeria, J.; Rodes, J.; Mato, J.M.

Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma. J. Hepatol. 2000, 33 (6), 907-914. Bottiglieri, T. S-Adenosyl-L-methionine (SAMe): from the bench to the bedside—molecular basis of a pleiotrophic molecule. Am. J. Clin. Nutr. 2002, 76 (5), 1151S—1157S. Tsukamoto, H.; Lu, S.C. Current concepts in the

pathogenesis of alcoholic liver injury. FASEB J. 2001, 75 (8), 1335-1349. 13:

Mato, J.M.; Alvarez, L.; Ortiz, P.; Pajares, M.A.

S-Adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol. Ther. 1997, 73 (3), 265-280.

14.

Pascale, R.M.; Simile, M.M.; De Miglio, M.R.; Feo,

F.

Chemoprevention

of hepatocarcinogenesis:

15%

S-adenosyl-t-methionine. Alcohol 2002, 27 (3), 193-198. Agency for Healthcare Research and Quality. S-Adenosyl-t-methionine for Treatments of Depression, Osteoarthritis, and Liver Disease, Evidence

Report/Technology Assessment No. 64. http://www. ahrq.gov/clinic/tp/sametp.htm (accessed 2002). 16.

Rambaldi, A.; Gluud, C. S-Adenosyl-L-methionine for alcoholic liver disease. Cochrane Database Syst. Rev. 2001, 4, CD002235.

i7.

Mato, J.M.; Camara, J.; Fernandez de Paz, J.; Caballeria, L.; Coll, S.; Caballero, A.; GarciaBuey, L.; Beltran, J.; Benita, V.; Caballeria, J.; Sola, R.; Moreno-Otero, R.; Barrao, F.; Martin-Duce, A.; Correa, J.A.; Parés, A.; Barrao, E.; GarciaMagaz, I.; Puerta, J.L.; Moreno, J.; Boissard, G.; Ortiz, P.; Rodés, J. S-Adenosylmethionine in alcoholic liver cirrhosis: a randomized placebocontrolled, double-blind, multicentre trial. J. Hepatol. 1999, 30 (6), 1081-1089.

Androstenedione Benjamin Z. Leder Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.

INTRODUCTION Androstenedione (chemical name: 4-androsten-3,17dione) is a steroid hormone produced primarily in the reproductive system and adrenal glands in men and women. It circulates in the bloodstream and is the immediate precursor to the potent anabolic/ androgenic hormone testosterone in the steroid synthesis pathway. Despite this well-known physiologic classification, as well as a growing body of evidence demonstrating that orally administered androstenedione is converted to more potent steroid hormones, the United States Food and Drug Administration has classified the hormone as a “dietary supplement.’’ As such, it is available to the general public without a prescription and can be easily purchased in health clubs, nutrition stores, and over the Internet.

GENERAL

DESCRIPTION

The seemingly contradictory classification above is based on the definition set forth in the 1994 Dietary Supplement Health and Education Act (DSHEA). According to the DSHEA, a substance is defined as a dietary supplement if it is a “product (other than tobacco) intended to supplement the diet that bears or contains one or more of the following dietary ingredients:

a vitamin, mineral, amino

has increased

dramatically.

however,

was

the media

report

that

the St. Louis

Cardinals baseball player Marc McGwire had used androstenedione in the 1999 season (during which he broke the record for most home runs in a season). The publicity that surrounded this supplement also prompted an increased interest in related “prohormones,’’ such as norandro stenedione and androstene-

diol. This then led to a proliferation of claims concerning the potential benefits of andro stenedione use. Presently, manufacturers credit it not only with promoting muscle growth and improving athletic performance, but also with increasing energy, libido, sexual performance, and general quality of life. Additionally, androstenedione is now often packaged in combination with other substances as part of an intensive nutritional approach to performance enhancement. An example of such a combination is shown in Fig. 1. Clearly, the use of androstenedione and related compounds is currently outpacing the accumulation of data that may or may not eventually provide a rational basis for their use.

acid, herb or

other botanical... or a concentrate, metabolite, constituent, extract, or combination of any ingredient described above.’’ Hence, because androstenedione can be synthesized from plant products, it falls under that umbrella. Furthermore, the DSHEA specifies that the Department of Justice cannot bring action to remove a product unless it is proven to pose “a significant or unreasonable risk of illness or injury”’ when used as directed. Not surprisingly, since the passing of the DSHEA, the use of dietary supplements

Initially, androstenedione use was primarily confined to athletes in strength and endurance-related sports, an interest that seems to have sprung from reports of its use in the official East German Olympic athlete doping program. The event that most dramatically sparked widespread curiosity in androstenedione,

In fact, by 1999,

the dietary supplement industry in the United States was generating annual sales of 12 billion dollars."

BIOCHEMISTRY AND PHYSIOLOGY Androstenedione is a steroid hormone that is produced primarily in the adrenals, testes, and ovaries. It is classified as a “weak androgen’’ because it binds to the body’s receptor for androgen hormones in a much less potent fashion than classic anabolic/androgenic steroids such as testosterone.”! It is synthesized from the precursor hormone dehydroepiandrosterone (DHEA—itself a dietary supplement) and is the direct precursor to testosterone. In normal physiologic circumstances, androstenedione can also be converted

Benjamin Z. Leder, M.D., is Assistant Professor, Endocrine Unit

at Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.

Encyclopedia of Dietary Supplements DOL: 10.1081 /E-EDS-120022910

Copyright © 2005 by Marcel Dekker. All rights reserved.

to potent feminizing hormones such as estrone and estradiol (both members of the “estrogen’’ class of androbetween relationship The hormones). enzymes the and stenedione, other steroid hormones,

Androstenedione

PROHORMONE FACTORS 4-Androstenedione: 100 mg 19-Nor-5-Androstenedione: 50 mg 5-Androstenediol: 50 mg

DHEA: 50 mg H/IGF FACTOR L-Arginine Pyroglutamate: 2500 mg L-Ornithine Alpha-Ketoglutarate: 1250 mg Taurine: 750 mg

Colostrum: 250 mg

LH BOOSTER Tribulus: 250 mg Acetyl-L-Carnitine: 250 mg L-Carnitine: 100 mg

DHT BLOCKERS Saw Palmetto: 200 mg Beta Sitosterol: 200 mg Pygeum Africanum: 50 mg

Uris

ESTROGEN BLOCKERS Kudzu: 100 mg Chrysin : 250mg

involved in the conversion of androstenedione to testosterone and estrogens is shown in Fig. 2. Importantly, the enzymes that convert androstenedione to potent hormones like testosterone and estradiol are active not only in endocrine glands, but also in many peripheral body tissues such as muscle, bone, liver, and brain.?! Thus, if orally administered

androstenedione has biological activity, it may act either directly or by conversion to these more potent agents.

ANDROSTENEDIONE

USE

There are no precise data concerning the prevalence of androstenedione use in the general population. Our best estimates are based on industry sales figures

and extrapolations from data on classic anabolic/ androgenic steroid use in specific populations. For example, in 1997, it was estimated that 4.9% of male

and 2.4% of female adolescents in the United States had used illegal anabolic steroids.) Because these substances are so readily available, there is concern that androstenedione use in this particularly susceptible population might greatly exceed these numbers. Recently, in fact, a study was published that seems to validate these concerns. In this study, a survey was administered in five health clubs in Boston, Massachusetts, and the results revealed that 18% of men and 3% of women respondents had used androstenedione or other adrenal hormone dietary supplements at least

Fig. 1 A typical combination dietary supplement product.

once. These percentages suggest that as many as 1.5 million U.S. health club members alone have used these substances.!!

PHARMACOKINETICS AND HORMONAL EFFECTS OF ANDROSTENEDIONE IN MEN Because so many of the claims that surround androstenedione are based on the premise that oral administration increases serum testosterone levels, it may be surprising to some that prior to 1999, there was only a single published study investigating the ability of orally administered androstenedione to be converted to more potent steroid hormones.'! In this study, 2 women were given a single dose of androstenedione, and the levels were subsequently measured over the next several

hours.

Since

1999, however,

numerous

small

studies (mostly in men) have investigated the effects of the supplement.°"*! In general, these studies report that serum androstenedione levels increase dramatically after oral administration and thus confirm that a significant portion of the supplement is absorbed through the gastrointestinal tract after ingestion. However the answer to the more important question, namely, whether it is then converted to more potent steroid

hormones

such as testosterone

and estradiol,

appears to be complex. In general, these studies suggest that the ability of oral androstenedione to increase estrogen and testosterone levels in men is dose

Androstenedione

0

HO Dehydroepiandrosterone

| 0 0-mg dose group 38-HSD

w 100-mg dose group a 300-mg dose group

HCOOH

|

CYP19

(aromatase

O

Estrone

4-Androstenedione

I

OH

HCOOH CYP19 (aromatase)

(0)

A

HO Testosterone

% changeinserum

% change in serum

estradiol

testosterone

178-HSD

178-HSD

Fig. 3 Percentage change in serum testosterone and estradiol in healthy men after a single androstenedione dose (as measured by 8 hr of frequent blood sampling). (Adapted from Ref.!4!.) (View this art in color at www.dekker.com. )

Estradiol-17 6

Fig. 2 Androstenedione’s relationship to other steroid hormones. Enzyme abbreviations: 3B-HSD, 3f-hydroxysteroid dehydrogenase; 17B-HSD, 17f-hydroxysteroid dehydrogenase.

dependent and is possibly related to the age of the study population as well. Specifically, the bulk of the research indicates that when androstenedione is administered to men in individual doses between 50 and 200mg, serum

estrogen levels increase dramatically. However, larger individual doses (e.g., 300mg) are required to increase serum testosterone levels. For example, King and colleagues studied the effects of a single 100-mg oral dose of androstenedione in 10 men between the ages of 19 and 29 and reported that while serum androstenedione and estradiol levels increased significantly, testosterone levels did not change.""*] These investigators then specifically measured the portion of circulating testosterone that is not bound to protein and considered the “‘bioactive’’ portion (called free testosterone) and similarly saw no effect of the supplement. In a separate study, Leder and colleagues gave 0, 100, or 300mg of androstenedione to normal healthy men between the ages of 20 and 40 for 7days and took frequent blood samples on days 1 and 7.!'4! As in the study by King, they also found that men receiving both the 100- and 300-mg dose of androstenedione experienced dramatic increases in serum estradiol that were often well above the normal male range. Another similarity was that 100mg did not affect serum testosterone levels. As shown in Fig. 3, however, the novel finding of this study was that 300 mg of androstenedione increased serum testosterone levels significantly, albeit by only a modest amount (34%).

Leder and colleagues further observed that there was a significant degree of variability among men with regard to their serum testosterone response after androstenedione ingestion. As shown in Fig. 4, some subjects, even in the 300-mg dose group, experienced relatively little change in testosterone levels, whereas

serum testosterone levels doubled in other men. This finding suggests that there may be individual differences in the way androstenedione is metabolized that could impact any one person’s physiological response to taking the supplement. Brown and colleagues investigated the hormonal response in a group of men between the ages of 30 and 56.9 In this study, subjects consuming 100mg of androstenedione three times daily experienced

1800 1600 1400 raN fo}Oo

1000

400

Sa

(ng/dl) testosterone serum

baseline

peak

Fig. 4 Individual variability in the peak serum testosterone level achieved after a single 300-mg dose of androstenedione in men. Each line represents one study subject. (Adapted from Ref.!"4!,)

Androstenedione

10

increases in serum estrogens but not serum testosterone. However, unlike in the study by King and colleagues discussed above, free testosterone did increase significantly (albeit again by only a small amount). Finally, several studies have compared the hormo-

nal effects of androstenedione with those of other “prohormone’’ dietary supplements. Broeder and colleagues studied the results of a 100-mg twice-daily dose of oral androstenedione, androstenediol (a closely related steroid hormone), or placebo in men between

the ages of 35 and 65.!1 They found that both compounds increased estrogen levels but neither affected total serum testosterone levels. Similarly, Wallace and

colleagues studied the effects of 50-mg_ twice-daily doses of androstenedione and DHEA in normal men and reported no increases in serum testosterone levels

with either.!"°

controlled substance Enforcement Agency.

METABOLISM IN MEN

by

the

United

States

Drug

OF ANDROSTENEDIONE

One of the consistent findings of the various androstenedione studies in men is the inefficiency of conversion of the supplements to testosterone. Leder and colleagues explored this issue further by investigating the pattern of androstenedione metabolism in healthy men.!'7] Specifically, they measured the concentration of inactive testosterone metabolites (also called conjugates) in the urine of subjects ingesting androstenedione and found an increase of over 10-fold compared to their baseline levels. This finding was in direct contrast to the much more modest changes in serum testosterone they had observed. It suggests that while much of the androstenedione that is absorbed after oral administra-

EFFECTS ON MUSCLE SIZE AND STRENGTH IN MEN The results of the studies discussed above suggest that androstenedione use in men would be less likely to promote the muscle building and performance enhancing effects associated with testosterone use and more likely to induce the undesirable feminizing effects associated with estrogens. Several studies have assessed the ability of androstenedione (with or without exercise) to increase muscle size and strength and have been uniformly disappointing.'”?-!?'>-!°] For example, Broeder and colleagues, in the study described above, also measured changes in body composition and strength in subjects taking 100mg androstenedione twice daily in combination with a 12-week intensive weighttraining program.” Despite using sensitive methods that can detect small changes in body composition, they found no differences in muscle mass, fat mass, or strength in the subjects receiving androstenedione compared to those receiving a placebo tablet. Importantly, however, in this study as well as all of these studies referenced above, the supplement was given

in doses that were not sufficient to increase testosterone levels. It thus remains unknown whether doses of androstenedione sufficient to increase testosterone levels will enhance muscle mass or athletic performance. This issue is particularly important because it is likely that many ingest doses that far exceed those used in research investigating “high’’ dose androstenedione. Additionally, the issue of whether androstenedione can increase muscle mass or strength has important regulatory ramifications. If androstenedione is shown to build muscle, it could be classified as an “anabolic steroid’? under the 1990 Anabolic Steroid Control Act and regulated as a

tion is converted to testosterone, it is then immediately further metabolized to inactive compounds in the liver. The investigators confirmed this hypothesis by directly measuring the concentration of one of these inactive metabolites (testosterone glucuronide) in the serum of these subjects. As expected, they found that testosterone glucuronide levels increased by 500—1000% (as opposed to the 34% increase in biologically active serum testosterone after a single 300-mg dose of oral androstenedione). Together, these findings demonstrate the effectiveness of the liver in inactivating steroid molecules when taken orally.

PHARMACOKINETICS AND HORMONAL EFFECTS OF ANDROSTENEDIONE IN WOMEN Since the initial report of androstenedione administration in 2 women in 1962,!° research into the effects of the supplement has focused largely on the hormonal response to oral administration in young men. Between 2002 and 2003, however, two studies on women were published. The first of these studies examined the effects of a single dose of either 0, 50, or 100mg of androstenedione in postmenopausal women.!!®] The findings of this study were surprising. In contrast to the effects observed in men, even these low doses increased testosterone levels significantly in

women (Fig. 5). Also, unlike the results seen in men, estradiol levels

were unaffected by androstenedione administration. In the other study, 100mg of androstenedione was administered to young, premenopausal, healthy women. Similar to postmenopausal women, these subjects experienced significant increases in serum testosterone levels after androstenedione administration

_

Androstenedione

11

ea eb Sie Sc 80 60 40 20

(ng/dl) testosterone serumSo

0

120

240 360 480 time (minutes)

600

720

Fig. 5 Serum testosterone levels during 12hr of frequent blood sampling in postmenopausal women. Circles represent control subjects receiving no supplement, triangles those receiving 50mg of androstenedione, and squares those receiving 100mg. (Adapted from Ref.!'®).)

(estradiol was not measured).""*! Importantly, in both of these studies, the peak testosterone levels achieved by the older and younger women taking androstenedione were often significantly above the normal range. Together, these results predict that the physiological effects of the supplement may be different in men and

women,

as might

their potential

toxicities.

EFFECTS AND TOXICITY

Ever since the publicity surrounding androstenedione exploded in 1999, many reports in the lay press have focused on the potential dangerous side effects. Nonetheless, with the exception of a single case description of a man who developed 2 episodes of priapism in the setting of androstenedione ingestion,?°! there have been no published reports of androstenedioneassociated serious adverse events. This fact should be only partially reassuring, however, because androstenedione’s classification as a dietary supplement (as opposed to a drug) allows manufacturers to avoid responsibility for rigorously monitoring any potential toxicity of their product. It is well known that oral administration of certain testosterone derivatives can cause severe liver diseases,

and anabolic steroid use in general is associated with anecdotal reports of myocardial infarction, sudden cardiac death, and psychiatric disturbances (“roid rage’). Nonetheless, despite androstenedione’s close chemical similarity to these substances, it is important to note that it is not a potent anabolic steroid; nor does it have a chemical structure similar to those specific compounds

androstenedione administration could, over time, lead

to gynecomastia (male breast enlargement), infertility, and other signs of feminization. In women, because the supplement increases testosterone levels above the normal range, it could cause hirsutism (excess body hair growth), menstrual irregularities, or male-like changes in the external genitalia. In children, increases in both testosterone and estrogen levels could cause precocious puberty or premature closure of growth plates in bone, thereby compromising final adult height.

To

date, however, there have been no published reports investigating the long-term physiological effects in women.

ADVERSE

potential of androstenedione to cause these particular serious side effects appears to be limited. Of more pressing concern to clinicians are the possible longterm effects in specific populations. In clinical trials, the supplement was generally well tolerated, though several studies did report that it reduces high-density lipoprotein (HDL, or “good cholesterol’’) levels in men. Importantly, however, even the longest of these studies lasted only several months. It thus remains quite possible that androstenedione use, especially at high doses, could cause subtle physiologic changes over prolonged periods that could directly lead to adverse health consequences. In men, for example, the dramatic increase in estradiol levels observed with

that cause liver problems. Thus, the

PURITY OF COMMERCIALLY AVAILABLE ANDROSTENEDIONE Androstenedione is available from multiple manufacturers and can be purchased as a tablet, capsule, sublingual tablet, or even nasal spray. Often, it is combined with other products that claim to limit its potential side effects (such as chrysin, for example, which is purported to decrease androstenedione’s conversion to estrogens). Because the manufacture of dietary supplements is not subject to the same regulations as are pharmaceuticals, the: purity and labeling of androstenedione-containing products may not be accurate.

Catlin and colleagues,

for example,

reported the surprise finding that urine samples from men treated with androstenedione contained 19-norandrosterone, a substance not associated with androstenedione

metabolism,

but rather with the use

of a specific banned anabolic steroid.?"! Further investigation revealed that the androstenedione product used contained a tiny amount of the unlabeled steroid “19-norandrostenedione.’’” Though the amount of 19-norandrostenedione was not physiologically significant, it was enough to cause a “‘positive’’ urine test for illegal anabolic steroid use when tested in the standard fashion. In fact, it is precisely this type of contamination that may explain the recent increase in competitive athletes testing positive for 19-norandrosterone and other banned substances in well-known standard testing methods.

Androstenedione

12

Catlin and colleagues also analyzed nine common brands of androstenedione and showed that there was considerable variation among products in terms of both purity and content (see Table 1). Thus,

it is obvious

that some

brands

of andro-

stenedione are grossly mislabeled. Furthermore, this mislabeling adds to the considerable uncertainty that already exists regarding the long-term effects of androstenedione use.

REGULATORY STATUS AND DETECTION As mentioned previously, androstenedione is currently available over-the-counter in the United States due to its classification as a dietary supplement. There is a good possibility, however, that this classification may soon change as legislation has now been introduced in the Unites States Congress aimed at reclassifying ““prohormone’’ steroid supplements (including androstenedione) as controlled substances. In the meantime, many sports organizations, including the National Football League (NFL), the National Collegiate Athletic Association (NCAA), and the International Olympic Committee (IOC) have banned androstenedione use due to concerns that it may offer some athletes a competitive advantage. Despite these prohibitions, detection of androstenedione has not been standardized. Specifically, the method used most often to detect testosterone use, measurement of the

urinary testosterone-to-epitestosterone ratio, has not proven to be reliable in establishing androstenedione use.?7] Further study will clearly be needed to define novel testing procedures that are able to detect androstenedione use reliably.

CONCLUSIONS Androstenedione is a steroid hormone and a popular over-the-counter dietary supplement. It is marketed as a legal alternative to traditional anabolic steroids and is purported to increase strength, athletic performance, libido, sexual performance, energy, and general quality of life. Studies indicate that when taken orally by men, small doses are converted to potent estrogens and larger doses to both testosterone and estrogens. Comparatively, there appears to be a much more physiologically important increase in estrogens compared with testosterone in men. In women, the effects are reversed. Studies have thus far failed to confirm any effect on muscle size or strength, though the dosing regimens were modest. While documentation of adverse side effects among users of androstenedione is scarce, there is considerable concern over potential long-term toxicity, especially in women and adolescents. Finally, the lack of purity in androstenedione- | containing products introduces a further level of potential concern over these long-term health effects.

REFERENCES 1. Anonymous. Herbal treatments: the promises and pitfalls. Consumer Reports 1999, 64, 44-48. 2.

Williams

93

100

83

100

103

100

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35

50

identified)

250

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Clin. Endocrinol.

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Yesalis, C.E.; Barsukiewicz, C.K.; Kopstein, A.N.; Bahrk, M.S. Trends in anabolic—androgenic

steroid use among adolescents. Arch. Adolesc. Med. 1997, 151, 1197-1206. 5.

Kanayama, Borowiecki,

Pediatr.

G.; Gruber, A.J.; Pope, H.G., Jr.; J.J.; Hudson, J.I. Over-the-counter

drug use in gymnasiums: an underrecognized substance abuse problem? Psychother. Psychosom. 2001, 70 (3), 137-140. 6.

Mahesh,

7.

conversion of dehydroepiandrosterone and androstenedione to testosterone in the human. Acta Endocrinol. 1962, 41, 400-406. Broeder, C.E.; Quindry, J.; Brittingham, K.;

168 (10mg of was also present)

Labrie, F.; Simard, J.; Luu-The, V.; Pelletier, G.;

451-474.

testosterone

(From Ref.?1))

Wilson,

Belghmi, K.; Belanger, A. Structure, regulation and role of 3 beta-hydroxysteroid dehydrogenase, 17 beta-hydroxysteroid dehydrogenase and aromatase enzymes in the formation of sex steroids in classical and peripheral intracrine tissues.

0 (no steroid compounds

of Endocrinology;

phia, PA, 1998; 517-664. 3.

Amount of androstenedione found (in mg)

100

Textbook

J.D., Foster, D.W., Kronenberg, H.M., Larsen, P.R., Eds.; W.B. Saunders Company: Philadel-

Table 1 Analysis of nine common brands of androstenedione supplements Amount of androstenedione listed (in mg)

Orth, D.N.; Kovacs, W.J. The adrenal cortex. In

V.B.;

Greenblatt,

R.B.

The

in vivo

Panton, L.; Thomson, J.; Appakondu, S.; Breuel,

Androstenedione

13

K.; Byrd, R.; Douglas, J.; Earnest, C.; Mitchell, C.; Olson, M.; Roy, T.; Yarlagadda, C. The

Andro Project: physiological and hormonal influences of androstenedione supplementation in men 35 to 65 years old participating in a high-intensity resistance training program. Arch. Intern. Med. 2000, 160 (20), 3093-3104. Brown, Kohut,

G.A.; Vukovich, M.D.; Martini, E.R.; M.L.; Franke, W.D.; Jackson, D.A.;

King, D.S. Effects of androstenedione-herbal supplementation on serum sex hormone concentrations in 30- to 59-year-old men. Int. J. Vitam. Nutr. Res. 2001, 7/7 (5), 293-301. Brown, G.A.; Vukovich, M.D.; Reifenrath, T.A.; Uhl, N.L.; Parsons, K.A.; Sharp, R.L.; King, D.S. Effects of anabolic precursors on serum testosterone concentrations and adaptations to

androstenedione administration and serum testosterone concentrations in young men. J.

L5:

protein anabolism in young healthy men. J. Clin.

16.

Brown, Kohut,

17.

rT.

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Kicman, A.T.; Bassindale, T.; Cowan, D.A.; Dale, S.; Hutt, A.J.; Leeds, A.R. Effect of androstene-

tial health risks. Clin. Chem. 167-169. Kachhi, P.N.; Henderson, S.O.

2003,

49

Priapism

(1), after

androstenedione intake for athletic performance enhancement. Ann. Emerg. Med. 2000, 35 (4), 391-393.

21

King, D.S.; Sharp, R.L.; Vukovich, M.D.; Brown, G.A.; Reifenrath, T.A.; Uhl, N.L.; Parsons, K.A.

Leder, B.Z.; Longcope, C.; Catlin, D.H.; Ahrens, B.; Schoenfeld, D.A.; Finkelstein, J.S. Oral

androstenedione

Endocrinol.

dione ingestion on plasma testosterone in young women; a dietary supplement with poten-

Ballantyne, C.S.; Phillips, S.M.; MacDonald, J.R.; Tarnopolsky, M.A.; MacDougall, J.D. The acute

Effect of oral androstenedione on serum testosterone and adaptations to resistance training in young men. J. Am. Med. Assoc. 1999, 281 (21), 2020-2028.

J. Clin.

5449-5454.

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J. Appl.

effects of androstenedione supplementation in healthy young males. Can. J. Appl. Physiol. 2000, 25 (1), 68-78.

men.

women. J. Clin. Endocrinol. Metab. 2002, 87 (12),

Endocrinol. Metab. 2000, 85 (11), 4074-4080. Earnest, C.P.; Olson, M.A.; Broeder, C.E.; Breuel, K.F.; Beckham, S.G. In vivo 4-androstene-3,17dione and 4-androstene-3 beta,17 beta-diol

supplementation in young men. Physiol. 2000, 8/7 (3), 229-232.

L.

Leder, B.Z.; Catlin, D.H.; Longcope, C.; Ahrens, B.; Schoenfeld, D.A.; Finkelstein, J.S. Metaboyoung

G.A.; Vukovich, M.D.; Martini, E.R.; M.L.; Franke, W.D.; Jackson, D.A.;

King, D.S. Endocrine responses to chronic androstenedione intake in 30- to 56-year-old men. J. Clin.

Endocrinol. Metab. 2000, 85 (1), 55-59. Wallace, M.B.; Lim, J.; Cutler, A.; Bucci,

Effects of dehydroepiandrosterone vs. androstenedione supplementation in men. Med. Sci. Sports Exerc. 1999, 37 (12), 1788-1792.

resistance training in young men. Int. J. Sport. Nutr. Exerc. Metab. 2000, 70 (3), 340-359.

10.

Am. Med. Assoc. 2000, 283 (6), 779-782. Rasmussen, B.B.; Volpi, E.; Gore, D.C.; Wolfe, R.R. Androstenedione does not stimulate.muscle

22

Catlin, D.H.; Leder, B.Z.; Ahrens, B.; Starcevic, B.; Hatton, C.K.; Green, G.A.; Finkelstein, J.S. Trace contamination of over-the-counter andro-

stenedione and positive urine test results for a nandrolone metabolite. J. Am. Med. Assoc. 2000, 284 (20), 2618-2621. Catlin, D.H.; Leder, B.Z.; Ahrens, B.D.; Hatton, C.K.; Finkelstein, J.S. Effects of androstenedione administration on epitestosterone metabolism in

men. Steroids 2002, 67 (7), 559-564.

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*

L-Arginine Mauro Maccario Emanuela Arvat Gianluca Aimaretti Valentino Martina Roberta Giordano Fabio Lanfranco Lisa Marafetti

Mariangela Seardo Matteo Baldi

Ezio Ghigo University of Turin, Turin, Italy

INTRODUCTION Arginine was first isolated in 1895 from animal horn. It is classified as a nonessential amino acid even if occuring in newborns, young children, or other circumstances characterized by accelerated tissue growth (e.g., infection, sepsis, trauma) when its production may be too slow and not sufficient to meet the requirements. Thus, in these conditions, arginine may be classified as “semiessential.’’"'! Arginine participates in protein synthesis in cells and tissues. It is essential for the synthesis of urea, creatine, creatinine, and pyrimidine

bases. It also strongly influences hormonal release and has an important role in vasculature dynamics, participating in the synthesis of nitric oxide (NO).

BIOCHEMISTRY Dietary arginine is particularly abundant in wheat germ and flour, buckwheat, oatmeal, dairy products (cottage cheese, ricotta cheese, nonfat dry milk, skimmed yogurt), chocolate, beef (roasts, steaks), pork,

nuts (coconut, pecans, walnuts, almonds, hazel nuts, peanuts), seeds (pumpkin, sesame, sunflower), poultry

(chicken, turkey), wild game (pheasant, quail), seafood (halibut, lobster, salmon,

Mauro Maccario, M.D., is Associate Professor at the Division of

Endocrinology, Turin, Italy.

Dept. of Internal Medicine,

University of Turin,

Emanuela Arvat, M.D., is Associate Professor at the Division of

Endocrinology, Turin, Italy. Gianluca

Endocrinology, Turin, Italy. Valentino

Dept. of Internal Medicine, Aimaretti,

M.D.,

Ph.D.,

Dept. of Internal

is

Medicine,

University of Turin, at

the

Division

of

University of Turin,

; Martina,

M.D.,

is Researcher

at the

Division

of

Endocrinology, Dept. of Internal Medicine, University of Turin, Turin, Italy. Roberta Giordano, M.D., is at the Division of Endocrinology, Dept. of Internal Medicine, University of Turin, Turin, Italy. Fabio Lanfranco, M.D., is at the Division of Endocrinology, Dept. of Internal Medicine, University of Turin, Turin, Italy. Lisa Marafetti, M.D., is at the Division of Endocrinology, Dept. of Internal Medicine, University of Turin, Turin, Italy. Mariangela Seardo, M.D., is at the Division of Endocrinology, Dept. of Internal Medicine, University of Turin, Turin, Italy.

Matteo Baldi, M_D., is at the Division of Endocrinology, Dept. of Internal Medicine, University of Turin, Turin, Italy.

Ezio Ghigo, M.D., is Professor at the Division of Endocrinology, Dept. of Internal Medicine, University of Turin, Turin, Italy.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022067 Copyright © 2005 by Marcel Dekker. All rights reserved.

shrimp, snails, tuna), chick

peas, and soybeans.”! L-Arginine, delivered via the gastrointestinal tract, is absorbed in the jejunum and ileum of the small intestine. A specific amino acid transport system facilitates the process and is also responsible for assisting with the transport of the other basic amino acids, L-lysine and L-histidine. About 60% of the absorbed L-arginine is metabolized by the gastrointestinal enterocytes, and only 40% reaches the systemic circulation intact. Deficient intake of arginine produces symptoms of muscle weakness, similar to muscular dystrophy." Deficiency of arginine impairs insulin secretion, glucose production, and liver lipid metabolism." Conditional deficiencies of arginine or ornithine are associated with the presence of excessive ammonia in the blood, excessive lysine, rapid growth, pregnancy, trauma, or protein deficiency and malnutrition. Arginine deficiency is also associated with rash, hair loss and hair breakage, poor wound healing, constipation, fatty liver, hepatic cirrhosis, and hepatic coma." Depending on nutritional status and developmental stage, normal plasma arginine concentrations in

15

L-Arginine

16 GLUTAMINE

NH‘4-GROUPS

CO2+NH4 GLUTAMATE 2ATP

2ADP + Piz

CARBAMOYL PHOSPHATE

"ty

ASPARTATE

CITRULLINE

ORNITHINE

ATP

AMP + Ppi+ H,0

ARGININO-.” SUCCINATE ae C

>

Fig. 1 L-Arginine and Krebs cycle m the renal tubule.

FUMARATE

(Fig. 1).°! Without this effective mechanism to handle

humans and animals range from 95 to 250 mol/L. Toxicity and symptoms of high intake are rare, but symptoms of massive dosages may include thickening

the byproducts of metabolism and without an appropriate L-arginine intake, ammonia would accumulate rapidly, resulting in hyperammonemia. L-Arginine undergoes different metabolic fates. NO,

and coarsening of the skin, muscle weakness, diarrhea,

and nausea. The proximal renal tubule accounts for much of the endogenous production of L-arginine from L-citrulline. In the tubule, arginine reacts via the Krebs cycle with

L-citrulline,

the toxic ammonia formed from nitrogen metabolism,

producing the nontoxic and readily excretable urea

Nitric Oxide

Agmatine

Aldehyde *! Biotin deficiency has also been reported or inferred in several other circumstances including Leiner’s disease,°°°*! sudden infant death syndrome, ©? hemodialysis,!°' ©! gastrointestinal diseases and alcoholism,"! and brittle nails.!°! Additional

studies are

needed to confirm or refute an etiologic link of these conditions to the vitamin’s deficiency. The mechanisms by which biotin deficiency produces specific signs and symptoms remain to be completely delineated. However, several studies have given new insights on this subject. The classic assumption for most water-soluble vitamins is that the clinical findings of deficiency result directly or indirectly from deficient activities of the vitamin-dependent enzymes. On the basis of human studies on deficiency of biotinidase and isolated pyruvate carboxylase, as well as animal experiments regarding biotin deficiency, it is hypothesized that the central nervous system effects of biotin deficiency (hypotonia, seizures, ataxia, and delayed development) are likely mediated through deficiency of brain pyruvate carboxylase and the attendant central nervous system lactic acidosis rather than by

Biotin

disturbances

a

in brain fatty acid composition.!°7©!

Abnormalities in metabolism of fatty acids are likely important in the pathogenesis of the skin rash and hair loss.!”! Exciting new work has provided evidence for a potential role for biotin in gene expression. These findings will likely provide new insights into the pathogenesis of biotin deficiency.!!7"! In 1995, Hymes and Wolf discovered that biotinidase can act as a biotinyl transferase; biocytin serves as the source of biotin, and histones are specifically biotinylated.'©! Approximately 25% of total cellular biotinidase activity is located in the nucleus. Zempleni and coworkers have demonstrated that the abundance of biotinylated histones varies with the cell cycle, that these histones are increased approximately twofold compared to quiescent lymphocytes, and are debiotinylated enzymatically in a process that is at least partially catalyzed by biotinidase.!’?-”4! These observations suggest that biotin plays a role in regulating DNA transcription and regulation. Although the mechanisms remain to be elucidated, biotin status has been shown to affect gene expression. Cell culture studies suggest that cell proliferation generates an increased demand for biotin, perhaps mediated by increased synthesis of biotin-dependent carboxylases.!”*! Velazquez and coworkers have reported that biotin deficiency in rats reduces messenger RNA levels of holocarboxylase synthetase, but has differential effects on the amount of mRNA and enzyme protein for the various carboxylases.!”>,’* Studies have been conducted in diabetic humans

and rats that carbohydrate glucokinase, (PEPCK), and

support an effect of biotin status on metabolism. Genes studied include phosphoenolpyruvate carboxykinase expression of the asialoglycoprotein

receptor on the surface of hepatocytes.!’”-””! The effect of biotin status on PEPCK expression was particularly striking when diabetic rats were compared to nondiabetic rats. However, most studies have been performed

on rats in which metabolic pathways have been perturbed prior to administration of biotin. Thus, the role of the vitamin in regulation of these genes during normal biotin status remains to be elucidated.

Table 2

Adequate intake for biotin

consumption Amount

Age

en ee MEPS a

(ug/day)

ee

7-12 mo

6

1-3 yr

8

4-8 yr

12

9-13 yr

20

14-18 yr

25

19->70 yr Pregnancy

30 —

Lactation

30

35

Values for males and females in all age groups were combined because they do not differ. (From Ref.8°,)

value for free biotin determined microbiologically (6ug/L) and an average consumption of 0.78 L/day by infants of age 0-6 mo, an AI of 5 g/day was calculated. The AI for lactating women has been increased by 5 g/day to allow for the amount of biotin secreted in human milk. Using the AI for 0-6mo infants, the reference body weight ratio method was used to extrapolate Als for other age groups (see Table 2).

TREATMENT OF BIOTIN DEFICIENCY If biotin deficiency is confirmed, biotin supplementation should be undertaken and effectiveness should be documented. Doses between 100 pg and | mg are likely to be both effective and safe on the basis of studies supplementing biotin deficiency during pregnancy, chronic anticonvulsant therapy, and biotinidase deficiency.

TOXICITY Daily doses up to 200 mg orally and up to 20 mg intravenously have been given to treat biotin-responsive inborn errors of metabolism and acquired biotin deficiency. Toxicity has not been reported.

INDICATIONS AND USAGE REFERENCES In 1998, the United States Food and Nutrition Board

of the National Academy of Sciences reviewed the recommendations for biotin intake.“ The committee concluded that the data were inadequate to justify setting an estimated average requirement (EAR). However, adequate intake (AI) was formulated (Table 2). The AI for infants was based on an empirical determination of the biotin content of human milk. Using the

1.

2.

Mock, D.M. Biotin. In Present Knowledge in Nutrition, 7th Ed.; Ziegler, E.E., Filer, L.J., Jr., Eds.; International Life Sciences Institutes— Nutrition Foundation: Washington, DC, 1996;

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D.; V.

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Chauhan, J.; Dakshinamurti,

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Daberkow, R.L.; White, B.R.; Cederberg, R.A.; Griffin, J.B.; Zempleni, J. Monocarboxylate

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Mock, D.M.; Stadler, D.; Stratton, S.; Mock, N.I.

Mock, D.M.; Stadler, D.D. Conflicting indicators

Mock,

status in patients on chronic anticonvulsant therapy. Int. J. Vitam. Nutr. Res. 1982, 52 (4), 375-385.

biotin

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39,

Am. J. Clin. Nutr. 2002, 76, 1061-1068. Mock, D.M.; Henrich, C.L.; Carnell, N.; Mock,

biotin status in anticonvulsant Neurol. 1982, 12, 485-486.

Mock, D.M.; Stratton, S.L.; Mock, N.I. Concen-

trations of biotin metabolites in human J. Pediatr. 1997, 731 (3), 456-458.

Am. J. Clin. Nutr. 1997, 65, 951-958. Mock, D.M.; Henrich, C.L.; Carnell, N.; Mock,

in protein-energy malnutrition. Nutr. 1988, 43, 169-173.

122, 546-552. 36.

P.J.; Bishop,

Stumbo, P.; Mock, N.I. 3-Hydroxypropionic acid and methylcitric acid are not reliable indicators of marginal biotin deficiency. J. Nutr. 2004, 134, 317-320.

D.M.;

S. Biotin uptake by basolateral memhuman placenta: normal characteristics of ethanol. Proc. Soc. Biol. Exp. Med. (4), 404408.

M.I.; Stumbo,

N.L.; Swift, L. Lymphocyte propionyl-CoA carboxylase and accumulation of odd-chain fatty acid in plasma and erythrocytes are useful indicators of marginal biotin deficiency. J. Nutr. Biochem. 2002, 13 (8), 462-470.

placenta. Am. J. Physiol. 1992, 262, C302—C308. Schenker, °S.; Hu, Z:; Johnson; R.F.; Yang, Y.; Frosto, T.; Elliott, B.D.; Henderson, G.I.; Mock,

D.M. Human placental biotin transport: normal characteristics and effect of ethanol. Alcohol. Clin. Exp. Res. 1993, 17 (3), 566-575.

N.I.; Malik,

N.I. Indicators of marginal biotin deficiency and repletion in humans: validation of 3-hydroxyisovaleric acid excretion and a leucine challenge.

fetus.

villous membrane vesicles, cultured trophoblasts and the isolated perfused cotyledon of the human

Mock,

W.P.; Mock, D.M. Increased urinary excretion of 3-hydroxyisovaleric acid and decreased urinary excretion of biotin are sensitive early indicators of decreased status in experimental biotin deficiency.

3p.

Said,

H.M.;

Redha,

R.; Nylander,

W.

Biotin

transport in the human intestine: inhibition by anticonvulsant drugs. Am. J. Clin. Nutr. 1989, 49, 127-131.

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56.

Leiner’s disease: a biotin deficiency. J. Pediatr. 1957, 51, 537-548.

a7.

70.

rats: cholesterol and lipoproteins. Am. J. Clin. Nutr. 1986, 43, 831-838. Mock, D.M. Evidence for a pathogenic role of w6 polyunsaturated fatty acid in the cutaneous manifestations of biotin deficiency. J. Pediatr.

Ta

Zempleni,

Nisenson, A. Seborrheic dermatitis of infants and

Nisenson,

A. Seborrheic

dermatitis

of infants:

treatment with biotin injections for the nursing

58.

mother. Pediatrics 1969, 44, 1014-1015. Erlichman, M.; Goldstein, R.; Levi, E,; Greenberg, A.; Freier, S. Infantile flexural sebor-

rhoeic dermatitis. Neither biotin nor essential fatty acid deficiency. Arch. Dis. Child. 1981, 567, 560-562.

593

Johnson,

A.R.; Hood,

R.L.; Emery,

Gastroenterol. Nutr. 1990, 10, 222-229.

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J.L. Biotin

and the sudden infant death syndrome. Nature 1980, 285, 159-160.

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61.

62.

N.

74.

undergoing

chronic

Nephron 1987, 46, 331-332. DeBari, V.; Frank, O.; Baker,

hemodialysis. H.; Needle,

US:

M.

Am.

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410-415. 64.

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H.;

Koutisicos,

D.;

Agroyannis,

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Papastephanidis, (C.; Frangos-Plemenos, M.; Delatola, Z. Biotin in the management of uremic neurologic disorders. Nephron 1984, 36, 183-186.

65.

66.

Braguer, D.; Gallice, P.; Yatzidis, H.; Berland, Y.;

69.

Zempleni, J.; Mock, D.M. Chemical synthesis of biotinylated histones and analysis by SDS-PAGE/ streptavidin peroxidase. Biomol. Eng. 2000, 16°(5); ESE. Stanley;

J.S.;~

Griffins

“U:B.;)

Zemplenk,

0. -

Rodriguez-Melendez, R.; Perez-Andrade, M.E.; Diaz, A.; Deolarte, A.; Camacho-Arroyo, L.; Ciceron, I.; Ibarra, I.; Velazquez, A. Differential

Rodriguez-Melendez, R.; Cano, S.; Mendez, S.T.; Velazquez, A. Biotin regulates the genetic

expression of holocarboxylase synthetase and mitochondrial carboxylases in rats. J. Nutr. 2001, 737 (7), 1909-1913.

mis

Chauhan,

J.; Dakshinamurti,

K. Transcriptional

starved

toxins. Nephron 1991, 57, 192-196. Colombo, V.E.; Gerber, F.; Bronhofer,

10,038. Dakshinamurti, K.; Desjardins, P.R. Lipogenesis

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Sander, J.E.; Packman,

regulation of the glucokinase gene by biotin in

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Suchy, S.F.; Rizzo, W.B.; Wolf, B. Effect of biotin

deficiency and supplementation on lipid metabolism in rats: saturated fatty acids. Am. J. Clin. Nutr. 1986, 44, 475-480. Suchy, S.F.; Wolf, B. Effect of biotin deficiency and supplementation on lipid metabolism in

rats. J. Biol. Chem.

in biotin deficiency.

1991, 266,

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1261-1267. 19:

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Stockert, R.J. Biotin-dependent expression of the asialoglycoprotein receptor in HepG2. J. Biol. Chem. 1988, 263 (23), 11,280-11,283. National Research Council. Dietary reference

S.; Townsend, J.J. Brain

pyruvate carboxylase and the pathophysiology of biotin-dependent diseases. Neurology 1982, 32, 878-880.

68.

| Nutrition Foundation: Washington, DC, 2001. Zempleni, J.; Mock, D.M. Chemical synthesis of biotinylated histones and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/streptavidin-peroxidase. Arch. Biochem.

Crevat, A. Restoration by biotin in the in vitro microtubule formation inhibited by uremic

Floersheim, G.L. Treatment of brittle fingernails and onychoschizia with biotin: scanning electron microscopy. J. Am. Acad. Dermatol. 1990, 23, 1127-1132.

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in

effects of biotin deficiency and replenishment on rat liver pyruvate and propionyl-CoA carboxylases and on their mRNAs. Mol. Genet. Metab. 1999, 66, 16-23.

Water soluble vitamins in granulocytes, erythrocytes, and plasma obtained from chronic hemodialysis patients.

Knowledge

Biotinylation of histones in human cells: effects of cell proliferation. Eur. J. Biochem. 2001, 268, 5424-5429. ;

Livaniou, E.; Evangelatos, G.P.; Ithakissios, D.S.; Yatzidis, H.; Koutsicos, D.C. Serum biotin levels

in patients

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73.

Yatzidis, H.; Koutsicos, D.; Alaveras, A.G.; Papastephanidis, C.; Francos-Plemenos, M.

Biotin for neurologic disorders of uremia. Engl. J. Med. 1981, 305 (13), 764.

In Present

Biophys. 1999, 37] (1), 83-88.

Heard, G.S.; Hood, R.L.; Johnson, A.R. Hepatic

biotin and the sudden infant death syndrome. Med. J. Aust. 1983, 2 (7), 305-306.

J. Biotin.

Nutrition, 8th Ed.; Bowman, B.B., Russel, R.M., Eds.; International Life Sciences Institutes—

intakes for thiamin, riboflavin, niacin, vitamin B-6, folate, vitamin B-12, pantothenic acid, biotin,

and choline. In Recommended Dietary Allowances, 11th Ed.; Food and Nutrition Board Institute of Medicine, Ed.; National Academy Press: Washington, DC, 1998, 374-389.

Black Cohosh (Cimicifuga racemosa) Daniel S. Fabricant Norman R. Farnsworth UIC/NIH Center for Botanical Dietary Supplements Research for Women’s Health, Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy (M/C-877), University of Illinois at Chicago, Chicago, Illinois, U.S.A.

INTRODUCTION

through neurotransmitter regulation of hypothalamic function.

Native to the eastern United States, black cohosh has been a mainstay of Native American ethnomedicine for many years. Recently, its popularity has increased, primarily for relief of menopausal symptoms because of the potential toxicity of hormone replacement therapies. A thorough description of black cohosh, its current

uses

and

effects,

a full review

and

of clinical

studies to date are presented.

CHEMISTRY Fifty-seven triterpene glycosides (Fig. 1) have been reported from the roots/rhizomes of C. racemosa,!"! of which 23-epi-26-deoxyactein is generally recognized as the major component. Evaluation of 23-epi-26deoxyactein (formerly 27-deoxyactein), cimiracemoside

BOTANICAL NOMENCLATURE Actaea racemosa L. syn. Cimicifuga racemosa Nutt. (Ranunculaceae—buttercup family).

GENERAL Black

(L.)

DESCRIPTION

cohosh

represents

the

thick,

knotted

roots/

rhizomes of C. racemosa (L.) Nutt. (appropriate botanical identification methods are described later). The plant is native to the eastern United States, with a rich

tradition of ethnomedical use by Native Americans. It has been used clinically for relief of climacteric symptoms for almost 50 years, and its popularity has increased in recent years due to the potential toxicity of classical hormone therapy (equine estrogens + progestin). While its mechanism of action remains unclear, evidence

is surfacing to indicate that black

cohosh does not operate through classical endocrine pathways, i.e., estrogen receptors (ERs), to alleviate climacteric symptoms; recent data suggest action

Daniel S. Fabricant, Ph.D., is at the UIC/NIH Center for Botanical Dietary Supplements Research for Women’s Health, Program for Collaborative Research in the Pharmaceutical

Sciences, College

of Pharmacy (M/C-877), University of Illinois at Chicago, Chicago, Illinois, U.S.A. Norman R. Farnsworth, Ph.D., is at the UIC/NIH Center for Botanical Dietary Supplements Research for Women’s Health, Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy (M/C-877), University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Encyclopedia of Dietary Supplements DOI: 10.1081/E-EDS-120022095 Copyright © 2005 by Marcel Dekker. All rights reserved.

F,

and

cimicifugoside,

and

their

respective

aglycons, for binding affinity toward ER-f revealed no significant affinity."! The roots/rhizomes also contain 18 aromatic acids." Of these, caffeic acid has shown pregnant mare antigonadotropin activity,e~! rat uterine antispasmodic activity,! and smooth muscle relaxant/antispasmolytic activity in the rat ileum"7! and guinea pig ileum."! Ferulic acid has demonstrated luteinizing hormone (LH) release inhibition,”! follicle stimulating hormone (FSH) release stimulation,!! antiestrogenic activity,"° prolactin stimulation in cows!

and

inhibition

in rats”! and

uterine

relax-

ant/antispasmolytic activity in rats.!'*! Fukinolic acid has an estrogenic effect on MCF-7 cells with reference to estradiol."*! These activities may correlate with, or prove useful in the determination

of, the mechanism

of action of black cohosh. Additionally a number of plant sterols and fatty acids generally regarded as ubiquitous in the plant kingdom, are contained in the roots/rhizomes, the biological activities of which, in all probability, do not relate to the mechanism of

action." The weakly estrogenic formononetin has been reported in the plant.!'*] However, recent studies using plant material collected from different sites in the eastern United States at different times of the year indicate that the plant does not contain formononetin.">'®!

BOTANICAL DESCRIPTION C. racemosa is an erect, smooth-stemmed, perennial 1-2.5m in height. Large compound leaves are alternately arranged and triternate on short, clasping petioles. Basal leaf petioles are grooved in young 41

Black Cohosh (Cimicifuga racemosa)

42

OH

OH OH

CO>H .

eg

oO

O

COjH

Fukinolic Acid e)

=

OH

HO OH

Caffeic Acid

OH

OH

:

:

Xyl-O

HO

O

CO.H

OH

%7 Z

2H OH

Fukiic Acid

Cimicifugoside

Fig. 1 Triterpenes and phenolics of biological interest.

specimens. This shallow, narrow sulcus disappears as the petiole enlarges, whereas it remains present throughout life in the related eastern North American species C. rubifolia Kearney and C. americana

strands; lower and lateral surfaces with numerous root scars and a few short roots; horny fracture; slight

odor; bitter and acrid taste.!!

Michx."” Terminal leaflets are acute and glabrous with sharp serrated margins often trilobate and occasionally bilobed. Fruits are ovoid follicles occurring sessile on the pedicel. The flowering structure, the raceme, is a long, wandlike structure with showy white flowers (Fig. 2). The flowers possess numerous characteristic stamens having slender filaments with distinctive white anthers.''*! The roots/rhizomes have the following features: branched and knotted structure; dark brown exterior; white and mealy or brown and waxy interior; upper surface with several buds and numerous large stem bases terminated, frequently, by deep, cup-shaped, radiating scars, each of which shows a radiate structure, or less frequently, by fibrous

EFFECTS ON CLIMACTERIC SYMPTOMS RELATED TO MENOPAUSE With a history spanning almost 50 years of clinical study, mainly in Europe,?°! black cohosh is one of the more popular alternatives to hormone replacement therapy (HRT). Most of the clinical research has been performed on the commercially available Remifemin®. The formulation and dosage of Remifemin used in human studies has changed over time, as shown in Table 1. However, other commercial formulations are available, as evident in Table 2. Most have still not been clinically studied.

Black Cohosh (Cimicifuga racemosa)

43

number of hot flashes decreased 27% in both placebo and black cohosh groups. No significant differences were observed between groups. Thus, black cohosh, on the basis of this study, was no more effective than

placebo in the treatment of hot flashes. The source and formulation of the extract used in this study was not specified. A more recent open-labeled study using

breast

cancer

survivors,

treated

with

either

tamoxifen or a combination of BNO 1055 (a proprieblack cohosh extract) with tary hydroalcoholic tamoxifen, suggests a satisfactory reduction in the number and severity of hot flashes in the combination treatment group.'?! In

another

placebo-controlled

randomized,

double-blinded,

clinical study, for a duration

12 weeks, black cohosh was compared

and

of

with standard

conjugated estrogen (CE) therapy (0.625mg daily). Patients’ physical and psychological symptoms were measured every 4weeks. The end result of the study was that the patients treated with black cohosh had a statistically significant lower index score compared with those on placebo, with both the Kupperman menopausal (KM) and the Hamilton menopausal (HAM-A) scales, indicating a decrease in severity and frequency of hot flashes. Additionally, this study showed an increase in the number of estrogenized cells in the vaginal epithelium. In 2003, a similar and confusing study compared two different preparations of BNO 1055 extract to

CE therapy (0.6mg daily).?°! The study parameters

Fig. 2

Black

cohosh

raceme.

(View

this art in color at

www. dekker.com. )

The results of clinical studies have been measured using a variety of parameters. Self-assessments, physician assessments, and physiological parameters are usually used in tandem when designing such studies to measure psychological, neurovegetative, somatic, and physiological markers of menopause or, in the case of the treatment groups, relief from its climacteric symptoms. More significant when evaluating studies is the study design: More weight should be placed on studies following good clinical practice. The gold standard is a randomized, double-blind, multicenter, placebocontrolled study design. With that in mind, three studies on black cohosh have been randomized, double

blinded and placebo controlled.”°*?*°! The findings of the Jacobson study, spanning only 60days of treatment, may be limited by the time factor.7 Additionally, the study participants all had a history of breast cancer. The outcome of the study was that the median

were: patient self-assessment [diary and menopause rating scale (MRS)], CrossLaps (to measure bone resorption), bone-specific alkaline phosphatase (marker of bone formation), and endometrial thickness (measured by ultrasound). Both BNO 1055 extracts were equipotent to CE therapy and significantly better than placebo at reducing climacteric complaints. Additionally, the study showed that both BNO 1055 preparations had beneficial effects on bone metabolism in serum, specifically an increase in bone-specific alkaline phosphatase, and no reduction in bone resorption, thus leading to an increase in bone formation. No change in endometrial thickness was observed in the BNO 1055 treatment groups, but it was significantly increased with CE therapy. However, an increase in superficial vaginal cells was observed in both CE and BNO 1055 treatment groups. The authors of this study hypothesized that the impact of the BNO 1055 preparations was similar to the effects of selective estrogen receptor modulating (SERM), e.g., Raloxifene®, therapy on bone and neurovegetative climacteric symptoms, without any uterotrophic effects.?°! A recent double-blinded, randomized study compliant with good clinical practice used two dosages (low: 39 mg; high: 127mg) of an unspecified hydroalcoholic

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JUDUISSasse-JfIs juaned pue uerrsc°! At 35 ug/ml, the lipophilic extract was able to activate transcription of the estrogen-regulated genes; the hydrophilic extract showed no such activity. A recent study measuring an extract at a low concentration (4.75 g/L) reported increased ER levels, an effect also produced by estradiol, in human MCF-7 cells.°! An unspecified black cohosh extract tested in a transient gene expression assay using HeLa cells cotransfected with an estrogen-dependent reporter plasmid in the presence of human ER-« and ER-B cDNA failed to show transactivation of the gene.&7! Plasma Hormone

Levels

The effect of black cohosh

on serum

concentrations

of FSH and LH has been studied extensively. Crude

49

Black Cohosh (Cimicifuga racemosa)

alcoholic extracts suppressed plasma LH, with no effect on FSH in ovx rats.*4°! Further fractionation of the crude fraction showed that the activity resided in the lipophilic fraction, with the aqueous soluble fractions devoid of this activity.*! A later study in rats using lipophilic and hydrophilic extracts at high doses (140 and 216mg/rat, ip.) resulted only in LH suppression with a single injection administration of the lipophilic extract“! Another study reported LH suppression in Ovx rats with an unspecified dose and extract type of C. racemosa.™*! A recent study compared the effect of C. racemosa (BNO 1055) with that of estradiol on LH levels.°°! Reduced levels were reported for the black cohosh treated animals at 60 mg/day administered subcutaneously for 7 days. However, another study reported no estrogen agonistic effects on FSH, LH, or prolactin levels in ovx rats (DMBA model) with daily administration for 7 weeks

of a 40% isopropanolic extract (Remifemin).4)

Uterine Weight/Estrus Induction Uterine and ovarian weight increase, cell cornification,

and an increased duration of estrus are generally considered evidence of endometrial estrogenic activity. However, it has recently been debated that uterine weight is a poor marker for endometrial effects.!! Three studies demonstrating that black cohosh extracts increased the uterine weight of ovx rats have been reported,?°*? 6!) two of which used an undetermined

root extract.P3°] One study on immature mice reported similar findings.?° By contrast, two studies on ovx rats,>°°! as well as four studies on immature

mice,

reported the converse.?®°*°*3] One of these studies found that despite no increase in uterine or ovarian weight, the duration of estrus was _ significantly

increased by black cohosh.'°?! A study by the authors and collaborators demonstrated no attenuation in uterine weight at variable doses (4, 40, and 400 mg/kg day) of a 40% isopropanol extract in ovx rats, [64]

Hormonal

Secretion

The effect on prolactin secretion in pituitary cell cultures was assayed using an unspecified ethanolic extract of C. racemosa”?! Basal and TRH-stimulated prolactin levels were reduced significantly at doses of 10 and 100pg/ml. This effect was reversed by the addition of haloperidol (D-2 antagonist) to the cell cultures, suggesting dopaminergic regulation of hormone secretion by C. racemosa.

Cell Proliferation

An unspecified black cohosh extract failed to induce growth of MCF-7 cells significantly when compared to untreated control cells.°*! A study using isopropanolic and ethanolic extracts also failed to demonstrate growth of MCF-7 cells.!©°!

CNS Effects and Neurotransmitter Binding Osteopenia Inhibition The BNO 1055 black cohosh extract (60 mg/rat, s.c.) has been shown to increase the expression of collagen I and osteocalcin in rats to a level equivalent to that in ovx rats treated with estradiol (8 pg).°° An additional study using the BNO 1055 extract demonstrated an osteoprotective effect—a reduced loss of bone mineral density in rat tibia after 3mo of administration.$° A study using an unspecified isopropanol extract of C. racemosa showed reduced urinary parameters of bone loss. The authors of this study suggested that this action was similar to that of the

SERM Raloxifene.©7! A follow-up study using BNO

1055 vs. CE therapy showed beneficial effects of the extract on bone metabolism in humans, specifically an increase in bone-specific alkaline phosphatase in serum.°! While no direct correlation between species has been established, it is of note that studies on Asian

species of Cimicifuga have demonstrated similar activity and may be of importance for further investigation

of this biological activity.°°>?!

A study using an unspecified extract (25-100 mg/kg, orally) to measure effects on mice body temperature and ketamine-induced sleep time, using bromocriptine (D-2 agonist) pretreated with sulpiride (D-2 blocker) as a positive control, suggested a receptor mediated dopa-

minergic effect.°°! A further study was carried out to characterize neurotransmitter levels in the striatum and hippocampus after pretreatment in mice with the extract for 21days.!! Serotonin and dopamine metabolic levels in the striatum were substantially lower in comparison with the control group. These studies have helped lead to the hypothesis that it is dopaminergic action, rather than estrogenlike activity, that is responsible for the success of black cohosh in reducing climacteric symptoms.! A study by the authors and collaborators has pointed to the effects of black cohosh being mediated by serotonin (5-HT) receptors.!“! Three different extracts (100% methanol, 40% isopropanol, 75% ethanol) bound to the 5-HT7 receptor subtype at ICso < 3.12yg/ml. The 40% isopropanol extract inhibited (°H]-lysergic acid diethylamide (LSD), binding to the 5-HT; receptor with

Black Cohosh (Cimicifuga racemosa)

50

greater potency than the synthetic (?H]-8-hydroxy-2(diN-propylamino)tetralin to rat 5-HT,,. Analysis of ligand binding data suggests that the methanol extract functioned as a mixed competitive ligand of the 5-HT7 receptor. Further testing of the methanol extract in

widespread use. Thus, the data are more regarding the safety of this particular product, rather than that of black cohosh unspecified extracts. In clinical trials, minor cases of nausea, vomiting, dizziness, and head-

ache have been reported.'°! A recent review of AERs

293T-5-HT, transfected HEK cells revealed elevated cAMP levels; these levels were reversed in the presence

concluded that, for black cohosh preparations, the events are rare, mild, and reversible.7! Furthermore,

of the 5-HT antagonist methiothepin, indicating a receptor mediated process and possible agonist activity local to the receptor!)

case reports citing acute hepatitis, convulsions, and cardiovascular and circulatory insult have been reviewed.'*7! In these reports, no effort was made to positively identify the botanical used as black cohosh. This, combined with underreporting, is a common factor with respect to AERs for botanical dietary supplements.!7!]

Miscellaneous

A black cohosh methanol extract protected S30 breast cancer cells against menadione-induced DNA damage at variable concentrations, and scavenged DPPH

free

radicals at a concentration of 99 1M.!©8!

USE IN PREGNANT/LACTATING

WOMEN

Despite an absence of mutagenic effects reported to date, the use of black cohosh during pregnancy is contraindicated according to WHO suggestions.!™! Data are inconclusive regarding the effects on lactation. Additionally, the American Herbal Products Association (AHPA) assigned black cohosh 2a and 2b classifications, which state that the herb is not to be used during pregnancy or nursing unless otherwise directed by an expert qualified in the use of the described

substance”!

However,

experimental

data

are not available to confirm these warnings.

DOSAGE"?70} e

e

Dried rhizome and root: One gram up to 3 times

daily. Tincture (1:10): 0.4 ml daily (40-60% alcohol v/v). Fluidextract (1:1): Twenty drops twice daily (60% ethanol v/v, equivalent to 40 mg dried herb). Tablet equivalence: Two tablets a day (equivalent to 40 mg dried fluid extract).

ADVERSE EFFECTS A majority of adverse event reports (AERs) for black cohosh have been for Remifemin products because of

“The Commission E Monographs also recommend that usage not be extended for more than 6mo due to a lack of long-term safety data. Experimental data are not available to validate this 6-mo limit. >There appears to be confusion whether 40 mg refers to extract or herb. Correspondence with Remifemin manufacturers by the authors has not clarified this matter.

COMPENDIAL/REGULATORY STATUS Black cohosh in the United the provisions Education Act Dried

products are regulated and marketed States as dietary supplements under of the Dietary Supplement Health and © (DSHEA) of 1994 (U.S.C. Sec. 321).

roots/rhizomes,

whole,

powdered,

and

as

extracts, are now officially included in the United States Pharmacopeia—National Formulary"*\In the European Union, they are approved as nonprescription phytomedicines administered orally in compliance with the German Commission E Monographs. [73]

CONCLUSIONS With the current fear of side effects related to classical hormone therapy, modulation of certain climacteric symptoms of menopause by both dopaminergic and serotonergic drugs is becoming a viable and frequent treatment option. A review of the clinical trials associated with black cohosh leads to the conclusion that women using hydroalcoholic extracts of the rhizomes/ roots of this plant gain relief from climacteric symptoms (e.g., hot flashes) in comparison with placebo. Confounding the review of these clinical trials are the different types of extracts administered. Early in vitro studies tended to report that black cohosh extracts acted on ERs, or have some sort of direct estrogenic effect. It is, however, becoming clearer now that, at least in humans, the beneficial effects in reducing hot flashes relate, at least in part, to serotonergic or dopaminergic mechanisms regulating hypothalamic control, possibly mediating estrogenic mechanisms. Again, the controversy surrounding a potential direct estrogenic mechanism of action may also be due to variance in the extracts assayed. Full safety evaluations, either in animals or in humans, have not been conducted to date. However,

side effects reported in clinical trials

seem to be minimal.

Black Cohosh (Cimicifuga racemosa)

51

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FURTHER

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Boron Curtiss D. Hunt

United States Department of Agriculture, Agriculture Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota, U.S.A.

INTRODUCTION The element boron is essential for all higher plants and at least some organisms in each of the other phylogenetic kingdoms Eubacteria, Stramenopila (brown algae and diatoms), and Fungi. Physiologic concentrations of the element are needed to support metabolic processes in several species in Animalia. For example, embryological development in fish and frogs does not proceed normally in the absence of boron. There is

in oceanic salts.'7! Weathering of clay-rich sedimentary rock is the major source of total boron mobilized into the aquatic environment.”! Undissociated boric acid (orthoboric acid) is the predominant species of boron in most

natural

freshwater

systems,?!

where

most

concentrations are below 0.4mg/L and are not lowered by typical treatments for drinking water. The most common commercial compounds are anhydrous,

and pigs, require physiological amounts to assist normal biologic processes including immune function

pentahydrate, and decahydrate (tincal) forms of disodium tetraborate (borax, Na2B,4O7), colemanite (2CaO-3B,03:5H,O), ulexite (Na,O-2CaO: 5B,03-16H,O), boric acid (H3BO3), and monohydrate and tetrahydrate forms of sodium perborate

and bone

(NaBO;)."4!

evidence

that higher vertebrates,

development.

apparent homeostatic immune function.

e.g., chickens,

In humans,

control

and

boron

rats,

is under

is beneficial

for

At typical physiological concentrations (6.0 x 107’

to ~9.0 x 10-* mol/L) in plants, animals, or humans, inorganic

boron

is essentially

present

only as the

monomeric species boric acid, 1.e., B(OH)3, and borate,

COMMON

i.e., B(OH)4~.©! Polyborate species can form near neutral physiological conditions (pH ~7.4), when

CHEMICAL FORMS

Boron is the fifth element in the periodic table, with a molecular weight of 10.81, and is the only nonmetal in Group III. Only organoboron compounds are apparently important in biological systems during normal physiological conditions and they are defined for this discussion as those organic compounds that contain B-O bonds." B-N compounds form a part of organoboron compounds, because B-N is isoelectronic with C-—C. These complexes are present in plants, and most likely in human tissues.

borate

concentrations

Ve

SPECIATION

Environmental

Forms

is 4.6mg/L, and it is the 10th most abundant element

Curtiss D. Hunt, Ph.D‘, is at the United States Department of

Agriculture,

Agriculture

Research

+

2H,0

Service,

Grand

Forks

Human

Nutrition Research Center, Grand Forks, North Dakota, U.S.A.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022406 Published 2005 by Marcel Dekker. All rights reserved.

an

Many groups,

=

H30*

sie B(OH), —

tO

Biochemical

Boron does not naturally occur free or bind directly to any element other than oxygen, but for certain exceptions, e.g., NaBF, (ferrucite) and (K,Cs)BF4 (avogadrite).""! Its average concentration in the oceans

~0.025mol/L,

B(OH),©. B(OH),

BORON

exceed

unusually high boron concentration in biological systems, but still lower than that found in the snap bean leaf (0.1 mol/L).!7 Within the normal pH range of the gut and kidney, B(OH); would prevail as the dominant species (pH 1: ~100% B(OH)3; pH 9.3: 50%; pH 11: ~0%).! Boric acid is an exclusively monobasic acid and is not a proton donor. Rather, it accepts a hydroxyl ion (a Lewis acid) and leaves an excess of protons to form the tetrahedral anion

Forms

or more

hydroxyl

and those with suitable molecular

structures

biomolecules

contain

one

can react with boron oxo compounds to form boroesters, an important class of biologically relevant boron species. Several types of boron esters exist. Boric acid reacts with suitable dihydroxy compounds to form corresponding boric acid monoesters (“partial’’ esterification) (e.g., Fig. 1) that retain the trigonal-planar configuration and no charge. 55

Boron

56

are

O

(a

Mt }(CH>),, l (CH2)n

We

wae

ae

B SG

CH2)n l

‘

Fig. 1 Boric acid may complex with a suitable dihydroxy ligand to form a boric acid monoester (“‘partial’’ esterification) that retains a trigonal-planar configuration and no charge.

Fig. 3. Boric combine with corresponding esterification) configuration

acid monoesters or borate monoesters can a suitable dihydroxy compound to form a borodiester _ (““complete”’ _spiro-cyclic that is a chelate complex with a tetrahedral and negative charge.

In turn, a boric acid monoester can form a complex

in a covalent bond!!?!>! or a boroester.!'% Presence

with a ligand containing a suitable hydroxyl to create a borate monoester (“partial’’ esterification; monocyclic) (Fig. 2), but with a tetrahedral configuration and a negative charge. A compound of similar configuration and charge is also formed when borate forms a complex with a suitable dihydroxy compound. The two types of boromonoesters can react with another suitable dihydroxy compound to give a corresponding spiro-cyclic borodiester (“‘complete’’ esterification) that is a chelate complex with a tetrahedral configuration and negative charge (Fig. 3).!'° A partially esterified tridentate cleisto complex (Fig. 4) may be formed when a ligand contains three suitably cis-oriented hydroxyl groups."'"! A variety of important biological complexes are formed when nitrogen acts as an electron-pair donor to fill the vacant boron p- orbital. For example, the N® of histidine-57 of «-lytic protease and the boron atom of a peptide boronic acid interact to form a covalent bond and give rise to a reversible complex (Fig. 5). Boric acid and boric acid-like structures, instead of borate, are most likely the species reactive with biological ligands, because it is probably easier for a diol to substitute for a relatively loosely bound water molecule associated with boric acid or a boric acid-like structure than it is for the diol to substitute for a hydroxyl ion in borate or a borate-like structure of differences in charge.!!”!

in these molecules is essential; in its absence, they no

Procaryotes

longer perform their normal physiologic functions. The element is a structural component of certain antibiotics produced by certain myxobacteria, a distinct and unusual group of bacteria. For example, tartrolon B (Fig. 6) is characterized by a single boron atom in the center of the molecule."7! Recently, another related anti-

biotic, boromycin, was discovered to be potent against human immunodeficiency virus (HIV)."” It strongly. inhibits the replication of the clinically isolated HIV1 strain and apparently, by unknown mechanisms, blocks release of infectious HIV particles from cells chronically infected with HIV-1. One more new finding was that of a boron-containing biomolecule produced by a bacterium that is not an

antibiotic,'*! but rather a cell-to-cell communication signal. Communication between bacteria is accomplished through the exchange of extracellular signaling molecules called autoinducers (AIs). This process, termed ‘“‘quorum sensing,’’ allows bacterial populations to coordinate gene expression for community cooperative processes such as antibiotic production and virulence factor expression. AI-2 is produced by a large number of bacterial species and contains one boron atom per molecule. Not surprisingly, it is derived from the ribose moiety of biomolecule S-adenosylmeth® nine (SAM). The gliding bioluminescent marine bacterium, Vibrio harveyi (phylum Proteobacteria), produces and also binds AI-2. In V. harveyi, the primary receptor and sensor for AI-2 is the protein LuxP, which consists of two similar domains

Boron is an integral component of several biomolecules, where it is thermodynamically stabilized

OH

ms

|

A

O

OH

ficheare seB teedeesmo

M™ |(CH2)n

Fig. 2 Borate may complex with a suitable dihydroxy ligand to form a borate monoester (“partial’’ esterification; monocyclic) with a tetrahedral configuration and a negative charge.

Nat

org

aus Cr

H

CH, Fig. 4 A partially esterified tridentate cleisto complex may be formed when a ligand contains three cis-oriented hydroxyl groups.

Boron

57 ie

Asp

Ss

P

mt

His®” ( er

6


Ap;A = NAD* > Ap,A > NADH & 5'-ATP >. 5/-ADP > 5'/-AMP > adenosine (ADS). Species without these moieties do not bind boron well: 3/-AMP = 2’-AMP = cAMP = adenine (ADN).

Recent evidence suggests that the predominant place of boron function in plants is in the primary cell walls, where it cross-links rhamnogalacturonan II (RG-II) (Fig. 9), a small, structurally complex polysaccharide of the pectic fraction. RG-Is have an atom of boron that cross-links two RG-II dimers at the site of the apiose residues to form a borodiester.?4) However, this function is not adequate to explain all boron deficiency signs in plants. Boron oxo compounds also form stable ionic complexes with the polyol ligands mannitol, sorbitol, and fructose in liquid samples of celery phloem sap and vascular exudate and phloem-

fed nectaries of peach.’°! In fact, current data suggest that no free boric acid or borate is present in these phloem saps or vascular exudates.

Dietary Supplements Boron speciation in dietary supplements varies widely,”* as does the relevant information provided by various dietary supplement manufacturers. It is sometimes listed only in a general manner (e.g., “borates’’ or “boron’’), and occasionally in a more specific way (e.g., “sodium borate’’ or “sodium tetraborate decahydrate’’). Several commercially available forms (e.g., “boron amino acid chelate,’’ ‘boron ascorbate,’’ “boron aspartate,’’ “boron chelate,’’ “boron citrate,’’ “boron gluconate,’ “boron glycerborate,’’ “boron glycinate,’’ “boron picolinate,’’ “boron proteinate,’ and “boron bonded with niacin’’) are not well characterized

in the scientific literature.

Most

often,

dietary boron supplements are provided in conjunction with other nutrient supplements.

BIOAVAILABILITY AND EXCRETION If plant and animal boron absorption mechanisms are analogous, the organic forms of the element per se are probably unavailable.?71 However, the strong association between boron and polyhydroxyl ligands (described below) is easily and rapidly reversed by dialysis, change in pH, heat, or the excess addition of another low-molecular polyhydroxy] ligand.”*! Thus, within the intestinal tract, most ingested boron is probably converted to orthoboric acid (common name: boric acid), B(OH)3, the normal end product of hydrolysis of most boron compounds." Gastrointestinal absorption of inorganic boron and subsequent urinary excretion”®! is near 100%. In humans, lack of boron accumulation and relatively small changes in blood boron values during a substantial increase in dietary boron support the concept of boron homeostasis.7*!

DIETARY BORON SOURCES AND INTAKES Dietary Recommendations The tolerable upper intake level (UIL) for boron varies by life stage (Table 1).?*! No estimated average requirement,

recommended

dietary allowance,

or adequate

intake has been established for any age-sex group.

Boron

59

and 0.148, respectively. The median intake from supplements in the U.S. population is approximately

0.135 mg/day.??! Nonfood

Personal Care Products

Boron is a notable contaminant or ingredient of many nonfood personal care products. For example, an antacid was found to have a high boron concentration (34.7 ug/g)" such that the maximum recommended daily dose would provide 2.0mg/B/day, two times the estimated daily boron consumption for the overall adult U.S. population.

Dietary Sources and Intakes Ten representative foods with the highest boron concentrations (ug/g) are distributed among several

food categories!*7!: raw avocado (14.3), creamy peanut Fig. 9 Schematic representation of two monomers of the pectic polysaccharide rhamnogalacturonan-II cross-linked by an atom of boron at the site of the apiose residues to form a borodiester. Multiple cross-links form a supramolecular

network. (From Ref.!*!.)

Dietary Supplements For adults, the amount of boron commonly provided

in a single dietary boron supplement is 0.15 mg.?° However,

the quantity

supplied

by one

dose

may

be as low as 0.15mg or as high as 40.0mg.°° The mean usual intake of boron (mg/day) from dietary supplements for children (1-8yr), adolescents (9-IS5yr), males (19+yr), females (19+ yr), and pregnant/lactating women is 0.269, 0.160, 0.174, 0.178,

Table 1 Upper limits for boron set by the 2001 Food and Nutrition Board of the National Academy of Sciences Life stage group

Children

Age (yr)

Upper limit (mg/day)

1-3 4-8 9-13

3 6 ili!

Adolescents

14-18

Ng

Adults

19-70 >70

20 20

Pregnancy

50 yr

1000 1200

1200 1450

Infants,

7-12 mo

Children 1-3 yr 4-8 yr

“Using an estimated 10% coefficient of variation requirements around the population mean. (From Ref.!'°))

of individual

Preparations The nutritional preparations of calcium include mainly salts with such anions as carbonate, citrate, phosphate, lactate, and citrate-malate (CCM). Additionally, salts

with gluconic acid may occasionally be found, and calcium chelates with amino acids are also marketed. The calcium content (i.e., “elemental’’ calcium) varies from 40% for the carbonate salt to ~13% for CCM. For phosphate binding in ESRD, the acetate salt is more commonly used. In the United States, most preparations come in the form of swallowable or chewable tablets, with calcium contents ranging from 200 to 600 mg per tablet. Bioavailability is approximately the same for all the leading salts, although CCM and the chelates tend toward the high end of the range and the gluconic acid salts toward the low end. Absorbability of the salt is only very weakly related to solubility, and gastric acid is not necessary for calcium absorption if the supplement is taken (as recommended) with meals. The most extensive, side-by-side comparisons have involved the

carbonate and citrate salts, and the bulk of the evidence there indicates approximately equal absorbability for the two sources, with perhaps a slight edge for the carbonate salt.!'*! Poor pharmaceutical formulation will impede disintegration and hence prevent absorption, a problem encountered with many generic calcium supplement products sold in the 1980s and early 1990s.!'*! For this reason, preference should be given to supplements that meet United States Pharmacopeia (USP) disintegration standards, and even better, to those that have demonstrated bioavailability. A growing variety of fortified foods have been available since about 1999. As noted, fortification tends to improve the nutritional value of low-calcium foods and, to some extent, it can be thought of as equivalent to taking supplements with meals. However, interactions between added calcium and various food

constituents during food processing and storage may alter the absorbability characteristics of the former. For example, it was noted during the early days of juice supplementation that CCM was well absorbed from orange and grapefruit juices, and even better from apple juice, but poorly from lemon juice. These differences could not have been predicted from what was known of food chemistry. Hence, with fortified foods as with supplements, actual bioavailability of the product reaching the consumer should be demonstrated. When calcium is added to beverages (such as orange juice or soy beverage), an additional problem arises. Solubility of the principal calcium salts is relatively low, and serving size portions of such beverages would not sustain in solution more than a small fraction of the calcium content of, say,

a comparable

serving of milk. Hence, such fortification almost always requires physical suspension of a particulate. In some

beverages,

this suspension

is so poor

that

the calcium settles as a dense sludge at the bottom of the beverage container and may, accordingly, not be

ingested at all.!'4!

Supportive Therapy as a Part of Antiosteoporosis Pharmacotherapy Current antiosteoporosis pharmacotherapy includes bisphosphonates, selective estrogen receptor modulators (SERMs), estrogen, and anabolic agents such as the fluoride salts and PTH derivatives, including teri-

paratide. All have as their goal at least stabilization of bone mass. Some of them, such as the bisphosphonates, can lead to slow steady state bone gain (0.5 —1.0% per year), and the anabolic agents can produce as much as 10-15% bone gain per year. To support this increase, especially for the anabolic agents, calcium intake from diet must usually be augmented by supplements. Optimal doses for calcium during pharmacotherapy have not been established. However, all the bisphosphonates and SERMs have been tested only with 500-1000mg supplemental calcium, while fluoride has been shown to produce bone hunger calling for as much as 2500 mg Ca/day. Only estrogen has been studied with and without supplemental calcium, and here the evidence is very clear: Bony effects of estrogen are augmented two- to threefold, and estrogen dose can be reduced if calcium intake is above 1000 mg/ day.'>!1 With the more potent anabolic agents, a calcium phosphate preparation may be preferable, so as to ensure an adequate intake of both of the components of bone mineral and to compensate for the intestinal binding of diet phosphorus by high dose calcium supplementation.

Calcium

70

Ancillary Therapy for Prevention or Treatment of Miscellaneous Disorders Hypertension, pre-eclampsia, colon cancer, renolithiasis, premenstrual syndrome, polycystic ovary syndrome, and obesity—all multifactorial disorders— have each been shown to have a calcium-related

said to be a consequence of taking calcium carbonate, the evidence

is scant,!!®! and in several randomized

controlled trials, the difference in degree of constipation between the calcium- and placebo-treated groups has generally been small and usually not statistically significant.

component,”! and for several of them, calcium supple-

mentation has been shown in randomized controlled trials to reduce incidence and/or expression. Optimal calcium intake for this protection has not been established for any of the disorders concerned, but several

threads of evidence indicate that total intakes of 1200-1800 mg of Ca per day may be sufficient. The role of calcium in these disorders has been described above (see sections “Intraluminal Effects’? and “OffLoop Effects’’). Specific pharmacotherapy of any of the disorders concerned should always be accompanied by an adequate calcium intake, using supplements if necessary.

CONTRAINDICATIONS There are few, if any, true contraindications to calctum

supplementation. In general, supplementation moves contemporary intakes into the range that would have been the Paleolithic standard, and hence helps to normalize modern diets. However, patients receiving calcitriol therapy or suffering from disorders such as sarcoidosis, in which calcium absorption may be high, should not take supplements except under medical supervision.

PRECAUTIONS AND ADVERSE

REACTIONS

Calcium supplements may bind with tetracycline antibiotics and hence reduce their absorbability. The element has also been reported to interfere slightly with thyroxin absorption. Hence, a person requiring both calcium and thyroid replacements should take them at different times of the day or have plasma thyroxin and thyroid stimulating hormone (TSH) levels checked to ensure that the thyroid dose produces the desired therapeutic effect. Both calcium salts and high calcium foods reduce absorption of nonheme iron ingested at the same meal in unprepared subjects. However, chronic supplementation studies show no long-term deterioration in iron status in adults and no interference with augmenting iron status during growth.'”] The single-meal tests that are used to demonstrate this interference could not have detected physiological upregulation of iron absorption. Adverse reactions tend to be extremely rare and mostly idiosyncratic. Although constipation is often

OVERDOSAGE In its 1997 recommendations, the Food and Nutrition Board of the Institute of Medicine set a tolerable upper intake level (TUIL) for calcium of 2500 mg/day.”! However, it is important to note that there has never been a reported case of overdosage of calcium from food sources, even at continuing intakes over 6000 mg/day. Supplement intakes above 2500 mg/day are occasionally associated with a syndrome similar to the milk alkali syndrome. The pathogenesis of the hypercalcemia seen in this condition is complex, but there is usually hypoperfusion of both the kidneys and skeleton, the two most important internal regulatory organs for calcium. The condition can usually be managed by giving attention to adequate hydration and maintenance of blood flow to these critical organs. Except as support for the most potent osteoporosis pharmacotherapy, or in management of the hyperphosphatemia of ESRD, there is no known reason to use supplements at a dose above 2500 mg Ca/day.

REGULATORY STATUS Calcium supplements are regulated as foods in the United States. Bioavailability is not a regulated characteristic of marketed supplement products. Nevertheless, because of pharmaceutical formulation and food matrix effects on absorbability, bioavailability of different preparations of the same salt (e.g., calcium carbonate) may vary over a twofold range.

REFERENCES 1.

Awumey, E.M.; Bukoski, R.D. Cellular functions and fluxes of calcium. In Calcium and Human

Health; Humana:

Weaver,

C.M.,

Heaney,

R.P.,

Eds.;

shifting?

Bone

Totowa, NJ. 2005.

2.

Heaney, R.P. Is the paradigm 2003, 33, 457-465.

3.

Heaney, R.P. Ethnicity, bone status, and the calcium requirement. Nutr. Res. 2002, 22 (1-2),

4.

153-178: Heaney, R.P.;

Nordin,

B.E.C.

Calcium

effects

on phosphorus absorption: implications for the

Calcium

71

prevention and co-therapy of osteoporosis. J. Am. Coll. Nutr. 2002, 27 (3), 239-244. Barger-Lux,

M.J.;

Heaney,

R.P.;

Recker,

R.P.;

Recker,

R.R.;

Weaver,

13.

Absorbability of calcium sources: the limited role

14.

LD.

170-173. Heaney, R.P.; Berner, B.; Louie-Helm, J. Dosing

regimen for calcium supplementation. Miner. Res. 2000, 75 (11), 2291. Nordin, Morris,

10.

41,

1B.e.C:. Polley. H.A.; Marshall,

J. Bone

16.

KJ... iNeed,,. A.G:; D. The problem of

calclum requirement. Am. J. Clin. Nutr. 1987, 45, 1295-1304. Food and Nutrition Board, Institute of Medicine. Dietary Reference Intakes for Calcium, Magnesium, Phosphorus, Vitamin D, and Fluoride; National Academy Press: Washington, me 61997.

17;

load on absorption Res. 1990, JJ (5),

M.S.;

Barger-Lux,

M.J.

as the carbonate

and

citrate salts, with some observations on method. Osteoporosis Int. 1999, 9, 19-23. Shangraw, R.F. Factors to consider in the selec-

Public Health Report 1989, S104, 46-49. Heaney, R.P.; Rafferty, K.; Bierman, J. Not all

beverages

are

equal.

Nutr.

Nieves, J.W.; Komar, L.; Cosman, F.; Lindsay, R.

Calcium potentiates the effect of estrogen and calcitonin on bone mass: review and analysis. Am. J. Clin. Nutr. 1998, 67, 18-24. Recker, R.R.; Davies, K.M.; Dowd, R.M.; Heaney, R.P. The effect of low dose continuous estrogen and progesterone therapy with calcium and vitamin D on bone in elderly women: a randomized controlled trial. Ann. Intern. Med. 1999, 130, 897-904. Ilich-Ernst, J.Z.; McKenna, A.A.; Badenhop, N.E.; Clairmont, A.C.; Andon, M.B.; Nahhas, R.W.; Goel, P.; Matkovic, V. Iron status, menarche,

and calcium supplementation in adolescent girls. Am. J. Clin. Nutr. 1998, 68, 880-887.

Heaney, R.P.; Weaver, C.M.; Fitzsimmons, M.L.

The influence of calcium fraction. J. Bone Miner. 1135-1138.

Dowell,

of calcium

calcium-fortified Today 2005.

Wastney, M. Absorption of calcium oxalate does not require dissociation in rats. J. Nutr. 1999, 129,

R.P.;

tion of a calcium supplement. Proceedings of the 1987 Special Topic Conference on Osteoporosis.

C.M.

of solubility. Calcif. Tissue Int. 1990, 46, 300-304. Hanes, D.A.; Weaver, C.M.; Heaney, R.P.;

Heaney,

Absorption

R.R.

Time course of calcium absorption in humans: evidence for a colonic component. Calcif. Tissue Int. 1989, 44, 308-311. Heaney,

2.

18.

Clemens, J.D.; Feinstein, A.R. Calcium carbonate

and constipation: a historical review of medical mythopoeia. Gastroenterology 1977, 72, 957-961.

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LCA-Carn

Oxidation

LCA-CoA

MCA-CoA betaOxidation

CO,

MCA-CoA

MCA-Carn

=

a Glucose

Pyruvate

——=Pyruvate

VLCA-CoA

Peroxisome

Cytosol

Mitochondrion Matrix

Fig. 2 L-Carnitine and acetyl-L-carnitine transport and function. Abbreviations: Carn, L-Carnitine; AcCarn, acetyl-L-carnitine; OCTN2, organic cation transporter 2; UnTr, unidentified transporter(s); LCA-CoA, long-chain acyl-coenzyme A; MCA-CoA, medium-chain acyl-coenzyme A; VLCA-CoA, very-long-chain acyl-coenzyme A; LCA-Carn, long-chain acylcarnitine esters; MCA-Carn,

medium-chain acylcarnitine esters; Ac-CoA, acetyl-coenzyme A; CPT I, carnitine palmitoyltransferase I; CPT II,

carnitine palmitoyltransferase I]; CAT, carnitine acetyltransferase; acylcarnitine translocase; PDH, pyruvate dehydrogenase complex.

esters exported from peroxisomes are transported into mitochondria by CACT, and the acyl moieties are transesterified to coenzyme A and oxidized (Fig. 2).”!

Modulation of the Acyl-CoA/CoA Ratio in Cellular Compartments Coenzyme A participates in many metabolic processes in the cellular cytoplasm and organelles. However, neither coenzyme A nor its thioesters can cross the membranes separating these compartments. Thus, in each compartment, sufficient nonesterified coenzyme A must be made available to maintain metabolic activities in that part of the cell. Because of its ability to be transported across organelle membranes and undergo rapid transesterification with coenzyme A, L-carnitine facilitates availability of adequate amounts of the deacylated coenzyme. In mitochondria, the amount of acetyl-CoA generated from rapid f-oxidation of fatty acids or carbohydrate utilization may exceed the

COT,

carnitine

octanoyltransferase;

CACT,

carnitine—

capacity of the citric acid cycle to release the coenzyme. Transesterification of acetyl units to L-carnitine, catalyzed by carnitine acetyltransferase (CAT), frees intramitochondrial coenzyme A for participation in subsequent cycles of substrate utilization.°4) Lowering of the acetyl-CoA/CoA ratio stimulates pyruvate oxidation, secondary to an increase in pyruvate dehydrogenase complex activity.) Acetyl-L-carnitine can be removed from the mitochondrion via CACT for use in the cytoplasm, for export and use in other cells or tissues, or to be excreted.°4! This function has long been viewed primarily as a means to dispose of acetyl units from mitochondria. However, export of acetyl-L-carnitine from some cells and tissues (e.g., liver and kidney) may be important for supply of this metabolite to other tissues (e.g., brain), where it may have specific functions in addition to its use as a substrate for energy production. Moreover, by modulating the intramitochondrial acetyl-CoA/CoA ratio, L-carnitine plays a significant role in regulating glucose metabolism in skeletal muscle and heart."*!

L-Carnitine and Acetyl-t-Carnitine

75

Membrane Phospholipid Remodeling

PHYSIOLOGY

L-Carnitine and extramitochondrial CPT are important modulators of long-chain fatty acid utilization for membrane phospholipid biosynthesis and remodeling. L-Carnitine acts as a reservoir of long-chain fatty acids for incorporation into erythrocyte membrane phospholipids during repair after oxidative insult,”! and for use in the synthesis of dipalmitoylphospha-

Dietary Intake and Biosynthesis

tidylcholine,

the major component

of surfactant,

in

lung alveolar cells.!

Meat, fish, chicken, and dairy products are rich sources of dietary t-carnitine. Plant derived foods contain very small amounts of the substance. Most commercially available infant formulas contain L-carnitine, either provided from the milk component, or supplemented, as in the case of soy-protein-based formulas. There is no recommended dietary allowance or dietary reference intake for L-carnitine. In

Other Reported Actions of L-Carnitine and/or Acetyl-.-Carnitine

mammals,

L-carnitine

and protein turnover"!

L-Carnitine may mimic some of the actions of glucocorticoids in vivo. In HeLa

cells, L-carnitine reduced

glucocorticoid receptor-« affinity for its steroid ligand, and triggered nuclear translocation of the receptor.! It suppressed glucocorticoid receptor-mediated tumor necrosis factor-« and interleukin-12 release by human primary monocytes stimulated with lipopolysaccharide ex vivo.?! All of these effects of L-carnitine were concentration dependent. In rats and mice, L-carnitine

markedly suppressed liposaccharide-induced cytokine production, improving their survival during cachexia and septic shock.©! In humans, L-carnitine supplementation of surgical and AIDS patients decreased serum tumor necrosis factor-x concentration.) Glucocorticoids also increase the expression and activity of urea cycle enzymes. Hyperammonemia associated with chronic valproic acid therapy and with several inborn errors of metabolism, including CACT deficiency and medium-chain acyl-CoA dehydrogenase deficiency, is attenuated by L-carnitine administration. Experimentally, L-carnitine supplementation protects against lethal ammonia intoxication in mice.!*! One suggested mechanism for these effects of L-carnitine is increased synthesis and activity of urea cycle enzymes, a process also responsive to glucocorticoids. L-Carnitine is a peripheral antagonist of thyroid hormone action in some tissues.!” It inhibits thyroid hormone entry into cell nuclei. In a controlled clinical trial, L-carnitine was shown to reverse or prevent some

symptoms of hyperthyroidism.!! Acetyl-L-carnitine may directly or indirectly reverse age-associated mitochondrial decay. It acts as a chaperone to protect macromolecules, including carnifrom structural alteration tine acetyltransferase, and/or loss of function. Acetyl-t-carnitine partially reverses age-associated loss of mitochondrial membrane potential and decline in membrane cardiolipin concentration, and protects against oxidative damage

to mitochondrial DNA."! In aging rats, it improves brain function and ambulatory activity.!

is

synthesized

from

é-N-trimethyllysine, which is derived from _ posttranslationally methylated lysine residues in proteins, In normal

humans,

the rate

of synthesis is estimated to be about 1.2 wmol/kg body weight/day."! The rate of L-carnitine biosynthesis is regulated by the availability of e-N-trimethyllysine. Thus, conditions that increase protein methylation and/or protein turnover may increase the rate of L-carnitine biosynthesis."

Bioavailability Dietary L-carnitine may be absorbed through active or passive mechanisms. Evidence from several in vivo and in vitro studies indicates that L-carnitine is actively transported from the small intestinal lumen into enterocytes.”! However, the preponderance of data suggests that intracellular L-carnitine in the intestinal mucosa does not cross serosal membranes by an active transport mechanism. Absorption of dietary L-carnitine and L-carnitine supplements appears to occur primarily by passive diffusion.?! In humans, approximately 54-87% of dietary L-carnitine is absorbed,

depending on the amount

in the diet. The

bioavailability of dietary supplements (0.64 g/day) is 15-20%.?:!°] Unabsorbed L-carnitine is degraded by micro-organisms in the large intestine. Major metabolites identified are trimethylamine oxide in urine and y-butyrobetaine in feces.?1°] Bioavailability of oral acetyl-L-carnitine has not been studied in normal healthy humans.

Distribution in Tissues, Fluids, and Cells

L-Carnitine and acylcarnitine esters are present in all tissues. In most tissues and cells, they are present in higher concentration than in the circulation. For example, in human

skeletal muscle and liver, respec-

tively, nonesterified L-carnitine is concentrated 76-fold

L-Carnitine and Acetyl-t-Carnitine

76

and 50-fold from that in serum (estimated from data

in Ref."'), Tissue Accumulation

L-Carnitine and acetyl-L-carnitine are concentrated in most tissues via the high-affinity, Na*-dependent organic cation transporter OGIN2"2. K «fone i= carnitine binding is 3-SuM; OCTN2 binds acetylL-carnitine and propionyl-L-carnitine with comparable affinity. This protein is highly expressed in heart, placenta, skeletal muscle, kidney, pancreas, testis, and

epididymis and weakly expressed in brain, lung, and liver. L-Carnitine entry into the liver occurs via a low-affinity (K, = 5mM) transporter, probably distinct from OCTN2. Several other L-carnitine transporters have been identified, including OCTN1,

OCTN3,

and

ATB°*”! Specific roles for these transporters in carnitine metabolism in humans have not been determined.

L-CARNITINE DEFICIENCIES

L-Carnitine deficiency is defined biochemically as abnormally low concentration (less than 20M) of nonesterified L-carnitine in plasma./! A concentration ratio of acylcarnitine esters/nonesterified L-carnitine of 0.4 or greater in plasma is also considered abnormal. Nutritional L-carnitine deficiency has not been shown to occur in the absence of other mitigating factors.” Primary L-carnitine deficiency occurs as a result of defects in the gene coding for the plasma membrane L-carnitine transporter OCTN2."3! Characteristic features of this disease are cardiomyopathy, hypoketotic hypoglycemia, and muscle weakness. Secondary deficiency occurs in genetic diseases of organic acid metabolism and fatty acid oxidation defects, end-stage renal disease requiring chronic hemodialysis, and chronic use of several drugs (including pivalic acid-containing prodrugs, valproic acid, cisplatin, ifosfamide, and zidovudine), and due to other genetic and iatrogenic ; factors.4-17]

Homeostasis, Renal Reabsorption, and Excretion

INDICATIONS AND USAGE Circulating L-carnitine concentrations are maintained at a fairly constant level of around 504M, predominantly through efficient reabsorption by the kidney.) At a filtered load of SOpmol/L, the efficiency of L-carnitine and acylcarnitine ester reabsorption is 90-98%. However, as the filtered load of L-carnitine increases,

as, for example,

after consumption

of a

dietary supplement or after intravenous infusion, the efficiency of reabsorption declines rapidly. Physiologically, the efficiency of L-carnitine reabsorption is sensitive to the amount in the diet and to differences in the macronutrient content of the diet. Clearance of acylcarnitine esters is often higher than that of nonesterified L-carnitine. Experimental studies have shown that in rats and humans, kidneys are able to synthesize acetyl-L-carnitine from L-carnitine and either acetoacetate or B-hydroxybutyrate, and that L-carnitine, acetyl-L-carnitine, and y-butyrobetaine (also synthesized in human kidneys) are secreted from mucosal cells into the tubular lumen.”! Because the kinetics of transport of these metabolites by the sodiumdependent

L-carnitine

transporter

are

not

different,

the relative proportions appearing in urine reflect not only those in the glomerular filtrate, but also those in the renal tubular epithelium that are secreted into the lumen. Thus, under conditions of rapid intracellular

synthesis of acylcarnitine esters or direct accumulation from the circulation, secretion of these species will lead

to a higher proportion of acylcarnitine esters in urine compared to that in the circulation. By inference, kidneys may be substantially involved in the regulation of circulating acylcarnitine ester concentrations.”

L-Carnitine, acetyl-L-carnitine, and/or propionylL-carnitine may be used for replacement therapy to restore normal carnitine concentrations and/or a normal nonesterified-to-esterified carnitine ratio. They may be used as supplements to increase the carnitine load of the body and/or increase the flux of carnitine among

compartments.

In

some

conditions,

both

replacement therapy and supplementation are appropriate. For primary and some secondary carnitine deficiencies (see above), L-carnitine is used for replacement therapy. L-Carnitine Replacement Therapy and Supplementation in End-Stage Renal Disease

Regular L-carnitine supplementation in hemodialysis patients can improve lipid metabolism, antioxidant status, and anemia requiring erythropoietin, and may reduce incidence of intradialytic muscle cramps, hypotension,

asthenia, muscle weakness,

and cardio-

myopathy."'”'®! The recommended dosage is 50 mg/kg body weight/day, to a maximum of 3 g/day. L-Carnitine and Propionyl-.-Carnitine Supplementation for Cardiac Ischemia, Congestive Heart Failure, Cardiomyopathy, and Peripheral Artery Disease Experimental studies have shown L-carnitine to be an effective antianginal agent that reduces ST segment

L-Carnitine and Acetyl-L-Carnitine

77

depression and left ventricular end-diastolic pressure during stress in patients with coronary artery disease.!'?! Cardioprotective effects of L-carnitine have been observed following aorto-coronary bypass grafting and following acute myocardial infarction. Carnitine administration initiated early after acute myocardial infarction attenuated left ventricular dilation and resulted in smaller left ventricular volumes.!!”! L-Carnitine deficiency syndromes sometimes present with dilated cardiomyopathy and are often effectively treated with t-carnitine.?"! Thus, it was suggested that cardiomyopathy progressing to congestive heart failure but not associated with inherited L-carnitine deficiency might respond to L-carnitine supplementation. A large scale clinical trial of L-carnitine supplementation versus placebo in 574 patients with heart failure produced promising results with regard to improvement in maximum duration of exercise, but other endpoints, including death and hospital admissions during the follow-up period, were not different between treatment groups.?° Use of L-carnitine supplements for congestive heart failure not associated with inherited L-carnitine deficiency remains debatable.?°7"] Peripheral arterial disease is a common manifestation of atherosclerosis and is associated with reduced arterial circulation in the lower extremities. PropionylL-carnitine supplementation (2 g/day) for 6 mo improved treadmill exercise performance and enhanced functional status in patients with claudication associated with peripheral arterial disease.?7!

L-Carnitine Supplementation for Exercise Performance and Weight Reduction

acid oxidation, altered glucose homeostasis, enhanced

production,

modification

of training

responses, and altered muscle fatigue resistance.'?*! A review of published studies has led to the conclusion that L-carnitine supplements do not improve exercise performance in healthy humans.??**! On the other hand, in conditions where the nonesterified L-carnitine

concentration of skeletal muscle may be significantly reduced,

such

as in peripheral

arterial

disease

an increment of L-carnitine will not increase the rate at which this reaction occurs. There is no scientific evidence that L-carnitine supplements facilitate weight loss in humans.

L-Carnitine and Acetyl-.-Carnitine Replacement Therapy and Supplementation for Chronic Fatigue As a dietary supplement, acetyl-L-carnitine may improve symptoms of fatigue in humans. Use of the cancer chemotherapeutic agents cisplatin and ifosfamide is associated with fatigue. In a prospective study, improvement of symptoms of fatigue was observed in 50 nonanemic patients following L-carnitine supplementation to the chemotherapeutic regimen of cisplatin

or ifosfamide.” L-Carnitine may also attenuate the nephrotoxicity associated with cisplatin treatment.?*! Chronic fatigue syndrome (CFS) in humans was found to be associated with low circulating acetyl-Lcarnitine concentration and decreased accumulation in several brain regions.'*! It has been suggested that acetyl-L-carnitine helps to maintain neuronal metabolic activity by promoting glucose and lactate uptake and utilization through its role as a precursor of glutamate in neurons.?"! In a randomized, open-label study of 30 patients with CFS, acetyl-L-carnitine and propionyl-t-carnitine showed beneficial effects on fatigue and attention concentration.2!!

Acetyl-.-Carnitine Supplementation for Depression and Cognitive Function in the Elderly

.-Carnitine supplementation has been suggested to improve exercise performance in healthy humans. Proposed mechanisms include enhanced muscle fatty acylearnitine

L-carnitine is sufficiently high for it not to be limiting for transesterification of fatty acids by CPT I. Adding

and

end-stage renal disease, L-carnitine supplementation has produced some benefit to muscle function and exercise performance.'?° Because of its role in facilitating fatty acid oxidation, L-carnitine has been suggested to aid in weight loss regimes. Two facts argue against this. First, there is no evidence that it facilitates, directly or indirectly, mobilization of fatty acids from adipose tissue. Second, in normal humans, the intracellular concentration of

Acetyl-L-carnitine appears to have specific and perhaps unique roles in brain metabolism. Animal studies and in vitro experiments suggest that this agent has promise in slowing or reversing memory and cognition decline and the decline in physical performance that normally occurs in the process of aging. In studies of the elderly, patients with depressive syndrome scored significantly lower on the Hamilton Rating Scale for Depression (modified for the elderly) following supplementation with acetyl-L-carnitine.°7] Elderly subjects with mild mental impairment had improved scores on cognitive performance tests following such supplemen-

tation.°7] A meta-analysis of the efficacy of acetylL-carnitine in mild cognitive impairment and mild Alzheimer’s disease included all identified doubleblind, placebo-controlled, prospective, parallel-group studies using treatment doses of 1.5-3.0g/day of acetyl-L-carnitine that were conducted between 1983 and 2000.4! This analysis showed a significant advantage for acetyl-L-carnitine compared to placebo, with

L-Carnitine and Acetyl-L-Carnitine

78

beneficial effects observed on both clinical scales and psychometric tests. The benefit was observed by 3mo of supplement use, and it increased over time. The typical usage recommended by vendors is 1-3 g/day.

1-Carnitine therapy.27 antiretroviral undergoing administration as part of antiretroviral therapy with either zidovudine or didanosine reduced lymphocyte apoptosis and oxidant stress compared to the antiretroviral regimens without L-carnitine.?*)

L-Carnitine Supplementation in Liver Dysfunction with Hyperammonemia Hyperammonemia occurs in some inborn errors of metabolism and as a result of drug- or toxicantinduced hepatotoxicity. Mortality and metabolic consequences of acute ammonium intoxication in mice are reduced by pharmacologic administration of L-carnitine.) The mechanism for this effect may have two components. L-Carnitine administration normalizes the redox state of the brain (perhaps by increasing the availability of B-hydroxybutyrate and/or acetyl-L-carnitine to the brain), and it increases the rate of urea synthesis in the liver, perhaps in part by activation of the glucocorticoid receptor. At least part of the protective effect is associated with flux through the carnitine acyltransferases, as analogs of L-carnitine that are competitive inhibitors of carnitine acyltransferases enhance the toxicity of acute ammonium administration.! Thus, it has been proposed that L-carnitine increases urea synthesis in the liver by facilitating fatty acid entry into mitochondria, leading to increased flux through the B-oxidation pathway, an increase of intramitochondrial reducing equivalents, and enhancement of ATP production.!! Carnitine supplementation may benefit individuals with hepatic dysfunction due to inborn errors of metabolism or chemical intoxication.

ADVERSE

EFFECTS

Transient

diarrhea,

COMPENDIAL/REGULATORY

2.

L-carnitine

.-Carnitine and Acetyl-L-Carnitine Replacement Therapy and Supplementation in Human Immunodeficiency Virus (HIV) Infection L-Carnitine and acyl-L-carnitine ester concentrations are below normal in some HIV-infected patients

Rebouche, C.J. Carnitine function and requirements during the life cycle. FASEB J. 1992,

6 (15), 3379-3386. Rebouche, C.J. Carnitine. In Modern Nutrition in Health and Disease, 10th Ed.; Shils, M.E., Shike, M., Ross, A.C., Caballero, B., Weinsier, R.L.,

administration,

concurrent with lower plasma concentration of lactate. These observations suggest that L-carnitine activates normally depressed pyruvate dehydrogenase activity in insulin-resistant patients.>! Intravenous administration of acetyl-L-carnitine increases glucose disposal in Type 2 diabetic patients.°*! Such administration appears to promote storage of glucose as glycogen, rather than increase glucose oxidation.?°

STATUS

REFERENCES

L-Carnitine infusion improves insulin sensitivity in insulin-resistant diabetic patients.°°! Glucose oxidaduring

abdominal ,

L-Carnitine is approved as a pharmaceutical by the U.S. Food and Drug Administration for treatment of primary systemic carnitine deficiency, and for acute. and chronic treatment of patients with inborn errors of metabolism that result in secondary carnitine deficiency (e.g., medium-chain acyl-CoA dehydrogenase deficiency, glutaric aciduria, Type 2 diabetes, methylmalonic aciduria, and propionic acidemia).&?! L-Carnitine is also approved as a pharmaceutical by the U.S. Food and Drug Administration for the prevention and treatment of carnitine deficiency in patients with end-stage renal disease who are undergoing dialysis.©"!

1.

is increased

vomiting,

L-carnitine.©?!

L-Carnitine and Acetyl-L-Carnitine Replacement Therapy and Supplementation in Diabetes

tion

nausea,

cramps, and/or “‘fish-odor syndrome’’ have been noted in rare cases after consumption of 2-6g of

3.

4.

5.

Cousins, R.J., Eds.; Lippincott, Williams & Wilkins: Baltimore. in press. Eaton, S. Control of mitochondrial B-oxidation flux. Prog. Lipid Res. 2002, 47 (3), 197-239. Lopaschuk, G.D. Carnitine and myocardial glucose metabolism. In Carnitine Today; De Simone, C., Famularo, G., Eds.; R.G. Landis Co.: Austin, 1997; 71-93. Alesci, S.; De Martino, M.U.; Mirani, M.; Benvenga, S.; Trimarchi, F.; Kino, T.; Chrousos,

G.P. L-Carnitine: a nutritional modulator of glucocorticoid receptor functions. FASEB J. 2003; express article 10.1096/f}.02-1024fje, published online June 17, 2003 (accessed online August 2003).

L-Carnitine and Acetyl-L-Carnitine

6.

79

O’Connor, J.-E.; Costell, M.; Grisolia, S. Protec-

tive effect of L-carnitine on hyperammonemia. FEBS Lett. 1984, /66 (2), 331-334. Benvenga, S.; Ruggeri, R.M.; Russo, A.; Lapa, D.; Campenni,

A.; Trimarchi,

F. Usefulness

21.

of

L-carnitine, a naturally occurring peripheral antagonist of thyroid hormone action, in iatrogenic hyperthyroidism: a randomized, doubleblind, placebo-controlled clinical trial. J. Clin. Endocrinol. Metab. 2001, 86 (8), 3579-3594.

pps

Hirsch, A.T.; Cooke, J.P.; Olin, J.W.; Gorbunov, G.N.; Isner, J.; Lukjanov, Y.V.; Tsitsiashvili,

Liu, J.; Atamna, H.; Kuratsune, H.; Ames, B.N.

Delaying brain mitochondrial decay and aging with mitochondrial antioxidants and metabolites. Ann. N. Y. Acad. Sci. 2002, 959, 133-166. Rebouche,

and

10.

its

C.J.; Seim, H. Carnitine

regulation

in

eeu

metabolism

microorganisms

and

mammals. Annu. Rev. Nutr. 1998, 18, 39-61. Rebouche, C.J. Kinetics, pharmacokinetics and

24.

regulation of L-carnitine and acetyl-L-carnitine metabolism. Ann. N. Y. Acad. Sci. 2004, 7033, 30-41.

tds

Rebouche, C.J.; Engel, A.G. Carnitine metabolism and deficiency syndromes. Mayo Clin. Proc. 1983, 58 (8), 533-540.

12.

Ramsay, R.R.; Gandour, R.D.; van der Lei, F.R.

3.

Molecular enzymology of carnitine transfer and transport. Biochim. Biophys. Acta 2001, 1546 (1), 21-43. Tein, I. Carnitine transport: pathophysiology and metabolism of known molecular defects. J.

23: 26.

Teak

Zi

16.

Boéhmer, T. Drug-induced carnitine deficiency. In Carnitine. Pathobiochemical Basics and Clinical

28,

aS),

Semin. Perinatol. 1999, 23 (2), 152-161.

17.

18.

Sundberg, E. The role of L-carnitine in treating anemia hyporesponsive to erythropoietin. Dial. Transplant. 2004, 33 (6), 326-333. D.; Calvani, M.; G.; Santoro, Bellinghieri, Mallamace, A.; Savica, V. Carnitine and hemo-

dialysis. Am. J. Kidney Dis. 2003, 4/ (1, Suppl. 1), S116-S122.

.

Stanley,

McCormack, carbohydrate ischaemic

Lopaschuk,

W.C.;

G.D.;

Hall,

20.

30.

J.L.;

J.G. Regulation of myocardial metabolism under normal and

conditions.

Cardiovasc.

Res.

1997,

33 (2), 243-257... Atar, D.; Spiess, M.; Mandinova, A.; Cierpka, H.; Noll, G.; Liischer, T.F. Carnitine—from cellular

Wachter, S.; Vogt, M.; Kreis, R.; Boesch, C.; Bigler, P.; Hoppeler, H.; Krahenbiihl, S. Long-

term administration of L-carnitine to humans: effect on skeletal muscle carnitine content and physical performance. Clin. Chim. Acta 2002, 318 (1-2), 51-61. Heinonen, O.J. Carnitine and physical exercise. Sports Med. 1996, 22 (2), 109-132. Brass, E.P.; Hiatt, W.R. The role of carnitine and

Graziano, F.; Bisonni, R.; Catalano, V.; Silva, R.; Rovidati, S.; Mencarini, E.; Ferraro, B.; Canestrari, F.; Baldelli, A.M.; De Gaetano, A.; Giordani, P.; Testa, E.; Lai, V. Potential role of

levocarnitine supplementation for the treatment of chemotherapy-induced fatigue in non-anemic cancer patients. Brit. J. Cancer 2002, 86 (12), 1854-1857.

Applications; Seim, H., Léster, H., Eds.; Ponte Press: Bochum, 1996; 167-176. Famularo, G.; Matricardi, F.; Nucera, E.; Santini,

G.; De Simone, C. Carnitine deficiency: primary and secondary syndromes. In Carnitine Today; De Simone, C., Famularo, G., Eds.; R.G. Landis Co.: Austin, 1997; 119-161. Scaglia, F.; Longo, N. Primary and conden alterations of neonatal carnitine metabolism.

M.S.; Zabelskaya, T.F.; Amato, A. PropionylL-carnitine improves exercise performance and functional status in patients with claudication. Am. J. Med. 2001, 7/0 (8), 616-622. Brass, E.P. Supplemental carnitine and exercise. Am. J. Clin. Nutr. 2000, 72 (2, Suppl. S), 618S6238S.

carnitine supplementation during exercise in man and in individuals with special needs. J. Am. Coll. Nutr. 1998, 17 (3), 207.

Inherited Metab. Dis. 2003, 26 (2), 147-169.

14.

mechanisms to potential clinical applications in heart disease. Eur. J. Clin. Invest. 1997, 27 (12), 973-976. Arsenian, M.A. Carnitine and its derivatives in cardiovascular disease. Prog. Cardiovasc. Dis. 1997, 40 (3), 265-286. Hiatt, W.R.; Regensteiner, J.G.; Creager, M.A.;

Salle

Chang, B.; Nishikawa, M.; Sato, E.; Utsumi, K.; Inoue, M. t-Carnitine inhibits cisplatin-induced

injury of the kidney and small intestine. Arch. Biochem. Biophys. 2002, 405 (1), 55-64. Kuratsune, H.; Yamaguti, K.; Lindh, G; Evengard, B.; Hagberg, G.; Matsumura, K.,; Iwase, M.; Onoe, H.; Takahashi, M.; Machii, T.; Kanakura, Y.; Kitani, T.; Langstro6m, B.; Watanabe, Y. Brain regions involved in fatigue sensation: reduced acetylcarnitine uptake into the brain. NeuroImage 2002, 17 (3), 1256-1265. Tanaka, M.; Nakamura, F.; Mizokawa, Matsumura, A.; Matsumura, K.; Watanabe,

S.; Y.

Role of acetyl-L-carnitine in the brain: revealed by Bioradiography. Biochem. Biophys. Res. Commun. 2003, 306 (4), 1064-1069. Vermeulen, R.C.; Scholte, H.R. Exploratory open label, randomized study of acetyl- and propionylcarnitine in chronic fatigue syndrome. Psychosom. Med. 2004, 66 (2), 276-282.

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80

a2:

L.; Calvani, M.; Ancona, L. L-Acetylcarnitine in depressed elderly subjects. A cross-over study vs. placebo. Drugs Exp. Clin. Res. 1987, XII (7), 417-423.

33.

children on antiretroviral treatment. Eur. J. Clin. Nutr. 2000, 53 (10), 1317-1322. S.; G.; Marcellin, S.; Famularo, Moretti, Boschini, A.; Santini, G.; Trinchieri, V.; Lucci, L.; Alesse, E.; De Simone, C. L-Carnitine reduces lymphocyte apoptosis and oxidant stress in HIV-1-infected subjects treated with zidovudine and didanosine. Antioxid. Redox Signal. 2002, 4 (3), 391-403. Physician’s Desk Reference, 57th Ed.; Thompson

Tempesta, E.; Casella, L.; Pirrongelli, C.; Janiri,

38.

Salvioli, G.; Neri, M. L-Acetylcarnitine treatment

of mental decline in the elderly. Drugs Exp. Clin. Res. 1994, XX (4), 169-176.

34.

35.

36.

Montgomery,

S.A.;

Thal,

L.J.;

Amrein,

R.

Meta-analysis of double blind randomized controlled clinical trials of acetyL-L-carnitine versus placebo in the treatment of mild cognitive impairment and mild Alzheimer’s disease. Clin. Psychopharmacol. 2003, 78 (2), 61-71.

39.

Mingrone, G.; Greco, A.V.; Capristo, E.; Benedetti, G.; Giancaterini, A.; De Gaetano, A.;

FURTHER

Gasbarrini, G. 1-Carnitine improves glucose disposal in type 2 diabetic patients. J. Am. Coll. Nutr. 1999, 78 (1), 77-82.

Carnitine Today; De Simone, C., Famularo, G., Eds.; R.G. Landis Co.: Austin, 1997. :

Giancaterini, A.; De Gaetano, A.; Mingrone, G.; Gniuli, D.; Liverani, E.; Capristo, E.; Greco,

A.V. Acetyl-L-carnitine infusion increases glucose disposal in type 2 diabetic patients. Metabolism 1999, 49 (6), 704-708.

31

Vilaseca, M.A.; Artuch, R.; Sierra, C.; Pineda, J.; Lopez-Vilches, M.A.; Munoz-Almagro, C.;

Fortuny, C. Low serum carnitine in HIV-infected

PDR: Montclair, NJ, 2003; 3152-3155.

Liu,

READINGS

J.; Atamna,

H.;

Kuratsune,

H.;

Ames,

B.N.

Delaying brain mitochondrial decay and aging with mitochondrial antioxidants and metabolites. Ann. N. Y. Acad. Sci. 2002, 959, 133-166. Pettigrew, J.W.; Levine, J.; McClure, R.J. Acetyl-.carnitine in mood, aging, and disease. In Diet—Brain Connections: Impact of Memory, Mood, Aging and Disease; Mattson, M.P., Ed.; Kluwer: Norwell, MA,

2002; 159-178.

B-Carotene Elizabeth J. Johnson Robert M. Russell Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, U.S.A.

INTRODUCTION B-Carotene (molecular formula C4gH/s¢) is a fat soluble plant pigment found in red, orange, and yellow vegetables and fruits. B-Carotene is converted to vitamin A (retinal, retinol, retinoic acid), when the body is in short supply. It is an antioxidant—a compound that blocks the action of activated oxygen molecules that can damage cells. Dietary intake of foods containing B-carotene has been associated with cancer prevention. However, there is not enough evidence to support this. In fact, B-carotene supplementation may increase the risk of lung cancer among people already at high risk, such as smokers.

BIOCHEMISTRY AND FUNCTIONS B-Carotene belongs to a large class of plant pigments referred to as carotenoids. It is made up of eight isoprene units that are cyclized at each end of the molecule. B-Carotene functions in plants and in photosynthetic bacteria as an accessory pigment in photosynthesis, and protects against photosensitization in animals, plants, and bacteria. In humans,

the

only known function of f-carotene is its vitamin A activity. Other possible actions in humans include antioxidant

activity,

mutagenesis

and

immunoenhancement,

transformation,

inhibition

inhibition

of

of pre-

malignant lesions, and decreased risk of some cancers and some cardiovascular events. In the skin, B-carotene

has been suggested radiation damage.

to be protective

against

solar

ABSORPTION AND METABOLISM Because it is fat soluble, B-carotene follows the same intestinal absorption pathway as dietary fat.

Release from the food matrix and dissolution in the lipid phase are the important initial steps in the absorption process. B-Carotene is thought to be absorbed by the small intestinal mucosa via a passive, diffusion process. It is taken up by the mucosa of the small intestine and packaged into triacylglycerol-rich chylomicrons, and is partly converted to vitamin A by a specific enzyme, B-carotene 15,15’-oxygenase, in the intestinal mucosa. Both B-carotene and vitamin A (primarily as retinyl esters) are incorporated into chylomicrons and secreted into lymph for transport to the liver. Additional random cleavage at several double bonds in the polyene chain of B-carotene can occur when there is not an adequate supply of antioxidants, e.g., vitamin E. However, enzymatic central cleavage plays the major role in B-carotene breakdown under normal conditions. In conditions of oxidative stress (e.g., smoking or diseases associated with oxidative stress) or when high blood or tissue concentrations of B-carotene are present, both central and random cleavage may occur (Fig. 1).""! The delivery of B-carotene to extrahepatic tissue is accomplished through the interaction of lipoprotein particles with receptors and the degradation of lipoproteins by extrahepatic enzymes such as lipoprotein lipase. B-Carotene is present in a number of human tissues, including adipose, liver, kidney, adrenal gland, and testes, and is one of the major carotenoids human diet, serum, and tissues.

in

In fasting serum, f-carotene is found primarily in low density lipoproteins (LDL), but appreciable amounts are also found in high density lipoproteins

(HDL).?+! B-Carotene, being lipophilic, is located in the core of lipoproteins, which may explain why there is little transfer among them. The concentration of B-carotene in human serum is highly variable and depends on a number of factors, including B-carotene intake, efficiency of absorption, and other components of the diet.

Elizabeth J. Johnson, Ph.D., is a Research Scientist, Jean Mayer

USDA Human Nutrition Research Center on Aging, and Assistant Professor, Tufts University, Boston, Massachusetts, U.S.A. Robert M. Russell, M.D., is Director, Jean Mayer

USDA Human Nutrition Research Center on Aging, and Professor, Tufts University, Boston, Massachusetts, U.S.A.

Encyclopedia of Dietary Supplements DOT: 10.1081 /E-EDS-120022041 Copyright © 2005 by Marcel Dekker. All rights reserved.

BIOAVAILABILITY The bioavailability of a carotenoid is considered to be the fraction of ingested carotenoid utilized for normal 81

B-Carotene

82

B-Carotene 14"

15'

SS

«12

Se

1

al

10'

8'

“ha

| eee

As

s';CHO ———>

is

——— > 1'cooH

Ma

’

14'COOH CHO

SS

Steeles

l

Retina

er

T

M

COOH

CH,OH

ELE

aes

SS

eS

Retinol

OAS

ee

RS

Retinoic Acid

Fig. 1 ®-Carotene conversion to vitamin A (retinal, retinol, retinoic acid). Central cleavage between bonds 15 and 15’ forms retinal directly. Cleavage at other bonds forms apo-carotenals (CHO). Apo-carotenals may be oxidized to apo-carotenoic acids (-COOH), which could form retinoic acid.

physiological functions or storage. Information on carotenoid bioavailability is based largely on serum levels after ingestion. The bioavailability of b-carotene from food, concentrated extracts, or synthetic products is quite variable. Several human studies have reported that the serum response of B-carotene from foods is more varied than the response from B-carotene supplements. Factors that affect B-carotene bioavailability include vehicle type (supplement vs. food; processed vs. unprocessed food) and dietary factors (amount of

Cooking and mechanical homogenization increase the bioavailability of carotenoids from foods. The mechanism by which this occurs is most likely the disruption of the food matrix to release the carotenoid from the matrix and from protein complexes. For example, the plasma response of B-carotene has been reported to be three times greater in spinach and carrots that were pureed and thermally processed than it was when these vegetables were consumed in raw,

B-carotene, fat, and fiber). ©!

Although dietary fat facilitates the absorption of B-carotene, the amount of dietary fat does not affect the postprandial increases in plasma _ f-carotene concentrations."'*! However, when B-carotene is given in the absence of fat, no detectable change in serum level occurs.!'® Studies involving daily supplementation with highdose f-carotene on plasma concentrations of other carotenoids for several years find no overall adverse effect on plasma concentrations of other carotenoids."7! The f-carotene: vitamin A (retinol) equivalency ratio of a low dose (2 weeks) may cause dependence and need for increased

doses, and an atonic colon with

impaired function.!'7! It may also lead to pseudomelanosis coli (harmless), and to an aggravation of constipation with dependence and possible need for increased dosages. Chronic abuse with diarrhea and consequent fluid and electrolyte losses (mainly hypokalemia) may cause albuminuria and hematuria, and

may result in cardiac and neuromuscular dysfunction." Anthraquinone

laxatives,

such as cascara,

should

not be administered to patients with intestinal obstruction and stenosis, atony, severe dehydration states with water and electrolyte depletion, or chronic constipation.""!7! Cascara should not be administered to patients with inflammatory intestinal diseases, such as appendicitis, Crohn’s disease, ulcerative colitis, and irritable bowel syndrome, or in children under

12yr of age."':'7] As with other stimulant laxatives, cascara is contraindicated in patients with cramps, colic, hemorrhoids, nephritis, or any undiagnosed abdominal symptoms such as pain, nausea, or vomiting?) Due to the pronounced action on the large intestine and insufficient toxicological investigations, products containing cascara should not be administered to pregnant women.!!?°l Furthermore, anthranoid metabolites

are

excreted

into

breast

milk.

Thus,

cascara should not be used during lactation, due to insufficient data available to assess the potential for pharmacological effects in the breast-fed infant.?°

USE

Contraindications and Precautions

Adverse Reactions

Products containing cascara should only be used if no effect can be obtained through a change of diet or use of bulk-forming laxatives. Patients should also be warned that certain constituents of the drug are

In single doses, cramp-like discomfort of the gastrointestinal tract may occur, which may require a reduction of dosage. Overdose can lead to colicky abdominal spasms and pain, as well as the formation

Cascara Sagrada (Rhamnus purshiana)

92

of thin, watery stools. Long-term laxative abuse may lead to electrolyte disturbances (hypokalemia, hypocalcemia), metabolic acidosis, malabsorption, weight loss, albuminuria, and hematuria.?!77] Weakness

and

orthostatic hypotension may be exacerbated in elderly patients when stimulant laxatives are used repeatedly. Secondary aldosteronism may occur due to renal tubular damage after aggravated use. Steatorrhea and protein-losing gastroenteropathy with hypoalbuminemia have also been reported in laxative abuse.3] Melanotic pigmentation of the colonic mucosa (pseudomelanosis coli) has been observed in individuals taking anthraquinone laxatives for extended time periods.''*??-4] The pigmentation is clinically harmless and usually reversible within 4-12mo after

the drug is discontinued.??7***! Conflicting data exist on other toxic effects such as intestinal—neuronal damage after long-term use.”7! Use of the fresh drug may cause severe vomiting, with possible spasms."*) Cases of allergic respiratory diseases after occupational exposure to cascara have been reported.” Cascara sagrada is an etiologic agent of IgEmediated occupational asthma and rhinitis. One case of cholestatic hepatitis, complicated by portal hypertension, has been attributed to the ingestion of cascara in one patient who was also known to abuse alcohol and take a number of other prescription medications.?®!

Dosage Forms and Dose Cascara sagrada is available as extracts, fluidextracts,

and tablets.”! The average daily dose (taken at bedtime, or one-half dose in the morning and at bedtime) of standardized preparations is 20-30 mg of hydroxyanthracene derivatives calculated as cascaroside A (dried aged bark, 0.25-1 g)."] Do not exceed recommended dose and do not use for more than 1—2 weeks continuously.

REFERENCES 1.

E.N.; Wirth,

E.H.

j

Gathercoal,

3.

Tyler, V.E.; Bradley, L.R.; Robbers, J.E..Pharmacognosy, 9th Ed.; Lea and Febiger: Philadelphia,

Pharmacognosy;

Lea and Febiger: Philadelphia, 1947; 411-416.

4.

5.

._

1988; 62-63. Youngken, H.W. Rhamnaceae (buckthorne family). Textbook of Pharmacognosy; The Blakiston Company: Philadelphia, 1950; 543-549. Leung, A.Y. Cascara sagrada—new standards are needed. Drug Cosmetic Ind. 1977, 12, 42.

10.

Rhamnus purshiana DC. African Pharmacopoeia, \st Ed.; Organization of African Unity, Scientific, Technical & Research Commission: Lagos, Nigeria, 1985; 192-194. European Pharmacopoeia, 2nd Ed.; Council of Europe: Strasbourg, 2002; 549. Pharmacopée Francaise; Adrapharm: Paris, 1996; 1-5. The United States Pharmacopeia 25; The United States Pharmacopeia Convention Inc.: Rockville, MD, 2002; 281-282. Bruneton, J. Pharmacognosy, Phytochemistry,

11.

Medicinal Plants; Lavoisier: Paris, 1995; 360-361. Cascara Sagrada Bark; Blumenthal, M. et al.,

7. 8. 9.

of safety and effectiveness (GRAS).”°! The FDA has

thranol, cascara fluidextract aromatic, cascara sagrada

bark, cascara sagrada extract, and cascara sagrada fluidextract) in OTC drug products are not generally recognized as safe and effective or are misbranded. According to the FDA, products containing aloe and cascara sagrada ingredients must be reformulated or discontinued; the stimulant laxatives must therefore be deleted or replaced. Reformulated products will also need to be relabeled. This final rule is part of FDA’s ongoing OTC drug product review. These decisions were based on lack of data and information and the failure of interested persons to submit any new data from carcinogenicity studies.??!

Monographs Publications:

Geneva, Switzerland, 2001; Vol. 2.

6.

now issued a final rule stating that the stimulant laxative ingredients aloe (including aloe extract and aloe flower extract) and cascara sagrada (including casan-

Cortex Rhamni Purshianae; WHO on Selected Medicinal Plants; WHO 2.

Current Regulatory Status Prior to June 1998, cascara sagrada was recognized by the Food and Drug Administration (FDA) as a category I over-the-counter (OTC) preparation (monograph). The agency has reclassified cascara ingredients to category II (nonmonograph) and is adding them to the list of stimulant laxative ingredients for which the data are inadequate to establish general recognition

Farnsworth, N.R.; Fong, H.H.S.; Mahady, GB.

12.

Eds.; The Complete German Commission E Council: Botanical Monographs; American Austin, TX, 1998. Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 9th ed.;. Hardman, J., Ed.; McGraw-Hill:

13.

14.

New York, 2001.

Bradley, P.R. Cascara sagrada. British Herbal Medicine, British Herbal Compendium: Dorset, 1992: Vol. 1652.54. Westendorf, J. Anthranoid derivatives—Rhamnus species. In Adverse Effects of Herbal Drugs; De Smet, P.A.G.M. et al., Eds.; Springer-Verlag: Heidelberg, 1993; Vol. 2, 70.

Cascara Sagrada (Rhamnus purshiana)

15.

16.

Li;

18.

de Witte, P. Metabolism and pharmacokinetics of the anthranoids. Pharmacology 1993, 47 (Suppl. 1), 86-97. National Toxicology Program. NTP Toxicology and Carcinogenesis Studies of EMODIN (CAS NO. 518-82-1) Feed Studies in F344/N Rats and B6C3F1 Mice. National Toxicology Program Technical Reports Series 2001; 493, 1-278. Mereto, E.; Ghia, M.; Brambilla, G. Evaluation of the potential carcinogenic activity of senna and cascara glycosides for the rat colon. Cancer

93

Zoe

tious

22.

et al. Anthraquinone

human cancer. 216-217.

26.

data study.

Borrelli, F.; Mereto, E.; Capasso, F.; Orsi, P.; Sini, D.; Izzo, A.A.; Massa, B.; Boggio, M.; Mascolo, N.

140-143. Dae

28.

1985, 80, 912-923.

1998.

Muller-Lissner, S.A. Adverse effects of laxatives:

facts and fiction. Pharmacology 1993, 47 (Suppl. 1), 138-145.

68,

Postgrad.

Med.

laxatives

and

J. 1989,

65,

from Z.

the

Melbourne

Gasteroenterol.

colorectal 1993,

31,

Giavina-Bianchi, P.F., Jr.; Castro, F.F.; Machado,

M.L.; Duarte, A.J. Occupational respiratory allergic disease induced by Passiflora alata and Rhamnus_ purshiana. Ann. Allergy Asthma

Physician’ Desk Reference; Medical Economics: Godding, E.W. Therapeutics of laxative agents with special reference to the anthraquinones. Pharmacology 1976, /4 (Suppl. 1), 78-108.

1968,

Kune, G.A. Laxative use not a risk for colorectal cancer

Montvale,

21.

Med.

Patel, P.M.

Lett. 1996, 701, 79-83.

Am. J. Gastroenterol.

Intern.

25.

cancer:

Lewis, J.H.; Weingold, A.B. The use of gastrointestinal drugs during pregnancy and lactation.

Ann.

24.

colon. Life Sci. 2001, 69, 1871-1877.

20.

diarrhoea.

839-852. Loew, D. Pseudomelanosis coli durch Anthranoide. Z. Phytother. 1994, 76, 312-318.

Effect of bisacodyl and cascara on growth of aberrant crypt foci and malignant tumors in the rat

19:

Heizer, W.D. Protein-losing gastroenteropathy and malabsorption associated with facti-

29%

Immunol. 1997, 79, 449-454. Nadir, A.; Reddy, D.; Van Thiel, D.H. Cascara

sagrada-induced intrahepatic cholestasis causing portal hypertension: case report and review of herbal hepatotoxicity. Am. J. Gastroenterol. 2000, 95, 3634-3637. Food and Drug Administration, Department of Health and Human Services. Status of certain additional over-the-counter drug category II and III active ingredients. Final rule. Federal Register 2002, 67, 31125-31127.

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.

a

i. '*! cyclic mastalgia,"7"! as well

is a small

as to treat hormonally induced acne?! In addition, vitex has been traditionally used to treat fibroid cysts and infertility, stop miscarriages caused by progesterone

shrubby tree, approximately 1-6 m in height and native

to the Mediterranean region and Asia."'7! The tree is also widely cultivated in warm temperate regions of the world. The name chasteberry is thought to be derived from the traditional belief that the plant promoted chastity.2! The fruit of V. agnus castus (VAC) was used in ancient Greece and Rome, as well as by the monks of the Middle Ages to suppress sexual desire.4! In the past, extracts of VAC have been used

for the treatment of gynecological disorders, such as endometrial hyperplasia, hypermenorrhea, and secondary amenorrhea, as well as endocrine-dependent dermatoses (dermatitis dysmenorrhea symmetrica, acne vulgaris, eczema, and acne rosacea). !°*]

Today, extracts of the dried ripe fruits of VAC are regulated in the United States as dietary supplements under the 1994 Dietary Supplement Health and Education Act. They are widely used as a botanical dietary supplement for the management of female

insufficiency,?"! and treat indigestion.2!

CHEMISTRY Commercial products of VAC are prepared from the dried, ripe fruit, containing not less than 0.4% (v/w) of volatile oil and at least an 8% water-soluble extractive.7] To date, although the active constituents of VAC remain unknown, the European Pharmacopoeia recommends a minimum content of 0.08% casticin in the dried plant material.?*! Two compounds are currently used as marker compounds for quality control, the iridoid glycoside agnuside and the flavonol casticin.?4) Most vitex preparations used in European medicine are nonstandardized fluid extracts, tinctures,

and/or native dry extracts. The “native’’ or “‘total’’ extract is an approximate 10:1 (w/w) drug-to-extract

ratio containing 0.6-1.0% casticin.”! Gail B. Mahady, Ph.D., is Assistant Professor

at the Department

of Pharmacy Practice, UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Brigit Dietz, Ph.D., is Research Associate at the Department of

Medicinal Botanical University Joanna Medicinal Botanical University

Chemistry and Pharmacognosy, UIC/NIH Center for Dietary Supplements Research, College of Pharmacy, of Illinois at Chicago, Chicago, Illinois, U.S.A. Michel is Research Assistant at the Department of Chemistry and Pharmacognosy, UIC/NIH Center for Dietary Supplements Research, College of Pharmacy, of Illinois at Chicago, Chicago, Illinois, U.S.A.

Jan Engle, Pharm.

D., is Clinical Professor at the Department

of Pharmacy Practice, UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Rosalie Sagraves, Pharm. D., is Dean at the Department of Pharmacy Practice, UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago,

Chicago,

Illinois, U.S.A.

Encyclopedia of Dietary Supplements DOKI: 10.1081 /E-EDS-120022132 Copyright © 2005 by Marcel Dekker. All rights reserved.

The ripe, dried vitex fruit yields 0.4-0.7% (v/w) essential oil, depending on distillation time and comminution size. The oil is mainly composed of bornyl acetate,

1,8-cineole,

limonene,

o-

and _ f-pinene,

6-caryophyllene, and a-terpinyl acetate.?>! Flavonoids, iridoids, and diterpenes represent major groups of

secondary

constituents

also found

in the fruit.2!

Casticin (up to 0.2%) is considered to be the major flavonoid, with chrysoplenetin, chrysosplenol D, cynaroside, 5-hydroxy-3,4’,6,7-tetramethoxyflavone, 6-hydroxykaempferol, isorhamnetin, luteolin, and luteolin 6-C-glycoside derivatives being other com-

pounds of this class.?°-*”! Major iridoids found include agnuside (p-hydroxybenzoylaucubin, 0.0014%) and aucubin (0.0013%). Diterpene constituents include vitexilactone (0.001-0.004%), 68,7B-diacetoxy-13hydroxylabda-8,14-diene,

rotundifuran,

vitexlabdines 95

Chasteberry (Vitex agnus castus)

vitexilactone

vitexilabdine A

vitexlactam A

Fig.

1

Compounds

from

Vitex

agnus f

castus fruits.

A-D,

and vitexlactam

A.?56]

The

structures

of the

above-mentioned components are presented in Fig. 1.

clinical trials have assessed the safety and efficacy of VAC extracts for the symptomatic treatment of

PMS,!!!-13.15.28.31-37] Of these investigations, only three were

THERAPEUTIC

Extracts from VAC fruits are primarily used for the symptomatic management of corpus luteum insufficiency,

hyperprolactinemia,”"!°!

randomized,

controlled

trials

and

double blinded.?*7°?71

INDICATIONS

PMS, !21-14.25,28]

cyclic mastalgia."”"'*! A few clinical studies have also indicated that VAC may also be a potential treatment for infertility due to hyperprolactinemia and luteal-

phase defect,?! insufficient lactation, and to prevent miscarriages due to progesterone insufficiency.?!

two

were

:

The Schellenberg study was a randomized, placebocontrolled trial.2° In this study, patients with PMS symptoms were treated with either a VAC extract (n = 86; one tablet daily) or a placebo (n = 84) for three consecutive menstrual cycles. A PMS diagnosis was made according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-III). The main efficacy variable was change from baseline to endpoint (end of cycle 3) in the patient’s self-assessment (PSA) of six PMS symptoms (irritability, mood alteration, anger, headache, breast fullness, and other indications

CLINICAL STUDIES Since the 1950s, over 32 human or clinical studies have

assessed the safety and efficacy of various VAC extracts and tinctures (53-70% ethanol) for the treatment of acne, corpus luteum insufficiency, cyclic breast

pain, hyperprolactinemia, menopausal symptoms, increasing lactation, PMS, uterine bleeding disorders, and miscellaneous menstrual irregularities. Most of these investigations are open, uncontrolled studies assessing the effects of VAC on menstrual cycleirregularities or PMS. The results from randomized, controlled clinical trials are also published.

including bloating). The secondary efficacy variable used was a change in the Clinical Global Impressions (CGI) score for the severity of condition, global improvement, and risk/benefit. Mean improvement in PSA was significantly greater in the treatment group compared with placebo (P < 0.001). CGI scores for each of the three factors also revealed significant superiority of the treatment relative to placebo (P < 0.001). The observed response rate (>50% reduction in symptoms) was 52% and 24% for the treatment and placebo groups, respectively. Adverse events reported included treatment (n = 4): acne, multiple abscesses, intermenstrual

bleeding, urticaria;

placebo (n = 3): acne, early menstrual

period, and

gastric upset.2° Premenstrual Syndrome

A

PMS refers to the regular occurrence of affective symptoms, such as depressive moods, irritability, anxiety, confusion, and social withdrawal,

as well as

somatic symptoms including breast tenderness or heaviness and breast pain (mastalgia), abdominal bloating, cravings, fatigue, and headache.?" The syndrome affects approximately 30-40% of menstruating women and is one of the most frequent complaints noted in gynecology practice.'?®! Approximately 12

randomized,

double-blind,

placebo-controlled

trial involving 217 women with self-diagnosed PMS according to a modified version of the Menstrual Distress Questionnaire (MDQ), a rating scale covering most of the important PMS symptoms, assessed the efficacy of the fruit in treating the syndrome.8” Subjects were treated with either a powder of VAC (300mg tablets; two tablets three times daily; n = 105) or a soy-based placebo (n = 112) fora period of 3 mo, after which they all completed the modified MDQ again. Other than a statistically significant difference in effect

Chasteberry (Vitex agnus castus)

oF

between the active powder and the soy placebo for the symptom of “feeling jittery and restless’’ (P = 0.05), no other significant results were reported.2” Unfortunately, soy was a poor choice for a placebo in this study, as it is not considered to be biologically inert. A multicenter, randomized, double-blind, controlled

clinical trial compared the activity of a dried ethanol extract of the fruit with that of pyridoxine (vitamin Be) treatment of women with PMS.”*! The intent-totreat population included 127 women: 61 subjects were given one capsule of extract plus one placebo capsule daily for three cycles, while 66 were given one capsule of placebo twice daily on days 1-15 of their cycle, followed by one capsule (100mg) of pyridoxine twice daily on days 16-35. Therapeutic response was assessed using the Premenstrual Tension Syndrome (PMTS) scale, the CGI scale, and by recording six characteristic symptoms of PMS (breast tenderness, edema,

inner

tension,

headache,

constipation,

and

depression). Therapeutic efficacy was assessed by both patients and physicians, at the end of the trial. Initial mean PMTS scores were higher in the chaste tree group (15.2) compared to the pyridoxine group (11.9). By the end of therapy, the mean absolute change in PMTS score in each group was 5.1, representing a reduction

of 10.1

and

6.8, for the chaste

tree

and

pyridoxine groups, respectively (P < 0.038, both groups, 95% CI —6.4261 to —0.1670). Therefore, no difference could be found between the two treatment groups. The CGI scale showed that 77.1% (chasteberry) and 60.6% (pyridoxine) of patients showed improvement. Adverse events were rare, but included gastrointestinal complaints, skin reactions,

and transient headache.*) Six postmarketing studies assessed the safety and efficacy of various extracts of the fruit in 8391 female patients with menstrual abnormalities or PMS symptoms. !11:12,15.32,33,35] Three open (uncontrolled) studies also assessed efficacy."*7!*4] The dose used ranged from 40 to 42 drops or one capsule daily, for lday to 9yr and the outcomes measured included the physician’s assesment and PSA. Elimination of symptoms was observed in 29-42% of patients improvement in 51-59%, and no change in 1-10%. Adverse events were reported in 1-5% of patients but were generally not stated to be serious. The difficulty with these studies includes the lack of a control group, besides most of them not distinguishing between PMS and other menstrual disorders.!'*?!*4I An open (uncontrolled) clinical trial involving 50 women (43 completed) with late-luteal phase dysphoric disorder (DSM-III) assessed the effect of an ethanol fruit extract on the management of PMS.8!! Thirteen of the subjects were concurrently taking oral contra_ceptives. After 2mo of baseline observation, one tablet

of the

extract

was

administered

daily

for

three

cycles, followed by a post-treatment phase that lasted three cycles. Treatment effectiveness was evaluated using both the MDQ and the visual analogue scale (VAS). The MDQ was filled out by patients at the end of the first cycle and during cycles 3 and 6. The VAS was completed twice per cycle, once in the late-luteal phase when symptoms peaked and the other after menstruation during the follicular phase. By the end of the third cycle, the MDQ scores were reduced by 42.5% (P < 0.001), with a 50% reduction in the score in 20/43 patients. By the end of the post-treatment period, the scores remained approximately 20% below baseline (P < 0.001). The main symptoms that improved following treatment were breast tenderness, behavioral changes, negative feelings, and edema. The average late-luteal phase VAS score was reduced by 47.2% during the 3mo treatment phase (P < 0.01), and remained at 21.7% below baseline (P < 0.001) during the post-treatment phase. By contrast, the follicular phase score did not significantly change. The number of days with PMS symptoms was reduced from 7.5 to 6days (P < 0.001), and the concomitant use of oral contraceptives had no significant effect on any of the parameters investigated. Twenty patients (47%) reported 37 adverse events during the treatment and post-treatment periods.°"! An open (uncontrolled) study involving 36 women

with PMS, assessed the effect of a 58% ethanol extract of the fruit for the management of PMS symptoms." The subjects were treated with 40 drops of the extract daily over three cycles and the outcomes measured were a reduction in physical symptoms such as headache, swollen breasts, breast tenderness, bloating,

fatigue, and psychological changes such as increased appetite, sugar craving, nervousness and restlessness, anxiety, irritability, lack of concentration, depression, crying spells, mood changes, and aggressiveness. The duration of the luteal phase was also determined. After 3mo

of treatment,

69%

of women

had a reduction

in physical symptoms, where 80% showed a decrease in psychological symptoms (P < 0.05). The duration of the luteal phase lengthened from 5.4 to 11.4 days."

Mastalgia Breast pain (mastalgia) is a common complaint and is usually classified as cyclical (associated with the menstrual cycle) or noncyclical (not related to the menstrual cycle). Mild premenstrual breast discomfort, lasting for 1-4 days prior to menstruation that resolves upon initiation, is considered cyclic mastalgia, and is a symptom of PMS. In addition to the experiments reported above, a number of open studies?®“4) and three randomized controlled clinical trials!'”!?*>! have

Chasteberry (Vitex agnus castus)

98

assessed the safety and efficacy of VAC extracts for the treatment of cyclic mastalgia. A

randomized,

double-blind,

placebo-controlled

clinical trial involving 104 women with cyclic breast pain (for at least three cycles) assessed the efficacy of a VAC tincture (10g tincture containing 2g of crude drug in 53% ethanol VAC) in treating it[491 The patients were treated with placebo, VAC tincture (30 drops twice daily), or VAC tablets (one tablet twice daily) for three cycles. The subjects assessed the intensity of breast pain once per cycle using a VAS, and recorded the presence of menstrual bleeding and the intensity of pain in a diary. Prolactin levels were measured during the premenstrual week of cycles | and 3. At the end of the third treatment cycle, a significant reduction in breast pain was observed in the treated patients as compared with placebo (VAC solution, P = 0.006; VAC tablets, P = 0.007). A significant decrease in prolactin levels (P = 0.039) was also noted in the treatment groups as compared with placebo.>! A second randomized, placebo-controlled, doubleblind study with a similar design compared VAC solution (30 drops twice daily for three cycles) with placebo in the treatment of 100 women (50 per group) who had breast pain at least 5 days prior to menses in the last cycle before the study.!'”! The treatment phase lasted three menstrual cycles (2 x 30 drops/ day = 1.8ml of VAC or placebo). Mastalgia for at least Sdays of the cycle before the treatment was the strict inclusion condition. For assessment of the efficacy, VAS was used. Altogether 97 patients were included in the statistical analysis (VAC: n = 48, placebo: n = 49). Intensity of breast pain diminished quicker in the VAC group. This study design and duration were similar to that of Wuttke et al.!4! The results of this experiment showed a decrease in the VAS scores in both the treatment and placebo groups. However, as compared with the placebo, the treatment group had significantly lower VAS values at the end of each cycle (P =

0.018, 0.006, and 0.064 for cycles 1, 2,

and 3, respectively). In

a

randomized,

effects of VAC

placebo-controlled

trial,

the

solution and placebo (double-blind)

were compared with that of gestagen (Lynestrenol®) in 160 women with mastalgia.!'?! A complete remission or improvement of symptoms was reported in 82.1%, 74.5%, and 36.8% of the patients in the Lynestrol, VAC, and placebo groups, respectively. The difference in effect between treatment groups and placebo was significant (P < 0.01), but no significant dicrepancy

was found between the two treatment groups.!!! Open studies have been used to assess the effectiveness of VAC solution for the treatment of over 1700 women with mastalgia.°***! All these investigations assessed the efficacy of one VAC solution, at a dose of 45-75 drops per day for 1-6 cycles. Two of

these studies compared VAC treatment with Lynestrenol (5mg daily on days 12-24 of each cycle). Elimination of symptoms was observed in 46-81.5% of the treated

women,

improvement

in 12-39.6%,

and

no

effect in 6.5-29%. Collective reported adverse events from these studies included circulatory disturbances,

acne, and weight gain.°*“4]

Menstrual Cycle Irregularity and Infertility Since 1954, at least 17 investigations have assessed the efficacy of VAC extracts for the treatment of menstrual cycle disorders including amenorrhea, oligomenorrhea, polymenorrhea, corpus luteum insufficiency, and infertility.7! Two double-blind placebo-controlled clinical trials and several observational studies have investigated the effect of various fruit extracts on corpus luteal-phase dysfunction and infertility."°7?**] The products tested were ethanol (53-70%: extracts ethanol), and the dose administered was 20 drops twice daily, 15 drops three times daily, 30 drops twice daily, or one to two tablets or capsules daily. In

the

first

randomized,

double-blind,

placebo-

controlled trial, the efficacy of a dried VAC fruit extract was assessed in infertile women.!!®! The objective of this study was to determine if elevated pituitary prolactin levels could be reduced by treatment with VAC, and if the deficits observed in the luteal-phase length and luteal-phase progesterone synthesis could be normalized. Blood was obtained for hormone analysis on days 5, 8, and 20 of the menstrual cycle, both before and after 3mo of VAC therapy. Latent hyperprolactinemia was analyzed by monitoring prolactin release 15 and 30min after intravenous administration of 200g of thyroid hormone. Thirtyseven cases (placebo: n = 20, treatment: n = 17) were included in the statistical analysis. After 3mo of treatment, prolactin release was reduced, a significant

increase in the length of the luteal phase (10.5 days; P < 0.05) was observed, and deficits in luteal progesterone synthesis were decreased. These changes only occurred in the treatment group and were not observed in the placebo group. All other hormonal parameters did not change with the exception of 17f-estradiol, which was observed to increase during the luteal phase in the treatment group. The overall length of the menstrual cycles did not change, suggesting that there was a corresponding shortening of the follicular phase. Two women in the extract group became pregnant by the end of the study. No adverse events were reported.!!°! In a second randomized, double-blind, placebocontrolled trial, the efficacy of a VAC fruit extract

was assessed in 96 infertile women.”*! The outcome criteria measured included pregnancy or menstrual bleeding in women with secondary amenorrhea or

Chasteberry (Vitex agnus castus)

99

improved luteal hormone concentrations. The subjects were administered 30 drops of the extract twice daily for 3mo. Sixty-six patients completed the study, and positive outcomes were observed in 47% of women overall, with 61% in the treatment group and 38% in the placebo group, although the results did not reach statistical significance (P = 0.069). In women with amenorrhea or luteal-phase dysfunction, pregnancy resulted twice as often in the treatment group (15%) versus the placebo group (7%); however, no statistical analysis was reported.??! In open (uncontrolled) trials involving 48 (45 completed) infertile women (due to luteal-phase dysfunction), the efficacy of a VAC fruit extract for the normalization of progesterone concentrations was determined.“°! Inclusion criteria were normal prolactin levels (below 20 ng/ml), normal results in prolactin and thyroid stimulating stimulation (TSH) tests, and an abnormally low serum progesterone level below

12ng/ml on the 20th day of the cycle. Treatment consisted of a fruit extract, 40 drops daily, without any other medication for 3mo. Forty-five patients completed the studies (three were excluded because of concurrent hormone use). The outcome of therapy was assessed by the normalization of the midluteal progesterone concentration and correction (lengthening) of any pre-existing shortening of the phases of the cycle. Treatment was deemed successful in 39 out of the 45 women. Seven subjects became pregnant. In 25 patients, serum progesterone was restored to normal (>12ng/ml), and in seven cases there was a trend toward normalization of progesterone levels. However, no statistical analysis was performed on_ the resultant data.!** Two larger postmarketing trials, involving 479 women, assessed the safety and efficacy of a VAC fruit extract for the treatment of oligomenorrhea or polymenorrhea.47! The subjects were treated with 30 drops of the extract twice daily and the outcome measured was the bleeding-free interval. A lengthening of the

Endocrine-Dependent Dermatoses

Two uncontrolled clinical studies and one observational report have assessed the effects of a VAC fruit extract on acne caused by a hormone imbalance.!**! In one open study, 118 cases of acne were treated with a VAC extract (20 drops twice daily for 4-6 weeks, then 15 drops twice daily for 1-2 yr) and compared with conventional acne treatments.!! Patients treated with the fruit extract reported a more rapid healing rate after 6weeks 70% of subjects complete healing.

MECHANISM

and after 3mo of therapy, while taking the VAC extract stated

OF ACTION

Several potential mechanisms of action have been proposed to explain the activity of VAC extracts,

including inhibition of prolactin dopaminergic,°!°7] and estrogenic

secretion,4*°"! effects.6°°4+°4!

Extracts have been shown to act as a dopamine agonist in vitro and in vivo. The binding of an ethanol VAC extract and various fractions of the extract to the dopamine D, and other receptors was evaluated by both radio-ligand binding studies and by superfusion experiments.>7! The extract bound to the dopamine D, and opioid (u and « subtype) receptors with a median inhibitory concentration ranges between 20 and 70 ug/ml. Binding was not observed for the histamine H,, benzodiazepine and OFQ receptors, or the serotonin transporter. Two diterpenes, isolated from a hexane fraction of the extract, rotundifuran and 68,7B-diacetoxy-13-hydroxy-labda-8,14-diene (Fig. 2),

ad

wal

rotundifuran

bleeding-free interval was observed for 35days in 187/287 women receiving treatment for oligomenorthea and 26days in 139/192 patients being treated for polymenorrhea.4/!

6,7-diacetoxy-13-hydroxylabda-8, 14-diene

6-a.-acetoxy-13-hydroxy labda-8, 14-diene

Fig. 2 2-a«-Acetoxy-13-hydroxylabdadiene, rotundifuran, and 6B,7B-diacetoxy-13-hydroxy-labda-8,14-diene.

Chasteberry (Vitex agnus castus)

100

exhibited inhibitory actions on dopamine D) receptor binding with a median inhibitory concentration of 45 and 79 pg/ml, respectively.27>7! While lipophilic fractions of the extract bound to the p and « opioid receptors, binding to delta opioid receptors was inhibited primarily by an aqueous fraction of the extract. In superfusion experiments, the aqueous fraction of a methanol extract inhibited the release of acetylcholine in a concentration-dependent

manner.

In addition,

the D> receptor antagonist spiperone antagonized the effect of the extract suggesting a dopaminergic action mediated by D> receptor activation. A labdane diterpene, «-acetoxy-13-hydroxylabdadiene (Fig. 2), isolated from a fruit extract, was found to displace '2°T_sulpiride from recombinant human D), receptor binding sites in a dose-dependent manner.” This group also demonstrated that rotundifuran, at a concentration of 100uM, significantly (P < 0.05) inhibited the secretion of prolactin from cultured rat pituitary cells. Several groups have demonstrated that extracts bind to the estrogen receptor and have weak estrogenic effects, suggesting that chasteberry may also affect the estrogen /progesterone balance.©°°4°°! A methanol extract

of the fruit bound

to both

ERa

and

ERB,

induced the expression of estrogen-dependent genes, progesterone receptor (PR), and presenelin-2 (pS2) in Ishikawa cells.°° Significant binding affinity for both ERo and ERB was observed, with a median inhibitory concentration of 46.3 and 64.0ug/ml, respectively. However, the binding affinity of the extract for ERa and ERB was not significantly different.>°! Based on bioassay-guided isolation, the “estrogenic’’ component from the fruit extract was identified as linoleic acid (LA), which also bound to ERa and ERB.©*! Similar to the extract, LA also induced the expression of the PR mRNA in Ishikawa cells, at a concentration of

1 ug/ml, indicating that binding produced a biological estrogenic effect in vitro. In addition, low concentrations of the extract or LA (10pg/ml) upregulate the expression of ERB mRNA in the ER+ hormonedependent T47D:A18

urticaria, and weight gain.) Minor side effects include

fatigue, hair loss, increased intraocular pressure, palpitations, polyurea, sweating, and vaginitis.?-*°! One case of multiple follicular development was reported in a female patient after self-medication with a VACcontaining product for infertility.©7! Although the potential estrogenic effects of VAC extracts are weak,?°°*! its use during pregnancy or in women with estrogen-dependent breast cancer should not be recommended. In addition, patients with ' a feeling of tension and swelling of the breasts or other menstrual disturbances should consult a healthcare provider for medical diagnosis. Although there are no drug interactions reported, the potential dopaminergic effects of VAC extracts may reduce the efficacy of dopamine-receptor antagonists.?+°3] Furthermore, because of possible hormonal effects, VAC may interfere with the effectiveness of oral contraceptives and hormone therapy.)

PRODUCTS AND DOSAGE There is a wide range of VAC extracts and products available to consumers. The following examples are a general list of products used in clinical trials and listed in reference texts. This list is not complete, and is not

intended as a recommendation

the products are not recommended for children. e

e

e

cell line, a further indication of

estrogenic activity.>"

ADVERSE EFFECTS In general, VAC products and extracts appear to be very well tolerated and there have been few accounts of adverse reactions (ARs). A review of 30 human studies, involving 11,506 subjects, reported a total of 246 adverse events, thus representing an AR

flow,

nausea,

skin

e

rate of

approximately 2%."! The major ARs included acne, cycle changes, dizziness, gastrointestinal distress, menstrual

Dry native extracts, 8.3-12.5:1 (w/w), ca. 1.0% casticin: one tablet, containing 2.6-4.2mg native extract. The tablets should be swailowed whole with some liquid each morning. Dry native extract, 9.58-11.5:1 (w/w): one tablet containing 3.54.2 mg native extract each morning with some liquid.?®! Dry native extract, 6.0-12.0: 1 (w/w), ca. 0.6% casticin: PMS: one tablet containing 20mg native extract daily with water upon awaking or just before bedtime, before from meals.

e

increased

of one product over

another. The dose as listed is intended for adults, and

reactions,

e

Fluid extract: 1:1 (g/ml), 70% alcohol (v/v): 0.5-1.0 ml. Fluid extract: 1:2 (g/ml): 1.2-4.0 ml. Tinctures, alcohol 58vol% (100g of aqueousalcoholic solution contains 9g of 1:5 tincture): 40 drops, one time daily with some liquid each morning. Tinctures, ethanol 19% v/v (100g of aqueousalcoholic solution contains 0.192-0.288 g extractive corresponding to 2.4g dried fruit): 40 drops, once daily. Hydroalcoholic extracts (50-70% v/v): corresponding to 30-40 mg dried fruit?"

Chasteberry (Vitex agnus castus)

101

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42:

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14.

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School of Public Health and School of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A.

INTRODUCTION

neurotransmission,

Choline, an essential nutrient for humans, is consumed

in many foods. It is a constituent of all cell membranes and is necessary for growth and development. Also, as the major precursor of betaine, it is used by the kidney to maintain water balance and by the liver as a source of methyl groups for the removal of homocysteine in methionine

formation.

Moreover,

choline

is used to

produce the important neurotransmitter (nerve messenger chemical) acetylcholine, which is involved in memory and other nervous system functions. Maternal diets deficient in choline during the second half of pregnancy in rodents caused decreased neuronal cell growth and increased cell death in the memory center of fetal brains. This resulted in lifelong biochemical, structural, and electrophysiological changes in brains, and permanent behavioral (memory) modifications in the offspring. Dietary deficiency of choline in rodents causes development of liver cancer in the absence of any known carcinogen. In humans, dietary deficiency of choline is associated with fatty liver and liver damage. With the recent availability of a food choline

transmembrane

signaling,

and

lipid-cholesterol transport and metabolism." Choline can be acetylated, phosphorylated, oxidized, or hydrolyzed. There are several comprehensive reviews of the metabolism and functions of choline! Cells necessarily require choline, and die by apoptosis when deprived of this nutrient. Humans derive choline from foods, and from the de novo biosynthesis of choline moiety via the methylation of phosphatidylethanolamine using S-adenosylmethionine as the methyl donor (most active in the liver). This ability to form choline means that some of the demand for choline can, in part, be met using methyl groups derived from 1-carbon metabolism (via methyl-folate and methionine). Several vitamins (folate, vitamin Bj, vitamin Be, and riboflavin) and the amino acid methionine interact with choline in 1-carbon metabolism (Fig. 2). There has been renewed interest in these pathways during the past several years, engendered by recent insights that indicate that modest dietary inadequacies of the above-mentioned nutrients, of a degree insufficient to cause classical deficiency syndromes, can still contribute to important diseases such as neural

content database, and with new recommended adequate intakes of choline in the human diet, further

tube defects, cardiovascular disease, and cancer.”!

epidemiological and clinical studies on this nutrient can be expected.

results in compensatory changes in the others.'! For

BIOCHEMISTRY AND RELATION WITH OTHER NUTRIENTS

Jiannan Song, M.D., is at the Department of Nutrition, School of Public Health and School of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A. Steven H. Zeisel, M.D., Ph.D., is at the Department of Nutrition,

and School

of Medicine,

example, methionine can be formed from homocysteine using methyl groups from methyl-tetrahydrofolate (THF), or from betaine that are derived from choline. Similarly, methyl-THF

Choline (Fig. 1) is needed for synthesis of the major phospholipids in cell membranes. Phospholipids are the structural building blocks of membranes and have hydrophilic (water-attracting) properties that create the double layer structure of membranes. Choline is also involved in methyl metabolism, cholinergic

School of Public Health

Perturbing the metabolism of one of these pathways

University of

North Carolina, Chapel Hill, North Carolina, U.S.A.

Encyclopedia of Dietary Supplements DOL: 10.1081 /E-EDS-120022035 Copyright © 2005 by Marcel Dekker. All rights reserved.

can be formed from

1-carbon

units derived from serine or from the methyl groups of choline via dimethylglycine. Choline can be synthesized de novo using methyl groups derived from methionine (via S-adenosylmethionine). When animals and humans

are deprived of choline, they use more

methyl-THF to remethylate homocysteine in the liver and increase dietary folate requirements. Conversely, when they are deprived of folate, they use more methyl groups from choline, increasing the dietary requirement for choline."! The availability of transgenic and knockout mice has made possible additional studies that demonstrate the interrelationship of these methyl sources.'4] When considering dietary requirements, it is important

to realize that methionine,

methyl-THF,

and choline can be fungible sources of methyl groups. 105

Choline

106 @ ks

ELT sel OnE

ets Soe

On oe

CH;

X oi

Choline

Betaine

ete

Acetylcholine

as

¢ Hy

OV—N-CH;

CH, O

Fig. 1 Chemical structures of choline and related compounds.

Phosphatidylcholine

As discussed

earlier, de novo

choline

synthesis is

catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT), which is primarily found in the liver. The PEMT knockout (pemt~/~) mice developed fatty liver and other signs of choline deficiency. This phenomenon can be reversed by adding extra choline to their diet.! Another interesting finding is that pemt~/~ females have trouble delivering normal litters without additional choline in their diet (unpublished data). Furthermore, the biochemistry and morphology of the fetal brain was altered in pemt~/— offspring. Overexpression of the pemt gene in cell culture systems downregulated PI3K/Akt signaling and induced apoptosis, perhaps explaining impaired prolifera-

tion induced by pem?2 transfection.©! These results

~ AdoHcy

y DNA methylation other methylations

Phosphatidylcholin

oh al

PtdEtn

:

So

Tetrahydrofolate

oan

methyl-THF

Homocysteine

Betaine Py

|

Acetylcholine

Fig. 2. Pathways of choline metabolism. Choline can be a methyl group donor and interacts with methionine and folate metabolism. It can be acetylated to form the neurotransmitter, acetylcholine, and can be phosphorylated to form membrane phospholipids such as _phosphatidylcholine (lecithin). Abbreviations: AdoHcy—S-adenosylhomocysteine; AdoMet—S-adenosylmethionine; PtdEtn—phosphatidylethanolamine; THF—tetrahydrofolate.

together with the previous findings that humans are subject to dietary choline deficiency under certain conditions during which endogenous production cannot meet the demand, lead to the postulation that people with a defective PEMT enzyme could be more suscep-. tible to choline deficiency. This enzyme is highly polymorphic: 98 single nucleotide polymorphisms (SNPs) were found in 48 Japanese people.!*! Whether any of these would lead to functional outcomes is currently under investigation.

PHYSIOLOGY Choline Metabolism

Choline is found in foods as free choline and as esterified forms such as phosphocholine, glycerophosphocholine, sphingomyelin, and phosphatidylcholine.!7 Lecithin is a term often used interchangeably with phosphatidylcholine, whereas the compound is a phosphatidylcholine-rich mixture added as an emulsifying agent in the food industry. Pancreatic enzymes can liberate choline from dietary phosphocholine, glycerophosphocholine, and phosphatidylcholine. Before choline can be absorbed in the gut, some is metabolized by bacteria to form betaine and methylamines (which are not methyl donors). There is no estimate for percentage absorption of the various forms of choline in humans. The watersoluble choline-derived compounds (choline, phosphocholine, and glycerophosphocholine) are absorbed via the portal circulation, whereas the lipid-soluble compounds (phosphatidylcholine and sphingomyelin) are absorbed as chylomicrons. Lecithin is the most abundant choline-containing compound in the diet. About half of the lecithin ingested enters the thoracic duct, and the remaining is metabolized to glycerophosphocholine in the intestinal mucosa and subsequent to choline in the liver. The liver takes up the majority of choline and stores it in the form of phosphatidylcholine and

Choline

107

sphingomyelin. Kidney and brain also accumulate choline. Although some free choline is excreted with urine, most is oxidized in the kidney to form betaine, which is responsible for maintaining the osmolarity in kidney. A specific carrier is needed for the transport of free choline across

the blood-brain

barrier, and the

capacity is especially high in neonates.

CHOLINE DEFICIENCY AND ORGAN FUNCTION Many animals species, including the baboon, fed a choline-deficient diet deplete choline stores and develop liver dysfunction. Within hours of a cholinedeficient diet, fat accumulation can be detected in the rat liver. Prolonged choline deficiency may result in liver cirrhosis. Renal dysfunction is another major choline deficiency sign in animal models. Massive tubular necrosis and interstitial hemorrhage are usually followed by complete renal failure. Animals fed a choline-deficient diet may also develop growth retardation, bone abnormalities, neural tube defects, and pancreatic damage. However, supplementation of choline to poultry reduced bird’s liver fat and enhanced immune function.'®! Some humans (male and female) fed total parenteral nutrition (TPN) solutions devoid of choline, but adequate for methionine and folate, have significantly lower levels of plasma-free choline and develop fatty liver and hepatic aminotransferase abnormalities. However, liver damage resolves when a source of dietary (or intravenous) choline is provided.”! Fatty liver occurs because choline is required to make the phosphatidylcholine portion of the VLDL lipoprotein particle.

In the

absence

of choline,

VLDL

is not

secreted, and triglyceride accumulates in hepatic cytosol.

Choline and Cardiovascular

Disease

The choline-containing phospholipid phosphatidylcholine has been used as a treatment to lower the cholesterol concentrations because lecithin—cholesterol acyltransferase has an important role in the removal of cholesterol from tissue. Betaine, the oxidized product of choline,

has been

used

to normalize

the plasma

homocysteine and methionine levels in patients with homocystinuria, a genetic disease caused by 5,10methylenetetrahydrofolate reductase deficiency. Therefore, dietary choline intake might be correlated with cardiovascular disease risk. Many epidemiologic studies have examined the relationship between dietary folic acid and cancer-or heart disease. It may be helpful to also consider choline intake as a confounding factor because folate and choline methyl donation can be interchangeable."

Choline Deficiency and Cancer

An interesting effect of dietary choline deficiency in rats and mice has never been studied in humans. Deficiency of this nutrient in rodents causes development of hepatocarcinomas in the absence of any known carcinogen.""*! Choline is the only single nutrient for which this is true. It is interesting that cholinedeficient rats not only have a higher incidence of spontaneous hepatocarcinoma, but also are markedly sensitized to the effects of administered carcinogens. Several mechanisms are suggested for the cancerpromoting effect of a choline-devoid diet. These include increased cell proliferation related to regeneration after parenchymal cell death occurs in the choline(alters deficient liver, hypomethylation of DNA expression of genes), reactive oxygen species leakage from mitochondria with increased lipid peroxidation in liver, activation of protein kinase C signaling due

to accumulation of diacylglycerol in liver, mutation of the fragile histidine triad (FHIT) gene, which is a tumor suppressor gene, and defective cell-suicide (apoptosis) mechanisms.!!°! Loss of PEMT function may also contribute to malignant transformation of hepatocytes.)

CHOLINE

AND

BRAIN

Choline and Adult Brain Function

Acetylcholine is one of the most important neurotransmitters used by neurons in the memory centers of the brain (hippocampus and septum). Choline accelerates the synthesis and release of acetylcholine in nerve cells. Choline used by brain neurons is largely derived from membrane lecithin, or from dietary intake of choline and lecithin. Free choline is transported across the blood-brain barrier at a rate that is proportional to serum choline level, while lecithin may be carried into neurons as part of an ApoE lipoprotein. Choline derived from lecithin may be especially important when extracellular choline is in short supply, as might be expected to occur in advanced age because of decreased brain choline uptake." Single doses of choline or lecithin in adult humans may enhance memory performance in healthy individuals, perhaps with greatest effect in individuals with the poorest memory performance. Studies in students showed that lecithin or choline treatment improved memory transiently for hours after administration."7! In humans with Alzheimer-type dementia, some studies report enhanced memory performance after treatment with lecithin,"*! while other studies did not

observe this. Buchman et al.!'*! recently reported that humans on long-term TPN may have verbal and visual

Choline

108

memory

impairment

be improved

which may

with

choline supplementation. If lecithin is effective, it is ina

special subpopulation in the early stages of the disease. Choline and lecithin have also been effectively used to treat tardive dyskinesia, presumably by increasing cholinergic neurotransmission.!'*!

Choline and Brain Development

Maternal dietary choline intake during late pregnancy modulated mitosis and apoptosis in progenitor (stem) cells of the fetal hippocampus and septum and altered the differentiation of neurons in fetal hippocampus."'*! Variations in maternal dietary choline intake (choline supplementation or choline deficiency) during late pregnancy also were associated with significant and irreversible changes in hippocampal function in the adult animal, including altered long-term potentiation (LTP) and altered memory.''7] More choline (about 4x dietary levels) during days 11-17 of gestation in the rodent increased hippocampal progenitor cell proliferation, decreased apoptosis in these cells, enhanced LTP in the offspring when they were adult animals,

and

enhanced

visuospatial

and

auditory

memory by as much as 30% in the adult animals throughout their lifetimes.''7! The enhanced maze performance appears to be due to choline-induced improvements in memory capacity. Indeed, adult rodents decrement in memory as they age, and offspring exposed to extra choline in utero do not show this “senility.’""'*! In contrast, mothers fed cholinedeficient diets during late pregnancy have offspring with diminished progenitor cell proliferation and increased apoptosis in fetal hippocampus, insensitivity to LTP

when

they were

adult

animals,

and

decre-

mented visuospatial and auditory memory."” Early postnatal choline supplementation significantly attenuated the effects of prenatal alcohol on a learning task, suggesting that early dietary interventions may

also influence brain development.''?! The mechanisms for these developmental effects of choline are not yet clear. We hypothesize that some of the genes that are regulators of cell cycling are, in turn, regulated by

epigenetic events that are modulated by choline.?°! Specifically, DNA methylation is an epigenetic event that is required for a proper gene regulation during

neurogenesis.”!! DNA methylation, especially at the CG sites, is associated with a reduction of gene expression. In brain and other tissues, a cholinemethyl-deficient diet directly alters gene methylation. Specifically in CpG islands within specific genes, global DNA was significantly undermethylated in brains of choline-methyl-deficient rats.°7! Rats fed a choline-deficient diet showed a poorer retention of nociceptive memory in the passive avoidance task,!?*!

suggesting that effects on brain may not be limited to the fetal period. Are these findings in animals likely to be true in humans? We do not know. Human and rat brains mature at different rates. In terms of hippocampal development, the embryonic days 12-18 in the rat correspond to approximately the last trimester of human. Rat brain is comparatively more mature at birth than is the human brain, but human hippocampal development may continue for months or years after birth. A pilot study on prenatal choline supplementation in humans is underway at this time.

HUMAN REQUIREMENT FOR CHOLINE Though males have a dietary requirement for choline”! human studies in women, children, or infants have not been completed. Thus, we do not know whether

choline is needed in the diet of these groups. Pregnancy " may be a time when dietary supplies of choline are especially limiting.

Gender and Choline Requirement Males have higher choline requirement than do females.'! Female rats are less sensitive to choline deficiency than are male rats perhaps because estrogen enhances females’ capacity to form the choline moiety de novo from S-adenosylmethionine.?*!

Pregnancy and Choline Requirement

Though female rats are resistant to choline deficiency, pregnant rats are as vulnerable to deficiency as are males.”°! During pregnancy, large amounts of choline are delivered to the fetus across the placenta and this depletes maternal stores. Choline concentration in amniotic fluid is 10-fold greater than that in maternal blood.

At birth, humans

and

other mammals

have

plasma choline concentrations that are much higher than those in adults.?*! This is accompanied by significant depletion of the maternal choline pool. In rats,

the liver choline concentrations in late pregnancy decreased to less than one-third that of nonpregnant females. It is not known whether the de novo synthesis of choline increases during pregnancy. Choline in Milk

Large amounts of choline are required in neonates for rapid organ growth and membrane biosynthesis. Choline administration significantly increased milk

Choline

109

production (and milk choline content) during the first month

of lactation in cows, but did not affect fat or

protein concentrations in milk.?”] Human_ infants derive much of their choline from milk. Mature human milk contains more phosphocholine and glycerophosphocholine than choline, phosphatidylcholine, or sphingomyelin. Human milk contains 1.5-2mM choline moiety per liter. The mother’s need for choline is likely to be increased during lactation because much must be secreted into milk. Lactating rats are more sensitive to choline deficiency than are nonlactating rats.?>! Choline in Plasma

Plasma choline concentration varies in response to diet and can rise as much as twofold after a two-egg meal. Fasting plasma choline concentrations vary from 7 to 20M, with most subjects having concentrations of 10 1M. Individuals who have starved for up to 7 days have diminished plasma choline, but levels never drop below 50% of normal. Plasma phosphatidylcholine concentration also decreases in choline deficiency,?*! but these values are also influenced by factors that change plasma lipoprotein levels. Fasting plasma phosphatidylcholine concentrations are approximately 1-1.5mM.

Food Sources

In foods there are multiple choline compounds that contribute to total choline content (choline, glycerophosphocholine, phosphocholine, — phosphatidylcholine, and sphingomyelin).?%! Foods highest in total choline concentrations per 100g were: beef liver (418 mg), chicken liver (290 mg), eggs (251 mg), wheat germ (152mg), bacon (125mg), dried soybeans (116mg), and pork (103 mg). Betaine in foods cannot be converted to choline but can spare the use of choline as a methyl group donor. The foods highest in betaine concentrations per 100g were: wheat bran (1506 mg), wheat germ (1395mg), spinach (725mg), pretzels (266 mg), shrimp (246mg), and wheat bread (227 mg). Both commercially available infant formulas and bovine milk contain choline and choline-containing compounds.®" Soy-derived infant formulas have lower glycerophosphocholine concentration, but have more phosphatidylcholine than do either human milk or bovine-derived formulas. Free choline is added to infant formulas when they are formulated.

Adverse

High

Effects

doses

excessive

of choline

cholinergic

have

been

stimulation,

associated

such

with

as vomiting,

salivation,

sweating,

and

gastrointestinal

effects.

In

addition, fishy body odor results from the excretion of trimethylamine, a choline metabolite from bacterial action." The tolerable upper limit for choline has

been set at 3 g/day.”!

;

DIETARY RECOMMENDATIONS The Institute of Medicine (IOM) recently made recommendations for choline intake in the diet.!! There were insufficient data to derive an estimated average requirement for choline; hence, only an adequate intake (AI) could be estimated. The IOM report cautioned, “‘this amount will be influenced by the availability of methionine and methyl-folate in the diet. It may be influenced by gender, and it may be influenced by pregnancy, lactation, and stage of development. Although Als are set for choline, it may be that the choline requirement can be met by endogenous synthesis at some of these stages.’’ The IOM recommendations are given in Table 1.

CONCLUSIONS Choline in the diet is important for many reasons. Humans deprived of it develop liver dysfunction. Also, parenterally nourished patients need a source of choline. As our understanding of the importance of folate

Table 1 IOMs recommended adequate intakes (Als) of choline for humans

Groups Amount (mg/day) Infants

0-6 mo

125, 18mg/kg

6-12 mo

150

Children 1-3 yr

200

4-8 yr

250

9-13 yr

375

Males

14-18

yr

550

19 yr and older

550

Females

14-18 yr

400

19 yr and older

425

Pregnancy (all ages)

450

Lactation (all ages)

550

(From Ref.”!,)

Choline

110

and homocysteine nutrition increases, there should be increased interest in studying how choline interacts with these compounds. Recent findings about choline in brain development should stimulate comparable studies in humans. The availability of food composition data now makes it possible to examine interactions

Buchman, A. et al. Choline deficiency: a cause of hepatic steatosis during parenteral nutrition that can be reversed with intravenous choline 1995, 22 (5), Hepatology supplementation. 3. 1399-140

10.

between choline, folate, and methionine when consid-

ering epidemiological data.

REFERENCES 1.

Zeisel, S.H.; Blusztajn, J.K. Choline and human nutrition. Ann. Rev. Nutr. 1994, 74, 269-296.

2.

Institute of Medicine and National Academy of Sciences USA. In Dietary Reference Intakes for Folate, Thiamin, Riboflavin, Niacin, Vitamin By, Panthothenic acid, Biotin, and Choline; National Academy Press: Washington DC, 1998;

Cohen, B.M. et al. Decreased brain choline uptake in older adults. An in vivo proton magnetic resonance spectroscopy study. JAMA 1995, 274, 902-907.

12,

Sitaram, N. et al. Choline: Selective enhancement

of serial learning and encoding of low imagery words in man. Life Sci. 1978, 22 (17), 1555-1560.

13.

Little, A. et al. A double-blind, placebo controlled trial of high-dose lecithin in Alzheimer’s disease. ” J. Neurol. Neurosurg. Psychiatry 1985, 48 (8), 736-742.

14.

Buchman, A.L. et al. Verbal and visual memory improve after choline supplementation in longterm total parenteral nutrition: a pilot study. J. Parenter. Enteral. Nutr. 2001, 25 (1), 30-35.

thes

Growdon,

Tos

4.

ils

Waite, K.A.; Cabilio, N.R.; Vance, D.E. Choline

deficiency-induced liver damage is reversible in

Pemt/~ mice. J. Nutr. 2002, 132 (1), 68-71. 5.

6.

7.

Saito, S. et al. Identification of 197 genetic variations in six human methyltransferase genes in the Japanese population. J. Hum. Genet. 2001, 46 (9), 529-537. Zeisel,

S.H.

et al. Concentrations

methionine,

and their combinations

on

the performance and immune response broilers. Br. Poult. Sci. 2000, 47 (1), 83-88.

choline

of

and

Meck, W.H.; Williams, C.L. Choline supplementation during prenatal development reduces proactive interference in spatial memory. Res.

Dev.

Brain

Res.

1999,

//8

(1-2),

18.

Meck, W.H.; Williams, C.L. Metabolic imprinting of choline by its availability during gestation: Implications for memory and attentional processing across the lifespan. Neurosci. Biobehav. Rev. 2003, 27, 385-399.

mo

Thomas, J.D. et al. Neonatal choline supplementation ameliorates the effects of prenatal alcohol exposure on a discrimination learning

of choline-

Swain, B.K.; Johri, T.S. Effect of supplemental

Choline

51-59.

containing compounds and betaine in common foods. J. Nutr. 2003, 733 (5), 1302-1307. 8.

A.J.

Albright, C.D. et al. Maternal choline availability

Brain

Zou, W. et al. Overexpression of PEMT2 downregulates the PI3K/Akt signaling pathway in rat hepatoma cells. Biochim. Biophys. Acta 2002, 1581 (1-2), 49-56.

Gelenberg,

alters the localization of p15Ink4B and p27Kip1 cyclin-dependent kinase inhibitors in the developing fetal rat brain hippocampus. Dev. Neurosci. 2001, 23 (2), 100-106.

Kim, Y.-I. et al. Folate deficiency causes second-

ary depletion of choline and phosphocholine in liver. J. Nutr. 1995, 124 (11), 2197-2203.

J.H.;

lecithin administration to patients with tardive dyskinesia. Trans. Am. Neurol. Assoc. (JC:w3w) 1978, 103 (95), 95-99.

Voll 3.

resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes. Carcinogenesis 1997, 18, 731-738.

11.

ACKNOWLEDGMENTS Support was received from the National Institutes of Health (AG09525, DK55865) and by grants from the NIH to the UNC Clinical Nutrition Research Unit (DK56350) and Center for Environmental Health Susceptibility (ES10126). Jiannan Song is a recipient of a Royster Fellowship from the University of North Carolina.

deficiency selects for

Zeisel, S.H. et al. Choline

task in rats. Neurotoxicol. Teratol. 2000, 22 te)

703-711. 20.

Niculescu, M.D.; Yamamuro, Y.; Zeisel, S.H. Choline availability modulates human neuro-

blastoma cell proliferation and alters the methylation of the promoter region of the

Choline

111

cyclin-dependent kinase inhibitor 3 gene. Neurochem. 2004, 89 (5), 1252-1259.

21:

22;

J.

PEs

Lachner, M.; Jenuwein, T. The many faces of histone lysine methylation. Curr. Opin. Cell Biol. 2002, 14 (3), 286-298.

Zeisel, S.H. et al. Pregnancy and _ lactation are associated with diminished concentrations of choline and its metabolites in rat liver. J. Nutr. 1995, 125, 3049-3054.

26.

Zeisel, 5. Jepstein, MF: Varina! Ree Elevated choline concentration in neonatal plasma. Life Sci. 1980, 26 (21), 1827-1831.

iAP

Pinotti, L. et al. Rumen-protected choline administration to transition cows: Effects on milk

Alonso-Aperte,

E.; Varela-Moreiras,

G.

Brain

folates and DNA methylation in rats fed a choline deficient diet or treated with low doses of methotrexate. Int. J. Vitam. Nutr. Res. 1996, 66 (3), 232-236.

23.

Nakamura, A. et al. Dietary restriction of choline

reduces hippocampal acetylcholine release in rats: In vivo microdialysis study. Brain Res. Bull. 2001, 56 (6), 593-597.

24.

production and vitamin E status. J. Vet. Med.— Ser. A 2003, 50 (1), 18-21.

Drouva, S.V. Estradiol activates methylating enzyme(s) involved in the conversion of phosphatidylethanolamine to phosphatidylcholine in rat pituitary membranes. Endocrinology 1986, 119 (6), 2611-2622.

28.

Zeisel, S.H. et al. Choline, an essential nutrient for

humans. FASEB J. 1991, 5 (7), 2093-2098.

20.

Zeisel,

S.H.

et al. Concentrations

of choline-

containing compounds and betaine in common foods. J. Nutr. 2003, 733 (5), 1302-1307.

30.

Holmes-McNary,

M. et al. Choline and choline

esters in human and rat milk and infant formulas. Am. J. Clin. Nutr. 1996, 64, 572-576.

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2 to CoQ was also decreased in these platelets, indicative

of the presence

of oxidative

metabolism.?*! Treatment of these patients with idebenone, an analog of coenzyme Q, reduced heart hypertrophy and improved heart muscle function. In several studies, patients with mitochondrial encephalopathy, lactic acidosis, and strokes (MELAS) displayed significant improvement after coenzyme Q or idebenone treatment.?°

Statin Therapy Statins are the drugs most commonly used for the treatment of hypercholesterolemia, and, in addition to efficient cholesterol lowering, they also have antiinflammatory activities. The basis for their use is that inhibition of HMG-CoA reductase decreases the farnesyl pyrophosphate pool to such an extent that squalene synthase, which catalyzes the terminal regulatory step in cholesterol synthesis, is no longer saturated, thereby inhibiting overall synthesis."'*! It appears, however, that the extent to which the farnesyl pyrophosphate pool is decreased by therapeutic doses of the drug also affects the saturation of trans- and cis-prenyltransferases in spite of the fact that these latter enzymes have a higher affinity for farnesyl pyrophosphate. Consequently, synthesis of both coenzyme Q and dolichol is inhibited. Rats treated with statins exhibit decreased levels of coenzyme Q, dolichol, and dolichyl phosphate in heart and muscle,

and the same

is probably

also

true in humans. In humans, statin treatment significantly decreases blood coenzyme Q concentration,?”! although the clinical significance of this phenomenon remains to be established. Various degrees of myopathy, myalgia, and rhabdomyolysis have been reported in statin-treated patients, and it is possible that these conditions are related to decreased muscle coenzyme Q content.

Given the widespread use of statins, it is

important that future studies address a possible causal link between these side effects of statin treatment and altered tissue coenzyme Q content.

stress.

Upon supplementation, the CoQ)9 content in the platelets increased and complex I activity was also elevated. In Huntington’s disease, magnetic resonance spectroscopy detected increased lactate concentration in the cerebral cortex. Administration of CoQ; caused a significant decrease in lactate that reversed upon discontinuation of the therapy. Deficiency of frataxin, a regulator of mitochondrial iron content, causes Friedrich’s ataxia. When patients with this disease ‘were treated with coenzyme Q and vitamin E for 6 mo, progression of their neurological deficits was slowed down, associated with an improvement in cardiac and skeletal muscle energy

Exercise

During endurance exercise training, the coenzyme Q concentration increases in rat muscle on a weight basis due to an increase in mitochondrial mass. After 4 days of high-intensity training, the coenzyme Q content in the exposed muscles of healthy persons is unchanged.”*! Supplementation (120 mg/day) doubles the coenzyme

Q concentration

in the plasma,

but

there is no change in the muscle content as judged by HPLC analysis of the tissue homogenate and isolated mitochondrial fraction in both control and trained subjects.

Coenzyme Qio

130

increased levels of ubiquinol-10 within circulating lipoproteins and increased resistance of human low-density lipoprotein to the initiation of lipid peroxidation. Biochim. Biophys. Acta. 1992, 1126, 247-254.

Dosage So far, no toxic or unwanted

side effects have been

described for CoQ9 supplements, not even after ingestion in gram quantities. In most studies, 100-200 mg has been given per day in two doses. In genetic disorders, in the case

of adults, the dose may

Turunen, M.; Wehlin, L.; Sjéberg, M.; Lundahl, J.; Dallner, G.; Brismar, K.; Sindelar, P.J. B2-

increase

to 300mg/day and in neurological diseases, up to 400 mg/day. In the latter case, in the frame of large multicenter trials, doses up to 1200mg have been supplied. A patent on the use of statins combined with coenzyme Q has expired recently, although this combined preparation has not been manufactured so far. Now it may be possible for the pharmaceutical industry to introduce capsules containing statins and coenzyme Q in order to decrease the potential for muscle damage. In this case, relatively low doses of CoQjio (e.g., 50 or 100mg/day) appear to be appropriate.

Integrin and lipid modifications indicate a nonantioxidant mechanism for the anti-atherogenic effect of dietary coenzyme Q10. Biochem. Biophys. Res. Commun. 2002, 296, 255-260.

10.

IB

Mosca, F.; Fattorini, D.; Bompadre, S.; Littarru,

3!

dilution step. Anal. Biochem. 2002, 305, 49-54. Griinler, J.; Ericsson, J.; Dallner, G. Branch-

point reactions in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins.

14.

Biochim. Biophys. Acta. 1994, 12/2, 259-277. Morré, D.J.; Morré, D.M. Cell surface NADH

oxidases (ECTO-NOX proteins) with roles in cancer, cellular time-keeping, growth, aging and neurodegenerative

diseases.

Free Radic.

Res. 2003,

hd:

37, 795-808. 4.

Fontaine,

P. A ubiqui-

Echtay, KS.;_ Winkler, E.;. Frischmuth, K,; Klingenberg, M. Uncoupling proteins 2 and 3

are highly active H + transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone). Proc. Natl. Acad. Sci. U.S.A. 2001, 95, 1416-1421. 6.

Mohr, D.; Bowry, V.W.; Stocker, R. Dietary sup-

plementation

Tomasetti, M.; Littarru, G.P.; Stocker, R.; Alleva,

Folkers, K.; Hanioka, T.; Xia, L.J.; McRee, J.T.; Langsjoen, P. Coenzyme Qjo increases T4/T8

ratios of lymphocytes in ordinary subjects and relevance to patients having the AIDS related complex. Biochem. Biophys. Res. Commun. 1991, 176, 786-791. Bentinger, M.; Dallner, G.; Chojnacki, T.;

with

coenzyme

Qjo

results

in

E. Distribution

and breakdown

of

16.

Ue

Nakamura, T.; Ohno, T.; Hamamura, K.; Sato, T.

Metabolism of coenzyme Q)o: biliary and urinary excretion study in guinea pigs. Biofactors 1999, 9, 111-119.

18.

Faust, J.R.; Brown, M.S.; Goldstein, J.L. Synth-

esis of delta 2-isopentenyl tRNA from mevalonate in cultured human fibroblasts. J. Biol. Chem. 1980, 255, 6546-6548.

19.

Low, P.; Andersson, M.; Edlund, C.; Dallner, G. Effects of mevinolin treatment on tissue dolichol and ubiquinone levels in the rat. Biochim. Biophys. Acta. 1992, 1165, 102-109.

20.

Aberg, F.; Zhang, Y.; Appelkvist, E.L.; Dallner,

Witting, K.; Pettersson, K.; Letters, J.; Stocker, R.

Anti-atherogenic effect of coenzyme Qo in apolipoprotein E gene knockout mice. Free Radic. Biol. Med. 2000, 29, 295-305. 8.

Turunen, M.; Olsson, J.; Dallner, G. Metabolism

labeled coenzyme Qj in rat. Free Radic. Biol. Med. 2003, 34, 563-575. Rustin, P.; Munnich, A.; R6tig, A.Mitochondrial respiratory chain dysfunction caused by coenzyme Q deficiency. Meth. Enzymol. 2004, 382, 81-86.

Stocker, R.; Bowry, V.W.; Frei, B. Ubiquinol-10

protects human low density lipoprotein more efficiently against lipid peroxidation than does a-tocopherol. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1646-1650. 7.

Watts, G.F.; Playford, D.A.; Croft, K.D.; Ward, N.C.; Mori, T.A.; Burke, V. Coenzyme Qio

Swiezewska,

E.; Ichas, F.; Bernardi,

none-binding site regulates the mitochondrial permeability transition pore. J. Biol. Chem. 1998, 273, 25,734-25,740. >,

R.D.

R. Coenzyme Qj9 enrichment decreases oxidative DNA damage in human lymphocytes. Free Radic. Biol. Med. 1999, 27, 1027-1032.

G.P. Assay of coenzyme Qj in plasma by a single

3.

R.; Olson,

and function of coenzyme Q. Biochim. Biophys. Acta. 2004, 1660, 171-199.

REFERENCES

2.

B.E.; Neuenschwander,

improves endothelial dysfunction of the brachial artery in type II diabetes mellitus. Diabetologia 2002, 45, 420-426.

pe

1.

Burke,

Randomized double-blind, placebo-controlled trial of coenzyme Q10 in isolated systolic hypertension. South Med. J. 2001, 94, 1112-1117.

G. Effects of clofibrate, phthalates and probucol on ubiquinone levels. Chem. Biol. Interact. 1994, OT =e:

|

Coenzyme Qj

ZAS

22

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Kalen, A.; Appelkvist, E.L.; Dallner, G. Agerelated changes in the lipid compositions of rat and human tissues. Lipids 1989, 24, 579-584. Folkers, K.; Vadahanavikit, S.; Mortensen, S.A.

26.

Biochemical rationale and myocardial tissue data on the effective therapy of cardiomyopathy with coenzyme Qj. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 901-904.

Za:

Sacher, H.L.; Sacher, M.L.; Landau, S.W.; Kersten, R.; Dooley, F.; Sacher, A.; Sacher, M.; Dietrick, K.; Ichkhan, K. The clinical and hemo-

dynamic effects of coenzyme

oy

Qjo in congestive

Beal, M.F. Coenzyme Qj as a possible treatment for neurodegenerative diseases. Free Radic. Res. 2002, 36, 455-460.

oN

Lodi, R.; Hart, P.E.; Rajagopalan, B.; Taylor, D.J.; Crilley, J.G.; Bradley, J.L.; Blamire, A.M.; Manners, D.; Styles, P.; Schapira, A.H.V.;

Cooper, J.M. Antioxidant treatment improves in

metry. J. Neurol. Sci. 1999, 162, 65-68. Ghirlanda, G.; Oradei, A.; Manto, A.; Lippa, S.; Uccioli, L.; Greco, A.V.; Littarru, G.P. Evidence

of plasma CoQ} -lowering effect by HMG-CoA reductase inhibitors: a double-blind, placebocontrolled study. J. Clin. Pharmacol. 1993, 33, 226-229.

cardiomyopathy. Am. J. Ther. 1997, 4, 66—72.

24.

vivo cardiac and skeletal muscle bioenergetics in patients with Friedreich’s ataxia. Ann. Neurol. 2001, 49, 590-596. Abe, K.; Matsuo, Y.; Kadekawa, J.; Inoue, S.; Yanagihara, T. Effect of coenzyme Qo in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and_ stroke-like episodes (MELAS): evaluation by noninvasive tissue oxi-

28.

Svensson, M.; Malm, C.; Tonkonogi, M.; Ekblom, B.; Sjédin, B.; Sahlin, K. Effect of Qio

supplementation on tissue Qo levels and adenine nucleotide catabolism during high-intensity exercise. Int J. Sport Nutr. 1999, 9, 166-180.

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Copper Leslie M. Klevay Grand Forks Human Nutrition Research Center, Agricultural Research Service, Grand Forks, North Dakota, U.S.A.

INTRODUCTION Since the discovery in 1928 that copper is an essential nutrient, hundreds of experiments to clarify its function have been conducted with several species of animals and, under very controlled conditions, with adult human volunteers. People respond to copper depletion similar to animals."!! The earliest experiments involved hematology, which preoccupied nutritional scientists for decades. Gradually, evidence for the adverse effects of copper deficiency on the cardiovascular and skeletal systems accumulated. Cardiovascular research related to copper deficiency, including associated lipid metabolism and cardiovascular physiology, now exceeds that on hematology. Early work on bone structure and function is being collected and extended. Methods for assessing nutritional status for copper are poorly developed. However, there are a sufficient number of reports of low activities of enzymes dependent on copper and low copper values in important organs to suggest that a considerable number of people may be too low in this element. These data complement measurements of dietary copper suggesting that the Western diet, which is frequently low in copper, may be the source of this abnormal biochemistry. Some people with abnormal gastrointestinal physiology may absorb too little copper as well.

GENERAL

DESCRIPTION

Copper is an essential and versatile nutrient that operates as the active site in 10 to 15 enzymes.''~! These proteins moderate the chemistry of this metallic element to enhance various metabolic processes related to oxidation. There also are several other copper-binding proteins of physiological importance, in addition

to some newly discovered proteins called metallochaperones."! The latter proteins act in the intracellular transport of metallic elements and help to ensure that free copper ion is nonexistent in the body.>-

ACTIONS, BIOCHEMISTRY, AND PHYSIOLOGY The essentiality of copper for mammals, including people, was discovered!” when rats fed a milk diet with adequate iron became anemic and grew poorly. Copper proved to be the active material in several foods that were curative and could prevent the condition. All the classic deficiency experiments with animals were done with milk diets. Adequate copper permits normal utilization of dietary iron. In addition to preventing anemia, it assists in blood coagulation,®7! crosslinking?!) of connective tissues of arteries, bones, and heart, defense against oxidative damage,

energy transformations, myelination of brain and spinal cord, reproduction, and synthesis of hormones."'!] Inadequate copper produces adverse effects!'?-'4] on the metabolism of cholesterol and glucose, blood pressure control and heart function, bone, mineralization, and immunity.

Hypercholesterolemia in copper deficiency has been found in at least 25 independent laboratories,!'*] most recently by Davis and Feng," Fields, Lewis, and Bureau,!!® and Wildman and Mao,!'7! since the

original observation."*! Glutathione is an effective regulator of 3-hydroxy-3-methylglutaryl coenzyme A activity.4?°] Copper deficiency disrupts glutathione metabolism,?") leading to increased activity of this enzyme??4! and contributing to the hypercholesterolemia that occurs.

In contrast, decreased

activities of

lecithin : cholesterol acyltransferase!”*! and lipoprotein lipase!**! also contribute to the hypercholesterolemia of deficiency. Electrocardiograms of animals deficient in copper reveal human cardiovascular risk factors such as branch block and abnormalities of the ST segment!3).

Leslie M. Klevay, M.D., S.D. in Hyg., is Professor at the Department of Internal Medicine, University of North Dakota, North Dakota, U.S.A.

;

The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity /affirmative action employer and all agency services are available without discrimination. Encyclopedia of Dietary Supplements DOT: 10.1081 /E-EDS-120022059 Copyright © 2005 by Marcel Dekker. All rights reserved.

other heart blocks and wave pathologies are numerous.'3] The heart blocks are probably caused by decreased activity of an ATPase isoform localized to the conduction system of the heart.!?7 Copper deficiency depresses vasodilation via alterations in nitric oxide physiology.8?! The mechanism 133

Copper

134

has been

reviewed?!3°!

and may

involve,

inter alia,

guanylate cyclase, which contains copper.21! There seems to be little doubt that copper deficiency can affect desaturase (and elongase) enzymes, but agreement is lacking on the details and directions of all the changes. Some of the data have been reviewed 1271,32.33] These enzymes can alter the number of double bonds in a fatty acid and can also increase its length. Prostaglandin metabolism is also affected.?1!

FOOD SOURCES AND SUPPLEMENTATION As far as is known, food source does not affect copper absorption, in marked contrast to iron and zinc, which are more easily absorbed from animal, than from plant, products. Higher concentrations of copper in many plant foods can compensate if fractional absorption is slightly lower. Vegetarian diets are high in copper.247>) Phytates either have no inhibitory effect on copper, or have a markedly smaller effect than that on zinc.8-!8] At intestinal pH, copper complexes with phytates are soluble whereas zinc complexes are not.

Phytates can thus enhance the utilization of copper.?*! Copper absorption at 55-75% is considerably higher than that of other trace elements; absorption occurs mainly in the upper small intestine, but stomach and colon may absorb the element as well."!! Thus, the concentration of copper in foods is an important characteristic that determines nutritional usefulness. In order of increasing concentration on a weight basis, fats and oils, dairy products, sugar, tuna, and lettuce

are low in copper; legumes, mushrooms, chocolate, nuts and seeds, and liver are high in copper.277*! Bread, potatoes, and tomatoes are consumed in suffi-

ciently large amounts by U.S. adults for these foods to contribute substantially to copper intake, although they are not considered to be high-copper foods.?! The Western diet typical of the United States, parts of Europe, and wealthy enclaves in the developing world is often low in copper. Approximately one-third of these diets are low in comparison with those used in successful depletion experiments of men and

women?!

under

controlled

conditions

and

in

comparison to the estimated safe and adequate daily dietary intakes (ESADDI)"*! and the newer estimated average requirement (EAR) and recommended dietary allowance (RDA) of the National Academy of Sciences (U.S.) (below). Estimations of dietary copper intakes based on calculations from the amount of copper in individual foods are too high in comparison to chemical analysis of the composite diets***”4%). the smallest error from calculation is an excess of 52%.*! It may be that people who provide dietary data report intakes that

are somewhat low, but it is unlikely that they are 50% too low; the reporting error is probably similar across experiments. The calculated 25th, median, and 75th percentiles for intakes of 51- to 70-yr-old men in a statistical sample of the U.S. population (Table C-15 in Ref) are 1.19, 1.47, and 1.81 mg copper daily. Corrections based on the mean excess in copper found by

calculation!**+*’**! decrease these estimates

to 0.44,

0.64, and 0.87 mg daily. Although younger men seem to eat more copper, women eat less! Data from several publications on dietary intakes of copper were pooled!?° 8] and a frequency distribution curve was derived for 849 analyzed diets. Approximately one-third of the diets contained less than 1 mg of copper daily. Further analytical confirmation of diets low in copper is available from men and women randomly selected in Baltimore. Thirty-six percent and 62% of the diets were below the respective dietary reference intakes for copper.°”! : Three approaches to supplementation are available. Diets below the EAR and the RDA can be improved by avoiding foods low in copper and by selecting foods

high in copper.8”] A copper-deficient salad (lettuce, mayonnaise, oil, tuna, etc.) can be improved by adding sunflower seeds, mushrooms, legumes, etc.5*! Soy products are increasingly popular and are high in copper,?"! as are nuts?! and chocolate!*! Beer enhances the utilization of copper in rats fed a deficient diet, resulting in a sixfold increase in longevity, with less cardiac damage and lower plasma cholesterol.&*! In contrast to iron, fortification of foods with copper is uncommon. Some new snacks and drinks promoted as products with exceptional nutritional properties are fortified with copper. A variety of tablets and capsules containing copper are available commercially. Copper gluconate is the only copper supplement listed by the United States Pharmacopeial Convention for oral use.©*! We have used copper sulfate effectively in experiments with animals!'*°° and human volunteers.!#°*?4] Others have used copper salts of amino acids.” It is not easy to identify the chemical form of copper in some of the available supplements. Cupric oxide is contained in some vitamin—mineral supplements; this form is no longer used in animal nutrition because the copper is utilized poorly.>®! Cupric oxide is used in the preparations with many ingredients because of its high concentration of copper, not because of demonstrated efficacy.

INDICATIONS AND USAGE The Western diet is associated with rapid growth in infancy, increasingly early sexual maturation, tall

Copper

135

adults, and low rates of infection. This diet is also associated with common diseases of affluence such as cancer, heart disease, obesity, and osteoporosis.!”! Numerous anatomical, chemical, and physiological characteristics of people with some of these latter illnesses have been found in several species of animals

deficient in copper.!!3:!41

No single indicator provides an adequate assessment of copper nutriture (nutritional status). Indices useful in experiments with animals have sometimes been helpful in depletion studies of people, but most do not seem to be altered by marginal deficiency. Circulating copper may not reflect the actions of enzymes inside cells in various organs where the metabolic processes affected by copper take place. Liver copper, generally impossible to assess in people, is the best indicator in animal experiments.°° Experiments with animals reveal that plasma copper can be normal or increased even though copper in liver or other organs may be low.©°-°8! Thus, normal or high plasma copper values in people may not be an accurate reflection of copper nutriture. Data on which to base dietary reference intakes for copper are elusive and, often, absent. Consequently, some of the values in Table | are rounded and values for males and females are combined. The adequate intake (AI) values are based on intakes of apparently healthy, full-term infants whose sole source of copper was human milk. Values for pregnancy are based on the amount of copper in the fetus and other products of conception. Those for lactation are the amounts needed to replace the average amount secreted in human milk. EARs are values estimated to meet the requirement of half of the healthy individuals of the group. Copper RDAs are based on the EAR plus an assumed coefficient of variation of 15%, which is larger than the 10% assumed for some other nutrients.!* It seems clear that there is little or no copper deficiency in the industrialized world if one relies on

Table 1 Daily adequate intake (AI), estimated average requirement (EAR), and recommended dietary allowance (RDA) for copper, mg Age

AI (mg)

0-6 mo

0.20 or 30 (ug/kg)

7-12 mo

0.22 or 24 (ug/kg)

EAR (mg)

RDA (mg)

traditional criteria of deficiency such as decreased plasma copper or ceruloplasmin. However, these markers are affected by the acute phase response and are easily increased by nondietary variables such as inflammation, oral contraceptives, and pregnancy. ‘Copper depletion experiments with men and women reveal unfavorable alterations in biochemistry and physiology with minimal or no changes in circulating copper - and without anemia (above). Copper deficiency is the leading nutritional deficiency of agricultural animals worldwide'®*!; can people be far behind? The recent report on dietary reference intakes'*”! and its predecessors, e.g., Ref.) summarize the rea-

sons why people may decide to take (or avoid) nutrient supplements. Growth and function are improved when nutrients are increased above levels just sufficient to prevent deficiency. There is little evidence that small surpluses

of nutrients

are

detrimental,

while

small

deficits will lead to deficiency over time. There is no evidence of unique health benefits from the consumption of a large excess of any one nutrient. Meeting recommended intakes for nutrients will not provide for malnourished individuals. There seems to be little or no anemia responsive to copper in the United States, although this phenomenon does not seem to have been studied adequately in the last half century. Copper deficiency can masquerade as the myelodysplastic syndrome, however.!”! Supplementation of middle-aged Europeans with copper protected their red blood cells from oxidative hemolysis in vitro,°’! indicating that extra copper improved the quality of the cells. Several of the classical risk factors for ischemic heart disease have been produced in animals deficient in copper. Similar changes have been found in more than 30 men and women in successful copper depletion experiments using conventional foods and have been reversed by copper supplementation.“°“! Copper intakes of 0.65—1.02 mg daily in these experiments were insufficient. Criteria of depletion included abnormal electrocardiograms°*) and blood pressure regulation,“ dyslipidemia,*) glucose intolerance,**) and hypercholesterolemia.“"! Two of the experiments were interrupted prematurely with early repletion with copper because of abnormal electrocardiography; all of the metabolic and physiological abnormalities disappeared with copper repletion. In contrast is a balance experiment using a formula diet that failed to confirm these results.'”"! Applesauce,

1-3 yr

0.26

0.34

4-8 yr

0.34

0.44

cheese, chicken, cornflakes, crackers, lettuce, margar-

9-13 yr

0.54

0.70

14-18 yr

0.685

19-70 yr

0.70

0.89 0.90

Pregnancy

0.80

1.00

1.00

1.30

ine, milk, orange juice, and rice provided less than 31-34% of dietary energy (calculated at 2400 kcal/ day).!’*! As actual energy intake ranged from 2415 to 3553 kcal,!’" the food part of the formula was probably about 26%. Because formula diets are known to

Lactation

lower

serum

cholesterol,!”*) the potential increase

in

Copper

136

cholesterolemia from the low copper intake may have been obscured. [74-80] Activities of enzymes dependent on copper and organ copper concentrations®!-°! have been found to be decreased in people with cardiovascular (mostly ischemic) diseases. There is a positive correlation between cardiac output and copper in heart tissue of patients with coronary heart disease.7! Decreased copper in organs and decreased enzyme activities are evidence of impaired copper nutriture.?'?7) No long-term copper supplementation has been done in patients with cardiac arrhythmia, dyslipidemia, glucose intolerance, hypercholesterolemia, or hypertension. However, some dietary regimens found to alleviate some of these conditions may have included an increase in copper intake as a hidden variable: for example, the Lifestyle Trial,°*! the protective effect of legumes on cholesterol, blood pressure, and diabetes,”*! and the benefit of whole

coronary

heart disease.°

Spencer?”

grain foods on

described

two

men and a woman whose premature ventricular beats,

which had persisted for years, were thought to be due to coronary heart disease. These premature beats disappeared after they ingested 4mg of copper (as copper gluconate) per day. Copper-deficient people have osteoporosis that can be cured with extra copper (reviewed in Ref.''*!). This phenomenon has been found mainly in young children. Adults may have skeletal pathology from low copper status as well. Copper is decreased in bone in both osteoarthritis and ischemic necrosis of the femoral

head.”*! Low serum copper in patients with fractures of the femoral neck"! or decreased lumbar bone density!'?:1°-1°!l may indicate covert copper deficiency. There is no epidemiologic evidence that low copper intakes produce the osteoporosis that occurs in late middle age. However, two double-blind, placebocontrolled trials have shown that trace element supplements including copper improved bone mineral density

in postmenopausal women.!107-103] Premature infants and people with extensive burns

may need extra copper. The former"! are sometimes born before their mothers can load them with copper in the last trimester.""°! Enzyme activities or improved physiology are more likely to be useful in assessing benefits of copper therapy than are measurements of circulating copper in prematurity. In analogy to vitamin Bj, deficiency, any disruption of the gastrointestinal tract has the potential to impair copper nutriture. For example, some people with cystic fibrosis or pancreatic insufficiency may need extra copper,''0°107] Copper-dependent enzyme activity and copper concentration have been found

to be decreased

in ulcerative

colitis biopsies.!!%®!

Supplementation of people with these conditions should be done under medical supervision. A potential

role for copper supplements in the treatment of rheumatoid arthritis and psoriasis has not been proved. Though adults may have unmet needs for copper to provide cardiovascular,

hematopoietic,

or skeletal

benefit, neither the dose nor the duration of therapy is clear. People in supplementation trials have tolerated 3-6 mg daily over their usual dietary amounts for weeks or months. There is probably no reason to exceed the tolerable upper intake level (UL) of 10mg daily (Table 2).

Potential Toxicity and Precautions All chemicals, including essential nutrients, are toxic if the dose is excessive. It seems that people have a 50- to 400-fold safety factor for copper considering usual dietary intakes and the tolerance level found with several species of experimental animals."°"! The UL connotes an intake that can, with high probability, be ~ tolerated biologically by almost all individuals. Gastrointestinal signs and symptoms such as nausea are prominent in the setting of this limit. A small, double-blind study has revealed that adults are unaffected in 12 weeks by a daily supplement of 10mg of

copper.'*! The UL values in Table 2 are based on this experiment; no value is available for infants less than lyr old. van Ravesteyn administered 38 mg of copper daily to people for as long as 14days; toxicity was

not mentioned."'° Copper supplements should be taken with food!'!''!7! and should not be taken by people with biliary disease, liver disease, idiopathic copper toxicosis or Wilson’s disease, or by people taking penicillamine or trientine. Although copper can interfere with zinc utilization, this phenomenon does not seem to be of practical importance to people. In contrast, copper deficiency has been induced in people (and in numerous species of pets and animals in zoos) by the ingestion of Table 2 Daily tolerable upper intake level (UL) for copper, mg

Age group

UL (mg)

Children 1-3 yr

1.00

4-8 yr

3.00 5.00

9-13 yr Adolescents

14-18 yr

8.00

Adults 19-70-+ yr

10

Pregnancy

8.00

Lactation

8.00-10.00

Copper

137

recently minted pennies (U.S.), which are almost pure zinc."''*! The dose of supplemental zinc that is excessive for adults is ill-defined, but the adult UL for zinc, 40 mg daily, is based on reduced copper nutriture from zinc in food, water, and supplements combined. A case of copper-responsive anemia has been reported in a patient with acrodermatitis enteropathica overtreated with zinc.!'!4! This potential exists for patients with Wilson’s disease treated with zinc, particularly children.!''*! Vitamin C is known to interfere with the utilization of copper, but its UL of 2¢ daily is not based on copper effects. Adverse effects on blood pressure regulation and copper utilization were found

O’ Halloran, T.V.; Culotta, V.C. Metallochaper-

ones, an intracellular shuttle service for metal ions. J. Biol. Chem. 2000, 275, 25,057-25,060. May,

Linder,

P.M.;

P.W.;

Williams,

D.R.

Ambivalent effect of protein binding on computed distributions of metal ions complexed by ligands in blood plasma. Experientia 1976, 32, 1492-1494. May,

Linder,

P.M.;

P.W.;

Williams,

D.R.

in women fed 1.5g vitamin C daily! Simple sugars

Computer simulation of metal-ion equilibria in biofluids: models for the low-molecular-weight complex distribution of calcium(II), magnesium(II), manganese(II), iron(III), copper(II), zinc(II), and lead(II) ions in blood plasma. J. Chem. Soc. Dalton Trans. 1977, 588-595.

such as fructose, glucose, and sucrose

Hart, E.B.; Steenbock, H.; Waddell, J.; Elvehjem,

interfere with

the utilization of copper!'!®!!71: It should be noted that high-fructose corn syrup is found in many processed foods and beverages. Copper supplements should not be used as emetics.

C.A. Iron in nutrition. VII. Copper as a supplement to iron for hemoglobin building in the rat. J. Biol. Chem. 1928, 77, 797-812. Lynch, S.M.; Klevay, L.M. Effects of a dietary copper deficiency on plasma coagulation factor activities in male and female mice. J. Nutr.

CONCLUSIONS

Biochem. 1992, 3, 387-391. Mann, K.G.; Lawler, C.M.; Vehar, G.A.; Church,

The Western diet often is low in copper. Statements to the contrary are based on dietary calculations, which are falsely high. The best way to ensure an adequate intake of copper is to minimize the intake of foods low in copper and to increase that of foods high in

W.R. Coagulation factor V contains copper ion. J. Biol. Chem. 1984, 259, 12,949-12,951. Linder, M.C.; Goode, C.A. 6.4 Specific copper components in special tissues. In Biochemistry of Copper; Plenum Press: New York, 1991; 212-220. Davis, G.K.; Mertz, W. Copper. In Trace Elements in Human and Animal Nutrition, Sth

it, such as cereals, grains, legumes, mushrooms,

nuts,

and seeds. Dietary copper can be increased by using the food pyramid as a guide. Only a few foods are fortified with copper. Copper gluconate is probably the best supplement. There seems to be little copper deficiency in Western society if one considers anemia as its only sign. However,

by

Ed.; Mertz, W., Ed.; Academic Press: San Diego,

12.

adults with diseases of the cardiovascular,

gastrointestinal, and skeletal systems have repeatedly been found to have low concentrations of copper in important organs and to have low activities of enzymes dependent on copper. These signs are consonant with deficiency. Anemia may therefore not be the most sensitive sign of deficiency in adults. Premature infants may also be deficient in copper. Large intakes of vitamin C or zinc can impair the proper utilization of copper in people.

A3..

Noyes Publications: Park Ridge, N.J., 1982; 1-205.

3.

Prohaska, J.R. Biochemical changes in copper deficiency. J. Nutr. Biochem.

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In

Advances in Experimental Medicine and Biology, Ist Ed.; Plenum Press: New York, 1989; Vol. 258. Klevay, L.M.; Medeiros, D.M. Deliberations and eva-

luations of the approaches, endpoints and paradigms for dietary recommendations about copper. J. Nutr. 1996, 126, 2419S—2426S. Lei, K.Y.; Carr, T.P. Role of Copper in Lipid Metabolism; CRC Press: Boca Raton, FL, 1990; 287.

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-?7! Hippuric acid does have antibacterial effects if present in acidic urine (pH 5.0) and at concentrations of 0.02-0.04 M. However, cranberry juice rarely can achieve the bacteriostatic concentrations by itself without the addition of exogenous hippuric acid to the diet.?®)

Cranberry (Vaccinium macrocarpon) Aiton

Antiadhesion

145

immobilized via

Urinary tract infection More recently, emphasis has been on the role of components that act by interference with bacterial adherence of Escherichia coli to uroepithelial cells.”?-°"! Several studies found antiadherence activity in mouse and human urine.??! Two compounds were identified that inhibited adherence. One was fructose and the other was a nondialyzed polymeric compound. While fructose in vitro inhibits adherence,???" it is unlikely to contribute to in vivo antiadhesion activity

the

human

sialic

gastric mucus

acid-specific

and erythrocytes

adhesin.

Therefore,

the

authors hypothesized that cranberry or its constituent may inhibit de novo adhesion. In vivo or clinical studies to demonstrate prevention or treatment of H. pylori infection have not yet been published in English.

Antioxidant

Antioxidant capacity is not restricted to a particular class of cranberry components but has been found in a wide range of fractions.©°! Polyphenols are reducing

in urine because it is metabolized before reaching the

agents, and together with others, such as vitamin C,

urinary tract. Later proanthocyanidins were identified as the compounds responsible for preventing uropathogenic E. coli from adhering to the urinary tract.°] Their chemical structure was subsequently elucidated.''7! Because of the poor bioavailability of this polymer, other mechanisms may be responsible for the antiadhesive effect of cranberry in UTI, dental plaque, and Helicobacter pylori infection.

they may protect the body’s tissues against oxidative stress. The antioxidant activity of the berry in vivo cannot be accounted for on the basis of increased vitamin C alone.8* Crude cranberry fruit extracts have significant antioxidant activity in vitro.°” The total antioxidant activity of 100g of cranberry was estimated to be equivalent to that of 3120 mg of vitamin C."" Isolated polyphenolic compounds from whole cranberries are comparable or superior to that of vitamin E in their activity."*! Cranberry ranks higher than apple, peach, lemon, pear, banana, orange, grapefruit, and pineapple,"°! as well as avocado, cantaloupe, melon,

Dental plaque Because of cranberry’s diverse range of biologically active compounds, it may affect the formation and pathogenicity of dental plaque by: 1) blocking adherence of bacteria to surfaces; 2) inhibiting enzymes associated with the formation of plaque (e.g., preventing biofilm formation); and 3) reducing acid tolerance and viability of cariogenic organisms. To examine the effect of cranberry juice on the coaggregation of oral bacteria, a high-molecular weight nondialyzable material of unknown molecular structure was isolated from the juice.°?! In vitro, this nondialyzable material dissociated coaggregates formed by bacteria. It acted preferentially on pairs of bacteria in which one or both members were gram-negative anaerobes. After 42 days, bacterial counts in media selective for Staphylococcus mutans from saliva samples of 30 volunteers using a standard mouthwash to which the nondialyzable material was added showed a two order of magnitude reduction in colony forming units in the experimental group compared to the placebo group.

nectarine, plum, and watermelon.!! 38]

Cranberries contain two phenolics, namely flavonoids and hydroxycinnamic acids, which have antioxidant potential. The contribution of individual phenolics to total antioxidant capacity is generally dependent on their structure and content in the berry. The highest antioxidant activity has been noted in peonidin-3-galactoside (21% of antioxidant capacity). Quercetin-3-galactoside, cyanidin-3-galactoside, and peonidin-3-arabinoside

each contribute about 10-11%. Different methods of assessment of antioxidant capacity, varying substrate systems, divergent ways of extraction, length of storage, and differential concentrations of active antioxidants confound the antioxidant activity-chemical structure relationship. Given the diversity and abundance of phenolic antioxidants in cranberry, considerable potential exists for cranberry products to prevent oxidative processes related to cardiovascular disease and cancer at the cellular level and in vivo.

Helicobacter pylori Atherosclerosis

Adhesins mediate adhesion of H. pylori to epithelial cells. Because cranberry or its constituents have been shown to inhibit adherence of E. coli to uroepithelial cells in vitro, the hypothesis that it would prevent adhesion of H. pylori to gastric mucus and cells was tested.641 A high-molecular weight nondialyzable material from cranberry juice was demonstrated to restrain the adhesion of three strains of H. pylori to

Consumption of flavonoids may decrease the risk of atherosclerosis.4°! One of the possible mechanisms by which they may protect against vascular disease is as antioxidants, which inhibits low-density-lipoprotein (LDL) oxidation."*! Others include: 1) inhibition of platelet aggregation and adhesion; 2) inhibition of the inflammatory response; 3) induction of

Cranberry (Vaccinium macrocarpon) Aiton

146

endothelium-dependent vasodilation; and 4) increase of reverse cholesterol transport and decrease of total and LDL-cholesterol. Data supporting these methods are preliminary. Evidence to support other ways by which cranberry or its constituents may decrease the risk of atherosclerosis is not available in the literature. Cancer

The antioxidant capacity alone of cranberry constituents may not account for the observed effects.b844 A soluble-free extract had the highest antiproliferative activity and maximum calculated bioactivity index for dietary cancer prevention compared to ten other fruits."°! Given the diversity of molecular structures and bioactivity among the classes of phytochemicals in cranberry, it is likely that they may fight cancer by several different mechanisms. These include: 1) induction of apoptosis; 2) slow initiation, promotion, and progression of tumors!*>784. and 3) inhibition of the inflammatory response." In vivo carcinogenesis studies will need to be performed to further confirm antitumor promotion activity and identify individual components and mixtures responsible for activity. Cytotoxicity of cranberry or its constituents toward tumor cells has not been reported.

symptomatic UTIs in women. For the five trials not included in the meta-analysis, one reported a significant result for symptomatic UTIs in women and another reported a significant result for asymptomatic UTIs in elderly women. For treatment, no trials meeting the inclusion criteria were found; only a few uncontrolled trials were found. The Cochrane Library concluded that there was no good quality or reliable evidence of the effectiveness of cranberry juice or other cranberry products for the treatment of UTI. For both prevention and treatment, the review authors concluded that more research was needed. Two other studies not included in the Cochrane reviews reported apparently contradictory results. The first was a study of adults with spinal cord injury who demonstrated a reduction in biofilm load,*! but

the importance of this effect for frank UTI was not determined. A small study of urostomy patients also found equivocal results. Many of the clinical study reports available in the literature suffer from major limitations. Many trials have not been controlled or randomized, and randomi-

zation procedures have not always been described. Crossover designs used in some research may not be appropriate for studies of UTI. Other limitations include

no blinding or failed blinding,

lack of con-

trolled diets or dietary assessment, use of convenience samples, and small numbers

Safety Studies No animal toxicology studies have been reported, nor have serious adverse events in humans consuming cranberry products for long periods been reported.

of subjects. Trials have

been faulted for the large number of withdrawals. Intention-to-treat analyses were not often applied. Most studies have been conducted in older or elderly patients. Very few have been conducted in younger patients,

with

or without

comorbidities,

or in men.

Primary outcomes have differed from study to study and have often included urinary pH, as well as rate of bacteriuria, biofilm* load, and urinary white and

CLINICAL STUDIES

red blood cell counts, rather than UTI. It is also not

Efficacy Urinary tract infection The

use

of cranberry

to prevent

or treat UTI

is

clear what is the optimum dosage or type of product. There is limited evidence of efficacy or safety for forms of cranberry product other than juice or juice cocktail. Finally, the published articles do not describe the quality and composition of the products tested.

common. The accumulating evidence from small, non-

controlled and controlled clinical trials suggests that the berry may relieve symptoms associated with UTI and may reduce the need for antibiotics. The Cochrane

Library“***! conducted separate reviews of the fruit for the prevention and treatment of UTI. Each review used similar search strategies and selection criteria. These included all randomized or quasirandomized controlled trials. Trials of at least 1mo to at least 5days were included for prevention and treatment, respectively. For prevention, seven trials met the inclusion criteria. Meta-analysis performed using data from the two better trials found that when consumed over a year, cranberry juice decreased the number of

Adverse Effects

The U.S. Food and Drug Administration granted generally recognized as safe (GRAS) status to cranberry foods and beverages. This means that their safety is well established. The few clinical studies assessing the adverse effects of cranberry juice cocktail have

“Biofilms are collection of proteinaceous material that cover the bacterial populations and often render them resistant to sterilization and antimicrobial treatments.

Cranberry (Vaccinium macrocarpon) Aiton

147

reported no or few side effects other than diarrhea and other gastrointestinal symptoms. The safety of cranberry capsules, tablets, and example, has not been established.

concentrates,

for

Observed Drug Interactions and Contraindications

There is insufficient reliable information available on cranberry dietary supplements or juice cocktail to assess their safety or their interaction with other dietary supplements, foods, medications, or laboratory tests. Because of its oxalate levels, cranberry may be a

causative factor in nephrolithiasis. The results of three small studies of juice cocktail and tablets are equivocal, showing differences in urine acidification, calcium and oxalate excretion, and other promoters

and inhibitors of stone formation.°47! There is one report of a 4-mo-old infant being hospitalized in Spain for cranberry juice intoxication and acidosis.“*! Five unsubstantiated reported cases suggest an interaction between cranberry juice and warfarin.! Theoretically, the juice could interfere with the copper-reduction glucose test since ascorbic acid (a reducing agent) and hippuric acid have each been reported to cause a false-positive reaction with the copper-reduction glucose determination in vitro. However, the results of two small studies are equivocal and inconclusive indicating that interference may be variable and

dependent on the type of reagent strip kit.°°°"! REGULATORY STATUS In the United States, cranberry is classified as a food when sold as juice, juice cocktail, and other conventional forms. Cranberry products, such as encapsulated powders, tablets, or tinctures, are regulated as “‘dietary

lack of appropriate standardized analytical methods. Consequently, the precise estimation of cranberry constituent intake is hampered. Furthermore, the bioavailability, metabolism, stability, purity, and composition of cranberry products tested in ‘clinical studies have not been established or published. Therefore, the ability to infer epidemiological relationships with health and disease can be confounded. Evidence for health benefit of cranberry is preliminary and inconclusive. Current evidence from in vitro and clinical studies has been conflicting. This could reflect differences among sources of cranberry or its constituents, form of product consumed, and level of intakes. In addition, clinical studies performed to date

have had many methodologic limitations and few have assessed safety. Nevertheless, results of clinical studies are encouraging for the relief of symptoms associated with and the prevention of UTI. The complex composition of cranberry creates problems in extrapolation of research results on dietary intake of individual constituents to intake of whole fruits or extracts of whole fruits. Synergistic effects of the whole may enhance the health benefits beyond what can be achieved by the individual constituents. The complex mixture of compounds could also protect against side effects. More research on potential synergistic and protective effects among the classes of compounds in cranberry and with other food constituents and pharmaceuticals is necessary. For these reasons, it is important to understand the composition of cranberry, determine the bioavailability and metabolism of its constituents in isolation and as part of the whole mixture, and rigorously examine the biological effects of cranberry on disease conditions in order to establish its potential for being safe and providing health benefit.

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! Cells were incubated for 5 days in medium with (12.5-1000 ug/ml) and without the extract. Compared to untreated control cell cultures, cell proliferation was enhanced at extract concentrations less than 125ug/ml (P < 0.05), whereas it was inhibited at concentrations greater than 250pug/ml (P < 0.05 at 1mg/ml). In addition, the activity of alkaline phosphatase, a protein synthesized by bone cells during osteogenesis, was increased significantly at all concentrations (P < 0.05).

Dang gui is one of the most important herbal drugs in TCM, primarily being used for blood tonification and the treatment of gynecological disorders. More recently, interest has focused on dang gui’s possible cardiovascular, hepatoprotective, hematopoietic, antioxidant, antispasmodic, and immunomodulatory effects. Despite its long tradition of use and current

Wound-Healing Effects One group of researchers found that a crude extract of dang gui (characterization and dosage not available) significantly, accelerated epithelial cell proliferation in wounds.”:?67] The activity was reportedly associated with an increase in DNA synthesis and epidermal growth factor (EGF) mRNA expression. The same researchers observed direct wound-healing effects of dang gui crude extract, with activity associated with increased ornithine carboxylase activity and increased c-Myc expression. Another study found that dang gui prevented bleomycin-induced acute injury to rat

lungs.”*! Alveolitis and the production of malondialde-

hyde (MDA) were all reduced (P < 0.01 or P < 0.001), suggesting immunomodulatory and antioxidant effects. Analgesic Effects Two uncontrolled clinical trials were found that addressed the traditional Chinese use of dang gui as an analgesic for pain due to “blood stasis’’: both used

injectable preparations. In one, an ethanol extract was administered (intramuscularly) on alternate days for a total of 20 doses into the pterygoideus externus of 50 patients with temporomandibular joint syndrome. A 90% cure rate was claimed.!??! Thirty cases of refractory interspinal ligament injury were treated by local injection of 2ml of 5% or 10% dang gui twice weekly for 2-3 weeks. Twenty-four (80%) of these patients reported a disappearance of pain, no tenderness, and ‘the ability to work as usual; 4 (13%) patients reported alleviation of pain; 2 (7%) reported no improvement.?"! These uses are consistent with the traditional use of dang gui in TCM. However, the effects of injectable preparations cannot be extrapolated to oral use of dang gui.

widespread

clinical utility, there has been very little

clinical work verifying the therapeutic efficacy of dang gui when used alone, primarily due to the fact that in TCM, botanicals are generally used in combinations rather than as single agents. Based on the literature available, and keeping many of its limitations for an English readership in mind, there is limited clinical support for the use of dang gui alone for the following indications: pulmonary artery and portal hypertension, acute ischemic stroke, dysmenorrhea, infertility, and pain due to injury or trauma. The use of dang gui for most of these indications is consistent with TCM. One trial on menopausal symptoms found no effect of dang gui on hormonal activity. Most of the trials available are of poor methodological quality. Clinical and preclinical studies provide some support for a wide variety of actions of dang gui. These include the promotion of circulation, vasodilation/relaxation, and the inhibition of platelet aggregation,

all of which

are

consistent

with

the

“blood

quickening’’ properties ascribed to dang gui in TCM. Similarly, the hematopoietic effect of dang gui is consistent with its use in TCM to “nourish blood.’’ Its smooth muscle (uterus, vessels, trachea) relaxant effects are consistent with its use for dysmenorrhea, asthma, and coughing. Dang gui may relax or stimulate the uterus depending on a variety of factors: In general, the volatile oil fraction appears to be a uterine relaxant, while the nonvolatile constituents appear to

stimulate contractions. There is some support for the traditional use of dang gui as an analgesic and vulnerary. The radiation protective effect of dang gui in animals is most likely due to its antioxidant activity. Assays for an estrogenic effect of dang gui have had mixed, but largely negative, results. The relevancy of many of these actions to the therapeutic use of dang gui in humans has not yet been demonstrated.

MEDICAL INDICATIONS SUPPORTED BY CLINICAL TRIALS The clinical data regarding the use of dang gui alone are scarce and of poor methodological quality. Based on the available data, it is best that dang gui be used

'

Dang Gui (Angelica sinensis)

within the context practitioners.

of TCM

163

and by qualified TCM

threatened miscarriage. For such uses, dang gui must be used according to TCM principles under the guidance of a qualified TCM practitioner.

DOSAGES"! Interactions

e

e

Crude drug: 6-12g daily to be prepared as a decoction. Liquidextract (1:1): 3-5 ml three times daily.

SAFETY

PROFILE

Side Effects

Based on a review of the available traditional and scientific data, dang gui is a very safe herb with a low probability of side effects when used within its normal dosage range. One review article that claimed to cover 200 reports on dang gui pharmacology stated

that dang gui had no major side effects.27! Individual case reports regarding the potential of dang gui to promote bleeding have been prepared. Contraindications

Based on a review of the available literature and the experience of practitioners, dang gui is contraindicated prior to surgery and in those, generally speaking, with bleeding disorders.

Precautions

Precautions regarding the use of dang gui and other botanicals used in traditional systems of medicine must be differentiated between those recognized in the scientific literature and those recognized traditionally. There is evidence suggesting an anticoagulant effect for dang gui, and there are two published reports on its ability to enhance the effects of chronic treatment with warfarin (see “Interactions’’). A few unpublished case reports suggest that high doses or chronic administration of dang gui alone during pregnancy may be associated with miscarriage. There are also anecdotal reports of dang gui alone causing increased blood flow during menses (Upton, personal communication, unreferenced). Therefore, patients should consult with a qualified health care professional prior to using dang gui if they have bleeding disorders, are using anticoagulant medications, or wish to use it during menses or in the first trimester of pregnancy. It must, however, be noted that in TCM, dang gui is specifically indicated for certain bleeding disorders that are due to an underlying diagnosis of blood stasis and in certain cases of

Two reports are available suggesting that dang gui can enhance the effects of the anticoagulant warfarin. According to one of these, a 46-yr-old woman with atrial fibrillation who had been stabilized on warfarin for almost 2 yr (5 mg daily) consumed a dang gui product (Nature’s Way) concurrently for 4weeks (565— 1130 mg daily). She experienced a greater than twofold elevation in prothrombin time (from 16.2 to 27 sec) and international normalized ratio (from 2.3 to 4.9). No other cause for this increase could be determined. Within 1 mo of discontinuing dang gui use, coagula-

tion values returned to acceptable levels.) An animal study investigated the interaction of dang gui and a single dose and a steady-state dose of warfarin.“ Six rabbits were administered a single dose of warfarin (2mg/kg s.c.). Seven days later, the same animals were given an aqueous extract of dang gui (2 g/kg p.o., twice of a 2 g/ml extract daily) for 3 days, after which they were again given a single dose of warfarin. Plasma warfarin concentrations were measured at intervals up to 72hr after each warfarin dose, and prothrombin time was measured daily during dang gui treatment and after the warfarin doses. Mean prothrombin time did not change significantly during the dang gui treatment period. However, when measured after coadministration of dang gui and warfarin, prothrombin time was significantly lowered at 24, 36, and 48hr compared to that with warfarin treatment alone (P < 0.05 or P < 0.01). No significant variations in the single dose pharmacokinetic parameters of warfarin were observed after treatment with dang gui. Hence, the mechanism of decrease in prothrombin time could not be correlated to the pharmacokinetics of warfarin. Another group of 6 rabbits was given 0.6mg/kg of warfarin s.c. daily for 7 days; a steady-state plasma concentration was achieved after day 4. On days 4, 5, and 6, the rabbits were treated as above with dang gui. Mean prothrombin time was again significantly increased after coadministration with dang gui (P < 0.01) and 2 rabbits died at days 6 and 7 after the dang gui treatment had begun. Plasma warfarin levels did not change after dang gui treatment. The authors suggested that these results indicate that precautionary advice should be given to patients who medicate with dang gui or its products while on chronic treatment with warfarin. One study reported that dang gui might enhance the antitumor effect of cyclophosphamide in mice with transplanted tumors.>!

Dang Gui (Angelica sinensis)

164

Pregnancy, Mutagenicity, and Reproductive Toxicity

Treatment of Overdose No data available.

Because of its blood-moving properties, dang gul should only be used in pregnancy while under the supervised care of a qualified health professional. According to TCM practice, dang gui is used in combination with other herbs in various stages of pregnancy."’7] Formulae traditionally used in pregnancy are prescribed within the context of specific diagnoses in which the use of dang gui in pregnancy is clearly

Toxicology

The following LDs values have been reported dang gui extract (8:1 or 16:1), 100g/kg p.o. in rats3?,401, dang gui aqueous extract, 100 g/kg i.v. in mice"); dang ' gui 50% ethanol extract, greater than 40 g/kg p.o. in

mice'*]. dang gui total acids, 1.05 + 0.49 g/kg ip. in

indicated. In the West, dang gui is often used alone out of this traditional medical context. Because of this,

mice.7]

several Western sources consider dang gui to be contraindicated in pregnancy. Data regarding the effect of dang gui preparations on the fetus are lacking.

approximately 410 mg/kg ip.“°] In a review of the toxicology literature on dang gui, it was reported that iv. injection of the volatile fraction of dang gui could cause kidney degeneration.) :

The

LDso

of ferulic acid i.v. in mice was

reported to be 856.6mg/kg,'"! and that of ligustilide,

Lactation

REGULATORY STATUS

There are three unpublished case reports of a rash in infants of lactating mothers who were taking dang gui. The rashes reportedly resolved upon discontinuation of the preparation by the mother. Specific details regarding the preparations used were lacking (Romm,

Regulated as a dietary supplement (USC 1994).

2002, personal communication to AHP, unreferenced).

REFERENCES

Dang gui is a member of the botanical family Apiaceae, a group of plants that contain many types of photoreactive compounds known to cause rashes.

Carcinogenicity Insufficient data are available with which to make a definitive determination regarding the carcinogenicity of dang gui. One animal study identified a possible antitumor effect of dang gui applied to mice with Ehrlich ascites tumors.°° Data are mixed regarding estrogen positive tumors; while one in vitro assay found that dang gui stimulated the growth of MCF-7 breast cancer cell lines 16-fold, with no measurable effect on estrogen receptors,?” another found a possi-

ble antitumor effect in T-47D and MCF-7 cell lines.2*! Data regarding the potential estrogenic effects of dang gui have been mixed.

1.

certain countries produce only positive results? A systematic review of controlled trials. Controlled 2.

3.

4.

5.

Influence on Driving No data available. Based on the experience of modern herbal practitioners, no negative effects are to be expected.

6.

Overdose

7. No data available.

Vickers, A.; Goyal, N.; Harland, R.; Rees, R. Do

Clin. Trials Des. Meth. Anal. 1998, 79, 159-166. Xu, J.Y.; Li, B.X.; Cheng, S.Y. Short-term effects

of Angelica sinensis and nifedipine on chronic obstructive pulmonary disease in patients with pulmonary hypertension. Zhongguo Zhongxiyi Jiehe Zazhi 1992, 1/2 (12), 707, 716-718. Tu, J.; Huang, H. Effects of Radix Angelicae sinensis on hemorrheology in patients with acute ischemic stroke. Gong Zazhi 1984, 4 (3), 225-228. Gao, S.W.; Chen, Z.J. Effects of sodium ferulate

on platelet aggregation and platelet thromboxane A> in. patients with coronary heart disease. Zhongxiyi Jiehe Zazhi 1988, 8 (5), 263-265, 269. Terasawa, K.; Imadaya, A.; Tosa, A.; Mitsuma, T.; Toriizuka, K.; Takeda, K.; Mikage, M.; Hattori, M.; Namba, T. Chemical and clinical evaluation of crude drugs derived from Angelica acutiloba and Angelica sinensis. Fitoterapia 1985, 56 (4), 201-208. Huang, Z.P.; Liang, K.H. Effect of Radix Angelicae sinensis On serum gastrin levels in patients with cirrhosis. Zhonghua Neike Zazhi 1994, 33 (6), 373-375. Huang, Z.P.; Guo, B.; Yuan, S.Y.; Ai, L.; Liang, K.H. Effects of Radix Angelicae sinensis on

Dang Gui (Angelica sinensis)

165

systemic and portal hemodynamics in cirrhotics with portal hypertension. Zhonghua Neike Zazhi 1996, 35 (1), 15-18. Xie, F.; Li, X.; Sun, K.; Chu, Y.; Cao, H.; Chen, N.; Wang, W.; Liu, W.; Mao, D. An experimental

study on drugs for improving blood circulation and removing’ blood stasis in treating mild chronic hepatic damage. J. Trad. Chin. Med. 2001, 21 (3), 225-231. Ne, YON, Lan ESeeLioy sSoHila; Chosic.c, Sheng, H.P.; Lee, S.S.; Cho, C.H. Protective effect

10.

it.

of polysaccharides-enriched fraction from Angelica sinensis on hepatic injury. Life Sci. 2001, 69 (6), 637-646. Ding, H.; Peng, R.; Yu, J. Modulation of Angelica sinensis polysaccharides on the expression of nitric oxide synthase and Bax, Bcl-2 in liver of immunological liver-injured mice. Zhonghua Ganzangbing Zazhi 2001, 9 (Suppl.), 50-52. Gao, Y.M.; Zhang, H.K.; Duan, Z.X. Treatment

of 112 cases of dysmenorrhea with jingyou pill. Lanzhou Daxue Xuebao 36-38.

12,

low-density lipoprotein without changing the lipoprotein Cu(2+)-oxidizability: possible explanation by phenolic location. Eur. J. Clin. Nutr. 1997, 51 (10), 682-690.

20); 211

22

Hirata, J.D.; Swiersz,

L.M; Zell, B.; Small, R.;

Ettinger, B. Does dong quai have estrogenic effects in postmenopausal women? A doubleblind, placebo-controlled trial. Fertil. Steril. 1997, 68 (6), 981-986.

14.

Eagon,

P.K.;

Hunter,

D.S.;

Elm,

M.S.;

23,

tS:

24.

20%

26.

De

gene

expression.

on

steroid Clin.

hormone-regulated

Chim.

Acta

2001,

28.

Soc.

Exp.

Biol.

Med.

29.

31/2,

1998,

SU:

ae

Ma, L.; Mao, X.; LiX.-Zhao,

Skt

18.

Angelica sinensis polysaccharides on mouse bone marrow hematopoiesis. Zhonghua Xueyexue Zazhi 1988, 9 (3), 148-149. Wang, Y.; Zhu, B. The effect of Angelica polysaccharide on proliferation and differentiation of hematopoietic precursor cells. Zhonghua Yixue Zazhi 1996, 76.(5), 363-366. Carbonneau, M.A.; Leger, C.L. Supplementation

33)

1

H. The effect of

with wine phenolic compounds increases the antioxidant capacity of plasma and vitamin E of

T.; Egashira, Y.; Sanada, H.

Hirabayashi, T.; Ochiai, H.; Sakai, S.; Nakajima, K.; Terasawa, K. Inhibitory effect of ferulic acid

Hu, H.J.; Hang, B.Q.; Wang, P.S. Anti-inflamma-

Yaoxue

Hu, H.J.; Hang, B.Q.; Wang, P.S. Anti-inflamma-

tory effect of the root of Angelica sinensis. Zhongguo Zhongyao Zazhi 1991, /6 (11), 684— 686. Yang, Q.; Populo, S.M.; Zhang, J.Y.; Yang, G.G.; Kodama, H. Effect of Angelica sinensis on the proliferation of human bone cells. Clin. Chim. Acta 2002, 324, 89-97. Ye, Y.N.; Koo, M.W. Angelica sinensis modulates migration and proliferation of gastric epithelial cells. Life Sci. 2001, 68 (8), 961-968. Ye, Y.N.;

Liu, E.S.D.

Xu, Q.Y4hiau;,

A mechanistic

Wee Lin, Y:He

in rats lung. Hubei

Zava, D.T.; Dollbaum, C.M.; Blen, M. Estrogen and progestin bioactivity of foods, herbs,

andspices. Proc. 217 (3), 369-378.

Ohta, T.; Nakano,

study

of

Radix

Angelica

sinensis prevents bleomycin-induced acute injury

213-219. 16.

acid.

proliferation induced by Angelica sinensis in a normal gastric epithelial cell line. Biochem. Pharmacol. 2001, 6/ (11), 1439-1448.

Rosenberg Zand, R.S.; Jenkins, DJA); Dimandis, E.P. Effects of natural products and

nutraceuticals

of ferulic

tory effect of ferulic acid. Zhongguo Daxue Zazhi 1990, 2/ (5), 279-282.

Tress,

N.B.; Eagon, C.L. Modulation of estrogen action. Proc. Am. Assoc. Cancer Res. 2001, 42 (March), pages unavailable.

potential

and isoferulic acid on murine interleukin-8 production in response to influenza virus infections in vitro and in vivo. Planta Med. 1995, 61 (3), 221-226.

Jiangsu Zhongyi 1988, 9 (1), 15-16. 12;

E. Antioxidant

Antioxidant activity of ferulic acid B-glucuronide in the LDL oxidation system. Biosci. Biotechnol. 1997, 61 (11), 1942-1943.

danggui 1988, /,

Pu YF Xia, -Y-K.; Shi, -Y.P:-Treatment iof 34 cases of infertility due to tubal occlusion with compound danggui injection by irrigation.

Graf,

Free Rad. Biol. Med. 1992, 73 (45), 435-438.

a2

Yike

Daxue

Xuebao

1997,

18 (1), 20-23. Tong, Y.F. Treatment of temporomandibular joint dysfunctional syndrome by injection of Angelica sinensis extract into pterygoideus externus: clinical analysis of 50 cases. Zhongyi Zazhi 1991, J2 (5), 293. Cui, L.X. Treatment of interspinal ligament injury with danggui injection. Shanghai J. Acupunct. Moxibust. 1989, 8 (1), 22. Pharmacopoeia of the People’s Republic of China; Chemistry and Industry Press: Beijing, 2000; Vol. 1. Mei, Q.B.; To, J.Y.; Cui, B. Advances

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sinensis.

Shanxi

Zazhi

'

Dehydroepiandrosterone (DHEA) Salvatore Alesci Clinical Neuroendocrinology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A.

Irini Manoli Endocrine Section, Laboratory of Clinical Investigation, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland, U.S.A.

Marc R. Blackman Endocrine Section, Laboratory of Clinical Investigation, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland, U.S.A.

INTRODUCTION

scientific evidence supporting the purported potential

DHEA is the acronym used to designate the hormone “dehydroepiandrosterone,’’ also referred to as “‘prasterone.’’ The chemical name for DHEA is 5-androsten-3f-ol-17-one (Fig. 1). As a dietary supplement, it is marketed under different trade names (e.g., Nature’s Blend DHEA, Nature’s

potential short- and long-term adverse risks of consuming exogenous DHEA. Moreover, variations in quality control and manufacturing practices of dietary supplements result in differences in concentrations and purity of the marketed compounds, and insufficient surveillance for side effects.

health

Bounty

DHEA,

DHEA

Max,

DHEA

benefits,

and little information

regarding

the

Fuel, etc.). A

pharmaceutical-grade preparation, currently available only

for

trade name GL701).

experimental

use,

has

been

Prestara (previously known

assigned

the

as Aslera or

BIOCHEMISTRY AND PHYSIOLOGY Biosynthesis and Metabolism DHEA is primarily produced in the zona reticularis of

HISTORICAL OVERVIEW AND GENERAL DESCRIPTION

the adrenal

Discovered in 1934, DHEA is the most abundant steroid hormone, and is produced by the adrenal glands in humans and other primates. It acts as a weak androgen and serves as a precursor of other steroids including more potent androgens and estrogens. To date, however, the exact functions of this hormone remain unknown.

DHEA

cortex.

In healthy

women,

the adrenal

gland is the principal source of this steroid, whereas in men, 10-25% of the circulating DHEA is secreted by the testes.' It can also be synthesized within the central nervous system (CNS), and can be considered a “neurosteroid.’’?! Pregnenolone, the immediate precursor of DHEA,

is derived from cholesterol through

the action of the cytochrome P450 side-chain cleavage

is broadly traded on the Internet,

under the claim of being a ‘‘marvel hormone.’’ Despite the growing popularity of its use, there is insufficient

Salvatore Alesci, M.D., Ph.D., is Staff Scientist at the Clinical Neuroendocrinology Branch, National Institute of Mental Health,

National Institutes of Health, Bethesda, Maryland, U.S.A. Trini Manoli, M.D., is Visiting Fellow at the Endocrine Section,

Laboratory of Clinical Investigation, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland, U.S.A. Marc R. Blackman, M.D., is Chief at the Endocrine Section,

Laboratory of Clinical Investigation, National Center for Comple-

mentary and Alternative Medicine, Bethesda, Maryland, U.S.A.

National

Institutes of Health,

Encyclopedia of Dietary Supplements DOT: 10.1081 /E-EDS-120022909 Copyright © 2005 by Marcel Dekker. All rights reserved.

HO Fig. 1 Chemical (DHEA).

structure

of

dehydroepiandrosterone

167

Dehydroepiandrosterone (DHEA)

168

Cholesterol CYPscc

Pregnenolone i

CYP17 Y

Fig. 2 Schematic diagram of DHEA/DHEAS biosynthesis and , CYP17 metabolism. Abbreviations: CYPscc, v cytochrome P450 side-chain cleavage |~@@™ 38-HSD -| DHEA |-17B-HSD =~—| Testosterone | enzyme; CYP17, cytochrome P450 17alpha hydroxylase; 3B/17B/3a-HSD, 3-beta /17-beta/3-alpha hydroxyCYParo steroid dehydrogenase; 5a-R, 5-alpha DHEASH DHEAST

170—Hydroxypregnenolone

Androstenedione

a

bd

|

reductase; DHEASH, DHEA sulfohydrolase; DHEAST, DHEA sulfotransferase; CY Paro, cytochrome P450 aromatase.

Androsterone

enzyme (CYPscc). It is converted into DHEA by cytochrome P450 17a-hydroxylase (CYP17), while hydroxysteroid sulfotransferase (DHEAST) catalyzes the transformation of DHEA into its 3-sulfated metabolite DHEAS (Fig. 2). This can be converted back to DHEA by the action of sulfohydrolases (DHEASH), located in the adrenal gland and peripheral tissues. Human plasma contains DHEA-fatty esters (DHEA-FA), which are formed from DHEA by the enzyme lecithin-cholesterol acyltransferase. Newly formed DHEA-FA are incorporated into very-lowdensity lipoproteins (VLDL) and low-density lipoproteins (LDL) and may be used as substrates for the synthesis of active oxidized and hydroxylated metabolites in the periphery, such as 7a/B-hydroxy-DHEA in the brain, and androstenedione, androstenediol, and androstenetriol in the skin and immune organs.&-4)

Regulation of DHEA Production Release of DHEA

by the adrenals is mostly synchro-

nous with that of cortisol, under the stimulus of the

hypothalamic corticotropic-releasing hormone (CRH) and pituitary adrenocorticotropic hormone (ACTH). However, the finding of dissociation between DHEA

and cortisol secretion during several physiologic and pathophysiologic states suggests that other non-ACTH mediated mechanisms may be involved in the modulation of DHEA secretion (Table 1). Estrogens, growth hormone, insulin, and prolactin stimulate DHEA secretion by human adrenal cells. However, these findings have not always been replicated in animal or clinical studies. A complex intraadrenal network involving vascular and nervous

systems,

local growth

and

immune

factors,

and

a

“‘cross-talk’’ between cells of the cortex and medulla,

the other component of the adrenal gland, also regulate DHEA secretion. The existence of a specific “adrenal androgen stimulating hormone’’ has also been postulated, but remains controversial.?!

Table 1 Dissociation of cortisol and DHEA/DHEAS secretion during physiological and pathological conditions Condition

DHEA

Physiological (age-related) Fetal stage Birth Infancy and childhood Adrenarche (6-8 yr) Puberty Adrenopause (50-60 yr) Pathological Anorexia nervosa Chronic/severe illness Burn trauma Cushing’s disease Congenital adrenal hyperplasia Ectopic ACTH syndrome Idiopathic hirsutism Partial hypopituitarism without ACTH deficiency End-stage renal diseases

Stress

a eh N = normal serum decreased serum levels.

(From Ref.!!)

jj t l |

N

DHEAS ii T Ih T il i!

N N N N N N or T

I | { N T

T T i T iy

IN, [; OF 1 NOR oct

i

{

Cortisol

ii

i i)

N N

l

if

1 ee See ee eae levels; { = increased serum

T ee levels; | =

Dehydroepiandrosterone (DHEA)

169

Adrenarche and Adrenopause

At

birth,

DHEAS

steroid. However,

is the predominant a dramatic

involution

circulating of the fetal

adrenal zone, starting in the first postnatal month and continuing through the first year of life, is paralleled by a sudden decrease in DHEA/DHEAS, which remains unchanged for the next 6yr. By the age of 6-8 yr, the adrenal gland matures, culminating in the

glucose-6-phosphate dehydrogenase (G6DPH), thus interfering with the formation of mitochondrial NADP(H) and ribose-6-phosphate and _ inhibiting DNA synthesis and cell proliferation.""! The steroid hormone has also been proposed to exert antiglucocorticoid, cytokine modulatory, potassium channel and cyclic guanyl monophosphate (cGMP) stimulatory, and thermogenic effects.

creation of the zona reticularis, followed by an abrupt elevation

in

DHEA

and

DHEAS

concentrations,

termed the “adrenarche.’’!! Peak concentrations of DHEA (180-800 ng/dl) and DHEAS (45-450 g/dl) are reached during the third decade of life. Subsequently, there is a progressive 2% decline per year in DHEA and DHEAS secretion and excretion, with concentrations equal to 20% of the peak by the age of 80, and values lower in women than in men.©-7! This marked decline has been termed ‘“adrenopause.”’ DHEA and DHEAS levels are higher in men than in women at all ages.

Mechanisms

of Action

PHARMACOKINETICS Absorption and Tissue Distribution While DHEA is marketed as an oral product, it has also been shown to be absorbed when administered by the transdermal,

intravenous,

subcutaneous,

and

vaginal routes. Crystalline and micronized formulations result in higher DHEAS serum concentrations, possibly due to an enhanced rate of absorption"?! After absorption in the small intestine, DHEA is mainly sulfated in the liver. The nonoral route averts first pass liver degradation, resulting in higher serum levels. DHEA concentrations are high in the brain, with

a brain-to-plasma

ratio

of 46.5

to

1,03] and

Despite the identification of high-affinity binding sites for DHEA in rat liver, T-lymphocytes, and endothelial cells, the search for a specific, cognate DHEA receptor has been unsuccessful. Multiple mechanisms of action have been proposed for DHEA. Most important among these are that DHEA can be metabolized into more potent androgens [testosterone and dihydrotestosterone (T and DHT)] and estrogens (estradiol and estrone) in the periphery, which can interact with specific androgen and estrogen receptors. DHEA itself can bind to the androgen and estrogen receptors, but its affinity is extremely low compared with those of the native ligands. It has been estimated that DHEA and DHEAS function as precursors of 50% of androgens in men, 75% of active estrogens in premenopausal women, and 100% of active estrogens in postmenopausal women."! Lipophilic DHEA, but not hydrophobic

follows a one-compartment model of disappearance. Its volume of distribution is 4.6 + 0.9L, while the half-life from that compartment is 13.7hr."*! In men, 77.8 + 17.3% of the DHEAS that enters the circulation

DHEAS,

will

can be converted

into both androgens

and

plasma, spleen, kidney, and liver concentrations follow in descending order. Cerebrospinal, salivary, and joint fluid levels are directly related to those of

serum. Bioavailability, Metabolism, and Clearance

Pharmacokinetic studies on DHEA reveal a clearance rate compatible with a two-compartment model. The initial volume of distribution is 17.0 + 3L. DHEA disappears from the first compartment in 17.2 + 6.2 min

and from the second in 60.2 + 12.3min'“! DHEAS

reappear

as

DHEA,

while

in

women,

it is

estrogens intracellularly in target tissues, by “‘intracrine’’ processes.4! This conversion depends on the levels of different steroidogenic and metabolizing enzymes, and on the hormonal milieu. For example, DHEAS and sulfatase are present in high concentrations in the prostate, and the resultant metabolism of DHEA to DHT accounts for up to one-sixth of the

60.5 + 8.2%. The opposite conversion of DHEA to DHEAS is much smaller, 5.2% + 0.7% in men, and 6.25% + 0.54% in women. Mean metabolic clearance rates (MCRs) were calculated using the constant infusion technique: The DHEA MCR is 2050 + 160L/day in men and

intraprostatic DHT.”!

for DHEAS is 13.8 + 2.7L/day in men and 12.5 + 1.0L/day in women. The differences in the clearance rates of DHEA and DHEAS are partly

DHEA can also function as a neurosteroid, by modulating neuronal growth, development, and excitability, the latter via interaction with y-aminobutyric acid (GABAa), N-methyl-p-aspartate (NMDA), and sigma receptors.!'! It is known to be a potent inhibitor of

2040

+

160L/day

in

women,

whereas

the

MCR

explained by different binding efficiencies with albumin. Circulating DHEA is primarily bound to albumin, with only minimal binding to sex-hormone

Dehydroepiandrosterone (DHEA)

170

binding globulin (SHBG); the remaining small amount is free. There is no known specific DHEA binding protein. In comparison, DHEAS is strongly bound to albumin

but

not

to

SHBG,

and

an

even

smaller

amount is protein free. Obesity results in increased MCR for DHEA from 2000 to 4000 L/day in women. A rise in MCR is also caused by insulin infusion in men.!'°

administration to patients with primary (Addison’s disease) and secondary adrenal insufficiency. In such cases, DHEA deficiency is usually not corrected. Despite optimal glucocorticoid and mineralocorticoid replacement, however, these subjects often experience chronic fatigue, reduced sense of well being, and lack of sexual interest. Oral administration of 50mg/day of DHEA for 4mo to 24 women with adrenal insufficiency increased serum

Supplementation

androstenedione,

levels of DHEAS,

one, and androstenediol overall well being, mood,

Considerations related to the metabolism of DHEA become more complicated when it is administered as a dietary supplement (in the United States) or as a drug (in some other countries). The steroid is usually given orally in a single morning dose, as its constant interconversion to DHEAS and the long half-life of DHEAS make multiple dosing unnecessary. In addition, morning dosing mimics the natural rhythm of DHEA secretion. Doses ranging from 25 to 1600 mg/day have been used in different studies. After an oral dose, the half-lives of DHEA/DHEAS are longer (24hr) than those reported in intravenous tracer

studies, which

may

reflect the conversion

of

DHEAS to DHEA”! Oral administration of 2050 mg of DHEA in patients with primary or secondary adrenal insufficiency restores serum DHEA and DHEAS concentrations to the range observed in normal young subjects, while a dose of 100-200 mg/day results in supraphysiological concentrations. Different metabolic pathways for exogenous DHEA in relation to gender and age have been reported. DHEA levels after oral administration of 25 or 50mg DHEA for 8 days were persistently higher in women versus men!!! Similarly, oral administration of 200mg of micronized DHEA in single or multiple doses for 15 days in healthy adult men and women resulted in higher serum concentrations and bioavailability (measured by DHEA Cyax and AUC) in women. The net increase in DHEAS levels was 21-fold in women and 5-fold in men."®! The metabolic fate of exogenous DHEA also differs by gender and age. While in pre- and postmenopausal women, DHEA is mostly transformed into androgens, in men it is prefer-

entially

metabolized

concentrations

into

of DHEA,

estrogens. testosterone,

Higher

serum

and estradiol

are achieved in elderly subjects."'7!

THERAPEUTIC APPLICATIONS DHEA Replacement in Adrenal Insufficiency The strongest evidence for a beneficial effect of DHEA replacement is provided by studies of its

testoster-

glucuronide,

and improved

sexual

activity.2"! In

and

another study of 15 men and 24 women with Addison’s disease, a 3-mo administration of 50 mg/day of DHEA corrected the hormonal deficiency and improved selfesteem, while it tended to enhance overall well being,

mood, and energy.?1J

-

DHEA Replacement in Adrenopause and

©

Age-Related Disorders As noted, aging is accompanied by a profound decrease in circulating levels of DHEA and DHEAS in both sexes.” Epidemiological studies suggest an association between DHEA and DHEAS decline and the adverse effects of aging, albeit with gender differ-

ences. One large prospective observational study showed a small,

but

significant,

inverse

correlation

between

serum DHEAS levels and risk of cardiovascular mortality in men at 19 yr of follow-up, whereas in women, high DHEAS levels were associated with an increased risk of cardiovascular death at 12 yr of follow-up, and this trend lost significance at 19yr of follow-up.) Similar results were reported in another study of 963 men and 1171 women aged >65yr: all-cause and cardiovascular mortality were highest in men with DHEAS levels in the lowest quartile, whereas no significant association between circulating DHEAS and mortality was found in women.”*! Other studies failed to demonstrate this inverse relationship in men.24 Positive correlations between low DHEAS levels and depressed mood and bone loss have been reported in aged women.”*! In comparison, DHEAS levels are reduced in men, with noninsulin dependent diabetes mellitus (NIDDM).”° Reports of an association between low DHEA levels and Alzheimer’s disease are conflicting. It remains uncertain as to whether the DHEAS decline is simply a biomarker of aging, or is causally related to morbidity and mortality in the elderly. One study on the effects of a 6-mo oral adminstration of 50mg/day of DHEA in 13 men and 17 women aged 40-70yr showed restoration of DHEA and DHEAS

Dehydroepiandrosterone (DHEA)

171

levels to young-adult values, with improvement in physical and psychological sense of well being, but not sexual

interest,

in both

genders.

These

effects

were

accompanied by increased serum levels of IGF-I, reduced IGF-I binding protein-1 (IGFBP-1), and a significant decrease in apolipoprotein Al and HDLcholesterol in women?” Another study using 100 mg/day of DHEA for 6mo in 9 men and 10 women aged 50-65 yr reported decreased fat mass and enhanced muscle

strength

in men,

whereas

increased

levels of

downstream androgens were detected in women.”®! In a more recent study of 39 elderly men treated for 3mo with

100mg/day

of oral DHEA,

no treatment

effect

on body composition or subjective well being was found, whereas a significant reduction in HDL-cholesterol was reported.??! In 280 men and women aged 60-79 yr treated for 12mo with 50 mg/day of oral DHEA, a slight but significant increase in bone mineral density (BMD) at the femoral neck and the radius, and an increase in serum testosterone, libido, and sexual function were observed

in women.?°! Similar changes were reported in 14 women aged 60-70 years who were treated for 12mo with a 10% DHEA skin cream. In addition to a 10-fold increase

in

DHEA

levels,

the

authors

described

increased BMD at the hip, and decreased osteoclastic and increased osteoblastic bone markers. Other changes included improved well being, a reduced skinfold thickness, and lower blood glucose and insulin levels,

with no adverse change in lipid profile.) DHEA replacement did not affect BMD in other studies.*! Little information is available about the effects of DHEA therapy on cardiovascular function and insulin sensitivity from interventional studies. DHEA facilitated fibrinolysis and inhibited platelet aggregations in humans.°?! In one study of 24 middle-aged men, administration of 25mg/day of oral DHEA for 12 weeks decreased the plasma levels of plasminogen activator inhibitor type 1 (PAI-1), and increased dilatation of the brachial artery after transient occlusion,"! In rodent models

of NIDDM,

dietary administra-

tion of DHEA consistently induced remission of hyperglycemia and increased insulin sensitivity. Some clinical studies in aged men and women have shown improved insulin sensitivity after DHEA replacement,/?!741 whereas

others have not confirmed

those

findings in women.?7?>! There are few studies on the effect of DHEA on cognitive function. DHEA administration improves memory and decreases serum levels of B-amyloid in aging mice. In contrast, treatment with 100 mg of oral

DHEA patients

did not

improve

with

Alzheimer’s

cognitive

performance

disease.°°!

Potentially Beneficial Effects of DHEA Supplementation

in

Moreover,

DHEA replacement did not affect memory in any of the controlled studies in healthy elderly, as previously discussed.

DHEA

administration, often in large doses, has been

proposed in the management of numerous disorders, including obesity, cancer, autoimmune diseases, AIDS, mood disorders, as well as in enhancing physical performance. The scientific evidence supporting the benefits of DHEA therapy in these conditions is, however, very limited. Pharmacologic treatment with DHEA in mice genetically predisposed to become obese reduced weight gain and fat cell size. In obese rats, the steroid decreased food intake by 50%. In humans, some obser-

vational studies indicate a relationship between circulating DHEA and DHEAS levels, body mass index (BMI) and weight loss, whereas others do not confirm this.” Similar inconsistencies are encountered in interventional studies. Administration of a high oral dose of DHEA (1600mg/day) decreased body fat and increased muscle mass, with no net change in body weight, in a small group of healthy young men.&*! As mentioned

above,

a reduction

in fat mass

was

also

reported in healthy elderly men after a 6mo administration of 100mg/day of DHEA,”*! whereas topical application of a 10% DHEA cream for 1 yr decreased femoral fat and skinfold thickness in postmenopausal women.?!! Other studies in healthy elderly individuals and in obese men failed to demonstrate changes in body fat after DHEA treatment.??*>! DHEA has been reported to exhibit chemopreventive activity in mouse and rat models, although it has also been found to be hepatocarcinogenic in rats. Epidemiological research has revealed increased DHEA levels to be associated with a rise in risk of ovarian cancer and breast cancer in postmenopausal women, whereas decreased levels are linked with increased risk of bladder, gastric, and breast cancer in premenopausal women. To date, we are unaware of clinical studies documenting the effects of DHEA intervention on

cancer initiation or propagation. Fluorinated DHEA analogs that cannot be converted into androgens or estrogens appear to have antiproliferative effects and have been tested in several preclinical cancers including bowel polyposis“?! and used without side effects in doses up to 200 mg/day orally for 4 weeks. 4° Increased antibody production in response to bacterial infections and decreased mortality from endotoxic shock were reported in DHEA-treated mice. In a study of 71 aged individuals, however, DHEA administration did not enhance antibody response to influenza vaccine.!! In contrast, 50mg/day of oral DHEA increased the activity of natural-killer cells by twofold, with a concomitant decrease in T-helper cells in postmenopausal women.?] In 28 women with mild-to-moderate systemic lupus erythematosus, oral

Dehydroepiandrosterone (DHEA)

172

body mass of 4.5kg was observed in healthy young men taking 1600mg/day of DHEA for 4 weeks? 4 A summary of the various potential therapeutic applications of DHEA replacement/supplementation is presented in Table 2.

treatment with 200mg/day of DHEA for 3-6mo improved well being and decreased disease activity and prednisone dosage requirements.'*7! The same DHEA dose administered for 16mo to patients with rheumatoid arthritis showed no beneficial effects. Serum DHEA and DHEAS levels'in patients infected with HIV are directly associated with CD4 cell counts and the stage and progression of the disease. This observation has stimulated self-administration of DHEA as an adjunct to antiviral treatment by AIDS patients. In the only published placebo-controlled trial of DHEA in patients with advanced HIV disease, treatment with 50 mg/day orally for 4 mo resulted in increased DHEAS

CLINICAL PHARMACOLOGY

AND TOXICOLOGY

Dietary/NonDietary Sources and Available Preparations There are no known dietary sources of DHEA, although it was suggested that the supplement chromium picolinate could stimulate endogenous DHEA secretion. The Mexican plant “wild yam’’ contains some natural DHEA precursors, which cannot be con-

levels and improved mental function, with no change

in CD4 cell count!4! These results were consistent with those from a previous open-label study in 32 HIV-positive patients treated with DHEA doses of

200-500 mg/day for 8 weeks.!*"!

verted into DHEA.

Studies in adults and adolescents with major depressive disorders have revealed a blunted DHEA circadian variation, with low DHEA and _ high

yam’’ (such as diosgenin and dioscorea) are used to produce various forms of synthetic DHEA, including tablets, capsules, injectable esters (Gynodian Depot, Schering), sublingual and vaginal preparations, topical creams, lozenges, and herbal teas. DHEA is usually sold in tablets of 5-5Omg. A pharmaceutical-grade preparation (Prestara) has been developed for potential use as a prescription drug. During the manufacture

cortisol/DHEA

ratio

at

8:00am.

In 22

patients

with medication-free or stable major depression, supplementation with 30-90mg/day of oral DHEA for 6weeks decreased Hamilton depression scale scores as much as 50%.!°! Similar results were reported in a well-controlled study in 15 patients aged 45-63 yr with midlife-onset dysthymia who, after a 3-week administration

of 90mg

of DHEA,

reported

of DHEA,

sterol extracts of ‘‘wild

other steroidlike compounds,

like andro-

stenedione, may be produced and could contaminate DHEA. Moreover, the real steroid content of the DHEA preparation sold over the counter may vary from 0% to 150% of the amount claimed.©°! In addition, there is a lack of information about comparability or bioequivalence among the many products on the market or information about lot-to-lot variability of any particular product in terms of characterization (content) and standardization (contaminants).

improvements

in depressive symptoms.7! DHEA is a popular dietary supplement among athletes. Nevertheless, at 150 mg/day orally, it did not improve body mass or strength in young male athletes and weight lifters.'4*?! In contrast, increased quadriceps and lumbar strength were reported in healthy elderly

men on DHEA replacement,”*! and an increase in lean Table 2

However,

Clinical conditions for which DHEA use has been proposed

Condition

Effect

References

Adrenal Insufficiency

Improved general well being, mood, sexual function No significant effects

17 18

Aging

Improved physical well being, bone mineral density or sexual function Decrease in HDL or no effects

24

Improved well being, fatigue, disease activity in SLE patients, enhanced immune response No effect in HIV patients

38-40 41, 42

Decreased fat- and increased muscle mass No changes

25, 35, 45, 46 17, 24, 26, 32, 45, 46

Autoimmune disease

Body composition Insulin resistance

25-27, 33

Improved insulin sensitivity

28, 31

No change

24, 32

Depression

Improved mood

43, 44

Alzheimer’s disease

No effect on cognitive function

53

Cardiovascular

CESS

SASIREEEE tenes

Improved endothelialAe function Somer asSelmer aia Se Eee ESIC

Ee

30

Dehydroepiandrosterone (DHEA)

173

Dosage and Administration

There is no consensus on a recommended dietary allowance (RDA) or optimal treatment dose for DHEA. Replacement with oral doses of 20-50mg/da for men and 10-30mg/day for women appears adequate to achieve DHEA/DHEAS levels similar to those in young adults, as suggested by most studies in subjects with adrenal or age-related DHEA/DHEAS deficiency, though oral doses as low as 5mg/day have been reported to be effective. Higher DHEA doses may be necessary for patients with very low endogenous DHEA levels secondary to steroid administration or chronic disease. Doses of 200-500 mg and 200mg/day have been used in patients with HIV and systemic lupus erythematosus, respectively. Serum DHEAS levels and its androgenic and estrogenic metabolites must be closely monitored during replacement, to enable appropriate dose adjustments. Rigorous dose ranging studies are needed to determine the optimal doses to achieve a beneficial effect.

Adverse Reactions, Long-Term Contraindications

Effects, and

DHEA appears to elicit few short-term side effects when used in the recommended doses. Women may experience mild hirsutism, increased facial sebum production, and acneiform dermatitis. Circulating levels of

downstream androgens rise above young-adult values in healthy elderly women treated with 100mg/day DHEA;

however,

the long-term consequences

of this

DHEA administration is not recommended for people under 40 yr of age, unless there is a documented deficiency state. It should be avoided during pregnancy, lactation, and in persons younger than 18 yr, since dosage and safety of the treatment under these conditions have not been evaluated.

Known

Drug Interactions

Drugs known to interfere with DHEA and/or DHEAS include various hormone preparations, drugs acting on the

CNS,

cardiovascular

drugs,

adrenergic

and

adrenergic blocking agents, and anti-infective agents. Synthetic glucocorticoids such as dexamethasone are the most potent suppressors of DHEA and DHEAS.

DHEA

and/or

DHEAS

levels

are

known

to be

decreased in patients taking aromatase inhibitors, oral

contraceptives, dopaminergic receptor blockers, insulin, troglitazone, and multivitamins, or fish oil. They are also decreased due to the induction of the cytochrome P450 enzymes, by carbamazepin, phenytoin, or rifampicin. Metformin (a biguanide antihyperglycemic drug) and calcium channel blockers are shown to increase DHEA and DHEAS levels. The effect of alcohol is controversial.?!

COMPENDIAL/REGULATORY

STATUS

In the early 1980s, DHEA was widely advertised and sold in U.S. health food stores as an “antiaging,” “antiobesity,’’ and “‘anticancer’’ nonprescription drug. However, on the basis of unknown potential long-term

increase are unknown. No significant changes in complete blood count, urinalysis, hepatic, and thyroid indices were found in women after 28 days of treatment with 1600mg DHEA per day.8°! A dose escalation study of 750-2250mg oral DHEA conducted in: 31 HIV-positive men revealed no serious dose-limiting toxicity.P4 Because of its potent androgenic and estrogenic effects, it would appear prudent to avoid DHEA replacement /supplementation in individuals with a personal

Olympic Committee, in 1985 the U.S. Food and Drug Administration reclassified DHEA as a prescription drug. In October 1994, the U.S. Dietary Supplement Health and Education Act allowed DHEA to be sold again as an over-the-counter dietary supplement, as long as no claims are made regarding therapeutic efficacy.

or family history of breast, ovarian, or prostate cancer.

CONCLUSIONS

risks,

and

following

the

ban

by the

International

This would seem especially important for postmenopausal women, considering the demonstrated direct correlation between DHEA levels and breast and ovarian cancers in this group,”?! and the reported increase with DHEA supplementation in free IGF-1.57! Caution may also be appropriate in HIV patients, since high DHEA levels have been implicated in the pathogenesis of Kaposi’s sarcoma. Moreover, increased insulin resistance and decreased cholesterol and high-density lipoproteins after administration of a high dose 1600mg/day of DHEA for 4 weeks have

ciency,

been reported in aged women.!*!

healthy aged people have been inconclusive, save for

Despite a large amount of research related to DHEA, and its alleged utility to sustain ‘“‘eternal youth,’’ several key questions remain unanswered. For example, the physiologic function(s), regulation, and mechanisms of actions

causal link and adverse Results from be beneficial

of DHEA

remain

unknown,

and

a

between age-related declines in DHEA effects of aging has not been proven. clinical studies suggest that DHEA may in some patients with adrenal insuffi-

whereas

those

from

intervention

studies

in

Dehydroepiandrosterone (DHEA)

174

some modest gender differences in selected outcome measures. Well-characterized and standardized products need to be evaluated for safety and efficacy, starting with dose ranging to determine an optimal dose. Because DHEA can be converted to estrogen and

testosterone,

its

potential

adverse

effects

LI;

Pashko,

we

Casson, P.R.; Straughn, A.B.; Umstot, ES.; Abraham, G.E.; Carson, S.A.; Buster, J.E. Deliv-

ery of dehydroepiandrosterone to premenopausal women: effects of micronization and nonoral administration. Am. J. Obstet. Gynecol. 1996, 174 (2), 649-653.

13.

Robel, P.; Baulieu, E.E. Dehydroepiandrosterone

(DHEA) is a neuroactive neurosteroid. Ann. N. Y. Acad. Sci. 1995, 774, 82-110. 14.

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Alesci, S.; Bornstein, S.R. Intraadrenal mechan-

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in

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Schwartz,

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Yen,

S.S.

The

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Wolkowitz, O.M.; Kramer, J.H.; Reus, V.L; Costa, M.M.; Yaffe, K.; Walton, P.; Raskind, M.; Peskind, E.; Newhouse, P.; Sack, D.; De Souza, E.; Sadowsky, C.; Roberts, E. DHEA treatment of Alzheimer’s disease: a randomized, double-

blind, placebo-controlled study. Neurology 2003, 60 (7), 1071-1076. Williams, J.R. The effects of dehydroepiandrosterone on carcinogenesis, obesity, the immune system, and aging. Lipids 2000, 35 (3), 325-331. Nestler, J.E.; Barlascini, C.O.; Clore, J.N.; Blackard,

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DHEA in aging and other disease states. Ageing Res. Rev. 2002, 7 (1), 29-41.

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Davidson, M.; Marwah, A.; Sawchuk, R.J.; Maki, K.; Marwah, P.; Weeks, C.; Lardy, H. Safety and

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W.G. Dehydroepiandrosterone reduces serum low density lipoprotein levels and body fat but does not alter insulin sensitivity in normal men. J. Clin.

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Morales, A.J.; Nolan, J.J.; Nelson, J.C.; Yen, S.S.

J. Clin. Endocrinol. Metab. 1994, 78 (6), 1360-1367. Morales, A.J.; Haubrich, R.H.; Hwang, J.Y.; Asakura, H.; Yen, S.S. The effect of six months

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M.; Ogawa, H. Dehydroepiandrosterone supplementation improves endothelial function and insulin sensitivity in men. J. Clin. Endocrinol. Metab. 2003, 88 (7), 3190-3195.

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Beer, N.A.; Jakubowicz, D.J.; Matt, D.W.; Beer, R.M.; Nestler, J.E. Dehydroepiandroster-

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J. Clin.

Pharmacol.

2002,

Echinacea Rudolf Bauer Karin Woelkart Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz, Graz, Austria

INTRODUCTION Echinacea is a herbal medicine that has been used for centuries, customarily as a treatment for common cold, coughs, bronchitis, upper respiratory infections (URIs), and inflammatory conditions. It belongs to Heliantheae tribe of the Asteraceae (Compositae) family, and the nine species of these perennial North American wildflowers have a widespread distribution over prairies, plains, and wooded areas.

BACKGROUND Three species of Echinacea are used medicinally: E. purpurea (Fig. 1), E. pallida (Fig. 2), and E. angustifolia (Fig. 3). In the United States of America, Echinacea preparations have been the best selling herbal products in health food stores. However, an analysis reveals that completely different preparations are sold under the name Echinacea."! The most investigated preparation, which is mainly available on the German market, contains the expressed juice of E. purpurea aerial parts. Besides this, hydroalcoholic tinctures of E. purpurea aerial parts and roots, as well as from E. pallida and E. angustifolia roots, can be found.*! In North America especially, it is also common to.sell encapsulated powders from aerial parts and roots of the three species. Despite the popularity of the herb and the fact that over 400 scientific papers have been published about its ability to “boost the immune system’’ both in vitro and in vivo, its molecular mode of action is still not

fully understood. At least it is evident that Echinacea preparations can enhance phagocytosis and stimulate

the production of cytokines in macrophages, meaning that they preferentially stimulate the nonspecific immune system. It is apparent that the multiplicity and diversity of parts of various plants, methods of extraction, and solvent used, as well as the components on which the extracts have been standardized, have

hampered recommendations regarding Echinacea usage. Several reviews on the evidence regarding the effectiveness of orally ingested Echinacea extracts in reducing the incidence, severity, or duration of acute URIs have been published. So far, nine controlled treatment trials and four prevention trials have been performed with Echinacea preparations. Seven of the treatment trials reviewed by Barrett reported generally positive results, and three of the prevention trials marginal benefit.4! More recently, the clinical effect of a preparation from E. purpurea, standardized on alkamides, cichoric acid, and polysaccharides, has been confirmed.

It is apparent from this study that early intervention with Echinacea could potentially reduce the indirect cost of a cold in terms of workplace absenteeism and performance."! Clinical efficacy has been approved by the German Commission E for the expressed juice of the aerial parts of E. purpurea in the adjuvant therapy of relapsing infections of the respiratory and urinary tracts, as well as for the alcoholic tincture of E. pallida roots as adjuvants in the treatment of common cold and flu! The regulatory status of Echinacea products is variable. In the United States of America, they are considered as dietary supplements; in Canada, as Natural Health Products (NHPs); and in several European countries, they have drug status.

HISTORICAL AND BOTANICAL ASPECTS OF ECHINACEA SPECIES Rudolf Bauer, Ph.D., is at the Department of Pharmacognosy,

The genus Echinacea (Asteraceae) is endemic to North

Karl-Franzens-University

America, where it grows in the Great Plains between

Institute of Pharmaceutical Graz, Graz, Austria.

Sciences,

Karin Woelkart is at the Department of Pharmacognosy, Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz, Graz, Austria.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022101 Copyright © 2005 by Marcel Dekker. All rights reserved.

the Appalachian Mountains in the east and the Rocky Mountains in the west.!”] The medicinal application of Echinacea can be traced back to the Native Americans, 177

Echinacea

178

19th century, European

settlers took over the use of

the plant. When the monograph on Echinacea in the National Formulary of the U.S. was published in 1916, the roots of both E. angustifolia and E. pallida were officially included, with the result that differentiation between these two species was often neglected later on. The use of the aerial parts of E. purpurea has primarily become prevalent in Europe over the last 60 yr. The taxonomy of the genus Echinacea has been studied intensively by McGregor,”! who accepted nine species and two varieties. Only recently, Binns et al. reported a taxonomic revision of the genus Echinacea mainly from a morphological view point." The fact that E. purpurea is quite different from the other species, led to the creation of a special subgenus “echinacea.” In addition, they assigned E. pallida, E.

atrorubens,

and

E.

laevigata

to

the

subgenus

“‘nallida.’’ The other species described by McGregor”! have been determined as varieties from E. pallida or E.

atrorubens.

However,

the

new

system

is not

widely accepted, and the taxonomic classification by McGregor is still used. Fig. 1 Echinacea purpurea. www.dekker.com. )

(View

this art in color at

who regarded it as one of the most favorable remedies for treating wounds,

ACTIVE PRINCIPLES, PHARMACOLOGICAL EFFECTS, AND STANDARDIZATION

snakebites, headache, and com-

mon cold. The territories of the tribes that most frequently used Echinacea show a close correspondence with the distribution range of E. angustifolia, E. pallida, and E. purpurea. But, possibly, other Echinacea species have also been used.'*! In the second half of the

The constituents of Echinacea, as in any other plant,

cover a wide range of polarity, ranging from the polar polysaccharides and glycoproteins, via the moderately polar caffeic acid derivatives, to the rather lipophilic polyacetylenes and alkamides. This makes it necessary

Fig. 2. Echinacea pallida. (View this art in color at www.dekker.com. )

Echinacea

179

Fig.3 Echinacea angustifolia. (View this art in color at www.dekker.com. )

to study separately the activity of different polar extracts of Echinacea, such as aqueous preparations, alcoholic tinctures, and hexane or chloroform extracts.

Polysaccharides and Glycoproteins

glycoproteins,

Systematic fractionation and subsequent pharmacological testing of the aqueous extracts of the aerial parts of E. purpurea led to the isolation of two polysaccharides (PS I and PS IJ) with immunostimulatory properties. They were shown to stimulate phagocytosis in vitro and in vivo, and enhance the production of oxygen radicals by macrophages in a dose dependent way. Structural analysis showed PS I to be a 4-O-methylglucuronoarabinoxylan with an average MW of 35,000 Da, while

PS II was

anti-inflammatory activity’?! In a Phase-I clinical trial, a polysaccharide fraction (EPO VIIa), isolated from E. purpurea tissue culture and injected at doses of 1 and 5 mg, caused an increase in the number of leukocytes, segmented granulocytes, and TNF-«."'*! Three

demonstrated

to be an

acidic arabinorhamnogalactan of MW 45,000 Da. A xyloglucan, MW.79,500 Da, was isolated from the leaves and stems of E. purpurea, and a pectin-like polysaccharide from the expressed juice.!'"! Polysaccharides from E. angustifolia have also been found to possess

MW _ 17,000,

21,000,

and

30,000

Da, containing about 3% protein, have been isolated from E. angustifolia and E. purpurea roots. The dominant sugars were found to be arabinose (64-84%), galactose (1.9-5.3%), and glucosamines (6%). The protein moiety contained high amounts of aspartate, glycine, glutamate, and alanine!'4! An ELISA method has been developed for the detection and determination of these glycoproteins in Echinacea preparations."'*! £. angustifolia and E. purpurea roots contain similar amounts of glycoproteins, while E. pallida has less."*! Purified extracts containing these glycoprotein—polysaccharide complexes exhibited B-cell stimulating activity and induced the release of interleukin-1 (IL-1), TNF-« and interferon-a«,f (IFN-«,8) in macrophages, which could also be reproduced in vivo in mice.!'4)

Echinacea

180

Recently, Alban et al.!© demonstrated stimulation of the classical pathway (CP) and alternative pathway (AP) of the complement system in human serum by an arabinogalactan-protein type II isolated from pressed juice of the aerial parts of E. purpurea. In a study performed later, the influence of an oral administration of a herbal product, consisting of an aqueous-ethanolic. extract of the mixed herbal drugs Thujae summitates, Baptisiae tinctoriae radix, E. pupureae radix, and E. pallidae radix, standardized on Echinacea glycoproteins, on cytokine induction and antibody response against sheep red blood cells was investigated by Bodinet et al.!'”] in mice. This administration caused a significant enhancement of the antibody response against sheep red blood

cells, inducing

an increase

in the numbers

Influenza A virus infection in Balb/c mice was also tested. The data show that the oral treatment with this aqueous-ethanolic extract induced a statistically significant increase in the survival rate, prolonged the mean survival time, and reduced lung consolidation and Recently,

Hwang,

Dasgupta,

and

Actor"?! showed that macrophages respond to purified polysaccharide and also alkamide preparations. Adherent and nonadherent mouse splenocyte populations were incubated in vitro with E. purpurea liquid extract (fresh Echinacea root juice, mature seed, fresh leaf, and fresh fruit juice extracted in 40-50% alcohol),

or with water or absolute alcohol (25 mg dry powder/ ml of solvent) soluble E. purpurea dried root and leaf extract preparations. Whole splenocyte populations were capable of producing significant amounts of IL-6 in response to E. purpurea water preparations. Likewise, the water soluble extract of E. purpurea was able to stimulate nonadherent splenocyte populations to produce

tumor

necrosis

factor-«

(TNF-a),

oO.

R'O

OH

OH

ele

HO

OH

1 Echinacoside

Glucoside (1,6-)

Rhamnose (1,3-)

2 Verbascoside

H

Rhamnose (1,3-)

Fig. 4 Main phenylpropanoid glycosides found in Echinacea species.

of

splenic plaque forming cells and the titers of specific antibodies in the sera of the treated animals.” The influence of the same extract on the course of

virus _ titer.!1®

HO

IL-10,

and macrophage inflammatory protein-la (MIP-1a) from nonadherent splenocytes. But only significant concentrations of TNF-a and MIP-la« mediators were produced from adherent populations at similar dose concentrations. The immune stimulatory ability of components contained within Echinacea extracts offers insight into possible therapeutic potential to regulate nonadherent lymphocytes in immune responses and activation events.

chromatography (HPLC) analysis.'! Also, capillary electrophoresis [micellar electrokinetic chromatography (MEKC)] has been successfully applied for the analysis of caffeic acid derivatives in Echinacea extracts and enables the discrimination of the species.°" The roots of E. angustifolia and E. pallida have been shown to contain 0.3-1.7% echinacoside (1).! Both species can be discriminated by the occurrence of 1,3- and 1,5-O-dicaffeoyl-quinic acids (3, 4), which are only present in the roots of E. angustifolia. Echinacoside has

antioxidant,?"

low

antibacterial,

antiviral

OH

Caffeic Acid Derivatives

Alcoholic tinctures of Echinacea aerial parts and roots are likely to contain caffeic acid derivatives (see Figs. 4-6). Extracts of different species and plant parts of Echinacea can be distinguished by thin layer chromatography (TLC) or high-performance liquid

and

activity, but does not show immunostimulatory effects.!""! It is also reported that echinacoside inhibits hyaluronidase”! and protects collagen type III from free radical induced degradation in vitro.°*! The aerial parts of E. angustifolia and E. pallida have been shown to contain verbascoside (2), a structural analog of echinacoside (1). The roots of E. purpurea do not contain echinacoside, but cichoric acid (2R,3R-dicaffeoyl tartaric acid; 6) and caftaric acid (monocaffeoyl tartaric acid; 5). Cichoric acid (6) is also the major polar constituent in the aerial parts of Echinacea species. Echinacoside (1) and cichoric acid (6) have also been produced in tissue cultures of E. purpurea and E. angustifolia."'"| The latter acid (6)

3 1,3-Dicaffeoyl-quinic acid (Cynarin)

4 1,5-Dicaffeoyl-quinic acid

Fig. 5 Quinic acid derivatives from Echinacea species.

Echinacea

181

ethanol/water and storage at 4°C, a decline to 0 was observed in the content of echinacoside (1) and cynarin (3) from 0.25mg/ml extract and 0.09mg/ml extract, respectively (see Fig. 7).8 In order to standardize Echinacea preparations and guarantee a consistent

COOH

:

)

RO

H

a OH

COOH

OH

content of caffeic acid derivatives, it is vital to control

this enzymatic activity. 5 2-O-Caffeoyl tartaric acid (Caftaric acid)

R=H

6 2,3-O-Di-caffeoyl tartaric acid

°

(Cichoric acid)

R=

OH

a

OH

Fig. 6

Tartaric acid derivatives from Echinacea species.

has been shown to possess phagocytosis stimulatory activity in vitro and in vivo, while echinacoside (1), verbascoside (2), and 2-caffeoyl-tartaric acid (5) did not exhibit this activity."!! Robinson described cichoric acid as an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase.”4! It is especially abundant in the flowers of all Echinacea species and the roots of E. purpurea (1.2-3.1% and 0.6-2.1%, respectively). Much less is present in the leaves and stems. E. angustifolia contains the lowest amount of cichoric acid.”! The content, however, strongly depends on the season and the stage of development of the plant and is highest at the beginning of the vegetation period and decreases during plant growth.”>! It undergoes enzymatic degradation during preparation of alcoholic tinctures and pressed juices of Echinacea.” Niisslein et al. found that polyphenol oxidases (PPO) are responsible for the oxidative degradation of exogenous and endogenous caffeic acid derivatives.?°?” Apart from cichoric acid, echinaco side (1) and cynarin (3) from E. angustifolia roots are also highly susceptible to enzymatic degradation and oxidation in hydroalcoholic solutions during the extraction process. During the 16 days after extraction of E. angustifolia roots with 60% (v/v)

Alkamides

Striking differences have been observed between the lipophilic constituents of E. angustifolia (alkamides; see Fig. 8) and E. pallida roots (ketoalkenynes; see Fig. 9). E. purpurea roots also contain alkamides, however, mainly with two double bonds in conjugation to the carbonyl group, while E. angustifolia primarily has compounds with a 2-monoene chromophore. The chief lipophilic constituents of E. pallida roots have been identified as ketoalkenes and ketoalkynes with a carbonyl group in the 2-position.”! The main components are tetradeca-8Z-ene-11,13-diyn-2-one (17), pentadeca-8Z-ene-11,13-diyn-2-one (18), pentadeca-8Z, 13Z-diene-11-yn-2-one (19), pentadeca-8Z,11Z,13Etriene-2-one (20), pentadeca-8Z,11F,13Z-triene-2-one (21), and pentadeca-8Z,11Z-diene-2-one (22). They occur only in trace amounts in E. angustifolia and E. purpurea roots. Therefore, they are suitable as markers for the identification of E. pallida roots. However, it has been observed that these compounds undergo auto-oxidation when the roots are stored in powdered form. Then, the hydroxylated artifacts, 8-hydroxy-9E-ene-11,13-diyn-2-one (25), 8-hydroxypentadeca-9E-ene-11,13-diyn-2-one (26), and 8-hydroxypentadeca-9EF,13Z-diene-11-yn-2-one (27), can be primarily found (see Fig. 10), often with only small residual quantities of the native compounds. Hence, the roots of E. pallida are best stored in whole form. About 15 alkamides have been identified as important lipophilic constituents of E. angustifolia roots. They are mainly derived from undeca- and dodecanoic acid, and differ in the degree of unsaturation and the

I

configuration of the double bonds (see Fig. 8). The main structural type is a 2-monoene-8,10-diynoic acid isobutylamide, and some 2'-methyl-butylamides have also been found. In E. purpurea roots, 11 alkamides have been identified with the isomeric mixture of

0.25

© ., % :

Zo

0.2

% 5

, A w Echinacoside

@ Cynarin

€ E015

dodeca-2E,4E,8Z,10E/Z-tetraenoic

oD

= E

nal Of

© G = 4009 oo oO ic

§

acid

isobutyl-

amides (11, 12) as the major compounds.""1] The aerial parts of all three Echinacea species contain alkamides of the type found in E. purpurea roots, and also with dodeca-2E,4E,8Z,10E/Z-tetraenoic acid

0 OOS

15)

Oo

2936-24" hours

4ey

141

191

233

391

Fig. 7 Content of echinacoside and cynarin in 60% EtOH E. angustifolia root extract, during storage at 4°C over 16 days.

isobutylamides (11, 12) as the main constituents.) When testing the alcoholic extracts obtained from the aerial parts and from the roots for phagocytosis stimulating activity, the lipophilic fractions showed the

Echinacea

182

==

RS

==

N

a 7

8

ee

Undeca-2Z,4E-diene-8,10-diynoic acid-isobutylamide

Dodeca-2E,4Z-diene-8,10-diynoic acid-isobutylamide

pe — na ae ©cy 9

Trideca-2E,7Z-diene-10,12-diynoic acid-isobutylamide

17 Tetradeca-8Z-ene-11,13-diyn-2-one

R=

18 Pentadeca-8Z-ene-11,13-diyn-2-one

R=

19 Pentadeca-8Z,13Z-diene-11-yn-2-one

R=

20 Pentadeca-8Z,11Z,13E-triene-2-one

R=

7

“y

a

Ppt Ann es

21 Pentadeca-8Z,11E,13Z-triene-2-one

oh og

R=

10 Dodeca-2E,4Z-diene-8,10-diynoic acid-2-methyl-butylamide 22 Pentadeca-8Z, 11Z-diene-2-one

9 ane

ey H

23 Heptadeca-8Z, 11Z-diene-2-one

11 Dodeca-2E,4E,8Z,10E-tetraenoic acid-isobutylamide 24 Pentadeca-8Z-ene-2-one

O

ee

Sa

Fig. 9 roots.

tA eli

12 Dodeca-2E,4E,8Z,10Z-tetraenoic acid-isobutylamide

and ketoalkynes found

in E. pallida

highest activity, indicating that they represent an active principle. Ethanolic extracts of the aerial parts of E. angustifolia and E. purpurea displayed immunomodulating activity on the phagocytic, metabolic, and bactericidal activities of peritoneal macrophages in mice.'''! Purified fractions from E. purpurea and E. angustifolia roots were shown to enhance phagocytosis in mice by a factor of 1.5-1.7.73] Alkamides also displayed moderate inhibitory activity in vitro in the 5lipoxygenase (porcine leukocytes) and cyclo-oxygenase

O

eee 13 Undeca-2E-ene-8, 10-diynoic acid-isobutylamide

O

Ketoalkenes

iat

14 Undeca-2Z-ene-8,10-diynoic acid-isobutylamide

O

O

Pe ele

OH

R

ey

15 Dodeca-2E-ene-8,10-diynoic acid-isobutylamide

O

se oe al

a

SS H

25

8-Hydroxytetradeca-9E-ene-11,13-diyn-2-one

26

8-Hydroxypentadeca-9E-ene-11,13-diyn-2-one

27

8-Hydroxypentadeca-9E, 13Z-diene-1lyn-2-one

R= R=

R=

16 Pentadeca-2E,9Z-diene-12,14-diynoic acid-isobutylamide

Fig. 8 Main alkamides found in Echinacea aerial parts and E. purpurea and E. angustifolia roots.

Fig. 10 8-Hydroxy-ketoalkenynes formed oxidation in powdered E. pallida roots.

: via

auto-

Echinacea

183

(microsomes from ram seminal vesicles) assays,2!) More recently, Goel et al. conducted an in vivo study to examine the immunomodulatory effects of various dose levels of cichoric acid, polysaccharides, and alkamides, isolated and purified from E. purpurea. Among the components, the alkamides significantly increased the phagocytic activity as well as phagocytic index of alveolar macrophages from Sprague-Dawley ats. They produced significantly more TNF-« and nitric oxide after stimulation with lipopolysaccharides (LPS) than the other components or the control. These results suggest that the alkamides are one of the active constituents of E. purpurea.?*! The immunomodulatory effects appear to be more pronounced in lungs than in spleen.0”! Determination of the alkamide content (dodeca2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides) in the different plant parts showed that it accumulates primarily in the roots and inflorescences, the highest

being found in E. angustifolia. E. pallida roots contain only trace amounts, roots of E. purpurea 0.004-0.039%, and those of E. angustifolia 0.009-

Close monitoring of the transport for 6hr revealed a nearly complete transfer to the basolateral side after 4hr and no significant metabolism. Transport experiments performed at 4°C showed only a slight decrease, which is a strong hint that dodeca-2F,4E,8Z,10E/Ztetraenoic acid isobutylamides (11, 12) cross biological membranes by passive diffusion. These data suggest that alkamides are more likely than caffeic acid conjugates to pass through the intestinal barrier and thus be available for influencing an immune response.?* From the above, it is obvious that not a single, but several constituents, like the alkamides, cichoric acid,

glycoproteins, and polysaccharides, are responsible for the immunostimulatory activity of Echinacea extracts, and the application of extracts appears to be reasonable.

However,

conformity of these extracts is

a must for generating consistent products and reproducible activity. It is desirable to have products like the preparation from freshly harvested E. purpurea plants

used in the studies by Goel et al.°.??°>”] standardized on all groups of compounds, alkamides, cichoric acid, and polysaccharides.

0.151%. The yield in the leaves is 0.001-0.03%.!" In a recent study, 62 commercial dried root and aerial samples of E. purpurea grown in eastern Australia were

analyzed for the medicinally active constituents. Total concentration in root samples was 6.2 + 2.4mg/g, and in aerial samples was 1.0 + 0.7mg/g. The proportion of individual alkamides in root samples was found to be consistent across all samples. The large range in levels suggests that quality standards for the marketing of raw material should be considered.) In 2001, Dietz, Heilmann, and Bauer!**! reported the presence of dodeca-2EF,4E,8Z,10E/Z-tetraenoic acid

isobutylamides (11, 12) in human blood after oral administration of an ethanolic extract of E. purpurea. More recently, a study showed that the absorption maximum (Cmax) of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (11, 12) is reached 30min after oral application. Due to these results, the mucous membrane of the mouth is most likely the major area of absorption.*! Jager et al. investigated the permeability of isobutylamides (11, 12) through Caco-2 monolayers. They found that the alkamides were almost completely transported from the apical to the basolateral side of the monolayer in 6hr by passive diffusion and that no significant metabolism occurred.!?°! Matthias et al.°° investigated the bioavailability of caffeic acid derivatives and alkamides also using The caffeic acid conjugates Caco-2-monolayers. (caftaric acid, echinacoside, and cichoric acid) perme-

ated poorly through the Caco-2 monolayers although one

potential

readily

with

metabolite,

an

apparent

cinnamic

acid,

permeability

diffused

(Papp)

of

(Nee ral Oa cm/s, while alkamides were found to diffuse

with Papp ranging from 3 x 10m tos3sbe

100% em/s.

CLINICAL EFFICACY A series of experiments have shown that E. purpurea extracts have significant immunomodulatory activities. Among the many pharmacological properties reported, macrophage activation has been demonstrated most convincingly. Phagocytotic indices and macrophagederived cytokine concentrations have been shown to be responsive to Echinacea in a variety of assays. Activation of polymorphonuclear leukocytes and natural killer cells has also been reasonably demonstrated. Changes in the numbers and activities of T- and B-cell leukocytes have been reported, but are less certain. Despite this cellular evidence of immunostimulation,

pathways leading to enhanced resistance to infectious disease have not been described adequately. Several dozen human experiments—including a number of blind randomized trials—have reported health benefits of Echinacea preparations. So far, clinical efficacy has been studied in randomized controlled clinical trials for cold pressed juice and hydroalcoholic tincture/extract of E. purpurea aerial parts, a standardized extract from E. purpurea roots (hydroalcoholic tincture/extract), and for the hydroalcoholic extract of E. pallida roots. For E. angustifolia root tinctures, only a pharmacological study plus a survey on healthy individuals have been performed, but a clinical double-blind experiment is in progress. All of them have used a variety of different Echinacea preparations and study designs. Most importantly, the phytochemical profile of the preparations was not determined or not reported in most of the earlier studies. Since different Echinacea preparations

Echinacea

184

have varying phytochemical profiles due to the use of diverse species, plant parts, and extraction procedures, the variation in reported clinical effectiveness may be due to discrepancies in the chemical profile. The most robust data come from trials testing E. purpurea extracts in the treatment for acute URI. Although suggestive of modest benefit, these experi-. ments trials are limited both in size and methodological quality. Hence, while there is a great deal of moderately good quality scientific data regarding pharmacological effects of E. purpurea, effectiveness in treating illness is still in discussion.47%! A recent Cochrane review”! summarized 16 randomized clinical trials of Echinacea for upper respiratory tract infection. The authors concluded that evidence is insufficient to recommend a specific Echinacea product. From the data, it is seen that E. purpurea may especially be efficacious, but they are still weak and inconclusive. Recently, a formulation prepared from freshly harvested E. purpurea plants and standardized on the basis of three known

active components,

alkamides,

cichoric acid, and polysaccharides (Echinilin™), was found to be effective for the treatment of a naturally acquired common cold. This study was conducted to determine the systemic immune response to Echinamide treatment during a cold, particularly to assess its effects on leukocyte distribution and neutrophil function. These effects of Echinacea were associated with a significant and sustained increase in the number of circulating total white blood cells, neutrophils, and natural killer cells, and a decrease in lymphocytes. These results suggest that possibly by enhancing the nonspecific immune response and eliciting free radical scavenging properties, this standardized formulation of Echinacea may have led to a faster resolution of the cold symptoms.?71 A randomized controlled trial of healthy children was performed by Taylor et al. to determine whether an E. purpurea pressed juice pre-

paration is effective in reducing the duration and/or severity of URI symptoms and to assess its safety in this population.“°! The E. purpurea preparation, as dosed in this study, was not effective in treating URI

symptoms in patients 2-11 yr old, and its use was associated with an increased risk of rash. In a recent study by Sperber et al., administration of E. purpurea pressed juice before and after exposure of healthy subjects to rhinovirus type 39 (RV-39) did not influence the rate

CLINICAL PARTICULARS Dosage Information

For internal use, the daily dose for aerial parts of E. purpurea for adults is 6-9ml of pressed juice or equivalent preparations or 3 x 60 drops of a tincture (1:5, ethanol 55% v/v). For dried roots, the recom-

mended daily dose is 3 x 300mg. For external use, semisolid preparations with a minimum of 15% of pressed juice are recommended. The dosage for children is a proportion of adult dose according to age or body weight. The duration of administration should not exceed 8 weeks.!7! For E. pallida roots, the recommended daily dosage of Commission E is tincture 1:5 with 50% (v/v) ethanol from native dry extract (50% ethanol, 7: 11:1), corresponding to 900 mg herb.

Adverse Effects and Toxicological Considerations

Clinical reports provide indications of a good tolerability of Echinacea preparations. No significant herb— drug interactions with Echinacea have been reported. Based

this system,

at

and

hepatic

and

intestinal

sites.

Therefore,

caution

should be exercised when it is coadministered with drugs dependent on CYP3A or CYPI1A2 for their elimination. The German health authorities list the following contraindications and possible side effects of Echinacea:

Contraindications:

e

double-blind,

e

clinical trial, a total

fexofenadine,

effect of 400mg E. purpurea root material administered four times daily with water was assessed on cytochrome P450 (CYP) activity in vivo by use of the CYP probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (hepatic and intestinal CYP3A). The extract reduced the oral clearance of substrates of CYP1A2 but not that of CYP2C9 and CYP2D6. The extract selectively modulated the catalytic activity of CYP3A

statistical hypothesis testing had relatively poor power to detect statistically significant differences in the frequency and severity of illness. In this randomized, placebo-controlled

such as itraconazole,

lovastatin.7! In a recent study by Gorski et al.'*! the

of infection. However, because of the small sample size,

of 92% of Echinacea recipients and 95% of placebo recipients were infected. Cold developed in 58% of the former and 82% in the latter.44

on in vitro studies, an ethanolic extract from

the roots of E. angustifolia may be a mild inhibitor of the cytochrome P450 3A4 enzyme complex system. This tends to increase levels of drugs metabolized by

Allergy against one of the ingredients or against Compositae plants. The Commission E warns for general reasons not to use Echinacea in case of progressive systemic diseases like tuberculosis, leukoses, collagenoses, multiple sclerosis, and other autoimmune diseases.

Echinacea

e

The Commission E warns not to use Echinacea in case of AIDS and HIV infection.

185

REFERENCES 1.

Side effects:

e

e

In rare cases, hypersensitivity reactions might occur. For drugs with preparations of Echinacea, rash, itching, rarely face swelling, shortness of breath, dizziness, and blood pressure drop have been reported. Incase of diabetes, the metabolic status may worsen."

Shivering and other “influenza-like’’ symptoms have been occasionally observed after intravenous administration. Brief fever can be the result of the secretion of IFN-a« and IL-1 (endogenous pyrogen) from macrophages, i.e., it always occurs when macrophages are stimulated. Rarely, acute allergic reactions can occur. Therefore, it can be concluded that there is a very low incidence of side effects associated with Echinacea the treatment, Also in long-term preparations. expressed juice of E. purpurea was shown to be well

tolerated.°!

2.

Brevoort, P. The booming U.S. botanical market. Herbalgram 1998, 44, 33-46. Bauer, R.; Wagner, H. Echinacea—Ein

Handbuch

3.

fiir Arzte,

Apotheker

und

andere

Naturwissenschaftler, Wissenschaftliche Verlagsgesellschaft: Stuttgart, 1990. Bauer, R.; Wagner, H. Echinacea species as potential immunostimulatory drugs. In Economic and Medicinal Plant Research; Wagner, H., Farnsworth, N.R., Eds.; Academic Press Limited,

1991; Vol..5,.253-321. 4.

Barrett, B. Medicinal properties of Echinacea: a

critical 66-86. 2.

6.

review.

Phytomedicine

2003,

/0

Goel. V::, Loviin, .R.; sChangsde=, Slama Barton, R.; Gahler, R.; Bauer, R.; Basu,

(1), wie T.K.

Efficacy of a standardized Echinacea preparation (Echinilin) for the treatment of the common cold: a randomised, double-blind, placebo controlled trial. J. Clin. Pharm. Ther. 2004, 29 (1), 75-83. Blumenthal, M. The Complete German Commission E Monographs, Therapeutic Guide to Herbal Medicines; American Botanical Council:

CONCLUSIONS 7. Echinacea, one of the most popular botanical supplements in North America, is employed as an immune modulator, antimicrobial, and (topically) antiseptic. So far, the therapeutic activity of Echinacea cannot be unambiguously attributed to any particular constituents. However, pharmacological effects related to immune functions have been demonstrated for both high- and low-molecular-weight constituents. Compounds from the classes of caffeic acid derivatives,

alkamides, polysaccharides, and glycoproteins are regarded as the most relevant constituents. Employing diverse species, plant parts, and extraction procedures, procedures results in different Echinacea preparations having varied phytochemical profiles. Clinical effectiveness may vary because of discrepancies in the chemical profile. Standardization of botanicals should guarantee that preparations contain therapeutically effective doses of active principles and should assure consistent batch-to-batch composition and stability of the active constituents. Also, clinical trials

should be performed only with well-characterized Echinacea preparations. In summary, these preparations can be effective for the enhancement of the body’s defense mechanisms. However, further investigations are necessary to find the optimum dosage, the molecular mode of action, and the best application form. Serious adverse effects from the use of Echinacea appear to be extremely rare, and the potential for important herb— drug interactions appears to be limited.

Austin, 1988. Bauer, R. The Echinacea story—the scientific development of an herbal immunostimulant. In Plants for Food and Medicine—Modern Treatments and Traditional Remedies; Prendergast, H.D.V., Etkin, N.L., Harris, D.R., Houghton, P.J., Eds.; The Royal Botanic Gardens: Kew,

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Moerman, D.E. Native American Ethnobotany; Timber Press: Portland, OR, 1998; 205-206.

9.

McGregor, R.L. The taxonomy of the genus Echinacea (Compositae). Univ. Kansas Sci. Bull. 1968, 48, 113-142.

10.

Binns,

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Della Logia, R. Anti-inflammatory activity of Echinacea angustifolia fractions separated on the basis of molecular weight. Pharm. Res. Comm. 1988, 20 (Suppl. V), 87-90. Melchart, D.; Worku, F.; Linde, K.; Flesche, C.; Eife, R.; Wagner, H. Erste Phase-I-Untersuchung von Echinacea-Polysaccharid (EPO VIIa/EPS) bei iv. Application. Erfahrungsheilkunde, 1993, 42, 316-323.

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Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin. Planta Med. 2002, 68, 1118-1124. Age

Bodinet,

C.;

Lindequist,

U.;

Teuscher,

Bodinet, C.; Mentel, R.; Wegner, U.; Lindequist, U.; Teuscher, E.; Freudenstein, J. Effect of oral

application of an immunomodulating plant extract on influenza virus type A infection in

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S.; Dasgupta, A.; Actor, J.K. Cytokine

production by non-adherent mouse splenocyte cultures to Echinacea extracts. Clin. Chim. Acta. 2004, 343, 161-166.

20.

Pietta, P.; Mauri, P.; Bauer, R.MEKC analysis of different Echinacea species. Planta Med. 1998,

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Maffei Facino, R.; Carini, M.; Aldini, C.; Marinello, C.; Arlandini, E.; Franzoi, L.; Colombo, M.; Pietta, P.; Mauri, P. Direct charac-

terization of caffeoyl esters with antihyaluronidase activity in crude extracts from Echinacea angustifolia roots by fast atom bombardment tandem mass spectrometry. Farmaco 1993, 48, 1447-1461. Maffei Facino, R.; Carini, M.; Aldini, G.; Saibene, L.; Pietta, P.; Mauri, P. Echinacoside and caffeoyl conjugates protect collagen from free radical-induced degradation: a potential use of Echinacea extracts in the prevention of skin photodamage. Planta Med. 1995, 6/, 510-514.

Niisslein, B.; Kurzmann, M.; Bauer, R.; Kreis, W.

Enzymatic degradation of cichoric acid in Echinacea purpurea preparations. J. Nat. Prod. 2000, 63, 1615-1618.

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Woelkart,

K.;

Gangemi,

J.D.;

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R.B.;

Bauer, R. Enzymatic degradation of echinacoside and cynarin in Echinacea angustifolia root preph),

parations. Pharm. Bio. 2004, 42, in press. “i Goel, V.; Chang, C.; Slama, J.V.; Barton, R.; Bauer, R.; Gahler, R.; Basu, T.K. Alkylamides

of Echinacea purpurea stimulate alveolar macrophage function in normal rats. Int. Immunopharmacol. 2002, 2, 381-387.

30.

Goel, V.; Chang, C.; Slama, J.V.; Barton, R.; Bauer, R.; Gahler, R.; Basu, T.K. Echinacea stimulates

macrophage

31:

32:

function

in the lung and spleen of

normal rats. J. Nutr. Biochem. 2002, /3, 487-492. Miiller-Jakic, B.; Breu, W.; Probstle, A.; Redl, K.; Greger, H.; Bauer, R. In vitro inhibition of

cyclooxygenase and 5-lipoxygenase by alkamides from Echinacea and Achillea species. Planta Med. 1994, 60, 7-40. Wills, R.B.H.; Stuart, D.L. Alkylamide and cichoric acid levels in Echinacea purpurea grown in

iv

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34.

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application

of

Echinacea

cokinetics and bioavailability of alkamides from the roots of Echinacea angustifolia in humans. Submitted for publication.

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Jager, H.; Meinel, L.; Dietz, B.; Lapke, C.; Bauer, R.; Merkle, H.P.; Heilmann, J. Transport of alka-

mides from Echinacea species through Caco-2 monolayers. Planta Med. 2002, 68, 469-471.

36:

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Robinson, W.E. t-Chicoric acid, an inhibitor of

human immunodeficiency virus type (HIV-1) integrase, improves on the in vitro anti-HIV-1 effect of Zidovudine plus a protease inhibitor (AG1350). Antiviral Res. 1998, 39, 101-111.

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2000, 74, 106-112. 2

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(Sonnenhutkraut). J. Appl. Bot——Angew.

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Freudenstein, J. Effect of an orally applied herbal immunomodulator on cytokine induction and antibody response in normal and immunosuppressed mice. Phytomedicine 2002, 9 (7), 606-613.

18.

25%

3a:

Goel, V.; Lovlin, R.; Barton, R.; Gahler,

Basu,

T.K.

A

Chang, C.; Slama, J.V.; R.; Goonewardene, L.;

proprietary

extract

from

the

-

Echinacea

187

Echinacea plant (Echinacea purpurea) enhances systemic immune response during a common cold. Phytother. Res. in press.

38.

Ernst,

E. The

risk—benefit

40.

41.

43.

Ed.; Thieme:

126-141. Budzinski, Arnason,

Melchart, D.; Linde, K.; Fischer, P.; Kaesmayr, J.

Echinacea for preventing and treating the common cold. Cochrane Database Syst. Rev. 2000, (2), CD000530.

ESCOP Monographs. Foundation for Herbal 2nd

profile of commonly

used herbal therapies: ginkgo, St. John’s wort, ginseng, Echinacea, saw palmetto, and kava. Ann. Intern. Med. 2002, 136, 42-53.

39:

42.

44.

In The Medicinal

Stuttgart,

New

Scientific Products; York, 2003;

J.W.; Foster, B.C.; Vandenhoek, S.; J.T. An in vitro evaluation of human

cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures. Phytomedicine 2000, 7, 273-282.

Taylor, J.A.; Weber, W.; Standish, L.; Quinn, H.;

Gorski, C.; Huang, S.M.; Pinto, A.; Hamman, M.A.; Hilligoss, J.K.; Zaheer, N.A.; Desai, M.; Miller, M.; Hall, S.D. The effect of echinacea

Goesling, J.; McGann, M.; Calabrese, C. Efficacy and safety of Echinacea in treating upper respiratory tract infections in children. J. Am. Med.

(Echinacea purpurea root) on cytochrome P450 activity in vivo. Clin. Pharmacol. Ther. 2004, 75, 89-100.

Assoc. 2003, 290, 2824-2830. Sperber, S.J.; Shah, L.P.; Gilbert, R.D.; Ritchey, T.W.; Monto, A.S. Echinacea purpurea for

prevention of experimental rhinovirus colds. Clin. Infect. Dis. 2004, 38, 1367-1371.

45.

Parnham,

M.J.

Benefit-risk

assessment

of the

squeezed sap of the purple coneflower (Echinacea purpurea) for long-term oral immunostimulation.

95-102.

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Ephedra (Ma Huang) Anne L. Thurn Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland, U.S.A.

INTRODUCTION

supplements containing ephedrine alkaloids. As defined by Congress in the Dietary Supplement Health

Ephedra (ma huang) is the common name for a herbal product used in traditional Chinese medicine. It comprises the aerial parts of three principal plant species: Ephedra sinica Stapf, E. equisetina Bunge, and E. intermedia var. tibetica Stapf."-*! The plant is a natural source of the alkaloids (—)-ephedrine and

and Education Act, which became law in 1994, a diet-

(+)-pseudoephedrine.!"! GENERAL DESCRIPTION E. sinica Stapf is a low evergreen shrub with small scaly leaves. It flowers in June and July and produces fruit late in the summer.! Approximately 45-50 species of ephedra have been described worldwide, includ-

ing in the temperate and subtropical regions of Asia, Europe, and the Americas.”°! Most of those native to North,

South,

and

Central

America,

such

of ephedrine,

the main

active constituent

in

the ephedra species known as ma huang.) Alkaloid content increases as the plant matures, with peak concentrations in the fall.!°! No ephedrine-type alkaloids are found in the roots, berries, or seeds of these plants,

and the green upper parts of the stems contain significantly more alkaloids than the woody parts." Traditionally, ephedra has been administered as a tea prepared by soaking 2g dried aerial portions in 8 fluid oz of boiling water for 10 min, ideally resulting in a content of 15-30mg ephedrine. In commercial products, it is usually a formulation of powdered aerial

portions or a dried extract.'”! The ephedrine content of such products ranges from 12 to 80mg per serving, with most of them being in the lower part of the range. In the United States, ephedra was sold as a dietary supplement until April 2004 when the U.S. Food and Drug Administration (FDA) banned the sale of dietary

Anne

L. Thurn,

Ph.D.,

is Director,

Evidence-Based

Supplements, at the Office of Dietary Program Institutes of Health (NIH), Bethesda, Maryland, U.S.A.

herbs or other botanicals, amino acids, and other sub-

stances) or their constituents; is intended to be taken orally as a pill, capsule, tablet, or liquid; and is labeled on the front panel as being a dietary supplement. Ephedra-containing dietary supplements are most often promoted as weight loss aids or to improve energy levels and athletic performance, even though there is little or no evidence for their efficacy. A 1998 survey of more than 14,500 people 18 yr or older in five states for weight loss reported that 7% used nonprescription products, while 1% consumed ephedracontaining preparations.”!

as E.

nevadensis (used to make Mormon tea), E. trifurca, and E. antisyphilitica, contain no alkaloids.©! Most commercial uses of ephedra are based on its content

ary supplement is a product (other than tobacco) that is intended to supplement the diet; contains one or more dietary ingredients (including vitamins, minerals,

Research

National

Encyclopedia of Dietary Supplements DOK: 10.1081 /E-EDS-120022915 Copyright © 2005 by Marcel Dekker. All rights reserved.

HISTORICAL USE Ephedra has a history of more than 5000 years of medicinal use in China and India, where it has been used to treat cold, fever, flu, chills, headaches, edema caused by

nephritis, nasal congestion, aching joints, coughing, and wheezing and to induce diuresis or perspiration. Evidence that its use predates even traditional Chinese and Indian medicine comes from the discovery of a species of ephedra found in a Neanderthal grave in Iraq dating from 60,000 B.c!*l Analysis of the plants found in the grave indicated that they contained bioactive ingredients and were presumably used medicinally. There is documentation of its use by Discorides, the renowned

Greek

herbalist,

in the Ist century

and in

Europe from the 15th to the 19th centuries." In the 1600s, Native Americans and Spaniards in the American

Southwest used ephedra (the nonalkaloid-containing species) as a treatment for venereal disease." Ephedrine, the principal active ingredient in most species, was first isolated in 1885 by Nagayoshi Nagai, a Japanese chemist trained in Germany."®! Other alkaloids, such as pseudoephedrine, norephedrine, and norpseudoephedrine, with similar but not identical properties, were subsequently found in various ephedra species.!1"! 189

Ephedra (Ma Huang)

190

Early studies on the pharmacologic effects of ephedrine were conducted between

1888 and 1917, but it

was not until the 1920s during studies of a variety of Chinese traditional herbs that ephedrine alkaloids became known to Western medicine. Ephedra, one of the herbs tested, gave significant results—an extract given intravenously to dogs resulted in a large increase . in blood pressure.!"7] Subsequent publication of a series of studies on the pharmacologic properties helped Western physicians understand its potential usefulness." These experiments demonstrated that ephedrine had three advantages over epinephrine: It could be administered orally, was longer acting, and had a lower toxicity.) As a result, the former alkaloid replaced the latter in the management of children with mild-to-moderate asthma. Subsequently, it became widely used as a nasal decongestant and central nervous system stimulant. Its ability to stimulate the central nervous system was recognized by the Japanese military, who administered it by injection to kamikaze pilots during World War 2 The herb ephedra was once recognized as an official drug in the United States, but widespread availability of synthetic ephedrine-type alkaloids has virtually eliminated its clinical use.!'?!

CONSTITUENTS AND ACTIONS Ephedra

stems

contain

0.5-2.5%!

alkaloids,

com-

posed mainly of L-ephedrine and p-pseudoephedrine, with ephedrine content ranging from 30% to 90% of total alkaloid content depending on the source. The content of the various ephedra alkaloids varies in commercial preparations, but ephedrine and pseudoephedrine generally make up 90-100%.!'4) (—)Ephedrine and five structurally related alkaloids, (+)-pseudoephedrine, (—)-N-methylephedrine, (+)-Nmethylpseudoephedrine, (+)-norpseudoephedrine, and (—)-norephedrine, are responsible for the medicinal properties of these alkaloid-containing ephedra species." Ephedrine acts indirectly by stimulating «-, %-, B)-, and B2-adrenergic receptors to release norepinephrine from sympathetic nerve endings and directly as a B,-, B2-, and possibly B3-agonist.*!>'© It promotes bronchodilation by stimulating {>-receptors in the lungs, which accounts

for its effectiveness in treating bronchial asthma."~!¢ However, its other adrenergic actions result in the generally undesirable effects of central nervous system stimulation including insomnia, irritability, hyperactivity,!'*! hypertension,!'*” and gastrointestinal symptoms (nausea and vomiting).!'"") These side effects have led to the development of selective B»-agonists and the discontinuation of ephedrine use for bronchodilation. Ephedrine increases heart rate and therefore cardiac output." It also causes peripheral vasoconstriction,

which increases peripheral resistance and can lead to a sustained rise in blood pressure.!'® In general, the increase in blood pressure is dose dependent,”®! but

doses under 50mg do not always produce such an effect.0417) Chronic use of ephedrine for the management of asthma results in tachyphylaxis, making it an unreliable long-term therapeutic agent.’ Frequent doses become less effective as a result of the depletion of norepinephrine stores as well as the release of adenosine into synaptic junctions and increased cellular phosphodiesterase activity, both of which suppress catecholamine release."*! To potentiate the effects

of ephedrine,

methylxanthines

(e.g., caffeine

and theophylline), which interfere with the inhibitory effect of adenosine on both norepinephrine release and phosphodiesterase activity, are often combined. with ephedra or ephedrine.!'?7"! In the United

States,

ephedra-containing

dietary

supplements were widely promoted as weight loss aids because of their ephedrine content. The alkaloid has been postulated to do this by two mechanisms. First, by stimulating thermogenesis in laboratory animals'!?:!6?1] and humans,!'>'®! ephedrine increases energy expenditure. In rats, it has been shown to directly stimulate thermogenesis in brown adipose tissue via B-adrenergic receptors.!'*! But studies in humans have provided evidence against the role of brown adipose tissue in ephedrine-induced thermogenesis,18] Although the site of action remains unclear, the

thermogenic action in humans has been widely confirmed and serves as the major rationale for its use as a weight loss aid. Second, the combination of ephedrine and caffeine acts as an appetite suppressant, reducing food intake. One study showed that about 75% of the effect of this combination on weight loss is the result of its anorectic properties, which may

affect the satiety center in the hypothalamus.!'377! Evidence from animal studies and some human experiments shows that the combination reduces body fat but not lean body mass.''373] However, because the human studies had few participants, larger studies would have to be carried out to confirm this finding. The use of ephedrine as a nasal decongestant in over-the-counter preparations has been replaced for the most part by more selective B.-bronchodilators, but it is still included as a component of some pediatric prescription cold and cough medications and is found in a nonprescription medication for asthma." The ephedra constituent pseudoephedrine is generally preferred as an oral decongestant because it is less potent and therefore less likely than ephedrine to cause central nervous system stimulation or hypertension.!"®! Intravenous ephedrine is still widely used for the prevention and treatment of hypotension caused by

Ephedra (Ma Huang)

spinal anesthesia, particularly during cesarean section"! Other uses have included treatment of chronic urticaria, diabetic neuropathic edema, nocturnal enuresis, motion sickness, spastic or hypermotile bowel,

and myasthenia gravis.°47>!

In addition to ephedrine alkaloids, some ephedra species contain other nitrogen-containing secondary metabolites with known neuropharmacologic activity.”! These include several cyclopropyl analogs of L-glutamate and methanoproline, and a cyclopropyl analog of L-proline, as well as common amino acids such as L-glutamate, L-glutamine, L-serine, and L-proline. The stems of some ephedra species also contain kynurenates, derivatives of tryptophan catabolism that may help protect the plant from birds or rodents, but their

physiological function is unknown.) Kynurenates in ephedra stems exhibit antimicrobial activity against some Gram-positive and Gram-negative bacteria, although significantly less than the antibiotic ciprofloxacin. Nonnitrogenous compounds found in some ephedra species include organic acids, phenolic compounds (flavonoids and tannins), and essential oils primarily composed of terpenoids (38.9%).?°! The tannin fractions have been shown to inhibit angiotensin converting enzyme activity, although it was much less than that of captopril. Extracts of the roots contain the spermine-type

191

prevention, reduction of uremic toxins in renal failure, and immune modulation, as well as for their antimicro-

bial, antidiabetic, anti-inflammatory, antioxidant, and antitussive activity.” Ephedrine has also been used in preclinical research to examine the efficacy and potential mechanisms of action of ephedra for weight loss.:"5]

CLINICAL TRIALS A systematic review of the literature was conducted by RAND for published and unpublished sources of controlled clinical trials on ephedra and ephedrine used for weight loss and athletic performance in humans."! Fifty-two controlled clinical trials of ephedrine or botanical ephedra used for weight loss or athletic performance enhancement in humans were included.

Efficacy for Weight Loss Forty-four controlled trials were identified that assessed ephedra and ephedrine alkaloids used in combination with other compounds for weight loss, and 20 of these met inclusion criteria for the meta-analysis. Five pairs of treatment regimens were compared:

alkaloids ephedradines A, B, C, and D, feruloylhista-

mine, and bisflavanols and result in hypotension when administered intravenously.?7° The roots are not used in commercial dietary supplement products and are only a minor constituent of formulations used in traditional Chinese medicine.

PHARMACOKINETICS

1.

2.

3.

Ephedrine is well absorbed after oral administration, is excreted largely unchanged in the urine, and has a

serum half-life of 2-3hr.''*?7! When ingested in the form of ma huang, it has a tmax (time of occurrence for peak drug concentration) of nearly 4hr compared with only 2hr for pure ephedrine!!! Peak ephedrine blood levels are similar regardless of whether the alkaloid is taken as a herbal preparation or in the pure form. Ingestion of 400mg ma huang containing 20 mg ephedrine resulted in blood concentrations of 81ng/ml, which is the same as the peak ephedrine levels observed after administering an

4.

5.

Ephedrine vs. placebo (5 studies): Ephedrine was associated with 1.3lb/mo weight loss greater than placebo for up to 4mo of use. Ephedrine plus caffeine vs. placebo (12 studies): Ephedrine plus caffeine was associated with

2.21b/mo weight loss greater than placebo for up to 4mo of use. Ephedrine plus caffeine vs. ephedrine (3 studies): Ephedrine plus caffeine was associated with 0.8lb/mo weight loss greater than ephedrine alone. Ephedrine plus herbs containing ephedra plus caffeine (3 studies): Ephedra plus herbs containing caffeine was associated with 2.1 Ilb/mo weight loss greater than placebo for up to 4mo of use. Ephedrine vs. other active weight loss products (2 studies): No conclusions could be drawn about ephedrine vs. other active weight loss products because the sample size in these studies was too small.

equivalent amount of pure ephedrine.!"7!

Only one study compared an ephedra-containing product without caffeine but with other herbs and a placebo. This product was associated with a weight

PRECLINICAL STUDIES

loss of 1.8lb/mo,

more

than that associated

with a

placebo for up to 3mo of use.

Ephedra and its constituents have been studied in animals for a wide range of indications, including ulcer

None of the studies lasted longer than 6 mo; hence

long-term weight loss and maintenance could not be

Ephedra (Ma Huang)

192

assessed. The results indicate that the use of ephedrine, ephedrine plus caffeine, or dietary supplements containing ephedra plus herbs containing caffeine was associated with a statistically significant increase in weight loss for up to 4mo. Efficacy for Athletic Performance Enhancement There are no controlled clinical studies for athletic performance enhancement. RAND identified eight controlled trials of the effects of synthetic ephedrine for athletic performance enhancement, usually along with caffeine. The studies could not be pooled for meta-analysis because of the wide variety of interventions used. A few studies assessing the effect of ephedrine plus caffeine showed a modest improvement in very short-term athletic tasks such as weight lifting (1-2hr after a single dose) and time to exhaustion for aerobic exercises. However, these trials were of very

short duration and were done in small numbers of healthy young men, mostly military recruits. The results therefore cannot be generalized to the general public. Because all the studies were done in the same laboratory, the ability of other investigators to confirm the results has not been tested.

dra have also not water or methanol extracts of ephe

demonstrated any mutagenic effects.P°°"

In Vivo Toxicity Several studies in mice have determined the oral LDso (median lethal dose) of water extracts of ephedra, which ranges from 4000 to 8000 mg/kg depending on the alkaloid content of the species used.?! Based on these measurements, the equivalent range of ephedrine was calculated to be 520-720 mg/kg. The NTP also evaluated the toxicity of ephedrine in B6C3F1 mice and F344 rats. During 2-yr studies, the animals were given diets containing 0, 125, or 250 ppm ephedrine/day. The mean body weight of the rats and mice receiving diets containing either dose of ephedrine was lower than those of controls, and survival was similar for the controls and exposed animals.??! A teratogenicity study of ephedrine showed a frequency of 8% malformed chick embryo hearts with exposure to 0.5 pmol ephedrine and 26% for 5.0 umol, 274

ADVERSE

EVENTS

TOXICOLOGY

The safety of ephedra-containing dietary supplements has been a controversial subject, resulting in a high

In Vitro Toxicity

level of interest from regulators, manufacturers,

The toxicity of eight water extracts of ephedra prepared from the entire plant using either ground or whole

herb, boiled

for 0.5 or 2hr, and with

either

one or two extractions, was tested in a human hepatoblastoma cell line (HepG2) and a variety of animal cell lines.'?*! Of the cell lines tested, only a neuronal cell line

(Neuro-2a) showed significant sensitivity to the cytotoxic effects of the extracts. Grinding increased the toxicity of the resulting extracts. Normalizing the results for ephedrine content showed that the toxicity of the ephedra extracts could not be accounted for solely by ephedrine content, indicating the presence of other cytotoxic constituents. Kynurenates and cyclopropyl amino acids, both of which cause central nervous system toxicities and have been isolated from some species of ephedra, could be associated with the additional toxicity.°! However, neurotoxicity is eliminated during the boiling of extracts that are used in commercial products. In studies performed by the National Toxicology Program (NTP), ephedrine was not mutagenic in four strains of Salmonella typhimurium and did not cause chromosomal aberrations in vitro in Chinese hamster ovary cells.”*! Other in vitro research of ephedrine or

and

the public. MedWatch, the Adverse Reaction Monitoring System of the FDA, has recorded many reports of side effects concerning ephedra-containing products. In 1997, as a result of the increasing number of adverse event reports, the FDA published a proposed rule on the use of dietary supplements containing ephedrine alkaloids.°?! It recommended that ephedracontaining dietary supplement products be limited to a total of 8mg ephedrine per serving, with a daily limit of 24mg, a duration limit of 7 days, and label warnings. The General Accounting Office audited the methods used by the FDA to develop this rule and reported that because the evidence was from case reports and not controlled clinical trials, it was not sufficient to support the suggested limits on dose and duration.°4! As a result, the FDA withdrew the parts of the rule that place limits on the amount of ephedrine-type alkaloids permitted. Ephedra-containing products were the subject of significant media coverage for quite a few years because of the deaths of several professional athletes who were allegedly taking these products."! As a result of these safety concerns, ephedra was prohibited by the International Olympic Committee and the National Football League."

Ephedra (Ma Huang)

193

A review of the adverse effects reported in 50 controlled clinical trials of ephedra, ephedrine with or without caffeine, or botanicals containing caffeine concluded that use of these substances was associated with 2-3 times the risk of nausea, vomiting, psychiatric symptoms, autonomic hyperactivity, and palpitations compared with placebo.°! No serious adverse events such as myocardial infarction, stroke, or death were reported in these studies, but the authors of the review noted that the small total number of patients in these experiments was not adequate to distinguish a serious adverse event rate of | in 1000 or higher. To evaluate the incidence of serious side effects, several research reviewed ephedra-related adverse event reports in MedWatch as well as case reports in the published literature. The latter were evaluated in the RAND review because the total number of participants in the clinical trials was not sufficient to adequately assess the possibility of rare outcomes. Although such adverse event reports are not conclusive evidence of a cause-and-effect relationship, they can indicate the potential for such a relationship. RAND searched the literature for published case reports and the MedWatch database for cases of serious adverse events that were idiopathic in etiology. If use of ephedra or ephedrine-containing products was well documented, then the possibility that ephedra or ephedrine caused the event was considered. Sentinel events were defined as adverse events associated with ingestion of an ephedra-containing product within 24 hr prior to the event and for which alternative explanations were excluded with reasonable certainty. Possible sentinel events were defined as adverse events that met the first two criteria for sentinel events but for which alternative explanations could not be excluded. RAND reviewed 71 cases reported in the published medical literature, 1820 case reports from the MedWatch database, and more than 18,000 consumer complaints reported to a manufacturer of ephedracontaining dietary supplements. The documentation for most reports was insufficient to support decisions about the relationship between the use of ephedra or ephedra-containing dietary supplements and the adverse event. Only 65 cases from the published literature, 241 from MedWatch,

and 43 from a manufac-

turer of ephedra-containing dietary supplements had documentation sufficient for them to be included in the adverse event analysis. Among these, RAND identified 2 deaths, 3 myocardial infarctions, 9 cardiovascular accidents, 3 seizures, and 5 psychiatric events as sentinel events and 9 deaths, 6 myocardial infarctions,

10 cardiovascular accidents, 9 seizures, and 7 psychiatric events as possible sentinel events.?*! About half of the sentinel and possible sentinel events occurred in individuals under 30 yr of age.

Another study identified 140 MedWatch reports of adverse events concerning ephedra-containing products between June 1, 1997 and March

31, 1999 and

concluded that 31% were definitely or probably related to use of these products and another 31% were possibly related."*! The events included death, stroke, hypertension, tachycardia, palpitations, and seizures. A third study reviewed the MedWatch database for stroke, myocardial

infarction,

or sudden

death asso-

ciated with the use of ephedra-containing products from 1995 to 1997.2" Of 926 cases of adverse event reports concerning the use of ephedra-containing products,

37

serious

cardiovascular

events

were

identified as being temporarily associated with the use of these products. In 2003, on the basis of new information, including the RAND report, the FDA reopened the comment period on the proposed rule for dietary supplements containing ephedrine alkaloids for 30 days. In April 2004, the FDA banned the sale of dietary supplements containing ephedrine alkaloids.

CONTRAINDICATIONS Because of the potentially dangerous effects of ephedrine on the heart and central nervous system, individuals with a history of cardiovascular disease; hypertension; hyperthyroidism; seizures; depression or other mental, emotional, or behavioral conditions;

glaucoma; or difficulty in urinating because of benign prostatic hypertrophy should avoid taking ephedracontaining products.?7! Ephedrine is often used for intraoperative hypotension and bradycardia, raising concerns about preoperative use of ephedra-containing products. Such application puts people anesthetized with halothane at risk because halothane sensitizes the myocardium to ventricular arrhythmias.""! The potential for serious side effects rises as serving size and frequency of use is increased.?! These risks may also be enhanced when ephedra-containing products are used with other sources of stimulants such as caffeinated beverages, over-the-counter drugs, and

other dietary supplements containing stimulants.

DRUG INTERACTIONS Ephedra-containing

products

should

with, or for 2 weeks, after monoamine

not

be taken

oxidase inhibi-

tors or with drugs for Parkinson’s disease, obesity, or weight control; methyldopa; or any product containing

ephedrine, pseudoephedrine, or phenylpropanolamine.?”)

Ephedra (Ma Huang)

194

Shekelle, P.; Hardy,

FUTURE RESEARCH

S.; Maglione,

W.A.; Gagne, J.; Rhodes, S.; McKinnon, E. Ephedra and Ephedrine for Weight Loss and Clinical Athletic Performance Enhancement:

To assess the safety of ephedra, a rigorous case-control study of ischemic vascular, cardiovascular,

M.; Morton,

M.; Suttorp, M.; Roth, R.; Jungvig, L.; Mojica, and heat

stroke events should be given a high priority. Other approaches to fill the gaps in knowledge concerning ephedra might include surveys or the addition of questionnaires to existing cohort studies to deter-— mine current patterns of ephedra use, including dose,

Efficacy and Side Effects, Evidence Report/ Technology Assessment No. 76 (Prepared by California-RAND Evidence-Based Southern

intake of concurrent medications, and characteristics

0001). AHRQ

of users. The information retrieved would provide data on events and utilization could be used to plan the design of future studies. Basic research is needed on pharmacokinetic drug interaction assessments, including identification of interactions with agents such as anabolic steroids or sympathomimetics, and on physiologic responses under conditions such as exercise and thermal stress. Clinical trials will be necessary to assess the risk— benefit ratio of ephedra for weight loss among overweight and obese individuals. A phase II study could be used to evaluate adverse events, weight loss, physiological responses, and optimal dosing. A randomized clinical trial of adequate sample size could then be used to characterize side effects and evaluate efficacy with regard to moderate or long-term weight loss and maintenance and relevant health outcomes. The research portfolio described above would be time consuming and expensive but would answer the questions of ephedra safety and efficacy definitively.

for Healthcare Research and Quality: Rockville,

However, it likely that as a result of the FDA’s

Practice

a multistate

LQ: Lk

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14.

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Chen, K.K.; Schmidt, C.F. Ephedrine and related substances. Medicine 1930, 9, 1-117. Robbers, J.E.; Tyler; V.E. Tyler's Herbs vof Choice, Haworth Herbal Press: New York, 1999;

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ph, Peat

|

=

(PGE) and thromboxane Bz (TXB) compared to those from control animals. However, the difference reached significance for PGE, only.°*! Larger and more important differences were observed for leukocytes from the fish oil-fed animals but not for those with virgin olive oil. These results show that EPO may be an effective anti-inflammatory agent since its long-term dietary presence reduces lymphocyte reactivity, possibly due to its y-linolenic acid constituent.?>!

EPO and Atopic Dermatitis

Atopic dermatitis, also known as atopic eczema, is an immune-mediated inflammatory skin disorder characterized by redness, itching, and oozing vesicular lesions that become scaly, crusted, or hardened.?* Corticosteroid and antipruritic treatments are common for addressing atopic dermatitis, but concerns regarding the side effects of these drugs have prompted search for alternative natural and less toxic treatments.?7 The effects of EPO supplementation on atopic dermatitis have received much attention because the fatty acid composition of the oil may interfere with the production of proinflammatory cytokines, which could potentially reduce the symptoms of this disease.?*! Furthermore, low A-6-desaturase activity leading to y-linolenic acid deficiency has previously been reported

as a contributing factor to atopic dermatitis,” making EPO (with its high y-linolenic acid content) the subject

of much interest in eczema therapy. Morse et al.?° performed a meta-analysis on nine placebo-controlled studies investigating the efficacy of EPO supplementation in the treatment of atopic dermatitis; the indivi-

dual studies were performed in eight different centers.°°! This analysis included two of the earliest large-scale studies on EPO and eczema as well as seven small ones (14-47 participants). In these studies, patients and doctors assessed the severity of eczema in experimental and placebo groups by scoring measures for skin symptoms including itchiness, dryness, and scaliness. Subjects in the experimental groups were provided with Epogam EPO capsules containing 10% y-linolenic acid. Results from all studies were pooled together to give a global clinical score for each assessment point. This early meta-analysis showed that

199

the clinical scores for patients receiving Epogam supplements were significantly better than those for placebo groups, particularly for the symptom of itch (P < 0.0001). Furthermore, there was a positive correlation between plasma dihomo-y-linolenic acid. levels and

clinical

score

improvement.22

However,

the

results of this meta-analysis have been questioned by experts for two main reasons: first it did not consider the relatively large study by Bamford, Gibson, and Renier,?"! which included 123 participants and did not demonstrate any therapeutic effect of EPO on eczema. Second, this meta-analysis as well as seven of

the studies referenced by it were sponsored by Scotia Pharmaceuticals, the manufacturer of Epogam. Some recent studies have failed to reflect the efficacy of EPO as treatment for atopic eczema, as exhibited in the placebo-controlled trial conducted by Hederos and Berg!*?! that included 60 eczemic children supplemented with Epogam for 16 weeks as well as in the larger placebo-based trial by Berth-Jones and Graham Brown"?! that included 123 patients. It has been postulated that EPO treatment may modify immunological parameters associated with atopic dermatitis such as plasma interferon-y (INF-y) and immunoglobulin-E (IgE)

levels,247>

Recently,

Yoon,

Leg

and

Lee?!

investigated this possibility in 14 children with atopic dermatitis. A group of 6 children without the disorder were used as normal controls. EPO was administered to participants with atopic dermatitis until their symptom scores remained below | for 2 weeks. EPO treatments lasted 75 + 58 days; all participants receiving EPO exhibited clinical improvements and >42% of this group were completely cleared of symptoms

of atopic

dermatitis.

Before

EPO

treatments,

the mean plasma INF-y levels in the experimental group were significantly lower than that of the control group (P < 0.01). But after treatments, INF-y levels in the experimental group increased to a level equal to that of the control group (P < 0.01).°! Plasma IgE levels of the experimental group were significantly greater than that of the control group both before and after the treatment. The results of this study imply that EPO is therapeutic in children with atopic dermatitis,

and the observed clinical improvements are likely due to the normalization of INF-y levels and perhaps other immunological parameters.*! Thus, no consensus exists among the results of different studies on the effects of EPO supplementation for atopic dermatitis. The heterogeneous nature of the patients, who can exhibit slight to very serious symptoms,

may

explain

the

observed

inconsistencies.2°

Recent reviews show that the current body of literature is too inconsistent, making it impossible to conclusively link EPO supplementation to improvements in eczema symptoms.” 7381 Larger-scale, placebocontrolled studies are needed to provide firm evidence

Evening Primrose (Oenothera biennis)

200

about the postulated beneficial atopic dermatitis symptoms.

EPO and Rheumatoid

effects

of EPO

on

Arthritis

Arthritis is a degenerative chronic disease that can affect any of the body’s joints. The term arthritis refers to the inflammation

of a joint, but not all arthritic

diseases involve inflammation. One very common type of the disease is rheumatoid arthritis, which is a chronic inflammatory disease involving the body’s immune system. According to Health Canada, 4 million Canadians suffer from arthritis. Rheumatoid arthritis is an autoimmune condition that occurs in younger adults and sometimes in children. It is due to the release of inflammatory cytokines into the fluid space between the bones of a joint (synovium), which causes chronic inflammation of the cartilage covering the ends of the bones. The most common symptoms

neither the olive oil nor EPO group exhibited any clinical improvements in rheumatoid arthritis symptoms.'°! Although these early studies did not provide evidence of EPO’s therapeutic effects, it is possible that the supplementation regimes were insufficient and that higher doses of the oil given over a longer period may improve rheumatoid arthritis symptoms. More recently,

Brzeski,

Madhok,

and

Capell*®! examined

the effects of supplementation of 6g of EPO or olive oil per day for 6mo on symptom management in 40 rheumatoid arthritis sufferers. While olive oil supplementation was supposed to act as a placebo, a significant improvement was however noted at the end of the 6-mo evaluation period compared to baseline, similar to that observed for participants in the EPO group. Thus, both olive oil and EPO supplementations were equally effective in improving the clinical scores of rheumatoid arthritis sufferers, and selection of an appropriate placebo must be carefully exercised in such studies so that the possible therapeutic effects of EPO |

are pain, stiffness, and, if left untreated, bone defor-

are not masked. Belch et al.!*”! studied the effects of

mity.'°! The Raynaud phenomenon and Sjégren syndrome are two inflammatory diseases commonly seen in rheumatoid arthritis patients.°?! Raynaud phenomenon is an immunologic syndrome characterized by vascular spasms and enhanced blood cell aggregation

EPO and EPO plus fish oil (approximately 6g per day) on rheumatoid arthritis symptoms in 49 participants over a 12-mo supplementation period. In this study, 16 patients were given EPO, 15 EPO plus fish oil, and 18 liquid paraffin as placebo.” Measures of disease activity and NSAID use were used to assess the effectiveness of the treatments. After the supple-

that leads to ischemia of the fingers, toes, ears, and

nose and causes severe pain and pallor in the affected extremities.“ Sjégren syndrome is an immunologic disease affecting predominantly women in their 30s and leads to the destruction of exocrine glands. The main symptoms include persistent cough and dry eyes and mouth.'*"! Ingestion of EPO enhances dihomo-ylinoleic acid levels'*”! and may promote the production of anti-inflammatory series-1 PG or reduce proinflammatory series-2 PG production, which may be of benefit to rheumatoid arthritis patients. To investigate this possibility, research has been performed on the effects of EPO on rheumatologic conditions. Two early studies investigating the effect of EPO supplementation on rheumatoid arthritis symptoms were conducted by Brown et al.) and Hansen et al.'4), Both studies involved only 20 or less participants who were supplemented with low daily doses of EPO ( using spontaneously hypertensive rats (SHR), revealed that dietary EPO intake alters blood lipid profiles’ and systolic pressures, thereby improving cardiovascular risk factors.°"! The dietary proportions and types of fats consumed can alter

ability of EPO to reduce blood platelet aggregation in response to inducers (adenosine diphosphate or collagen) using a total of 40 male white New Zealand rabbits fed normal and atherogenic diets. These researchers also measured platelet thromboxane synthesis and malondialdehyde (MDA) production to assess blood platelet reactivity. Two control groups were used in this study: one group received a low fat diet (2.1%, w/w) with normal lipid composition (normal diet group), while the other was fed a high fat (12.2%, w/w) diet containing primarily saturated fatty acids (atherogenic diet group). The diets of experimental animals were supplemented with EPO to get a total fat content between 19.8% and 20.3%. These feeding trials lasted 6 weeks; on the last day, the animals were anesthetized, and blood samples were obtained for

subsequent analysis. Compared to the normal control diet, the atherogenic control diet resulted in hyperlipidemia (hypercholesterolemia and hypertriglyceridemia) with a concomitant decrease in high-density lipoprotein (HDL) levels. Platelet aggregation and platelet production of thromboxane Bz (TXB) and MDA were several times greater in the atherogenic diet group compared to controls. Supplementation of EPO in normal diets did not alter blood lipid and platelet parameters compared to normal controls. However, EPO significantly reduced total blood cholesterol and triacylglycerols, while increasing HDL levels in animals fed atherogenic diets, and reducing blood lipid aggregation level to that of normal control values (P < 0.05-P < 0.01). Treatment also reduced TXB> and MDA production by platelets from animals fed atherogenic diets, although these values were still significantly higher than those of the control animals fed on a normal control diet (P < 0.05-P < 0.01). Diets rich in saturates promoted the onset of cardiovascular risk factors such as platelet reactivity and aggregation, and EPO treatment normalized these parameters substantially in male New Zealand white

rabbits.8! More recently, De la Cruz et al.?! assessed the antioxidant potential of EPO in 40 male white New Zealand rabbits that were fed atherogenic diets (50% fat, w/w) or normolipidic diets (20% fat, w/w) containing 15% EPO (w/w); controls were fed either atherogenic or normolipidic diets without EPO enrichment. The duration of the feeding period was 6 weeks, after which all animals were sacrificed, and samples of liver, brain, heart, aorta, and blood platelets from each ani-

mal were collected to test MDA and glutathione levels. The results of this study showed that in normolipidic diets, EPO treatment did not alter MDA production

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or glutathione levels. However, atherogenic diets promoted MDA production (both induced and noninduced TBARS) and glutathione oxidation in all tissues and platelets. Meanwhile, tissues of animals receiving atherogenic diets plus EPO exhibited a lesser induction of MDA production and glutathione oxidation (P < 0.05-P < 0.01). Thus, EPO supplementation may reduce the production of lipid oxidation products in tissues, which are implicated in the onset

of atherosclerosis.°?! The cardioprotective effects of EPO may be due to the presence of antioxidative components. Charnock! recently evaluated the effects of dietary EPO, sunflower oil, borage oil, and saturated fat against ventricular fibrillation in male HoodedWistar rats fed normal chow diets supplemented with 6% EPO (w/w) for 32 weeks. At the end of the feeding

period,

the

animals

were

anesthetized

and

ventilated, followed by surgical ligation (occlusion) of the left descending coronary artery to induce ventricular fibrillation and cardiac arrhythmia. In this study, incidence and duration of ventricular fibrillation were lowest in the EPO group. Interestingly, EPO exerted a protective effect against ventricular fibrillation, which exceeded that of borage oil (containing three times more y-linolenic acid than EPO). This implies that y-linolenic acid may not be the therapeutic component of EPO; some other compounds present in EPO may exert cardioprotective effects in this

animal model.!©°! Very few investigations on the cardioprotective effects of EPO have been carried out in humans. Abraham et al.!°") examined changes in the fatty acid composition of adipose tissue, serum triacylglycerol and cholesterol ester levels, and lipoprotein profiles in men with low dihomo-y-linolenic acid levels supplemented with safflower oil or EPO for four mo. Participants in this study were split into four groups (6-9 men per group) receiving 10, 20, or 30ml per day of EPO or 20 ml per day of safflower oil. Adipose and blood samples were obtained from participants before and after the treatment period. Treatment with EPO at levels >20ml per day increased adipose dihomo-y-linolenic acid levels (P < 0.01); safflower oil did not increase adipose dihomo-y-linolenic acid content. Within individual participants, similar trends were observed between adipose fatty acid compositions and serum triacylglycerol and cholesterol ester fatty acid compositions. Safflower oil and EPO treatments did not significantly lower LDL

or raise HDL

levels,

implying that EPO may not exert strong antiatherogenic effects in humans, contrary to the findings from the animal models.°'! However, Abraham et al.!°"! used a highly select group of men most likely possessing low A-6-desaturase activity, which may have affected the outcome of this study. More recently, Khan et al.!°] performed a double-blind, placebo-based

study to examine the effects of an 8-mo treatment with food oils on vascular tone and endothelial function in 173 healthy volunteers (118 males and 55 females). The placebo oil used was 25:75 (w/w) mixture of soybean oil and coconut oil, administered at 10g per day to the placebo group. Participants in the EPO group received 5g EPO plus Sg placebo oil per day. The results for the EPO group showed that it did not alter the vasodilator responses to acetylcholine (“general nitroprusside or sodium function’) endothelial (“endothelium-independent vasodilation’’) in healthy volunteers!) The majority of results from human and animal studies exhibit different findings with respect to the cardioprotective effects of EPO, as certain invasive tests cannot be performed

on humans.

In animal models,

specific cardiovascular conditions can be induced, thereby allowing researchers to more accurately measure specific responses to EPO. More research involving human subjects and possibly trials involving recovering cardiovascular disease patients already tak- : ing medication need to be done in order to assess whether EPO exerts any cardioprotective effects in medicated patients.

EPO AND CANCER Cancer is a general term for more than 100 diseases that are characterized by uncontrolled and abnormal growth of cells (neoplasia) derived from normal tissues. Cancerous cells may develop into tumors that could produce toxins or spread locally or through the bloodstream and lymphatic system to other parts of the body (metastasize). The initial steps that lead to the transformation of normal cells into cancerous cells are not well understood. But many lines of evidence implicate the involvement of mutated genes in cells progressing toward cancer. Cancer cells are incapable of apoptosis, a property that makes them potentially ‘immortal.’ Two recognized treatments for cancer are chemotherapy, which is the administration of drugs (alkylating agents, e.g., vinblastine and chlorambucil) and radiation therapy in which cancerous tumors are subjected to ionizing radiation. Both treatments cause irreparable damage to the DNA of cancer cells, thereby promoting cell apoptosis. Currently, much research is being conducted to identify new cancer therapies with less adverse side effects and greater specificity toward cancer cells. Experimental and epidemiological studies have demonstrated that the composition of dietary fat affects the incidence and progression of some

cancers, “41 Several cancerous cell lines have been developed from animal and human malignant tumors. They serve as excellent in vitro models for cancer studies because

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203

the biochemical processes that occur within these cells are remarkably similar to those within their parent tumors. Early reports using cancerous cell lines have shown that y-linolenic acid and metabolites of this compound suppress cell proliferation.!°*! The fact that EPO is a dietary source of y-linolenic acid has prompted ‘several research groups to investigate its anticancer potential. Gardiner and Duncan"! examined the effects of EPO and safflower oil on the growth of precultured BL6 melanoma tumors implanted in

human subjects has been small scale and is quite prone to errors due to interpatient variability. Larger-scale human studies with subject populations that exhibit similar disease symptoms and severity are needed to provide strong support for the anticarcinegenic potential of EPO.

mice. After implantation, the mice were fed diets con-

Several adverse health effects have consistently been observed in menopausal and postmenopausal women. Menopausal hot flashes, postmenopausal osteoporosis, and chronic breast mastalgia (chronic breast pain) are examples of adverse health states affecting women.!"! The etiologies of these conditions are not well under-

taining 6% EPO or safflower oil for 4 weeks after which tumor growth and A-6-desaturase activity were measured in each animal. Results showed that EPO promoted in vivo tumor growthto a greater extent than safflower oil and that the A-6-desaturase activities of the in vivo BL6 tumors were lower than that of in vitro BL6 tumors. These results collectively imply that BL6 tumor growth in vivo is enhanced in the presence of dietary y-linolenic acid, which may ameliorate the antigrowth effects of low A-6-desaturase activity since ylinolenic acid is a product of this enzyme’s activity. Ramesh and Das'®”? examined the effects of topical EPO or fish oil application on skin carcinogenesis in male Swiss albino mice. This study employed a multistage skin carcinogenesis model involving two clearly defined steps: initiation and promotion using noxious chemicals topically applied on the skin of mice. Treatments of 10mg EPO or fish oil were used twice daily for 14weeks, after which papilloma (benign skin tumors) counts and histopathological assessments of excised skin samples were performed. Both fish oil and EPO significantly reduced papilloma formation compared to the control animals receiving no topical EPO

or

fish

oil

treatment

(P < 0.05).

However,

neither EPO nor fish oil could influence skin cell proliferation in this model. These results imply that EPO may interfere with the development of papillomas but cannot influence the metastatic properties of skin tumors in this model.!°7! Booyens et al.!°*! studied liver cancer cell proliferation in 6 patients taking EPO supplements and reported a decrease in tumor sizes in 3 patients as well as considerable reductions in plasma levels

of cancer

markers.!*!

More

recently,

Kollias

et al.!°*) studied the effects of EPO (4g per day) on breast fibroadenoma (benign solid growth) in 20 female subjects. The treatment period lasted 6 mo, after which clinical and ultrasound measurements of previously diagnosed fibroadenomas were compared. Results of these measurements showed that EPO treatment did not alter fibroadenoma size compared to matched controls.!**! Data from animal models imply that EPO may exert anticarcinogenic effects when consumed as a dietary component, and some human studies support these findings. However, the subset of studies involving

EPO AND WOMEN’S

stood, but one

HEALTH

common

factor has been

associated

with their declining estrogen level.!” It has been speculated that alterations in hypothalamic activity may underline the clinical aspects of adverse menopausal conditions; however, this view is not accepted widely.!71! For most women, estrogen replacement therapy (ERT) is an effective treatment for adverse menopausal conditions. But several concerns regarding the long-term safety of ERT have prompted the search for natural, nonhormonal substitutes, and EPO in this

regard has received some positive attention.” Chenoy et al [721 performed a randomized, doubleblind, placebo-based trial to evaluate the effect of oral

EPO consumption or paraffin supplementation (4 g per day for 6mo) in the treatment of hot flashes among 56 menopausal women. Results were based on reports given in diaries of all 56 participants, which showed no significant difference between EPO and placebo treatments despite numerous claims of its efficacy.!”7! Menopause is known to precede bone mineral density loss leading to osteoporosis that significantly weakens bones making them prone to fractures after light traumas. Adequate premenopausal calcium and vitamin D intakes are negative risk factors for osteoporosis, while smoking and back-to-back pregnancies are promotional risk factors. Bassey et al.!”*! recently performed a randomized placebo-controlled trial to investigate changes in bone density markers in response to Efacal®, an EPO plus eicosapentaenoic acid (EPA), and calcium supplement. This study included 43 preand postmenopausal women supplemented with either 4g Efacal, 1g calcium per day, or placebo. Final assessments were made after 12mo. Completed food frequency questionnaires were used to assess background calcium intake. The results of this study showed that Efacal treatments did not significantly alter bone mineral density or any other osteoporosis markers, which may

indicate the lack of therapeutic

efficacy of Efacal in osteoporosis treatment or the already optimal nutritional state of participants in this

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204

study.!”7! Breast mastalgia is the most commonly diagnosed breast problem and can lead to severe pain that interferes with daily life. Effective medications are available, but the high frequency of side effect occurrence with these drugs has stemmed interest in alternative remedies for breast mastalgia."! Recently, Blommers et al.'’*! investigated the effects of fish oil and EPO on breast mastalgia in 120 premenopausal women. The participants were divided into four groups receiving either 3g EPO plus 3g fish oil per day, 3g EPO plus 3g corn oil per day, 3 g fish oil plus 3 g corn oil per day, or 6g corn oil per day (the control group). The treatments were given for 6mo and symptom questionnaires were sent to participants 3 and 6mo into the trial and were compiled for subsequent symptom analysis. The results of this study showed no significant differences between the three experimental groups and the control group with respect to the number of days with active mastalgia, indicating that EPO supplementation may not be an effective treatment for breast mastalgia.!”*! Premenstrual syndrome affects millions of premenopausal women and exerts a wide variety of symptoms including abdominal pain, breast tenderness, headache, depression, fatigue, and mood swings that usually subside within 72 hr after the onset of menstrual flow.!”>! The syndrome occurs more frequently in women with

atopic allergies!’*! and in those with a high saturated fat intake,’ which implicates abnormal fatty acid metabolism or dietary PUFA deficiency as promotional factors for premenstrual syndrome. Some reports have indicated that EPO may be effective in the management of premenstrual syndrome symptoms. However, few placebo-controlled trials have been con-

ducted to investigate this possibility. Khoo, Munro, and Battistutta!’*! examined the therapeutic effectiveness of EPO (Efamol®) in the relief of symptoms of premenstrual syndrome. This was a_ prospective, randomized,

double-blind,

placebo-controlled

trial

involving 38 women supplemented with 4g per day of EPO or placebo for 6mo (6 menstrual cycles). No significant differences in premenstrual syndrome symptoms were observed between the EPO and placebo groups throughout this study. Yet both groups did exhibit improvements in overall symptoms (psychological, fluid retention, and breast pain) implying that the improvement observed in the EPO group was due to the placebo effect.’*! Budeiri, Li-Wan-Po, and Dornan'”! analyzed the results of 7 placebo-controlled trials that investigated the effects of EPO on premenstrual syndrome symptoms (meta-analysis). The differences that existed between these studies made a rigorous meta-analysis impossible. EPO was shown to exert moderate beneficial effects compared to placebo for breast pain associated with premenstrual syndrome. Yet, it displayed no overall evidence of

effectiveness in the management of premenstrual syndrome.!”! A link between pregnancy complications and low maternal PUFA status has been established. Marine diets rich in n-3 PUFA are known to reduce the incidence of gestation-induced hypertension, *"! a condition characterized by increased platelet aggregation and vasoconstriction leading to thrombosis in nonhypertensive women. If in addition to hypertension high urinary protein levels develop (proteinuria), the condition is called pre-eclampsia. Zielinski et al. studied the effects of EPO against hypertension development in pregnant rabbits. In this study, pregnant and nonpregnant rabbits were supplemented with 50mg/kg per day EPO for 10days. Pregnant and nonpregnant rabbits on regular diets were used as controls. After the supplementation period, angiotensin II, a blood clotting factor, was administered to animals to induce

hypertension, and the pressor response (increase in blood pressure) was measured. Results showed that. EPO-supplemented pregnant rabbits exhibiteda significantly lower systolic and diastolic response to angiotensin II compared to the control pregnant rabbits. No significant difference in the pressor response to angiotensin II existed between nonpregnant rabbits of both groups. These results may be attributable to the high levels of y-linolenic acid that may have enhanced PGE, (antiaggregatory) synthesis. Coadministration of PGE; with angiotensin II reduces vascular responsiveness to angiotensin II.°") Moodley and Norman”!

performed

a randomized,

placebo-based

trial to study the effects of 4 g/day EPO administration in 47 women with established pre-eclampsia, showing that no difference in pre-eclampsia symptoms (blood pressure, and blood aggregability) existed between the two groups.®7! One of the largest clinical trials investigating the effect of EPO treatment on women with pre-eclampsia was performed by D’Almeida et al?! A total of 150 women in their first trimester of pregnancy were supplemented with a mixture of EPO, EPA, and docosahexaenoic acid (DHA) or placebo for 6mo. Some were treated with magnesium oxide. Participants supplemented with the EPO mixture exhibited significantly better cardiovascular function (reduced incidence of edema, P < 0.004) and reduced pre-eclampsia, implying that EPO in combination with EPA and DHA may be helpful in the prevention of pre-eclampsia.*! Early studies have shown that EPO is an effective natural remedy for some female specific adverse health conditions.) But more recent studies disagree with some of these findings, showing that EPO exerts no significant change beyond a placebo effect in controlled human trials. More investigations are needed in order to conclusively resolve these discrepancies. In addition, patient assessments

should be based on clinical

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205

evaluations and not on symptom frequency questionnaires in order to reduce the level of uncertainty in results and increase the significance of experimental findings.

EPO AND DIABETES MELLITUS-INDUCED NEUROPATHY Insulin is a peptide-based pancreatic hormone that stimulates most body cells to absorb plasma glucose necessary for energy and cellular metabolism. Several mammalian glucose transporters require insulin for activation and cell membrane incorporation (receptor requirement). Diabetes mellitus is a disease caused by a relative or complete lack of insulin action that leads to detrimental alterations in carbohydrate metabolism. “Diabetes

mellitus’?

is Latin

for

“to

flow

sweet,’’

which refers to the large volume of urine produced daily with high glucose content. There are two forms of diabetes mellitus: juvenile onset and adult onset diabetes. Juvenile onset diabetes (Type-I diabetes) is a genetic disease believed to be caused by an autoimmune reaction early in life in which the pancreatic B-cells that secrete insulin into the bloodstream become nonfunctional, thereby causing insulin deficiency that can only be treated with insulin injections. Hence the term insulin-dependent diabetes mellitus (IDDM). Adult onset diabetes (Type-II diabetes), by far the most prominent form, is characterized by normal-to-high B-cell activity and insulin levels but markedly decreased insulin sensitivity, and tends to

affect obese individuals.®*! Both Type-I and Type-II diabetes result in high plasma glucose levels that can alter plasma proteins such as hemoglobin, disrupt microvascular circulation by increasing blood coagulation that may require the amputation of limbs and can lead to neuropathy. Diabetics have been shown to have diminished A-5- and A-6-desaturase activity, causing alterations in tissue fatty acid profiles and deficiencies in some long-chain PUFA.°! Membrane fatty acid composition is known to affect the cellular response to insulin. Increases in membrane PUFA levels enhance cellular insulin sensitivity and receptor recruitment, while decreasing the levels has the opposite effect, decreased insulin responsiveness. ®*! Since essential PUFA are known to improve insulin responsiveness at the cellular level, several studies have been done to investigate whether oils rich in PUFA,

such

as EPO, can improve diabetes management. Most of the investigations on the effects of EPO on diabetes mellitus have been carried out using animal models in which the administration of diabetogenic agents or pancreatectomy is done to induce the disorder. These models closely resemble Type-I diabetes and are particularly useful in the study of diabetic neuropathy.

Vascular changes that occur during diabetes mellitus may cause reductions in nerve perfusion leading to impaired nerve function and neuropathy. Cameron et al.” examined whether EPO supplementation could help overcome diabetic nerve conduction deficits in diabetic rats. The animals were treated with streptozotocin (a diabetogenic agent) and fed normal diets for Imo to allow for the accumulation of nerve conduction deficits. After this initial feeding period, experimental animals were fed diets supplemented with 10% EPO for 1mo to assess whether this treatment could reduce the extent of diabetes-induced abnormalities. Control diabetic and nondiabetic rats were fed normal diets for 2mo. Results of this study showed that after the initial 1 mo feeding period, diabetic rats exhibited deficits in motor and sensory nerve conduction velocity, which were maintained over a 2 mo period. In EPO-treated diabetic rats, deficits in nerve conduction velocity were restored to the level of nondiabetic control rats.'°’! These findings suggest that EPO is an effective treatment for diabetes-induced neuropathy in this animal model. sunflower oil, EPO,

More recently, the effects of and two structured y-linolenic

acid-containing triacylglycerols were examined on nerve conduction deficits in streptozotocin-induced diabetic rats.8*! Animals were fed diets supplemented with sunflower

oil, EPO,

dilinolein mono-y-linoleate

(DLMG), or tri-y-linoleate for 6 weeks after diabetes induction to examine whether the stereospecific distribution of y-linolenic acid in triacylglycerols could influence diabetes-induced neuropathy. All diets were prepared to contain equal amounts of y-linolenic acid, except for sunflower oil-supplemented diets that served as control diets. Results showed that all three y-linolenic acid-containing diets restored nerve conduction velocity, while sunflower oil-treated controls showed no such improvements, implying that the stereospecific distribution of y-linolenic acid in dietary triacylglycerols did not influence its therapeutic effects in this animal model.'*! Ford et al.) examined effects of treatments with EPO, «-lipoic acid, or sunflower oil on peripheral nerve conduction and vascular parameters in streptozotocin-induced diabetic rats. For 6weeks after the induction of diabetes mellitus, the animals were fed normal diets; normal untreated

rats were placed on the same diet for use as controls. After the initial feeding period, control and diabetic animals were treated daily with 300mg/kg body weight o-lipoic acid, 10g/kg body weight EPO, or 10g/kg body weight sunflower oil for 27 weeks. EPO treatments for 2weeks significantly improved both motor and sensory nerve conduction velocity in diabetic rats (P < 0.05 or less) and resulted in the restoration of normal nondiabetic control values in these animals. However, no significant vascular improvements were observed in the EPO-treated

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206

effects were observed for patients treated with MaxEPA fish oil. Animal studies show that EPO cyclosporincan significantly reduce treatments o et al.°! Yoshimot >! icity.047 A-mediated nephrotox improved tly significan s treatment showed that EPO comacid fatty plasma skin symptoms and abnormal health These patients. sis positions of hemodialy effects of EPO seem promising. However, the amount of existing literature is small making it hard to draw

diabetic group. o-Lipoic acid treatments resulted in significant improvements in both nerve conduction velocities and vascular parameters in diabetic rats (P < 0.05 or less). Sunflower oil-treated diabetic rats showed no significant differences in vascular and neuronal conduction parameters compared to control diabetic rats.” Findings from animal models show that EPO is an streptozotocin-induced in agent antineuropathic diabetes and may be of benefit in the prevention of neuropathy in diabetic humans. To date, however, no published trials have investigated the effects of EPO on diabetic neuropathy in humans.

most of these studies have been small scale with few having more than 40 subjects; large-scale trials involving humans are needed (Table 1).

OTHER HEALTH EFFECTS OF EPO

CONCLUSIONS

Animal and human studies have investigated the effects of EPO on mental health,?°° inflammatory

EPO might modify several health factors that could in turn modify the onset of disease states. It may exert therapeutic effects in inflammatory and immune- -

bowel

disease,”!!

and

renal health.!?7! Joy, Mumby,

clear

conclusions

about

these

effects.

Furthermore,

and Joy! systematically searched several scientific

mediated diseases, cardiovascular diseases, cancer, and

databases for randomized trials investigating the effects of EPO on schizophrenia. In some trials, EPO supplementation improved symptoms, while others showed no such effects.°° Greenfield et al.?*! showed that EPO treatment improved stool frequency in ulcerative colitis patients after 3mo of supplementation; no such

some conditions associated with menopause. These effects may in part be due to the oil’s y-linolenic acid constituent that could promote the production of antiinflammatory dihomo-y-linolenic acid-derived eicosanoids and/or reduce proinflammatory arachidonic acid-derived eicosanoid production, thus affecting

Table

1 Summary of research findings on the effects EPO in health and disease

Disease/condition studied

Type of evidence (References)

Overall findings

Atopic dermatitis

Small-scale human trials pee

EPO may be effective treatment for some clinical symptoms

Rheumatoid arthritis

Small-medium human trials

Inconclusive, several trials possess faults in their study protocol

Cardiovascular disease

Animal studies!!! and small-scale human studies ead

Cancer

scale

In vitro/animal studies [65-68] me and

EPO is effective in animals, no evidence suggests effectiveness in humans

small-scale human trials

Animal studies suggest EPO is antiproliferative, some human studies agree

Menopausal hot flashes

Small-scale human trials! vel

Inconclusive findings

Osteoporosis

Small-scale human studies ee

Ineffective in reducing bone density loss and other markers of osteoporosis

Breast mastalgia

Small-scale human studies a

Few studies indicate

therapeutic effectiveness Premenstrual syndrome

Small-scale human trials 78,79]

No clinical evidence of effectiveness

Pregnancy-induced hypertension/pre-eclampsia

Animal studies'°™!

Both animal and human studies implicate EPO as therapeutic

Diabetes. mellitus-induced neuropathy

Animal studies!®’*9!

and small-scale

human trials

EPO is an antineuropathic agent in diabetic animal models

Evening Primrose (Oenothera biennis)

207

several aspects of cellular biology and physiology such as immune hyper-reactivity, vascular function, and cellular proliferation. However, it is quite possible that some other components or group of compounds present in EPO may be responsible for its health effects. Incorporation into normal human diets would provide adequate levels of y-linolenic acid and vitamin E, thereby reducing the likelihood of their deficiency. Thus, EPO may exert several beneficial effects on human

11.

sion in human colo-rectal adenomas and adenocarcinomas. Gastroenterology 1994, 107, 11831188.

12;

health and disease states. Nonetheless, further studies

health/disease states and EPO can be made.

3?

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Folate Pamela Bagley Biomedical Libraries, Dartmouth College, Hanover, New Hampshire, U.S.A.

Barry Shane University of California, Berkeley, California, U.S.A.

INTRODUCTION Folate is an essential dietary B-complex vitamin required for human health. It functions as a cofactor in the synthesis of nucleotides required for DNA synthesis and in amino acid metabolism. The classic symptom of folate deficiency is megaloblastic (large cell) anemia, which is a reflection of defective DNA synthesis. Poor folate status is also a risk factor for cancer. The recent finding that periconceptional folate supplementation greatly reduces the incidence of birth defects in humans had lead to fortification of the food supply with folic acid in the United States and other countries.

Practically all tissue folates are polyglutamate forms in which the glutamate tail is extended through an unusual peptide bond via the gamma-carboxyl of glutamate. Glutamate chain lengths can vary from about 4 to 10 in human tissues. Metabolism of folates to polyglutamate forms is required for their biological activity, as the polyglutamate forms are much more effective substrates for folate-dependent enzymes than are

the

monoglutamate

derivatives,

which

are

the

transport forms of the vitamin." Conversion of folates to polyglutamates of chain length greater than 3 is also required for effective retention of folate by tissues.”

BIOCHEMISTRY AND FUNCTIONS

STRUCTURE AND CHEMISTRY Folate is used as a generic term for a family of chemically and functionally related compounds based on the folic acid structure (Fig. 1). Folic acid (molecular weight 441.4), also known as pteroylglutamate, is a chemically oxidized, synthetic form of the vitamin and consists of

a pterin (2-amino-4-hydroxypteridine) ring linked to para-aminobenzoic acid, which is conjugated to a molecule of t-glutamic acid. Folic acid is readily reduced within the cell to the metabolically active 5,6,7,8-tetra-

hydrofolate form. Folates in tissues act as donors and acceptors of one-carbon units in metabolic reactions known as one-carbon metabolism. These units can be at the oxidation level of methanol (5-methyltetrahydrofolate), formaldehyde (5,10-methylenetetrahydrofolate), or formate (5- or 10-formyltetrahydrofolate or 5,10methenyltetrahydrofolate). The predominant coenzyme forms are listed in Table 1.

Folate coenzymes are involved in three major interrelated metabolic cycles in the cytosol of cells. These cycles are required for the synthesis of thymidylate and purines, precursors for DNA

and RNA synthesis, and

for the synthesis of methionine from homocysteine and the interconversion of serine and glycine (Fig. 2). 5,10-Methylenetetrahydrofolate plays a central role in these cycles as it can be used directly for thymidylate synthesis, or reduced to 5-methyltetrahydrofolate in the methionine synthesis cycle, or oxidized to 10-formyltetrahydrofolate for use in purine synthesis. Although these synthetic cycles are located in the cytosol, mammalian cells also contain a large

0

H

of

Ye’

O~

| CHe——CHs——C.

marie

No

9

Pamela

Bagley,

M.S.,

Ph.D.,

Services Librarian at Biomedical Hanover, New Hampshire, ESA:

is Information Libraries,

and

Dartmouth

Education College,

Barry Shane, Ph.D., is Professor at the Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California, U.S.A.

Encyclopedia of Dietary Supplements DOL: 10.1081 /E-EDS-120022044 Copyright © 2005 by Marcel Dekker. All rights reserved.

pterin

aminobenzoic acid

glutamic acid

Fig. 1 Structure of folic acid (pteroylglutamate). Onecarbon substituents can be at the N-5 and/or N-10 positions of the reduced tetrahydrofolate molecule. 219

Folate

220

Table 1

Metabolically active forms of folate*

Unsubstituted folates 7,8-Dihydrofolate (reduced at positions 7 and 8) 5,6,7,8-Tetrahydrofolate

One-carbon group —CH; —CHO

Substituted folates 5-Methyltetrahydrofolate 5-Formyltetrahydrofolate (folinic acid, leukovorin) 5-Formiminotetrahydrofolic acid 10-Formyltetrahydrofolate 5,10-Methylenetetrahydrofolate 5,10-Methenyltetrahydrofolate

—CHNH —CHO —CH)—CH-

2Active forms are polyglutamate derivatives.

mitochondrial folate pool, which is also involved in the provision of one-carbon precursors for cytosolic one-carbon metabolism.&4!

majority of one-carbon units required for cytosolic one-carbon metabolism.! 5,10-Methylenetetrahydrofolate generated via the mitochondrial SHMT reaction is oxidized to 10-formyltetrahydrofolate and then hydrolyzed to formate. The formate produced exits the mitochondria and is the one-carbon reincorporated into the cytosolic one-carbon pool as 10-formyltetrahydrofolate. 10-Formyltetrahydrofolate can be reduced to 5,10-methylenetetrahydrofolate in the cytosol reversibly by the action of a Cl synthase enzyme. The tissue distribution of the cytosolic isozyme of SHMT is more limited than that of the mitochondrial enzyme; the cytosolic isozyme is highly expressed in liver and kidney and in rapidly replicating cells. A major role of the hepatic cytosolic SHMT enzyme may be to synthesize serine from glycine for use in gluconeogenesis. Mammalian cell mutants that lack mitochondrial SHMT but express cytosolic SHMT are glycine auxotrophs."7}

Serine—Glycine Interconversion

Nucleotide Synthesis

Serine is the major provider of one-carbon units for folate-dependent one-carbon metabolism." It donates its B-carbon to tetrahydrofolate to generate glycine

Folate is required for the synthesis of thymidylate, a nucleotide required specifically for the synthesis of DNA. Thymidylate synthase catalyzes the transfer of the one-carbon group from 5,10-methylenetetrahydrofolate to the 5’-position of (UMP and its reduction to a methyl group to generate dTMP. The folate molecule also provides the reducing component in this reaction, and the tetrahydrofolate is oxidized to dihydrofolate. The dihydrofolate generated has to be reduced back to tetrahydrofolate before it can be reutilized in onecarbon metabolism in a reaction catalyzed by dihydrofolate reductase (DHFR). Thymidylate synthase

and

5,10-methylenetetrahydrofolate.

The

reaction,

which is catalyzed by the pyridoxal phosphate (PLP)dependent enzyme serine hydroxymethyltransferase (SHMT), is at near equilibrium and can be easily reversed. Mammalian tissues contain two distinct isozymes of SHMT, one cytosolic and one mitochondrial, encoded by different genes.! Recent studies suggest that the mitochondrial isozyme, which is expressed in

all tissues, is responsible for the generation

Homocysteine

of the

Methionine

fixe

By.

Fig. 2. The major metabolic cycles of folatedependent one-carbon metabolism in the cytoplasm of cells. The cycles use reduced tetrahydrofolate (THF) polyglutamates as substrates. SHMT, serine hydroxymethyltransferase; MTHER, methylenetetrahydrofolate reductase; MS, methionine synthase; TS, thymidylate

MS 5-methylConte Ne

he Oy

CYCLE

MTHFR

Serine

MT.

Glycine

dUMP

NADP

synthase;

dTMP

5,10-anethylene-THF =—

NADPH

Glycine

PURINE

THY MIDYLATE CYCLE

CYCLE Serin e

NADPH

, NADP

roan

dihydrofolate

reductase;

5,10-methenyl-THF

ase;

a 10-formyl-THF

dTMP, deoxythymidine monophosphate; FAICAR, formylaminoimidazole carboxamide

CYH WeFormate.

7 We GAR

FAICAR

DHFR,

MTHFD, methylenetetrahydrofolate dehydrogenase; CYH, methenyltetrahydrofolate cyclohydrolase; GART, GAR formyltransfer-

AICAR

ee

AICART,

AICAR

ea

formyltransferase;

eee

ee

ribonucleotide; AICAR, aminoimidazole carboxamide ribonucleotide; GAR, glycinamide ribonucleotide; FGAR, formylglycinamide

ribonucleotide; B12, vitamin B12 (cobalamin).

Folate

221

activity is only expressed in replicating cells and is highest in fast growing cells. Consequently, drugs targeted to DHFR, such as methotrexate, have proven to be effective chemotherapeutic agents as they are selective inhibitors of rapidly growing cells.!*7! Folate coenzymes are also used in two steps of de novo cytosolic purine biosynthesis. The C-8 and C-2 positions of the purine ring are derived from 10-formyltetrahydrofolate in reactions catalyzed by glycinamide ribonucleotide transformylase and 5amino-4-imidazolecarboxamide ribonucleotide transformylase. Methionine Synthesis The methylation of homocysteine to produce methionine uses 5-methyltetrahydrofolate as the methyl donor in a reaction catalyzed by methionine synthase, one of only two vitamin By,-dependent enzymes in mammals (Figs. 2 and 3).!'! 5-Methyltetrahydrofolate is generated from 5,10-methylenetetrahydrofolate in a reaction catalyzed by the flavoprotein methylenetetrahydrofolate reductase (MTHFR). Methionine can be metabolized to S-adenosylmethionine, which acts as the methyl donor in many reactions, including the methylation of DNA, histones

and

other

proteins,

neurotransmitters,

and

phospholipids, and the synthesis of creatine. These methylation reactions play important roles in development, gene expression, and genomic stability. S-Adenosylhomocysteine, the product of methylation reactions, is a potent inhibitor of many methyltransferases and is catabolized by hydrolysis to adenosine and

erine

sane

NG cytosine

a

Xx

AdoHcy

os

P CH;X CH;-cytosine

homocysteine barr

PHYSIOLOGY Absorption, Transport, and Bioavailability Folic acid does not occur in nature and is rarely found in unfortified foods; it is not an active form of the coenzyme. However, it is the most common form of

folate used in supplements and in fortified food products because it is highly bioavailable and chemically stable and is readily reduced to tetrahydrofolate, the active coenzyme form of folate. Food folates typically occur in reduced, polyglutamyl form. Before absorption, they are cleaved to their monoglutamyl forms by a brush border glutamylhydrolase, sometimes called in the proximal small intestine by a saturable, pHsensitive transporter that transports oxidized and reduced folates. Most dietary folate, and folic acid in

MS } B,, CH,-THF

its carbon skeleton, and homocysteine is not normally

present in the diet. It is often the most limiting amino acid in human diets. Methylation reactions account for a large proportion of the methyl group intake in humans, and the methionine synthase reaction allows salvage of its backbone after its use for methylation. The folate-dependent methionine cycle is sensitive to folate status. When this is poor, the decreased ability to remethylate cellular homocysteine results in an increased plasma homocysteine level; the plasma total homocysteine level is an indirect indicator of folate insufficiency.

intestinal folate conjugase."") Folates are absorbed

saris

SHMT

homocysteine. In liver and kidney, homocysteine can be further metabolized to cysteine via the transsulfuration pathway. In most other tissues, homocysteine is exported to the circulation or is reconverted to methionine via the folate-dependent methionine synthase reaction. Methionine is a dietary essential amino acid as mammals lack the ability to synthesize homocysteine,

SAHH adenosine export

Fig. 3 The folate-dependent methionine synthesis cycle and the transmethylation cycle. THF, tetrahydrofolate; FAD, flavin adenine dinucleotide; PLP, pyridoxal phosphate; Bj, vitamin B,>; AdoMet, adenosylmethionine; AdoHcy, adeno-

sylhomocysteine; SHMT, serine hydroxymethyltransferase; MTHER, methylenetetrahydrofolate reductase; MS, methionine synthase; MATII, methionine adenosyltransferase; SAHH, AdoHcy hydrolase.

the diet, is metabolized to 5-methyltetrahydrofolate during its passage across the intestinal mucosa. When high doses of folic acid or other folate forms are consumed, a part is absorbed by nonsaturable, passive

diffusion, and appears in the circulation unchanged. The bioavailability of folic acid is close to 100% when it is consumed on an empty stomach. Although information on the bioavailability of food folate and folic acid taken with food is limited, the current best estimates are 50% (aggregate food folate) and 85%

(folic acid)."7! Very large doses of folic acid and other folates are well absorbed and can result in very high plasma vitamin levels. However, these fall quite rapidly as the renal threshold is exceeded, and much of the dose

Folate

222

is excreted within 24 hr. Although plasma folate levels

in excess of 100 times the normal can be achieved, tis-

sue folate levels increase only marginally, often less than two-fold, due to the limited ability of tissues to metabolize these large doses to the polyglutamate forms required for folate retention. Folate monoglutamate in plasma is transported into tissues via the reduced folate carrier, the same plasma membrane protein responsible for folate transport in

the gut.'*] For reasons not currently understood, the

properties of this protein differ between tissues. The liver carrier effectively transports both oxidized and reduced folate, while the carrier in most peripheral tissues has a very poor affinity for folic acid, and is specific for reduced folates. A second distinct folate transporter known as folate binding protein or the folate receptor is expressed in a more limited range of tissues. High levels of folate binding protein are expressed in the choroid plexus, kidney proximal tubes, and placenta and in a number of human tumors, while lower levels have been found

in a variety of other tissues. This transporter is responsible for reabsorption of folate in the kidney by a receptor mediated endocytotic process and is believed to play a similar role in folate transport in other

tissues."4] An additional distinct cellular transporter responsible for the transport of reduced folate monoglutamates into the mitochondrion has recently been identified."*!

Tissue Retention and Turnover

Folate monoglutamate transported into cells is metabolized to polyglutamate forms by the enzyme folylpolyglutamate synthase. Mammalian cells contain mitochondrial and cytosolic isozymes encoded by a single gene."® In the cytosol, much of the entering folate is metabolized to the 5-methyltetrahydrofolate derivative, which is a poor substrate for folylpolyglutamate synthase. This has to be metabolized to tetrahydrofolate via the methionine synthase reaction before effective polyglutamylation and tissue retention is achieved. As the flux of 5-methyltetrahydrofolate through the methionine synthase reaction is quite limiting, particularly when the cell contains high levels of 5-methyltetrahydrofolate polyglutamate, much of the newly absorbed folate is not retained by the tissue and appears in the circulation predominantly as 5-methyltetrahydrofolate. Under normal conditions of dietary intake and status,

whole

body

folate

turns

over

quite

slowly,

with a half-life in excess of 100 days." Urinary excretion of intact folate accounts for only a very small proportion of this turnover. Over 99% of tissue folate is in the polyglutamate form. The actual mechanism of

but primarily is poorly understood catabolism to generate bond C9-N10 the involves cleavage at moiety.""®! pterin a and tamates p-aminobenzoylpolyglu ed to hydrolyz are tamates The p-aminobenzoylpolyglu e hydrolas glutamyl l lysosoma monoglutamate by a N-methyl as urine in excreted and acetylated, and then is moiety pterin The mate. aminobenzoylmonogluta feces. the in excreted in bile and appears Feces contain very high levels of folate, but most, if not all, of this arises from bacterial synthesis in the lower gut. Studies in rodents have shown that some of this bacterially synthesized folate is bioavailable.) Whether folate synthesized by gut bacteria in humans is also bioavailable has not been determined.

FOLATE DEFICIENCY Megaloblastic Anemia Folate deficiency is usually due to a dietary insufficiency, although it can arise from other causes such as malabsorption syndromes. The classic symptom of folate insufficiency is megaloblastic anemia, a condition reflecting deranged DNA synthesis in the erythropoietic cells. This disorder is fairly common in pregnancy. Blood cells are enlarged and often multinucleated. Megaloblastic changes occur in all fast growing tissues such as the marrow and the gut epithelia. Affected cells contain close to twice the normal DNA content and the DNA is partially fragmented. Many are arrested in the G2 phase just prior to mitosis. Cells that divide often undergo apoptosis. The defect in DNA synthesis in folate deficiency has been ascribed to defective thymidylate synthesis under these conditions, with a resulting increase in uracil misincorporation into DNA. Removal of uracil by the repair enzyme uracil DNA glycosylase, and a decreased repair of the gaps produced by this enzyme, leads to an increase in double-stranded DNA breaks under these conditions.?° Megaloblastic anemia, or pernicious anemia, is also

a classic symptom of impaired Bj, status. This condition, which is quite prevalent in the elderly, is seldom due to a dietary deficiency but usually results from malabsorption of B,>. However, the anemia that results is identical to that of folate deficiency. In B)> deficiency, the Bj,-dependent methionine synthase enzyme is inactive, and cytosolic folate is ““trapped’’ as 5-methyltetrahydrofolate at the expense of other folate coenzyme forms required for one-carbon metabolism, such as thymidylate synthesis, leading to a functional folate deficiency in the cell.!!! As 5-methyltetrahydrofolate is a poor substrate for folylpolyglutamate synthase, the ability of tissues to accumulate folate is reduced and the functional folate

Folate

223

deficiency is compounded by a drop in cellular folate levels. As the defective DNA synthesis in pernicious anemia due to Bj)» deficit is caused by an induced secondary folate deficiency, high levels of folate cause a hematological response in patients. However, folate is ineffective in preventing the severe neurological pathologies associated with Bj» deficiency.

increased dietary folate not only lowers homocysteine in both groups but also eliminates the differences between the genotypes.'*! Although elevated homocysteine is associated with increased vascular disease risk and increased folate intake decreases plasma homocysteine levels, it remains to be established whether increased folate intake reduces vascular disease risk.

Vascular Disease

Neural Tube Defects

Severe genetic conditions that result in marked hyperhomocysteinemia are associated with a variety of clinical symptoms, including early onset occlusive cardiovascular and cerebrovascular disease. These genetic diseases include deficiency of methylenetetrahydrofolate reductase and cystathionine B-synthase, enzymes involved in the homocysteine remethylation and trans-sulfuration pathways, respectively. Lowering of homocysteine with high doses of folate and betaine improves the clinical picture in these patients, strongly suggesting that homocysteine is the causative agent

In the early 1990s, double-blind, randomized trials confirmed that supplemental folic acid given prior to conception and during early pregnancy significantly reduced (by over 70%) the incidence of neural tube defects

(NTDs),

the most

common

birth defects

in

been found consistently in prospective studies. Fasting homocysteine levels have been inversely correlated

humans.”°°l This was most likely not due to correction of a simple folate deficiency. Instead, it is thought that some women and/or their offspring have a higher requirement for folate due to genetic reasons. The current fortification of the food supply with folic acid in the United States and some other countries arose because those individuals at risk of having an NTD child cannot be identified at the moment (see below). A large number of folate candidate genes have been screened as risk factors for NTDs. Homozygosity of the 677T (Val) allele of methylenetetrahydrofolate reductase has been shown to be a risk factor in a number of studies, with the risk increasing very significantly in comparisons of infants of mothers with poor folate status. However, carrying this particular allele can only account for about 15% of the population-

with both plasma folate and food folate intake, and

based attributable derived risk.?7

of these conditions, although the actual mechanism is

not known.24 More recently, epidemiological studies have suggested that chronic mild hyperhomocysteinemia is a risk factor for occlusive vascular disease. In many case control studies, plasma homocysteine concentrations were higher in patients with vascular disease than in matched

controls. However,

this relationship has not

increased folate intake lowers the mean homocysteine of groups, with the greatest effect on those with the highest plasma homocysteine levels. If mildly elevated homocysteine is a causative risk factor for vascular disease, then it may be that a simple dietary intervention can lower the risk. A common polymorphism (677C — T, Ala to Val) in the flavoprotein methylenetetrahydrofolate reductase that results in a “heat-labile’’ enzyme and decreased enzyme activity in tissues has been implicated as one reason for the folate responsiveness of a subset of hyperhomocysteinemic subjects.?7) The incidence of the Val/Val homozygosity (around 10% in the U.S. population) is significantly higher in subjects with the highest deciles of homocysteine levels. The valine substitution reduces the affinity of the enzyme for its FAD cofactor, and loss of FAD leads to decreased protein stability. Folate binding “‘locks’’ the FAD cofactor in place on the enzyme and stabilizes

the protein.?*] Comparisons of Val/Val individuals with Ala/Ala subjects have demonstrated that differences between these groups are most obvious in subjects with poor folate and riboflavin status and that

Cancer

Epidemiological studies have shown that poor folate status is associated with an increased risk for certain types of cancer, including colon cancer.?8! The mechanism behind this is not known, but uracil misincorporation arising from defective thymidylate synthesis has been suggested as one possibility. As changes in folate status influence the remethylation of homocysteine to methionine and alter adenosylmethionine-to-adenosylhomocysteine ratios, it has also been proposed that the increased cancer risk in folate deficiency may be due to hypomethylation of DNA and/or histones. Changes in DNA and histone methylation have been observed in many

tumors,

and it has been demonstrated

that

methionine deficiency causes hypomethylation of DNA. Subjects homozygous for the methylenetetrahydrofolate reductase 667T allele demonstrate a decreased cancer risk, which is most pronounced in comparisons with subjects of good folate status. It has been speculated that this polymorphism directs more of the

Folate

224

one-carbon flux into thymidylate synthesis and away from methionine synthesis, although this remains to be proven.

folate from foods should be encouraged to supplement their diets with folic acid.

Treatment of Folate Deficiency

INDICATIONS AND USAGE

Folate deficiency has traditionally been diagnosed as a low serum or red blood cell folate level ( DADS > DAS.®?*4l Likewise,

the presence of the allyl group generally enhances protection over that provided by the propyl moiety.!’?*4!

the phosphorylation of multiple substrates, including histone H1. The G2/M phase arrest induced by DADS has been found to coincide with the suppression in p34° kinase activity." Overall, the ability of DADS to inhibit p34°*° kinase activation appears to

occur as a result of a decreased p34°/cyclin B(1) complex formation and a change in p34°™ hyperphosphorylation.?“! Several of the allyl sulfur compounds from garlic have also been reported to induce apoptosis.8?!-?7) DADS,

SAMC,

and ajoene have been shared to acti-

vate caspase-3."!7] More recently, allicin was claimed to induce the formation of apoptotic bodies, nuclear condensation, and a typical DNA ladder in cancer cells. Furthermore, these studies demonstrated that allicin leads to the activation of caspases-3, -8, and -9

Cell proliferation and apoptosis Several lipid- and water-soluble organosulfur compounds have been examined for their antiproliferative efficacy.47418>.85] Some of the more commonly used lipid-soluble allyl sulfur compounds in tumorigenesis research

are

ajoene,

DAS,

DADS,

and

DATS.

A

breakdown of allicin appears to be necessary for achieving maximum tumor inhibition. Studies by Scharfenberg, Wagner, and Wagner'®” found that the EDs) for lymphoma cells was two times lower for ajoene than for allicin. Previous

studies

reported

that lipid-soluble

DAS,

DADS, and DATS (100M) were more effective in suppressing canine tumor cell proliferation than isomolar water-soluble SAC, S-ethyl-cysteine, and S-propyl-cysteine.®**°! While treating human colon tumor cells (HCT-15) with 100uM DADS completely blocks growth, approximately 200M S-allyl mercaptocysteine (SAMC) is required to lead to a similar depression.'®>! No changes in growth were observed with concentrations of SAC up to 500uM. Undeniably, not all allyl sulfur compounds from garlic are equally effective in retarding tumor proliferation. Bae Evidence exists that these allyl sulfur compounds preferentially suppress neoplastic over nonneoplastic cells.83:37 Adding DATS (10M) in vitro to cultures of A549 lung tumor cells inhibited their proliferation by 47%, whereas it did not influence nonneoplastic MRC-5 lung cells.8”! The antiproliferative effects of allyl sulfides are generally reversible, assuming that apoptosis has not occurred. eran SAMC, DAS, and DADS have ae been reported to increase the percentage of cells blocked within the G,/M phase. (85,86.88.89] The ability of garlic to block

this phase is not limited to in vitro studies. p34ce? kinase is a complex that governs the progression of cells from the G» into the M phase of the cell cycle. Activation of this complex promotes chromosomal condensation and cytoskeletal reorganization through

and cleavage of poly(ADP-ribose) polymerase.!7) DADS has been also been reported to restrain the growth of H-ras oncogene transformed tumors in nude mice.“! This inhibition correlated with that of p21Hras membrane association in the tumor tissue. This group also demonstrated the importance of the allyl group in leading to a depression in ras.?°! Recently, allicin was found to induce activation of extracellular signal-regulated kinases 1 and 2 (ERK 1 and 2) in human peripheral mononuclear cells, and also in wild-type Jurkat T cells (114). It however failed to activate ERK 1 and 2 in Jurkat T cells that express p21(ras), in which Cys118 was replaced by Ser. Since these cells are not susceptible to redox-stress modification and activation,

the authors

postulated

that the

immune stimulatory effect of allicin is mediated redox-sensitive signaling such as activation p21(ras).?°!

by of

Cell differentiation

Lea, Randolph, and Patel?” suggest that at least part of the ability of DADS to induce differentiation in DS19 mouse erythroleukemic cells relates to its ability to increase histone acetylation. DADS caused a marked increase in the acetylation of H4 and H3 histones in DS19 and K562 human leukemic cells. Similar results were also obtained with rat hepatoma and human breast cancer cells. In 2002, Lea et al.®) provided evidence that DADS administered to rats could also increase histone acetylation in liver and a transplanted hepatoma cell line. The evidence suggested an increase in the acetylation of core histones and enhanced differentiation. Allyl mercaptan was a more potent inhibitor of histone deacetylase than DADS. In contrast to the effect on histone acetylation, there was a decrease in the incorporation of phosphate into histones when DS19 cells were incubated with 25 uM

SAMC”*I

Garlic (Allium sativum)

236

Mei, X.; Lin, X.; Liu, J.; Lin, X.Y.; Song, P.J.;

Dietary Modifiers of Garlic and Allyl Sulfur Efficacy

Hu, J.F.; Liang, X.J. The blocking effect of garlic on the formation of N-nitrosoproline in humans. Acta Nutr. Sin. 1989, 7/7, 141-145. Hsing, A.W.; Chokkalingam, A.P.; Gao, Y.T.; Madigan, M.P.; Deng, J.; Gridley, G.; F raumeni,

The influence of garlic on the cancer process cannot be considered in isolation since several dietary components can markedly influence its overall impact. Among the factors recognized to influence the response to

garlic

are

total

fat,

selenium,

methionine,

J.F., Jr. Allium vegetables and risk of prostate cancer: a population-based study. J. Natl. Cancer Inst. 2002, 94 (21), 1648-1651.

and

vitamin A.2-°9:! Selenium supplied either as a component of the diet or as a constituent of the garlic has been reported to enhance the protection against DMBA mammary carcinogenesis over that provided by garlic alone. Suppression in carcinogen bioactivation, as indicated by a reduction in DNA adducts, may partially account for this combined benefit of garlic and selenium. Both selenium and allyl sulfur compounds are recognized to alter cell proliferation and induce apoptosis.

Brenner, S.; Ruocco, V.; Wolf, R.; de Angelis, E.; Lombardi, M.L. Pemphigus and dietary factors. In vitro acantholysis by allyl compounds of the genus Allium. Dermatology 1995, 190 (3),

197-202. Perez-Pimiento, A.J.; Moneo, I.; Santaolalla, M.; de Paz, S.; Fernandez-Parra, B.; Dominguez-

Lazaro,

vivo.

to young

:

Free

Radical

Biol.

Med.

2003,

34

(9),

1200-1211. 10.

Peng, C.C.; Glassman, P.A.; Trilli, L.E.; HayesHunter, J.; Good, C.B. Incidence and severity

of potential drug—dietary supplement interactions in primary care patients: an exploratory study of 2 outpatient practices. Arch. Intern. Med. 2004, 164, 630-636.

of the health benefits, since preclinical studies indicate

water-soluble SAC provides comparable benefits to those compounds that are linked to odor. It is probable that garlic and its associated water- and lipid-allyl sulfur compounds influence several key molecular targets in cancer prevention. While most can savor the culinary experiences identified with garlic, some individuals because of their gene profile and/or environmental exposures may be particularly responsive to more exaggerated intakes.

reaction

Munday, R.; Munday, J.S.; Munday, C.M. Comparative effects of mono-, di-, tri-, and_ tetrasulfides derived from plants of the Allium family: redox cycling in vitro and hemolytic activity and phase 2 enzyme induction in

CONCLUSIONS Garlic may well have significance in enhancing health. Since it has relatively few side effects in most people, there are few disadvantages associated with its enhanced use, except for its lingering odor. However, odor does not appear to be a prerequisite for many

A.R. Anaphylactic

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45.

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Lea, M.A.; Rasheed, M.; Randolph, V.M.; Khan, F.; Shareef, A.; desBordes, C. Induction

of histone acetylation and inhibition of growth of mouse erythroleukemia cells by S-allyl mercaptocysteine. Nutr. Cancer 2002, 43 (1), 90-102.

Singh, S.V.; Pan, S.S.; Srivastava, S.K.; Xia, H.; Hu, X.; Zaren, H.A.; Orchard, J.L. Differential

induction of NAD(P)H: quinone oxidoreductase by anti-carcinogenic organosulfides from garlic. Biochem. Biophys. Res. Commun. 1998, 244 (3), 917-920. Singh, S.V. Impact of garlic organosulfides on p21(H-ras) processing. J. Nutr. 2001, /3/ (3s), 1046S-1048S.

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acetylation of histones induced by diallyl disulfide and structurally related molecules. Int. J. Oncol. 1999, 75 (2), 347-352.

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,

Ginger (Zingiber officinale) Tieraona Low Dog University of Arizona Health Sciences Center, Tucson, Arizona, U.S.A.

INTRODUCTION

PHARMACOKINETICS

Ginger is a popular spice and the world production is estimated

at 100,000tons

annually,

of which

80%

is

grown in China.'" In addition to its long history of use as a spice, references to ginger as a medicinal agent can be found in ancient Chinese, Indian, Arabic, and

Greco-Roman texts. Ginger has been used for a variety of conditions, but it is chiefly known as an antiemetic,

anti-inflammatory, digestive aid, diaphoretic, and warming agent. In the year 2000, ginger sales ranked 17th among those of all herbal supplements sold in U.S. mainstream retail stores.7!

Gingerol, when administered intravenously to rats, demonstrated a half-life of 7.23 min.©! It is not clear how this relates to the pharmacokinetics of whole rhizome on oral administration in humans. Scientific studies are currently underway to determine the pharmacokinetics of ginger when administered orally; however, the results from these inquiries have not yet been published.

PHARMACODYNAMICS NAME AND GENERAL

Antiemetic Activity

DESCRIPTION

The Zingiberaceae family consists of 49 genera and 1300 species, of which there are 80-90 species of Zingiber and 250 species of Alpinia. This entry will focus primarily upon the scraped or unscraped rhizome of common ginger, Zingiber officinale Roscoe, a reedlike plant grown in numerous subtropical areas of the world,

including

Jamaica,

India,

China,

and

Africa.

CONSTITUENTS Ginger rhizome contains 4-10% oleoresin composed of nonvolatile, pungent constituents (phenols such as gingerols and their related dehydration products, shogaols); nonpungent fats and waxes; 1.0-3.3% volatile oils of which 30-70% are sesquiterpenes, mainly B-bisabolene, (—) zingiberene, {-sesquiphellandrene, and (+) arcurcumene; monoterpenes, mainly geranial and neral; 40-60% carbohydrates, mainly starch; 9-10%

proteins and free amino

acids; 6-10%

lipids

composed of triglycerides, phosphatidic acid, lecithins,

and free fatty acids; vitamin A; niacin; and minerals."

Tieraona

Low Dog, M.D., is Clinical Assistant Professor in the

Department of Medicine and Program in Integrative Medicine, University of Arizona Health Sciences Center, Tucson, Arizona,

U.S.A. Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120024802 Copyright © 2005 by Marcel Dekker. All rights reserved.

Numerous human clinical trials have addressed the antiemetic effects of dried ginger root in the treatment of hyperemesis gravidarum,! motion sickness, postoperative nausea,®! and chemotherapy-induced nausea and vomiting”! The mechanism of action and constituent(s) responsible for the antiemetic activity of ginger are not completely understood. A class of antiemetics found to be clinically effective in the treatment of chemotherapy-induced and postoperative nausea and vomiting are the 5-hydroxytryptamine (5-HT) antagonists, specifically 5-HT3. Several components of ginger, viz., 6-gingerol, 6-shogaol, and galanolactone, have shown anti-5-HT activity in isolated guinea pig ileum. Galanolactone is a competitive antagonist predominantly at ileal 5-HT3 receptors.“° A study in rats found that an acetone extract of ginger and ginger juice effectively reversed the cisplatin-induced delay in gastric emptying typically seen when the drug is administered. The reversal produced by the ginger acetone extract was similar to the effect seen with the 5-HT3-receptor antagonist ondansetron; ginger juice, at doses of 2 and 4ml/kg orally (p.o.), was superior to the drug." Other researchers have demonstrated that ginger increases gastrointestinal motility, reducing the feedback from the gastrointestinal tract to central chemoreceptors,4!! though a double-blind crossover trial of 16 healthy volunteers who were randomly allocated to receive either 1g of dried ginger or placebo found no effect on gastric emptying.!'7} 241

Ginger (Zingiber officinale)

242 Motion sickness

Human studies evaluating the effects of experimentally induced motion sickness?! human clinical trials evaluating the use for motion sickness have been published. randomized,

ginger on and four of ginger The first

placebo-controlled

double-blind,

study

was published in 1988. Eighty Danish naval cadets (ages 16-19 yr) were randomized to receive either 1 g of dried ginger powder or placebo. Symptoms of seasickness were evaluated over the four following hours. Participants who received ginger powder experienced less seasickness than those in the control group (p < 0.05). No power calculation was included in the report. A

1994

randomized,

double-blind,

non-placebo-

controlled study of 1475 volunteers (age 16-65 yr) traveling by sea compared the efficacy of 7 antiemetic medications: Touristil® (cinnarizine 20mg, clomperidone 15mg), Marzine® (cyclizine 50 mg), Dramamine® (dimenhydrinate 50mg, caffeine 50mg), Permesin™ (meclizine 25mg, caffeine 20 mg) Stugeron® (cinnarizine 20mg), Scopoderm TTS® (scopolamine 0.5 mg) and Zintona® (product standardized to minimum 1.4% volatile oils and minimum 2.0mg_ gingerols and shogaols in capsule containing 250mg ginger rhizome). Stugeron and Scopoderm TTS were administered the evening prior to departure, with a second dose of Stugeron being given the morning of sea travel. The other medications were administered 2 hr prior

to

departure,

with

Touristil

and

motion sickness.''*! Side effects were significantly less in the ginger group (13.3%) than in those receiving dimenhydrinate (40%). Comorbid conditions were not ruled out and no power calculation was included in the report. Another

1999

randomized,

double-blind

1/2hr before the trip and, if necessary,

Ahr. Children randomized

took

12.5-25mg

1/2hr

500 mg every

to receive dimenhydrinate

before

trip

the

and,

if

necessary, 25mg every 4hr. Physicians’ rating of the therapeutic effectiveness showed highly significant difference between the treatment groups (p < 0.00001). Results were good in 100% of treatment | cases in the ginger-group, while in the dimenhydrinate group, they were modest in 69.2% and good in only 30.8%. All subjects in the ginger group reported symptom reduction within 30min of taking the extract, while 69.2% in the dimenhydrinate group reported a reduction in 60min (p < 0.00001). No patient in the ginger group reported any side effects, while most (84.6%) of the dimenhydrinate patients suffered

mouth in the 0.001). all of

from

side effects, including

dryness

of the

(69.23%) and vertigo (23.07%). The difference treatment group was highly significant (p ginger extract > placebo for VAS scores on pain and the Lequesne index, but no significant difference was seen when comparing ginger extract and placebo directly.°*! The lack of positive effects may have been due to inadequate trial length and/or insufficient dose. A

randomized,

double-blind,

placebo-controlled

study enrolled 261 (n = 247) patients with OA

of

Ginger (Zingiber officinale)

the knee as diagnosed by the American College of Rheumatology classification criteria.“*! The primary efficacy variable was the proportion of responders experiencing a reduction in “‘knee pain on standing,”’ using an intent-to-treat analysis. A responder was defined by a reduction in pain of >15mm on a visual analog scale. During the 6-week treatment period, patients ingested 1 capsule twice daily of 255mg ginger extract (EV.EXT 77, extracted from 25004000 mg of dried ginger rhizomes and 500-1500 mg of dried galanga rhizomes) or placebo. The percentage of responders experiencing a reduction in knee pain on standing was superior in the ginger extract group compared with the control group (63% vs. 50%; p = 0.048). Analysis of the secondary efficacy variables revealed a consistently greater response in the ginger extract group compared with the control group, when analyzing mean values: reduction in knee pain on standing (24.5 vs. 16.4mm; p = 0.005) and reduction in knee pain after walking 50ft (15.1 vs. 8.7mm; p = 0.016). One group of adverse events showed a significant difference between treatment groups: Gastrointestinal (GI) adverse events were more common in the ginger extract group [116 events in 59 patients (45%)] compared with the placebo group [28 events in 21 patients (16%)]. None of the GI adverse events were considered serious by the investigators. Both of these studies suggest the strong need for dose escalation studies that can determine the most efficacious dose that it is still safe and well tolerated. Studies of longer duration are also required before more definitive conclusions can be drawn about the safety, tolerance, and effectiveness of ginger for osteoarthritis.

245

Ginger inhibits platelet aggregation in vitro, acting as a potent inhibitor of arachidonic acid, epinephrine, adenosine diphosphate (ADP), and collagen. A placebo-controlled study of 8 healthy males found that ingestion of 2 g of ginger caused a dose-dependent reduction of thromboxane synthetase and _prostaglandin synthetase; however, no differences were found in bleeding time, platelet count, or platelet function between the placebo and control groups. In patients with coronary artery disease (CAD), powdered ginger administered in a dose of 4g/day for 3mo did not affect ADP- and epinephrine-induced platelet aggregation. However, a single dose of 10g produced a significant reduction in platelet aggregation induced by the two agonists.°?!

Gastrointestinal

Effects

Ginger has long been valued in traditional medicine for a wide variety of gastrointestinal complaints. Researchers are beginning to explore possible scientific explanations for these historical uses. In vitro research indicates that constituents present in ginger have antiulcer activity.“!) Animal research demonstrates that ginger reduces the occurrence of gastric ulcers induced by nonsteroidal anti-inflammatory drugs

(NSAIDs) and hypothermic restraint stress.) Cholagogic activity has been documented

Helicobacter pylori (HP) is the primary etiological agent associated with dyspepsia, peptic ulcer disease, and development of gastric cancer. Novel, inexpensive, and safe approaches to the eradication of H. pylori are currently being sought. A methanol extract of the dried, powdered ginger rhizome, fractions of the extract, and the isolated constituents,

Cardiovascular

Effects

In vitro research has shown that constituents in ginger have an inhibitory effect upon cholesterol biosynthesis.21 Animal studies have demonstrated lipidlowering activity via enhancement of the activity of hepatic cholesterol-7a-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, thereby stimulating conversion of cholesterol to bile acids, an important mechanism for eliminating cholesterol from the body.87! A study in rabbits found that an orally administered ethanolic extract of ginger (200 mg/kg) reduced lipids after 10 weeks feeding of a cholesterol rich diet. The authors found that, at this dose, ginger

produced results similar to those of gemfrimbrozil.?*!

In contrast to the in vitro and animal data, a study of patients with coronary artery disease found that 3mo ingestion of 4g/day dried ginger powder failed to

lower blood lipids.”!

in rats with the

acetone extract of ginger.!*?!

6-, 8-, and 10-

gingerol and 6-shogaol, were tested against 19 strains

of HP.4! The extract inhibited the growth of all 19 strains in vitro with a minimum inhibitory concentration (MIC) range of 6.25-50 g/ml. One fraction of the crude extract, containing the gingerols, was active and inhibited the growth of all HP strains with a MIC range of 0.78-12.5 pg/ml.

DOSE As with many botanicals, dosage ranges vary widely in research and, especially, in the marketplace. The following are a few doses (serving sizes) found in the literature: e e

Fresh or dried rhizome: 2-4 g daily.*"! Fluidextract: 1:1 (g/ml) 0.25-1.0 ml 3 times daily; tincture 1:5 (g/ml) 1.25—5.0 ml 3 times daily.!*!

Ginger (Zingiber officinale)

246

ADVERSE

EFFECTS

Patients treated with ginger have reported increased flatulence and heartburn compared to those on placebo.

CONTRAINDICATIONS

in the ginger group,'*! 1 spontaneous abortion of 27 in the crossover design study,'*) and 3 spontaneous abortions of 60 in the ginger group,7 although one of these occurred in a woman who had not begun taking the treatment. Though the total number of women in these clinical trials is small, the rate of spontaneous abortion is not any greater than that seen in the general population.

Due to its cholagogic effect, those with active gallstone disease should avoid ginger.

REFERENCES HERB-DRUG

1.

INTERACTIONS

None are known. There have been anecdotal and speculative warnings about ginger and warfarin; however, there are no documented cases in the literature. Standardized ginger extract had no significant effects on coagulation parameters or on warfarin-induced changes in blood coagulation in rats.4°! Though evidence is lacking for a direct interaction between warfarin and ginger,” it is probably still wise for practitioners and patients alike to be cautious about the use of doses greater than 4g/day of dried ginger in conjunction with antiplatelet/anticoagulant medications.

TOXICITY

Langner, E.S.; Griefenberg, S.; Gruenwald, J. Ingwer: eine Heilpflanze mit Geschichte [Ginger:

a medicinal plant with a history]. Balance Z. Prax. 2. 3.

4.

Wiss. Komplement. Ther. 1997, 7, 5-16. Blumenthal, M. Herb sales down 15% in mainstream market. HerbalGram 2001, 5/, 69. Evans, W.C. Trease and Evans Pharmacognosy, 15th Ed.; W.B: Saunders: London, England,

2002; 277-280. British Herbal

Pharmacopoeia

(BHP); British

Herbal Medicine Association: Exeter, U.K., 1996.

5.

Ding,

G.H.;

Naora,

K.;

Hayashibara,

Katagiri, Y.; Kano, Y.; Iwamoto,

6.

M.;

K. Pharmaco-

kinetics of [6]-gingerol after intravenous administration in rats. Chem. Pharm. Bull. (Tokyo) 1991, 39, 1612-1614. Portnoi, G.; Chng, L.A.; Karimi-Tabesh, L.; Koren, G.; Tan, M.P.; Einarson, A. Prospective

There is little Acute toxicity adverse effects up to 2.5 g/kg sing the dose mortality.{*8!

risk of toxicity when used as a spice. tests in mice found no mortality or when ginger extract was given at doses (by lavage) over a 7 day period. Increato 3.0-3.5g/kg resulted in 10-30%

comparative study of the safety and effectiveness of ginger for the treatment of nausea and vomiting in pregnancy. Am. J. Obstet. Gynecol. 2003, 189 (5), 1374-1377. 7.

8.

USE IN PREGNANCY Two studies have been published examining the effect of ginger in pregnant rats. One found that ginger tea (20 or 50 g/L) administered from gestation days 6-15 and then sacrificed at Day 20 significantly increased early embryonic loss and increased growth in surviving fetuses.'*"! No gross morphologic malformations were seen in the treated fetuses. Teratogenic studies on ginger extracts at doses of 100-1000mg/kg failed to observe any toxic effects or early embryonic loss." Researchers at the Hospital for Sick Children in Toronto, Canada, studied 187 pregnant women who used some form of ginger in the first trimester. They report that the risk of these mothers having a baby with a congenital malformation was no higher than that in a control group.! Of the published human studies, there was

Mowrey,

D.B.; Clayson,

D.E.

Motion

sickness,

ginger, and psychophysics. Lancet 1982, 7, 655—

1 spontaneous

abortion out of 32

9.

657. Phillips, S.; Ruggier, R.; Hutchinson, S.E. Zingiber officinale (ginger)—an antiemetic for day case surgery. Anesthesia 1993, 48 (8), 715-717. Sontakke, S.; Thawani, V.; Naik, M.S. Ginger as

an antiemetic in nausea and vomiting induced by chemotherapy:

10.

11.

12.

a randomized, cross-over, double

blind study. Indian J. Pharmacol. 2003, 35, 32-36. Huang, Q.R.; Iwamoto, M.; Aoki, S. et al. Anti-5effect of galanolactone, hydroxytryptamine-3 diterpenoid isolated from ginger. Chem. Pharm. Bull. (Tokyo) 1991, 39, 397-399. Sharma, S.S.; Gupta, Y.K. Reversal of cisplatininduced delay in gastric emptying in rats by ginger (Zingiber officinale). J. Ethnopharmacol. 1998, 62 (1), 49-55. Phillips, S.; Hutchinson, S.; Ruggier, R. Zingiber officinale does not affect gastric emptying rate. A randomised, placebo-controlled, crossover trial. Anaesthesia 1993, 48 (5), 393-395.

Ginger (Zingiber officinale)

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Lien, H.C.;

247

Sun, W.M.;

Chen,

Y.H.;

Kim,

H.;

Hasler, W.; Owyang, C. Effects of ginger on motion sickness and gastric slow-wave dysrhythmias induced by circular vection. Am. J. Physiol. Gastrointest. Liver Physiol. 2003, 284 (3), G48 1-G489,

14.

16.

els

18.

19;

20.

blind study comparing ginger (Zintona®) and dimenhydrinate in motion sickness. Healthnotes Rev. Complement. Integr. Med. 1999, 6 (2), 98101(Reviewed and edited by Fulder, S. and Brown, D.). Carredu, P. Motion sickness in children: results of a double-blind study with ginger (Zintona®) and dimenhydrinate. Healthnotes Rev. Complement. Integr. Med. 1999, 6 (2), 102-107. O’Brien, B.; Naber, S. Nausea and vomiting during pregnancy: effects on the quality of women’s lives. Birth 1992, 19, 138-143. Fischer-Rasmussen,

W.;

Kjaer,

S.K.;

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ons

trial. Obstet. Gynecol. 2001, 97 (4), 577-582. Willetts, K.E.; Ekangaki Abie Eden, J.A. Effect of

Mustafa, T.; Srivastava, K.C.; Jensen, K.B. Drug

oe

development: report 9. Pharmacology of ginger, Zingiber officinale. J. Drug Dev. 1993, 6, 25-89. Penna, S.C.; Medeiros, M.V.; Aimbire, F-.S.; Faria-Neto,

PRES.

Dermatol.

Nurs.

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Skinner, C.M.; Rangasami, J. Preoperative use of herbal medicines: a patient survey. Br. J. Anaesth. 2002, 89, 792-795. Ernst, E.; Pittler, M.H. Efficacy of ginger for nausea and vomiting: a systematic review of randomized

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randomized, placebo-controlled, cross-over study of ginger extracts and ibuprofen in osteoarthritis. Osteoarthritis Cartilage 2000, 8 (1), 9-12.

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Srinivasan, K.; Sambaiah, K. The effect of spices

on cholesterol 7 alpha-hydroxylase activity and on serum and hepatic cholesterol levels in the rat. Int. J. Vitam. Nutr. Res. 1991, 6/ (4), 364-369.

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Bhandari, U.; Sharma, J.N.; Zafar, R. The protec-

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Arfeen, Z.; Owen, H.; Plummer, J.L.; Isley, A.H.; Sorby-Adams, R.A.; Doecke, C.J. A double-blind randomized controlled trial of ginger for the

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Clark, B.M.; Dieppe, P.A.; Griffin, M.R. et al. Guidelines for the medical management of osteoarthritis: part II. Osteoarthritis of the knee.

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Zingiber officinale (ginger) used to prevent 8nausea.

Eberhart, L.H.; Mayer, R.; Betz, O. et al. Ginger

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Dissertations Abstr. Int. 1987, 47, 3297-3298. Meyer, K.; Schwartz, J.; Crater, D.; Keyes, B.

MOP.-associated

669-671. Pongrojpaw, D.; Chiamchanya, C. The efficacy of ginger in prevention of post-operative nausea and vomiting after outpatient gynecological laparoscopy. J. Med. Assoc. Thail. 2003, 86 (3), 244-250. does not prevent postoperative nausea and vomiting after laparoscopic surgery. Anesth. Analg. 2003, 96 (4), 995-998.

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Asping, U. Ginger treatment of hyperemesis gravidarum. Eur. J. Obstet. Gynecol. Reprod. Biol. 1990, 38 (1), 19-24. Vutyavanich, T.; Kraisarin, T.; Ruangsri, R.A. Ginger for nausea and vomiting in pregnancy: randomized double-masked placebo-controlled

Anaesth. Intens. Care 1995, 23 (4), 449-452. Bone, M.E.; Wilkinson, D.J.; Young, J.R.; McNeil, J.; Charlton, S. Ginger root—a new

antiemetic. The effect of ginger root on postoperative nausea and vomiting after major gynaecological surgery. Anaesthesia 1990, 45 (8),

Riebenfeld, D.; Borzone, L. Randomized, double-

a ginger extract on pregnancy-induced nausea: a randomised controlled trial. Aust. N. Z. J. Obstet. Gynaecol. 2003, 43 (2), 139-144. Zee Pace, J.C. Oral ingestion of encapsulated ginger and reported self-care actions for the relief of chemotherapy-associated nausea and vomiting.

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Schmid, R.; Schick, T.; Steffen, R.; Tschopp, A.; Wilk, T. Comparison of seven commonly used

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fenugreek (Trigonella foenumgraecum L.) on blood lipids, blood sugar and platelet aggregation in patients with coronary artery disease. Prostaglandins Leukot. Essent. Fatty Acids 1997, 56 (5), 379-384. Lumb, A.B. Effect of dried ginger on human platelet function. Thromb. Haemost 1994, 77, 110-111. Yoshikawa, M.; Yamaguchi, S.; Kunimi, Matsuda, H.; Okuno, Y.; Yamahara,

K.; J.;

Murakami, N. Stomachic principles in ginger. III. An anti-ulcer principle, 6-gingesulfonic acid, and three monoacyldigalactosylglycerols, gingerglycolipids A, B, and C, from Zingiberis Rhizoma originating in Taiwan. Chem. Pharm. Bull. 1994, 42 (6), 1226-1230.

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al-Yahya, M.A.; Rafatullah, S.; Mossa, J.S.; Ageel, A.M.; Parmar, N.S.; Tariq, M. Gastro-

protective activity of ginger Zingiber officinale Rosc., in albino rats. Am. J. Chin. Med. 1989, 17 (1-2), 51-56. Yamahara, J.; Miki, K.; Chisaka, T. et al. Cholagogic effect of ginger and its active constituents. J. Ethnopharmacol. 1985, 73 (2), 217-225. Mahady, G.B.; Pendland, S.L.; Yun, G.S.; Lu,

Z.Z.;

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American Botanical Council/Integrative Medicine Communications: Austin, TX/Newton, MA, 2000;

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153-159. Weidner, M.S.; Sigwart, K. The safety of ginger , extract in the rat. J. Ethnopharmacol. 2000, 73 (3), 513-520. Vaes, L.P.; Chyka, P.A. Interactions of warfarin with garlic, ginger, ginkgo, or ginseng: nature of

the evidence. Ann. Pharmacother. 2000, 34 (12), 1478-1482. 48.

Mascolo, N.; Jain, R.; Jain, S.C. et al. Ethnophar-

macologic investigation of ginger (Zingiber officinale). J. Ethnopharmacol. 1989, 27 (1-2), 129-140.

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Toxicol. 2000, 74, 507-512. Weidner, M.S.; Sigwart, K. Investigation

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teratogenic potential of a Zingiber officinale extract in the rat. Reprod. Toxicol. 2001, 15, 75-80.

Ginkgo biloba Kristian Stremgaard Stine B. Vogensen The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark

Koji Nakanishi Columbia University, New York, New York, U.S.A.

INTRODUCTION The tree Ginkgo biloba L. has a long history of use in traditional Chinese medicine. The ginkgo tree is classified in its own family and order, and holds a special position in evolutionary plant history because it provides a connection between the seedless vascular plants and seed plants. In recent years, the leaf extract of G. biloba has become one of the most widely used herbal remedies and is sold as a phytomedicine in Europe and as a dietary supplement worldwide. G. biloba extracts are used for the treatment of cerebral dysfunction and circulatory disorders, and have been studied in several animal experiments and clinical trials. There are a wide variety of chemical constituents in the extract, with the principal components being terpene trilactones (ginkgolides and bilobalide) and flavonoids.

G. biloba L., or the maidenhair tree (Fig. 1), is the only surviving member of its family (Ginkgoaceae) and order (Ginkgoales), underscoring its unique phylogenetic status. Fossil records show that the Ginkgo genus was present some 180 million years ago. The Ginkgoaceae peaked 130 million years ago, with numerous widespread species, but gradually gave way to modern angiosperms. Today, only one species, G. biloba, survives, and it occurs naturally only in

Stromgaard,

between | and 4m, and can reach an age of more than

1000 yr. Vertical growth generally slows down with the onset of sexual maturity at around 25yr. The appearance of the tree varies from slim and conical to full and rounded, with gray bark deeply furrowed on old trees. The ginkgo tree has characteristic green, leathery, fan-shaped leaves that turn goldenyellow in autumn. In young specimens, the leaves are divided into two distinct lobes, and hence the notation biloba (from Latin bi, double, Joba, lobes).

BACKGROUND

Kristian

eastern parts of China. The morphology of the ginkgo tree itself appears to have changed very little over 100 million years, and for this reason it is often called a “living fossil.’ The ginkgo tree takes its name from ginkyo in Japanese and yinhsing in Chinese; both words translate to “‘silver apricot,’’ referring to the appearance of the ginkgo nuts. The term “‘ginkgo’’ was first used by the German physician and botanist Engelbert Kaempfer in 1712, but Linnaeus provided the terminology “Ginkgo biloba’ in 1771. The ginkgo tree can grow up to 40m high, with a stem diameter

Ph.D.,

is Associate

Professor

at

the

Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark. Stine B. Vogensen, Ph.D., is Currently a Medicinal Chemist at MedChem Aps, Copenhagen, Denmark. at the Professor Ph.D., is Centennial Koji Nakanishi, York, New University, Columbia Chemistry, Department of New York, U.S.A.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022107 Copyright © 2005 by Marcel Dekker. All rights reserved.

The Ginkgo species is dioecious, having separate male and female trees. Among seed plants with a reproductive system, the ginkgo is a primitive tree, and its reproductive organs resemble those of seedless vascular plants such as ferns. G. biloba provides an important evolutionary connection between seedless vascular plants and seed plants, as discovered by Japanese botanist Sakugoro Hirase more than a hundred years ago. In the spring, before the leaves emerge, male G. biloba trees produce catkins rich in pollen, while female trees produce 2-3 mm long ovules. Each ovule secretes a small mucilaginous droplet that catches the airborne pollen and transports it inside the ovule, where multiflagellated spermatozoids are produced. A spermatozoid fertilizes the female egg cell, and the seed is shed from the tree approximately Imo after fertilization. Fully mature ginkgo seeds, also known as ginkgo nuts, have a pungent smell due to the presence of butanoic and hexanoic acids in the fleshy sarcotesta surrounding the

seed (Fig. 2).!! 249

Ginkgo biloba

250

The ginkgo tree can persist in conditions of low light and nutrient scarcity and is highly resistant to bacteria, fungi, and viruses. Furthermore, it is resistant to air

pollution; this has made G. biloba a popular roadside tree in urban areas of Japan, Europe, and northern

America. In China, the ginkgo tree is cultivated partly to meet demands for ginkgo nuts, a delicacy in Chinese and Japanese cuisine alike. The kernel is obtained by boiling the nuts until the hard shell cracks open; this kernel is subsequently boiled with sugar or roasted. Unfortunately, raw ginkgo nuts contain the toxin — 4-O-methylpyridoxine, which can result in serious food poisoning.

Ginkgo biloba Extract (GBE) For pharmaceutical purposes, an extract of G. biloba leaves was first introduced in Western countries in 1965 by the German company Dr. Willmar Schwabeunder

the

trade

name

Tebonin.

Later,

Schwabe

established a collaboration with the French company Beaufour-Ipsen, and together they developed a standardized G. biloba extract (GBE) termed EGb 761 (Extrait de Ginkgo biloba 761), which was sold under Fig. 1 A Ginkgo biloba tree. (View this art in color at www.dekker.com. )

Cultivation and History of Use

trade

names

such

as

Tanakan,

R6kan,

and

Tebonin forte. Other G. biloba products have entered the markets, and GBE is now among the best selling dietary supplements worldwide. Rising demand for GBE has spurred the increased harvesting of G. biloba leaves, and today, more than 50 million G. biloba trees are grown, especially in China, France, and the United

The ginkgo tree has been cultivated in China for several thousand years and, according to some of the earliest written ginkgo tree references dating back to the Song dynasty of the early 11th century, the tree was appreciated for its beauty and for its edible nuts. The tree was introduced into Japan from China in the 12th century, and some 500 years later into Europe and North America. The use of G. biloba for medicinal purposes was first mentioned in 1505 A.D. in a book by Liu Wan-Tai. In the Chinese materia medica Pen Tsao Ching from 1578, G. biloba is described as a treatment

for senility in aging members

of the royal court.

States, producing approximately 8000tons of dried leaves each year. The yellow or green leaves are harvested in mid- to late summer and then dried and pulverized. Through various extraction procedures, the active constituents are concentrated and undesired constituents such as organic acids are discarded. The composition of the leaf extract varies considerably and is related to the age of the plant, growth conditions, and time of harvest. To ensure the quality of GBE, the concentration of flavonoids and terpene trilactones, the presumed active constituents, has been standardized.

In these old records, it is mainly the use of nuts that

is described. Raw ginkgo nuts without the fleshy sarcotesta are described in traditional Chinese medicine as a treatment for a variety of lung-related ailments, including

asthma

and

bronchitis,

as

well

as

some

CHEMISTRY AND PREPARATION OF PRODUCT

kidney and bladder disorders. The use of G. biloba leaves has played only a minor role in traditional

G. biloba contains a wide variety of phytochemicals,

Chinese medicine, but in the modern Chinese Pharma-

including alkanes, lipids, sterols, benzenoids, caroten-

copeia, the leaves are considered heart and lungs.

beneficial for the

oids, phenylpropanoids, carbohydrates, flavonoids, and terpenoids, particularly terpene trilactones.7!

Ginkgo biloba

251

Fig. 2

Leaves and nuts of G. biloba. (View

this art in color at www.dekker.com. )

The major constituents are flavonoids, polyphenolic compounds that are widely distributed in the plant kingdom and are found in all green plants. Flavonoids are pigments responsible for the colors yellow, orange,

biflavonoids, such as bilobetin, ginkgetin, and isoginkgetin (Fig. 3) and proanthocyanidins such as procyanidin and prodelphinidin have also been isolated from G. biloba.

;

and

The terpene trilactones (TTLs) comprise 5 diter-

are also present in wine and tea. Currently, more than

penes named ginkgolide A, B, C, J, and M and the ses-

30 flavonoids have been found in G. biloba; the diver-

quiterpene bilobalide (Fig. 4). These compounds have unique structures only found in the ginkgo tree. The ginkgolide cage structure consists of six 5-membered rings, including 3 lactones, a tetrahydrofuran ring, and a spiro[4.4Jnonane skeleton. The ginkgolides differ in the positions and numbers of hydroxyl groups on the spirononane framework. In bilobalide, rings A

and red in autumn

leaves

and various

flowers,

sity arises from different glycoside substitutions of the flavonol aglycone. The flavonoids of GBE are almost exclusively flavonol-O-acyl-glycosides, including mono-, di-, or triglycosides of the flavonol aglycones quercetin and kaempferol primarily substituted at the 3-position (Fig. 3). Additionally, nonglycosidic

R3

R

kaempferol quercetin isorhamnetin

H OH © OCH3

amentoflavone bilobetin ginkgetin isoginkgetin

OH OCH3 OCH3 OCH3

Ro

Rs

OH OH OCH3

OH OH OH

OH

OCHs

Fig. 3. Structures of flavonoids.

Ginkgo biloba

252

O

R3 ginkgolide A (GA)

H

ginkgolide B (GB)

OH

ginkgolide C (GC) ginkgolide J (GJ) ginkgolide M(GM)

OH H OH

Fig. 4

H leh OH OH OH

O

OH

Bilobalide (BB)

OH Ory OH OH H

extracts.

PRECLINICAL STUDIES

Structures of ginkgolides and bilobalide.

and F are absent and the tetrahydrofuran ring of the ginkgolides (ring D) is replaced by a lactone (Fig. 4). The ratios of TTLs vary by season and between different parts of the tree. The TTLs are unique constituents of the ginkgo tree and have attracted great interest due to their complex structures and reported biological activities.“! Ginkgolides were first isolated from the root bark of G. biloba by S. Furukawa in 1932, and their structures were elucidated in 1967. The structure of bilobalide was determined in 1972. The ginkgolides have inspired many studies, particularly as targets for complex total synthesis, as templates for structure— activity relationship studies, and as terpenes with a surprising and novel biosynthetic pathway. Prior to these studies, it was thought that all terpenes were biosynthesized through the mevalonate pathway, but by examining the biosynthesis of ginkgolides, Arigoni and coworkers proved that terpenes can be synthesized through the deoxyxylulose phosphate or non-mevalonate pathway."!

Formulation and Analysis

A vast number of preclinical studies have investigated the in vitro and in vivo effects of G. biloba extract as well as the individual components of GBE, particularly the TTLs ginkgolides and bilobalide. The major components of all GBEs are flavonoids and TTEs, and it is believed that these 2 classes of compounds are responsible for the biological effects of GBE. In most cases, GBE has been investigated in in vitro or in vivo assays, or the extracts have been screened in

DNA arrays. The other primary approach has been to investigate single chemical components, such as the flavonoids, ginkgolides, and bilobalide, in a wide variety of assays. Although many biological effects can be explained by the individual components, it has often been suggested that GBE acts by a synergistic mechanism. The effects of GBE can be grouped into 3 related categories: 1) effects related to antioxidant activity; 2) effects on gene expression; and 3) direct effect on protein function, with these effects sometimes

being overlapping or related. The antioxidant effects of GBE are well documented, particularly using in vitro studies. These effects are most likely due to the flavonoids, which are well-known free-radical scavengers and antioxidants. Specifically, it has been shown that GBE can scavenge nitric oxide (NO), protect against lipid peroxidation of low-density lipoproteins (LDL), and inhibit the formation of oxygen radicals. It is believed that numerous disease states are related to free radicals. Therefore, it has been speculated that the antioxidant

Standardized extracts (dry extracts from dried leaves, extracted with acetone and water) contain 22-27% flavone glycosides and 5—7% terpene lactones, of which approximately 2.8-3.4% is ginkgolides A, B, and C and 2.6-3.2%

of terpene trilactones (ginkgolides and bilobalide) is by HPLC or gas-liquid chromatography. Certain commercial products such as EGb 761 do not contain biflavones, and the level of ginkgolic acids should be below 5mg/kg, because of their allergenic potential. Coated tablets and solutions for oral administration are prepared from these standardized, purified

is bilobalide.

Qualitative

and quantitative determination of flavonoid glycosides is carried out after hydrolysis to the aglycones kaempferol, quercetin, and isorhamnetin. The qualitative presence or absence of biflavones is determined by high-performance liquid chromatography (HPLC). Qualitative and quantitative determination

effects of GBE could be used to treat diseases such as atherosclerosis and cancer, as well as a number of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The effects of GBE on gene expression have been the subject of several investigations in recent years, and a general consensus has emerged that this effect is critically important when looking at the clinical effects of GBE.©! Gohil and coworkers used high-density oligonucleotide microarrays to study neuromodulatory effects in mice that had EGb 761supplemented diets.'©! Twelve thousand genes and

Ginkgo biloba

253

expressed gene tags from the hippocampus and cerebral cortex of the mice were analyzed for changes in gene expression, and of these, 10 were found to change more than threefold as a result of EGb 761 administration. Several of these 10 genes may be relevant to neurodegenerative disorders. In another approach, the expression of peripheral benzodiazepine receptor (PBR), a protein involved in cholesterol transport and many other biological events,

was

studied.)

It was

shown

that

GBE

treatment decreased expression of PBR in a timeand concentration-dependent manner, and that this effect is most likely due to the ginkgolides. The clinical relevance of this result is not entirely clear, but it might be related to cancer or neurodegeneration.®! Most clinical studies of GBE have looked at effects on various forms of neurodegenerative disease, particularly as potential treatment of Alzheimer’s disease (AD). Together with the accumulation of intracellular neurofibrillary tangles, deposition of B-amyloid plaques is the primary indication for AD, and it is believed that an increase of B-amyloid plaques is

present in GBE are almost entirely flavonol-glycosides, it is expected that these compounds or their metabolites play a key role in these events; however, as mentioned above, their bioavailability might be a limiting factor. The number of studies on the biological effects of ginkgolides increased dramatically in 1985, when it was reported that ginkgolides, particularly ginkgolide B (GB), are antagonists of the plateletactivating factor (PAF) receptor. The clinical application of GB (BN 52021) as a PAF receptor antagonist was investigated, but, as is true of all other antagonists of the PAF receptor, GB was never registered as a drug, primarily due to a failure to demonstrate

efficacy. The

clinical studies, however,

showed that GB was well tolerated and showed very few, if any, side effects. A large number of ginkgolide derivatives that have been prepared and tested for their ability to antagonize the PAF receptor and several derivatives showed increased potency in comparison to the native ginkgolides. Together,

these studies have led to a clearer under-

they act as antioxidants, free-radical scavengers, enzyme inhibitors, and cation chelators. They also show anti-inflammatory, antiallergic, anti-ischemic,

standing of the structural features required for PAF receptor antagonism.!?! Recently, it was found that ginkgolides are potent and selective antagonists of glycine (Gly) receptors. The Gly receptors are found primarily in the spinal cord and brain stem, but also in higher brain regions such as the hippocampus. They are, together with y-aminobutyric acid (GABA4g) receptors, the main inhibitory receptors in the CNS. Electrophysiological studies showed that GB antagonizes Gly receptors in neocortical slices!''! and hippocampal cells,!] and suggested that GB binds to the central pore of the ion channel, acting as a noncompetitive antagonist. Molecular modeling studies showed a striking structural similarity between picrotoxinin, an antagonist of both GABA, and Gly receptors, and ginkgolides."'! Thus, ginkgolides are highly useful pharmacological tools for studying the function and properties of Gly receptors. However, the physiological importance of this antagonism remains to be investigated. Several studies have shown that ginkgolides, particularly ginkgolide A (GA) and GB can modulate peripheral benzodiazepine (PB) receptors. These receptors are located mainly in peripheral tissues and glial cells in the brain, and are distinct from the benzodiazepine site on GABAa receptors. PB receptors are typically located on the outer membranes of mitochondria. The function

immunomodulatory, and antitumoral action"! The pharmacological effects of flavonoids in GBE have mainly been attributed to their utility as antioxidants and free-radical scavengers. Since the flavonoids

steroidogenesis, cell proliferation, and stress and anxiety disorders has been suggested. The primary action of GB is the inhibition of the expression of PB receptors.!"7!

central

to

the

pathogenesis

of AD.

Therefore,

the

recent finding that GBE seems to inhibit B-amyloid aggregation may be significant in explaining the effects of GBE in relation to AD.! The two major components of GBE are flavonoids and TTLs, and in contrast to the wealth of studies that have been performed using GBEs, far fewer investigations have looked at the effect of the individual components of these extracts. However, flavonoids and TTLs are thought to be responsible for most of the pharmacological properties of GBEs. An important consideration when looking at the effects on the CNS is the bioavailability, including penetration of the blood-brain barrier (BBB), of these components. It has been assumed that the bioavailability of flavonoids is low, whereas TTLs, in particular the ginkgolides, are nearly completely bioavailable. Very recent studies indicate that ginkgolides can penetrate the BBB, although only in limited amounts. Although such studies cannot predict which of the components of GBE are efficacious, bioavailability is obviously a critical parameter when evaluating the physiological effects

of GBE. Flavonoids possess many biological activities, and

of PB receptors is not entirely clear, but involvement in

Ginkgo biloba

254

Several studies have indicated that ginkgolides protect against various damaging CNS events, such as ischemia and other cerebrovascular and traumatic brain injury,

as well as inflammation. The mechanisms behind these effects are not entirely clear, and are probably multifaceted. Bilobalide (BB) is the predominant TTL found in GBE, and although no specific target has been established or pursued, a wealth of pharmacological evidence indicates that BB might be a very important compound when looking at neuromodulatory properties of G. biloba constituents. Several studies have shown that BB affects the major neurotransmitters

in the brain,

glutamate and GABA. Recently, it was demonstrated that BB is an antagonist of GABA, receptors; in neocortical rat brain slices, BB was a weak antagonist oB)yY2. (ICso = 46 uM), "1°! and at recombinant potent reasonably was BB GABA, receptors,

(ICsq = 4.6M)."41 Since antagonists of inhibitory receptors, particularly GABA, receptors, are known convulsants, the result of BB acting on GABAg receptors could pose a risk to patients ingesting GBE. In confirmation, a study of two epileptic patients showed an increased frequency of seizures with GBE administration. This increase was reversed when the patients stopped taking the extract.!’°! These results indicate that people with a low seizure threshold, such as epileptic patients, should be cautious when taking G. biloba

doses of 120-240mg used. Generally,

clinical

EGb

761 were most commonly

studies

have

shown

that

GBE can lead to an improvement in the symptoms associated with cerebral insufficiency, such as memory

loss, depression, and tinnitus. In Germany, GBE is registered as an herbal medicine to treat cerebral insufficiency. This is a diagnosis covering a range of conditions, as illustrated.by the list of indications

E: “disturbed , Commission the German from performance in organic brain syndrome within the regimen of a therapeutic concept in cases of demential syndromes with the following principal symptoms: disturbances deficits, memory depressive emotional condition,

in concentration, dizziness, tinnitus,

and headache. The primary target groups are dementia syndromes, including primary degenerative dementia, vascular dementia, and mixed forms of both724yip In two seminal clinical studies, a total of 549 AD

patients were evaluated for effects of EGb 761 treat-

ment.!'7!8] In both studies, EGb

761 significantly

slowed the loss of cognitive symptoms of dementia, and regression on certain data points was delayed by 7.8 mo, which is comparable to the currently available AD treatments, Aricept (donepezil, 9.5 mo) and Exelon

(rivastigmine, 5.5mo), both acetylcholinesterase inhibitors. Kleijnen and Knipschild reviewed 40 GBE clinical studies, which examined the efficacy of GBE in cere-

extract.

bral insufficiency.""*! In the studies, the standard dose

In contrast to these findings, other studies have shown the potential neuroprotective effect of BB in reducing glutamate release and phospholipid breakdown. Potential medicinal applications of BB have been described in patents, including use of BB for the protection of neurons from ischemia, as an anticonvul-

was 120 mg/day for at least 4—6 weeks. Of the 40 trials, only 8 were considered acceptable. The problems with many of the studies included small patient numbers, inadequate description of randomization procedures, inadequate patient characterization, and insufficient data presentation. Essentially, all the 8 trials reported positive results, and no serious side effects were reported. It was concluded that future studies could provide a detailed efficacy assessment of GBE treatment. A more recent review by Knipschild and colleagues summarized 55 additional clinical studies, which also looked at the effect of GBE on cerebral insufficiency. Knipschild reports that although there is good

sant, and for treatment

of tension

and anxiety.

BB

inhibits brain phospholipase A> activity, leading to a neuroprotective effect, and several studies have shown

that BB preserves mitochondrial respiration, especially under ischemic conditions.

evidence for a GBE effect, this evidence was obtained

CLINICAL STUDIES A vast number of clinical trials have been conducted using

GBEs

and,

in most

cases,

these

trials

have

examined effects related to dementia. Specifically, changes in memory, thinking, and personality in aging people were studied. In almost all of these investigations, the standardized extract EGb 761 was administered and,

although various dosing regimens were employed, daily

from an excessively small patient population and further, larger trials are required.?°! A meta-analysis systematically reviewed over 50 clinical studies on GBE for the treatment of dementia and cognitive malfunctions associated with AD. Only 4 of the 50 studies met the inclusion criteria for the evaluation; these 4 studies included more than 400 patients. It was concluded that administration of 120-240 mg of GBE for 3-6 mo had a small but significant effect on objective measures of cognitive function

Ginkgo biloba

255

in AD, without significant adverse effects in formal clinical trials.P4 Recently, two clinical studies have cast doubt on the positive clinical effect of GBE seen in almost all previous investigations. Both of these new studies include a larger number of patients, are carefully

in evaluating and determining the effects of GBE in relation to dementia. A primary function of GBE is to improve blood flow and to inhibit platelet aggregation, the latter through inhibition of the PAF receptor by ginkgolides.

designed,

naturally invite examination, and a number of clinical trials have looked at effects of GBE treatment in relation to peripheral vascular conditions. A meta-analysis of clinical trials investigating the effect of GBE on intermittent claudication, an early symptom of periph-

and

are

randomized,

double-blind

and

placebo-controlled. Knipschild et al. completed a clinical trail with 214 patients, who received GBE for 24 weeks. The patients suffered from either dementia or age-associated memory impairment (AAMI), and

no GBE treatment-related improvement was seen.?7!

Therefore, the vascular effects of GBE administration

In another study with 203 people over 60 yr who were

eral arterial disease, was carried out by Pittler and Ernst.?°! Of 12 clinical trials conducted, 8 were

given GBE for 6 weeks, no beneficial effect from the GBE treatment was observed"; the results of this

included in this analysis, and the authors concluded that GBE is superior to placebo in the symptomatic

investigation have been heavily debated. In a very recent evaluation from the Cochrane Library, Birks and Evans have critically reviewed 33 clinical studies, which were all randomized and doubleblind.?! The duration of the studies varied from 3 to 52 weeks, although the majority were conducted for 12 weeks. The participants were all diagnosed with

treatment of intermittent claudication. However, it was noted that the overall magnitude of the treatment effect was modest and its clinical relevance was uncertain.

either dementia or AAMI,

The effect of GBE on healthy people has also been examined. Several clinical studies indicate improved cognitive functions. This effect is still con-

although some of the older

troversial, as illustrated by a recent evaluation of the

studies did not fully verify the diagnoses. In their conclusion, Birks and Evans state that GBE appears

clinical trials studying GBE efficacy in healthy persons.?°! Canter and Ernst found nine placebo-

to be safe and with no

controlled, double-blind

side effects compared

to

placebo, and that there is promising evidence for improved cognition and function with GBE treatment. However, the authors also note the results of recent trials that did not show

GBE-related

improvement,

and therefore suggest that further clinical trials are required. Currently, at least two major clinical trials are ongoing. The U.S. National Institutes of Health 1s sponsoring the Ginkgo Evaluation of Memory (GEM) Study, which has enrolled more than 3000 elderly people from four medical centers in the United States. The goal is to find out whether medicine made from G. biloba can prevent or delay the changes in memory, thinking, and personality that can occur as people get older. Doctors refer to these changes as ‘“dementia,’’ the most well known type being Alzheimer’s disease. Half of the patients are taking pills that contain Ginkgo biloba, and the other half are on a placebo. After Syr, when the study has been completed, the two groups will be compared to see whether there are differences in how memory, thinking, and personality have changed, and to see whether G. biloba has been effective in preventing these changes. In France, the pharmaceutical company Ipsen is sponsoring another clinical trial, the GuidAge study, which aims to examine prevention of Alzheimer’s disease in patients over the age of 70 with memory impairment. The results from these two studies will obviously be of major importance

trials, which were generally

of acceptable methodological quality. None of these short-term (!

cancer, ''*! cardiovascular diseases,!'”] and sexual func-

American ginseng is a _ slow-growing perennial, aromatic, shade-loving herb, 60-80 cm tall, belonging to the Araliaceae family. It has a bifurcated root with compound, verticillated, oval-to-oblong leaves. Indigenous to North America, American ginseng was first

tion.!"*! It may help reduce the healing period required in patients with chronic bronchitis who are taking antibiotic drugs,'!*! and maintain blood sugar levels in diabetic patients.?°!

discovered in Quebec, Canada,"! by Father Lafitau in

the early 18th century, and has generated a lot of interest, particularly from China ever since.?*! Canada is one of the major producers in the world, with an annual yield of approximately 5 million pounds of 4-yr-old ginseng root (personal communication from grower’s associations). Besides having adaptogenic effects for increasing resistance to environmental stress, ginseng also has tonic, stimulant, and diuretic effects. Recently, cultivation of American ginseng in China has progressed rapidly due to the public demand for its yin (cool) characteristics instead of the yang (hot) of Asian ginseng.*! Among ginseng species, it has comparable,

but different, ginsenoside

content, which may account for the different therapeutical effects.'°”1 American ginseng has shown

Thomas, S.C. Li, Ph.D., is a Research Scientist at Agriculture and Agri-Food Canada, Pacific Agri-Food Summerland, British Columbia, Canada.

Research

Center,

Encyclopedia of Dietary Supplements DOL: 10.1081 /E-EDS-120024879 Copyright © 2005 by Marcel Dekker. All rights reserved.

CHEMISTRY AND PREPARATION

OF PRODUCT

There is considerable literature on the chemical compounds of ginseng.”'! However, the major active constituents of American ginseng are now generally accepted to be dammarane saponins, commonly referred to as ginsenosides Ra, Rb;, Rb», Rc, Rd, Re,

Rf, Rg;, Rgo, Rg3, Rhy, and Rh,.'7! A total of 28 ginsenosides have been extracted from ginseng roots.*! Six

major

ginsenosides

(Rb;,

Rbz,

Re,

Rd,

Re,

and Rg) are present, which are widely regarded as the chemical constituents mainly responsible for its bioactivity.“ Individual and total ginsenoside contents vary substantially among Panax species. 4 Soldati and Sticher?*! reported a total of 1.71% ginsenosides (Rb;, Rbo, Re, Rd, Re, Rf, Rgi, and Rg>) in the roots of American ginseng (P. quinquefolium). Ma et al.241 found that total ginsenosides (Rb, Rb2, Rc, Rd, Re, 259

Ginseng, American (Panax quinquefolium)

260

Rf, and Rg;) extracted from P. quinquefolium root ranged from 1.26% to 8.60%. These compounds also exist in leaves?’?*! and berries (Li, unpublished data). In addition to saponins, ginseng contains a high-molecular-weight polysaccharide and many other phytochemicals, including phytosterols, oils, acids, carbohydrates,

flavonoids,

nitrogen-containing

com-

pounds, vitamins A and Bj», and inorganics.??7"! There are many chemical components, ranging from organic acids, such as maltol (3-hydroxyl-2-methyl-r-pyrone), to glucose, sodium, magnesium, peptidoglycans, and volatile oils. It was reported that new C,7- and C;s-polyacetylenes and two known polyacetylenes, ginsenoyne G and acetylpanxydol, were isolated from dry ginseng roots.°!*"] Recently, phytosterols were extracted from American ginseng seed oil,??! which can be used in the

manufacture of dietary supplements, or as supplemental ingredients in foods. These sterols can be used to reduce serum cholesterol and low-density-lipid cholesterol levels in normal and mildly hypercholesteremic subjects.)

NUTRITIONAL AND MEDICINAL VALUES American ginseng is a much favored herb, used regularly by millions of healthy people to stimulate and maintain energy levels and achieve a sense of wellbeing. It was reported that ginseng may be able to help maintain metabolic balance by eliciting a homeostatic effect,**! which may not be effective in the absence of stress, but can return

the body processes

Ginseng is able to lower cholesterol levels in the blood by stimulating either cholesterol transport or an enzyme involved in cholesterol metabolism.°*! The decrease may also be due to an increased conversion of cholesterol into bile acids and/or the direct excretion of cholesterol.4°! The effects of saponins and monomer saponins from American ginseng on lipid metabolism were eval- , uated recently. It was found that at 0.5 mg/L, saponins

inhibited the activity of pancreatic lipase by 90%, while at 0.5mg/ml, ginsenosides Rc, Rb, and Rb» prevented

it by 100%, 96%, and 97%, respectively. This finding suggests the potential use of saponins as drugs for antiobesity.4"! In an evaluation of antihyperglycemic effects of American ginseng berry extracts in diabetic ob/ob mice, Xie et al.'**) reported that the extract may prove to be of clinical importance in the prevention and treatment of Type 2 diabetes. Reductions in. fasting blood glucose and prolonged elevation of postprandial glycemia (PPG) are important in blood sugar control following meals. Vuksan et al.?°! reported that in a placebo study of 10 men and women with Type 2 diabetes, the participants were randomly administered 0, 3, 6, or

9g of American ginseng root powder 120, 80,

40, or Omin before a 25g oral glucose challenge. They concluded that 3g of ginseng powder administered within 2 hr of the challenge may be sufficient to achieve reductions of PPG in Type 2 diabetic individuals.

Effect on Memory (Preclinical)

to normal

when there is stress or damage irrespective of the source.?°! Yuan and Dey®* conducted a study on the multiple clinical effects of American ginseng on patients with stomach cramps, loss of appetite and weight, hypertension, insomnia, and irregular menstrual cycles. They found that all patients were cured after taking a daily dose of 2g of American ginseng for 2 weeks.

Antioxidant Activity (Preclinical)

Extracts of ginsenoside have demonstrated the antioxidant properties of American ginseng. The extracts have been reported to exhibit effective antioxidant activity in lipid and aqueous media by both chelation of metal ions and scavenging of free radicals,!37! Ginseng extracts, specifically Rb, and Rb», protected human low-density lipoprotein against cupric ionmediated oxidation. Similar activity was observed against peroxyl radical-induced supercoiled DNA

breakage.8!

Effect on Metabolism (Preclinical and Clinical)

It was reported that ginsenoside Rb; could improve memory deficits induced by anticholinergic drug treatment and facilitate acetylcholine release from rat brain hippocampal slices.'“*! This specific effect of Rb; on cholinergic functions may warrant its further study for enhancing short-term memory acquisition. and retention in senile dementia.! It was stated that many patients with amyotrophic lateral sclerosis used natural or traditional therapies of proven benefit. One such therapy is American ginseng root. It improved learning ability and memory in mice.°!

Effects on Stress and Fatigue (Preclinical) The effects of ginseng on stress and fatigue have been expounded by Chinese herbalists for millennia. It is well known for its antistress and adaptogenic properties, but only recently have these effects been scientifically examined. Ginseng is traditionally reputed to regularize bodily functions and relieve many ailments resulting from physiological stress. Beneficial effects are thought to be due to a nonspecific influence on

Ginseng, American (Panax quinquefolium)

261

production and use of regulatory hormones. Various compounds in ginseng have been shown to increase nonspecific resistance to physical, chemical, and biological stresses, and relieve fatigue.°! Some of the manifestations include a decrease in body temperature, relaxation of muscle tone, and analgesia.!'7) Awang!“! indicated that the pharmacological profile of American ginseng supported the treatment of stress-related conditions in humans. Effect on the Immune System (Preclinical) Ginseng saponin can act as an immune stimulant or a supportive agent,7! although the mechanisms by which these saponin compounds exert their immunostimulant effects are not well understood. It was reported that ginseng is able to act prophylactically as an anti-inflammatory agent in humans. Ginseng is also able to increase antibody levels, stimulate natural killer cells and the release of the chemical messenger, interferon, which can activate the immune system.'*®! An aqueous extract, mainly oligosaccharides and polysaccharides from American ginseng, showed immuno-

modulating activities in vitro, which may be used clinically for the modulation of immune responses in humans.*?!

Effect on Cancer (Preclinical and Clinical) Potentially significant research has demonstrated recently that ginseng has an inhibitory effect on cancer development. It was suggested that ginseng should be recognized as a functional food for cancer prevention"! In a case-control study at the Korea Cancer Center Hospital, Yun and Choi!'®! reported, without mentioning the mechanisms

involved, that the risk of

oral, pharyngeal, stomach, and liver cancers associated with smoking was reduced with ginseng intake. It is believed that ginseng exhibits a toxic effect against cancer cells by inhibiting the biosynthesis of macromolecules.!'7! In vitro use of American ginseng and the breast cancer therapeutic agent, estradiol, has been

reported to synergistically inhibit cancer cell growth.P°!

In a study of the antiproliferative activity of ginsenosides using human prostate carcinoma LNCaP cell line, ginsenoside Rg3 displayed growth inhibitory

activity.P!) Many in vitro studies have been conducted recently on the effects of saponins extracted from ginseng on

cancer cells,>7! and the results indicated that it can

inhibit cancer cell proliferation and invasion, usually

at 10-180 1M.©?! Animal studies have also been conducted

on

the antitumor

effect

of saponins,

which

can decrease tumor growth and metastasis and improve survival.>+*° Oral administration of crude

ginseng extracts, at 50-500mg/kg body weight, for 10 days significantly reduced tumor growth in melanoma- and sarcoma-bearing mice.” Jia et al.5* did an intracranial glioma rat model study with a specially formulated ginseng product Careseng®, containing over 80% of Rh, and Rhp-like ginsenosides, against breast, prostate, lung, pancreatic, and brain cancer cell lines in vitro. It was concluded that Careseng® has provocative and novel anticancer properties. A few studies have been conducted with humans using ginseng extracts in China,>?l

SAFETY AND POSSIBLE INTERACTION WITH PRESCRIPTION DRUGS Ginseng has been used by humans for decades, which is a good indication of the safety of this unique herb. Brekhman and Dardymov'®") reported that the LD5o for ginseng ranges from 10 to 30g/kg body weight in mice. Correct oral ingestion of American ginseng is considered safe.!©*°! Daily doses of 0.25-0.5g and 0.4-0.8 g of dried root were recommended for young and elderly people, respectively.! Higher levels have been associated with insomnia.'*! Siegel! suggested avoidance of large doses because of possible side effects,

e.g., morning

diarrhea,

skin

eruptions,

sleeplessness, nervousness, hypertension, euphoria, and edema.”'! There are no standards or guidelines set by the National Institute for Occupational Safety and Health (NIOSH or OSHA) for occupational exposure to, or workplace maximum

allowable levels of,

ginseng (http: //www-ntp-server.niehs,nih.gov/htdocs/ chem_Background/ExecSumm/Ginseng/) (accessed March 2004). Ginseng was not on the American Conference of Governmental Industrial Hygienists (ACGIH) list of compounds for which recommendations for a threshold limit value (TLV) or biological exposure index (BEI) are made. The Dietary Supplement Health and Education Act (DSHEA) requires no proof of safety for dietary supplements on the market prior to October 15, 1994, which includes ginseng, since its products have been available for over 30 yr

(http://www.quackwatch.org/02consumerProtection/ dshea.html) (accessed in March 2004). Interactions with Drugs American ginseng can lower blood glucose.!©*! Theoretically, concomitant use with antidiabetes drugs might enhance blood glucose lowering effects and possibly cause hypoglycemia. It was reported that American ginseng can interfere with antipsychotic and stimulant drugs, and hormone therapy,!°” monoamine oxidase inhibitors, and warfarin.'©8-°?!

Ginseng, American (Panax quinquefolium)

262

Taylor, D. Getting to the root of ginseng: questions about the herb’s health benefits haven’t cooled the red-hot market in wild American ginseng. Smithsonian 2002, 33, 1—5.

Interactions with Food

It has been shared that concomitant use of ginseng can potentiate the stimulant effect of coffee and tea [7° It may also be important to take ginseng with a meal to avoid a hypoglycemic reaction, especially in patients with Type 2 diabetes. !**!

Liu, C.X.; Xiao, P.G. Recent advances on ginseng

research in China. J. Ethnopharmacol. 27-38. Kim,

Y.R.;

Lee,

S.Y.;

Shin,

B.A.;

1992, 36,

Kim,

K.M.

Panax ginseng blocks morphine-induced thymic apoptosis by lowering plasma corticosterone

Interactions with Diseases or Conditions

level. Gen. Pharmacol. 1999, 32, 647-652. Assinewe, V.A.; Arnason, J.T.; Aubry, A.; Mullin, J.;

American ginseng has been reported to decrease blood coagulation and adversely affect cardiac conditions, 4) increase the risk of hypoglycemic episodes, ©"! and have estrogenic effects.!7"!

Lemaire, I. Extractable polysaccharides of Panax quinquefolius L. Phytomedicine 2002, 9, 398404. Lin, J.H.; Wu, L.S.; Tsai, K.T. Effects of ginseng

on the blood chemistry profile of dexamenthasone-

QUALITY CONTROL AND REGULATORY STATUS

treated male rats. Am.

Quality control of any herbal product is a very important step. This will build up consumer confidence and make the herbal industry sustainable. There are many ginseng products, such as whole or sliced roots, cap-

American market. Consumers have been purchasing ginseng in its various forms for health benefits. However, researchers have long suspected that some of the products claiming to contain ginseng may be misand/or adulterated.”7]

Le

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7a

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Ginseng, Asian (Panax ginseng) Fabio Soldati Pharmaton SA, Head of Research and Development, Bioggio, Switzerland

INTRODUCTION Panax ginseng is one of the most investigated medicinal plants and is cultivated in China and Korea. In the Eastern world, ginseng roots have been used for more than 2000 yr as a tonic and a prophylactic to combat psychophysical tiredness and asthenia. The plant contains more than 200 identified chemical compounds, and among them ginsenosides are considered some of the most pharmacologically important constituents and characteristic markers. The United States Pharmacopeia—National Formulary (USP-NF) describes its attributes. Consistent efficacy and safety require constantly uniform composition, a condition which the raw material (roots) can scarcely fulfill. Standardization is the prerequisite for a constant pharmacological answer. High-performance liquid chromatography (HPLC) methods were developed for the quantification of ginsenosides. Numerous pharmacological studies have been performed on P. ginseng roots and extracts. Among other activities, the following are relevant: free radical scavenging effect, immunological effects, action on the central nervous system, and metabolic effects. These studies show not only the activity but also demonstrate that there is a certain relation between dose and response. Moreover, some experiments reveal that by increasing the dose of the extracts or the ginsenosides beyond certain limits, the desired pharmacological effects are reversed. This could explain the sometimes controversial results observed for the P. ginseng activity. The ginsenosides are considered prodrugs, and in the acidic medium of the stomach are immediately hydrolyzed; further metabolism occurs in the intestine. In humans, the hydrolysis products were detected in plasma and urine after oral administration. Toxicological studies (acute, subacute, and chronic toxicity, teratogenic activity) performed in mice, rats, rabbits, pigs, and dogs, and mutagenicity tests (Ames, DNA-repair, mouse-bone micronucleus, chromosomal

aberration in human lymphocytes), as well as the safety assessment on the cardiovascular and hormone system,

Fabio Soldati, Ph.D., is at Pharmaton SA, Head of Research and Development, Bioggio, Switzerland.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022108 Copyright © 2005 by Marcel Dekker. All rights reserved.

demonstrate that a standardized P. ginseng extract has a very large therapeutic index. Clinical experiments have shown major activity, particularly in the central nervous system (cognitive function) and on the immune system (stimulation of natural killer (NK) cells, antibody title, and antiviral

activity), but the results of the metabolic effect on physical endurance are controversial. The therapeutic dose of the dried roots is 0.5-2.0g, and doses of extracts

should be calculated accordingly. No contraindications are known. Data from clinical trials suggest that the incidence of adverse side effects with P. ginseng preparations is rare. In animals, no effect on fetal development has been observed; but since no human data are available, ginseng should not be used during pregnancy or lactation. The indications of P. ginseng root and standardized extracts supported by clinical data are for the enhancement of mental and physical capacities, and increased resistance against infections, in cases of weakness, exhaustion, tiredness, loss of concentration, and during

convalescence. In traditional medicine, it is also used in the treatment of impotence and prevention of hepatotoxicity.

BACKGROUND Panax ginseng C.A. Meyer Vernacular names: Asian, Chinese, Korean, or Orien-

tal ginseng. P. ginseng C.A. Meyer; Chinese, Korean, Asian, or Oriental ginseng. This species is a member of the plant family Araliaceae and is widely cultivated in China and Korea. Methods of cultivation, botanical

characteristics, and authentication of this medicinal plant have been extensively described.!'-7)

The Panax ginseng Root The ginseng root (described in detail in the USP-NF monograph) is among the most important traditional Chinese medicines. It has been used in China since antiquity to combat fatique and weakness. The earliest known mention of ginseng in Europe goes back to 1711 when a Jesuit, Father Jartoux, who worked in Chinese missions, sent a letter to the general procurator in Paris, 265

Ginseng, Asian (Panax ginseng)

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describing this plant, which he had never seen before, as having immense therapeutic properties. The ginseng plant then remained practically unknown to the scientific Western world until the early 1960s when researchers started to investigate its pharmacological and therapeutic properties. One of the major difficulties was the lack of demonstration of its healing power following protocols accepted by Western medicine, especially since ginseng is not considered to be part of it.4) Currently, more than 4600 publications on ginseng are referenced in the chemical abstracts. Radix ginseng has been used traditionally in China for the enhancement of mental and physical performance,

20-gluco-ginsenosides Rf, Rgi, Rgo, and Rh,. Ginsenoside Ro is a derivative of oleanolic acid. The most important of the ginsenosides are considered to be Rb;, Rb», Rc, Rd, Re, Rf, Rgi, and Rgp, with Rb;, Rb», Re, and Rg; being the most abundant. The total ginsenoside content of a 6-yr-old main root varies between 0.7% and 3%. The lateral roots can contain two to three times more saponins than the main root, while the slender roots can even contain up to

10 times more.) Formulation and Analysis

convalescence.*!

HPLC methods developed for the quantification of ginsenosides contained in the roots and in the finished

CHEMISTRY AND PREPARATION

poeia and European Pharmacopoeia for the definition of P. ginseng roots and P. ginseng extracts. Crude plant materials, powdered herbal drug, and .

in case of weakness, exhaustion, tiredness, and during

products! are also included in the U.S. PharmacoOF PRODUCTS

extracts

are used in tablets, hard and soft capsules,

Source Materials

effervescent tablets, lozenges, and liquid preparations. The development from a medicinal plant to a phyto-

The USP-NF gives a detailed description of the botanical and chemical characteristics as well as the purity criteria of the root in the monograph on Asian ginseng.)

medicine is a long process.

Proposed Active Constituents

So far, approximately 200 substances have been isolated and characterized from P. ginseng, both from primary and secondary metabolism of the plant. These include 30 ginsenosides, 40 ether-soluble compounds, eight nitrogen-containing compounds, 32 volatile compounds, 13 carbohydrates/polysaccharides, 9 glycans, 56 lipids and fatty acids, and 9 trace elements. The ginsenosides are characteristic substances of ginseng. They are commonly used as_ chromatographic “markers’’ in the assessment of chemical content and quality control of ginseng roots. They exert pharmacological activities [free radical scavengers and release of nitric oxide (NO)], but they are not solely responsible for the activities of P. ginseng, such as the immunological effects, free radical scavenging effects, action on the central nervous system, and metabolic effects.!! The ginsenosides are triterpene saponins of the dammarane type, which are derivatives of either protopanaxadiol or protopanaxatriol. Their structures were elucidated by Prof. S. Shibata and Prof. O. Tanaka at the Tokyo university and University of Hiroshima. Examples of protopanaxadiol saponins are ginsenosides Ra;, Raz, Ra3; Rb;, Rbo, Rb3; Re and Rd, and malonyl-ginsenosides Rb;, Rb2, Rc and Rd. Examples of protopanaxatriol saponins are ginsenosides Re, Rf;

In fact, in 1980, analysis

of the content of ginsenosides in different products on the market purported to contain roots of P. ginseng showed considerable variation from one batch to another of the same product, with none being detected in one product labeled as containing pure P. ginseng”! The variety of ginseng and the methods of cultivation are very important. There are various species of ginseng that, from a morphological point of view, are similar, but as far as their content of substances is concerned they are not identical. Also, the method of

preparation of the roots and their drying procedure can

differ considerably.

In the Far

East,

the roots

(which in nature are white) are sometimes treated with steam at about 100°C for differing times in order to protect them from microbiological pathogens. This high-temperature treatment results in the reddening of the epidermis of the roots producing the so-called “red ginseng.’’ Thus, the red ginseng is simply white ginseng, modified by heat treatment. Not only are the sugars in the epidermis of the root caramelized by the heating, but the ginsenosides are also partially chemically modified and/or destroyed. In addition, the distribution of the ginsenosides is different in the various parts of the plant, both in total content and

in the relative ratio of the different ginsenosides. In 1984, in cooperation with Prof. O. Tanaka from

the University of Hiroshima, we documented a relationship between the age of the plant and the content of ginsenosides. The major development of the roots occurs between the fourth and fifth year of growth, during which time the root doubles its weight, and the optimum yield of ginsenosides occurs at the fifth

Ginseng, Asian (Panax ginseng)

267

year.'®! China is the largest producer, but the average yield of ginseng per acre is only two-thirds of what could be reasonable expected, due almost entirely to diseases that attack the crop. Fungal diseases like

and the solvents employed, different extracts can be obtained, which can exert varying pharmacological effects.! Some of the relevant results from over 4600 published papers are discussed below.

Alternaria panax, Fusarium spp., Phytophtora cactorum, and Cylindrocarpon destructans can devas-

tate ginseng crops. A Syr growth span, of course, implies the use of insecticides and pesticides, but thanks to the implementation of controls in the country of origin, and information provided to the farmers (selective and rational use of pesticides), it is possible to lower the pesticide concentrations to safe levels. As mentioned above, the roots of wild P. ginseng are not uniform in their content of ginsenosides (markers and active ingredients of the plant) and the content

of pesticides can be too high in some

cases,

making them unsuitable for the production of pharmaceutical

products.

Therefore,

the

use

of cultivated

plants (with a rational use of pesticides) and standardized extraction methods is basic to ensuring the uniformity of an extract and thus its consistent pharmacological and toxicological effects. The USP gives a detailed description of the quality of a standardized Asian ginseng extract: “Powdered Asian Ginseng Extract is prepared from Asian Ginseng by maceration, percolation, or both processes performed at room temperature with suitable solvents such as alcohol, methanol, water, or mixtures of these

solvents, eratures material contains

and by concentrating the fluid extract at tempbelow 50°. The ratio of the starting crude plant to Powdered Extract is between 3: 1 and7: 1. It not less than 3.0 per cent of ginsenosides Rg},

Re, Rb;, Rc, Rb,

and Rd combined,

calculated

on

the anhydrous basis. It may contain other added substances.’’ Moreover, the USP gives detailed information on the methods for identification [thin layer chromatography (TLC)] and content determination (HPLC) of the ginsenosides as well as limits for microbial

contamination,

water,

pesticide

residuals,

heavy metals, organic volatile impurities, and alcohol

content. PRECLINICAL STUDIES Pharmacodynamic

Properties

Numerous pharmacological studies have been performed on P. ginseng extracts. Among other activities, the following are relevant: free radical scavenging effects, immunological

effects, action

on the central

nervous system, and metabolic effects. The exercise of such a range of pharmacological actions may be attributed to the fact that P. ginseng contains more than 200 different compounds that exert different activities. Depending on the method of extraction

Free Radical Scavenging Effects Studies with hearts from rats exposed to hyperbaric oxygen (HBO) and to HBO after treatment with 10mg/ml ginseng extract in their drinking water showed that ginseng prevents myocardial ischemia/ reperfusion damage and the impairment of endothelial functionality induced by reactive oxygen species arising from HBO exposure. These effects may be attributed to antioxidant intervention. In experiments with perfused rabbit lungs, the extract inhibited vasoconstriction induced by the thromboxane analog U46619 or by acetylcholine after exposure to free radicals generated by electrolysis. This effect appears to be due to the release of NO from the pulmonary endothelium. The extract had activities superior to the pure ginsenosides.!'° The extract of P. ginseng potentiated the relaxation induced by electrical stimulation or nicotine in monkey cerebral arterial strips denuded of the endothelium and partially contracted with prostaglandin F2alpha. The response to electrical stimulation was blocked by tetrodotoxin, whereas that to nicotine was suppressed by hexamethonium, while NG-nitro-L-arginine blocked both of these effects. Atropine, however, did not alter

the potentiating effect of P. ginseng extract, and relaxations induced by exogenous NO were unaffected. The enhancement of the neurogenic response appears to be associated with increments in the synthesis or release of NO from the perivascular nerve. Blockage of muscarinic prejunctional inhibition, superoxide scavenging action, and phosphodiesterase inhibition is not involved.!!!! A crude and a standardized P. ginseng extract of different saponin compositions were tested as to their efficacy in reducing lipid peroxidation, inflammation, and release of myocellular proteins after eccentric contraction exercise on a rat treadmill. Plasma creatine kinase (CK) levels were significantly reduced by approximately 25% after ingestion of both extracts. The two extracts reduced lipid peroxidation by approximately 15% as measured by malondialdehyde levels. Beta-glucuronidase concentrations and glucose6-phosphate dehydrogenase (G6PDH) levels, which can be considered

as markers

of inflammation,

were

also significantly decreased. The values of beta-glucuronidase were increased approximately 40% in vastus and in rectus muscles. The extracts appeared to be equally effective in reducing injuries and inflammation caused by eccentric muscle contractions.!!7!

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268

To clarify the relationship between the structures of ginsenosides and their properties, 11 individual ginsenosides, along with their core structures, protopanaxadiol, and protopanaxatriol, were used in 2,2’-azobis (2-amidinopropane hydrochloride) (AAPH)-induced hemolysis of human erythrocytes, a good experimental model

to determine

free radical-induced

membrane.

damage and to evaluate the antioxidative or pro-oxidative activities of various antioxidants.!'*! It was found that the core structures of ginsenosides, either protopanaxadiol or protopanaxatriol, play a pro-oxidative role in AAPH-induced hemolysis of erythrocytes. As to the individual ginsenosides, if there are no sugar moieties

minimum inhibitory concentration of 250pg/ml. Digestion of the fraction with pectinase resulted in a lower molecular weight oligosaccharide fraction, which

was noninhibitory at a concentration of 4mg/ml. " A high output nitric oxide synthase (iNOS) was shown in mice administered intraperitoneally with the acidic polysaccharide from ginseng. Newly synthesized iNOS protein was also observed in peritoneal macrophages cultured with interferon-gamma and the acidic polysaccharide. Spleen cells from acidic polysaccharide-treated mice did not proliferate in response to concanavalin

A, but responsiveness

was

restored

by the cotreatment of NG-monomethyl-L-arginine (NMMA) with concanavalin A. The treatment of mice

attached to the 20-position of the triterpene dammarane, the ginsenoside acts as a pro-oxidant, as with Rg;, Rhy, and Rg». A glucose attached to the 6-position instead of the 20-position sugar moieties can make the ginsenoside an antioxidant, as with Rh,. The antioxidants among ginsenosides follow two different mechanisms that can be expressed mathematically by the Boltzmann equation, as is the case with Rc and Rb;, and a polynomial equation, as with Re, Rd, Rg), Rb3, and Rh,. The orders of antioxidative ability are Rc > Rb; and Re > Rd > Rg, > Rb3 > Rh, respectively.!'3}

alleviated the acidic polysaccharide-induced suppression of antibody response to sheep red blood cells. Present results suggest that the immunomodulating activities of the acidic polysaccharide were mediated by the production of NO." Recently, it was demonstrated that P. ginseng extract and purified ginsenosides exert an adjuvant effect on the immune responses against porcine parvovirus, Erysiphelothrix rhusiopathie and against Staphylococcus aureus in dairy cattle.!'*!

Immunological Effects

Action of the Central Nervous System

The effect of oral administration of a standardized ginseng extract to mice for four consecutive days (10mg/day) on immune response was investigated. The extract enhanced antibody plaque forming cell response and circulating antibody titer against sheep erythrocytes. This finding was confirmed by oral administration of an extract with defined ginsenoside content to mice at doses of 10, 50, or 250mg/kg body weight daily for 5-6 days, which resulted in enhanced immune responses in a battery of six ex vivo tests including primary and secondary immune responses against sheep red cells, natural killing activity, mitogen-induced proliferation, interferon production, and T-cell-mediated cytotoxicity." A standardized extract from ginseng roots and several fractions of the extract were found to possess anticomplement and mitogenic activities in mice spleen cell cultures, with the strongest anticomplement activity being observed in the crude polysaccharide fraction. The polysaccharide with the major anticomplement activity consisted of arabinose, galactose,

A study in rats using learning and memory retention tests and determination of '*C-phenylalanine transport across the blood-brain barrier after oral administration of a standardized ginseng extract was undertaken." Learning and memory retention improved (5 of 7 tests) after an oral dose of 20mg extract/kg for 3 days, but remained unchanged or even decreased after an oral dose of 100 mg extract/kg for 3 days. The '*C-phenylalanine transport across the blood-brain barrier increased after oral dosing of 30 mg extract/kg or 5 days. Biochemical analysis of brain stem and brain cortex for concentration of monoamines and 3’,5/-cyclic adenosine monophosphate (AMP) and for activity of phosphodiesterase and adenylate cyclase after intraperitoneal injection of the standardized ginseng extract was also studied. The phosphodiesterase activity remained unchanged after intraperitoneal (i-p.) treatment with 50mg extract/kg for 5days, while the adenylate cyclase activity (with or without NaF activation) was decreased after i.p. treatment with 30 and 200mg extract/kg for Sdays, except in the case of 30 mg extract without NaF activation, where adenylate cyclase activity was increased. The 3/,5'-Cyclic AMP concentration decreased after ip. treatment with 200mg extract/kg body weight intraperitoneally for Sdays. The i.p. treatment with 50 mg extract/kg for

and glucose, and small amounts

of galacturonic acid,

glucuronic acid, and rhamnose. Its molecular weight was estimated to be 3.68 x 10°kDa.4°°0 An acidic polysaccharide fraction containing galactose, arabinose, and uronic acids showed inhibition of

Helicobacter pylori-induced hemagglutination with a

with

aminoguanidine,

a

specific

iNOS _ inhibitor,

Ginseng, Asian (Panax ginseng)

Sdays

increased

concentrations

the

dopamine

269

and

in brain stem, whereas

noradrenaline the serotonin

concentration was decreased in brain stem and increased in brain. These studies thus revealed the influence of ginseng extract on complex neurological processes such as learning and memory as well as on several aspects of brain metabolism.!"7! Using a two-way active avoidance with punishment (electric shock) reinforcement (shuttle box), the effects of a standardized ginseng extract were investigated on learning and memory in 2-, 10-, and 22-mo-old rats and in rats of the same age (5-mo old), which, after preliminary training with the same method, had been classified as “‘good,’’ “poor,’’ or ‘‘satisfactory’’ learners. In experiments on rats of different ages, the extract was administered orally daily for 10 consecutive days before training at increasing doses of 3, 10, 30, and 100mg/kg. The animals were trained for 5 days, and the retention test was given 14 days after the last administration of the extract (10 days after the last training session). In experiments on rats with different learning capabilities, the extract was administered orally at a dose of 10mg/kg for 10days after shuttle box training. The retention test was performed on the day following the last treatment. It was found that the extract exerted the most favorable effects on learning and memory in cases where these processes had decayed as a result of either senescence or individual specificities.'*! When the same extract was admi-

SN-K-SH cells were grown in the absence or presence of mitochoridrial permeability transition pore (MPTP) or beta-amyloid to assess neuroprotection. Both Rb; and Rg; reversed MPTP-induced cell death. Betaamyloid-induced cell death was not reversed by either ginsenoside, but Rg; produced a modest enhancement of cell death in this model. These results suggest that these two ginsenosides have neurotrophic and selective neuroprotective actions that may contribute to the purported enhancement of cognitive function.!!”! In addition, the cognition enhancing effects of ginsenosides Rb; and Rg; were investigated. Mice were trained in a Morris water maze following injection (i.p.) of Rb; (1mg/kg) or Rg; (1 mg/kg) for 4 days. Both Rb,- and Rg)-injected mice showed enhanced spatial learning compared to control animals. The hippocampus,

but not the frontal cortex, of the treated

mice contained higher density of a synaptic marker protein, synaptophysin, compared to the control mice. Electrophysiological recordings in hippocampal slices revealed that Rb; or Rg, injection did not change the magnitude of paired-pulse facilitation or long-term potentiation. The results suggest that Rb, and Rg, enhance spatial learning ability by increasing hippocampal synaptic density without changing plasticity of individual synapses.?°!

Metabolic Effects

nistered orally at doses of 3, 10, 30, 100, and 300 mg/kg

for 10 days to rats using the “shuttle-box’’ method for active avoidance, the most pronounced effect on learn-

ing and memory was obtained at the dose of 10 mg/kg. Using the “‘step-down’’ method for passive avoidance, the dose of 30 mg/kg significantly improved retention. In the staircase maze training with positive (alimentary) reinforcement test, only the dose of 10mg/kg significantly improved learning and memory. The dose of 100mg/kg greatly increased the locomotor activity of mice. These results show that ginseng at appropriate and _ physical doses improves learning, memory, capabilities. Bell-shaped dose-effect curves, reported with other nootropic drugs, were obtained." In a study designed to examine the cellular neurotrophic and neuroprotective actions of two pure ginsenosides in two-model systems, PC12 cells were grown in the absence or presence of nerve growth factor (NGF) as a positive control, and different concentrations of Rb, or Rg;. To assess neurotrophic

properties, neurite outgrowth was quantified for representative fields of cells. After 8days in culture, both ginsenosides enhanced neurite outgrowth in the pres-

ence of a suboptimal dose of (2ng/ml) NGF, but did not significantly stimulate it in the absence of NGF. However, after 18 days in culture, both ginsenosides increased the outgrowth in the absence of NGF.

The effect of a standardized ginseng extract on enzymatic activities, myotypological composition, capillaries, and mitochondrial content was studied in the skeletal muscle of male Wistar rats.’ The animals were divided into four groups, and were administered doses of 50mg/kg, while simultaneously performing exercise for a period of 12 weeks. After 24h of inactivity, the muscles of the hindlimb were extracted. With

regard to the enzymatic activities of the citrate synthase (CS) and lactate dehydrogenase (LDH), CS levels increased

with exercise, while the LDH

levels

showed no major variations, either due to the exercise or the treatment. Treatment with ginseng extract increased the capillary density and the mitochondrial content of the red gastrocnemius muscle. These results suggest that prolonged treatment with the standardized ginseng extract increases the capillary density and the oxidative capacity of the muscles with greater aerobic potential in a manner similar to the performance of physical exercise. When exercise and treatment are combined, the effects that are obtained separately are not potentiated.?4 The effect of prolonged treatment with a standardized P. ginseng extract on the antioxidant capacity of the liver was investigated. For this purpose, the extract was orally administered to rats at different doses for

Ginseng, Asian (Panax ginseng)

270

3mo, and untreated control rats were subjected to exhaustive exercise on a treadmill. A bell-shaped dose-response on running time was obtained, and the results showed that the administration of the extract significantly increases the hepatic glutathione peroxidase (GPX) activity and the reduced glutathione (GSH) levels in the liver, with a dose-dependent reduction of the thiobarbituric acid reactant substances (TBARS). After the exercise, there is reduced hepatic lipid peroxidation, as evidenced by the TBARS levels in both the controls and the treated animals. The GPX and superoxide dismutase (SOD) activities are also significantly increased in the groups receiving the extract, compared with the controls. The hepatic trans-

aminase levels, alanine-amino-transferase (ALT) and aspartate-amino-transferase (AST), in the recuperation phase 48 hr after the exercise, indicate a clear hepatoprotective effect related to the administration of the extract. At the hepatic level, the extract increases

the antioxidant capacity, with a marked reduction of the effects of the oxidative stress induced by the exhaustive exercise.7! The effect of a standardized P. ginseng extract on D-glucose uptake by Ehrlich ascites tumor cells was examined. Measurements were carried out using [°H]2-deoxy-b-glucose, a nonmetabolizable glucose analog, and indicated that it was transported from the medium into the cells and phosphorylated, but was neither further metabolized nor eliminated. Thus, the amount taken into the cells was a measure of D-glucose uptake. The results showed that the extract stimulated D-glucose transport, and that the maximum effect was obtained at a concentration of 2.01.g/ml representing an increase of about 35% above basal activity.) A study was performed to determine the effect of ginseng extract and ginsenosides (total saponin, panaxadiol, and panaxatriol) on jejunal crypt survival, endogenous spleen colony formation, and apoptosis in jejunal crypt cells of mice irradiated with high- and low doses of gamma-radiation. The radioprotective effect of ginseng was compared with the effect of diethyldithiocarbamate (DDC). The jejunal crypts were protected by pretreatment with ginseng extract (i.p.: 50mg/kg of body weight at 12 and 36hr before irradiation, p < 0.005). Ginseng extract (p < 0.005), total saponin (p < 0.01), or panaxadiol (p < 0.05) administration before irradiation (i.p.: 50mg/kg of body weight at 12 and 36 hr before irradiation) resulted in an increase in the formation of the endogenous spleen colony. The frequency of radiation-induced apoptosis in the intestinal crypt cells was also reduced by pretreatment with the extract of whole ginseng (p < 0.05), total saponin (p < 0.005), or panaxadiol

(p < 0.05) (ip. at 12 and 36hr before irradiation). The radioprotective effect on the jejunal crypts and apoptosis in the DDC-treated group appeared similar

to that in the ginseng-treated groups. Treatment with DDC showed no significant modifying effects on the formation of the endogenous spleen colony. In the experiment on the effect of ginsenosides, the result indicated that panaxadiol might have a major radioprotective effect.?7] In Asia, ginseng is commonly included in herbals used for the treatment of sexual dysfunction. Recent studies in laboratory animals have shown that ginseng enhances libido and copulatory performance. These effects may not be due to changes in hormone secretion, but to direct effects of ginseng, or its ginsenoside components, on the central nervous system and gonadal tissues. Indeed, there is good evidence that ginsenosides can facilitate penile erection by directly inducing the vasodilatation and relaxation of penile corpus cavernosum. Moreover, the effects of ginseng on the corpus cavernosum appear to be mediated by the release and/or modification of release of NO from endothelial cells and perivascular nerves. Recent © findings that ginseng treatment decreased prolactin secretion also suggested a direct NO-mediated effect at the level of the anterior pituitary. Thus, animal studies lend growing support for the use of ginseng in the treatment of sexual dysfunction and provide increasing evidence for a role of NO in the mechanism of ginsenosides.?*!

Pharmacokinetics

and Metabolism

The performance of studies on pharmacokinetics and metabolism on plant extracts is a very difficult task, since the extracts contain numerous substances. The only possibility is to do such investigations on some active ingredients of the plant extracts. The ginsenosides are considered prodrugs, and in the case of P. ginseng, ginsenosides Rg, (a representative of the protopanaxatrol derivatives) and Rb, (an example of the protopanaxadiol derivatives) are shown to be metabolized at the gastrointestinal level. In the acidic medium of the stomach, they are immediately decomposed into different ginsenoside artifacts whose chemical structures have been partially determined. The same hydrolysis of the ginsenosides also occurs in vitro under milder acidic conditions. At least five metabolites are formed from each ginsenoside. Thus,

considering that approximately 13 ginsenosides are contained in the root, at least 65 metabolites are obtained at the gastrointestinal level. The latter are formed in very small quantities and are especially difficult to detect in blood and urine. The amounts of nonmetabolized intact Rg, and Rb, absorbed by the gastrointestinal tract of the rat are about 1.9% and

0.1% of the doses, respectively. Whole-body autoradi-

ography was used to demonstrate the absorption and

Ginseng, Asian (Panax ginseng)

distribution of the radioactively labeled ginsenoside Rg; and its metabolites after oral administration. A study performed with mini-pigs showed, after intravenous administration, that the derivatives of protopanaxatriol, such as Rg), have a one-compartment pharmacokinetic profile, and a_ half-life of about 30min. On the other hand, for those of protopanaxadiol, such as Rby, the half-life is much longer (about 16hr), and their pharmacokinetics are described by a two-compartment model."! Using a sensitive mass spectrometric method, which is specific for the identification of ginsenosides in complex biological matrices, the degradation pathway of ginsenosides in the gastrointestinal tract of humans could be elucidated following the oral administration of a standardized P. ginseng extract. Within the framework of a pilot study, human plasma and urine samples of two subjects were screened for ginsenosides and their possible degradation products. In general, the urine data coincided well with the plasma data, and in both volunteers, the same hydrolysis products, which are not originally present in the extract ingested, were identified. It was shown that two hydrolysis products of the protopanaxatriol ginsenosides, namely G-Rh, and G-F;, may reach the systemic circulation. In addition, compound-K, the main intestinal bacterial

metabolite of the protopanaxadiol ginsenosides, was detected in plasma and urine. These products are probably responsible for the action of ginseng in humans. In contrast to previous reports, G-Rb, was identified in the plasma and urine of one subject.?4)

Preclinical Safety Data The results of several toxicity studies in animals with a P. ginseng extract have been reviewed."*!

271

histopathological effects were not noticed in beagle dogs after oral administration of 1.5, 5.0, and 15mg/kg for 90 days.

Reproduction toxicity No decrease of growth rate or reproduction and no treatment-related hematological or histopathological findings were seen in rats for 33 weeks in a twogeneration study with daily oral administration of 1.5, 5.0, and 15 mg/kg.

Embryo, fetal, and perinatal toxicity No abnormalities of fetal development have been detected in rats after daily oral administration of 40mg ginseng extract/kg on days 1-15 after mating, or in rabbits after daily oral administration of 20 mg/kg on days 7-15 after mating. In an in vitro study using whole rat embryo culture model, ginsenoside Rb, induced teratogenicity.? The significance of this study is uncertain due to the concentration of Rb, used, and to the fact that it is known

that ginsenosides that are not metabolized by the acidic medium and intestinal flora exert hemolytic activities (as is generally observed with saponins).

Genotoxicity No genotoxicity was observed in the hepatocyte-DNArepair test using concentrations of 0.1-10mg/ml of ginseng extract with or without ginsenosides or using 1-50 pg/ml of ginsenoside Rg;. Neither has mutagenicity been observed in Salmonella typhimurium and Chinese Hamster V79 cells.

Single dose toxicity

CLINICAL STUDIES

The LDso after oral administration is >5 g/kg in the rat, >2g/kg in the mini-pig, and >1 g/kg in mice, and after ip. administration, it is >1 g/kg in rats and mice. No noticeable changes of cardiovascular parameters such as electrocardiogram (ECG), pulse, blood pressure, cardiac output, and stroke volume were observed after single dose oral administration of 0.25, 0.5, and 2.0 g/kg in mini-pigs.

Performance

Repeated dose toxicity No hematological or histological abnormalities were observed in rats after 20 days of daily oral administration of 4.0g/kg. Treatment-related hematological or

In a double-blind crossover study, 12 student nurses working night shifts (34 consecutive nights followed by 3days of rest) were given 1.2 g of ginseng roots or placebo for the first three consecutive nights and tested on the morning after the third night. Crossover medication was given after an interval of at least 2 weeks. A third series of tests was carried out during normal daytime working, after no medication and following a good night’s sleep (GNS). The subjects assessed their mood, physical well being, and degree of lethargy by means of linear self-rating scales. Two psychophysiological performance tests and hematological tests were also carried out.

Ginseng, Asian (Panax ginseng)

272

The detrimental effects of night shifts were clearly seen. A constant trend in favor of ginseng compared to placebo was noted. Ginseng ratings were favorable for mood criteria, but not for physical well-being symptoms. Ginseng restored blood glucose levels raised by night shift stress. A small but consistent antifatigue activity of ginseng was concluded.?”! Various tests of psychomotor performance were carried out in a cohort of 16 healthy male volunteers given a standardized ginseng extract (100mg ginseng extract twice a day for 12 weeks) and in a similar group given placebo under double-blind conditions. A favorable effect of ginseng relative to baseline performance was observed in attention (cancelation test), processing (mental arithmetic, logical deduction), integrated sensory-motor function (choice reaction time), and auditory reaction time. However, end performance of the ginseng cohort was only statistically superior (p < 0.05) to the placebo group in mental arithmetic. No difference between ginseng and placebo was found in tests of pure motor function (tapping test), recognition (digit symbol substitution), and visual reaction time? In a double-blind, placebo-controlled, crossover study, 43 top triathletes received either placebo or 200mg of a standardized ginseng extract per day for periods of 10 weeks, respectively. Significant differences (p < 0.05) in various endurance parameters were only seen after the second treatment phase. It was concluded that ginseng improves endurance (resistance against end of season stress), but not optimum performance.) Twenty top class male athletes received 200mg standardized ginseng extract per day for 9 weeks. In the bicycle ergometer exercise test lasting 8 min, the post-treatment values were higher for maximal oxygen absorption and lower for blood lactate level and heart rate during exercise compared to pretreatment values. The differences were significant (p < 0.001),8% A double-blind study involved 30 athletes who received daily either placebo (n = 10), 200 mg ginseng extract standardized to 7% ginsenosides (n = 10), or 400 mg vitamin E and 200 mg ginseng extract standardized to 4% ginsenosides (n = 10) for 9 weeks. The same bicycle ergometer test was used and statistically significant variations in heart rate (p < 0.05), blood lactate (p < 0.01), and maximal oxygen absorption (p < 0.01) after exercise between either of the two ginseng preparations and placebo were found. Differences between the two ginseng preparations were not statistically significant. The levels of testosterone and luteinizing hormone in plasma, and free cortisol in urine, were unchanged

periods,.81)

after all treatment

A further double-blind, placebo-controlled study with 28 top class male athletes examined the persistence

of the effects of 9 weeks’ treatment (placebo or 200 mg ginseng extract with 4% ginsenosides) beyond the treatment period. Ginseng resulted in a significant improvement of maximal oxygen uptake during exercise (p < 0.01), heart rate at maximal exercise (p < 0.001), forced expiratory volume (p < 0.01), forced vital lung capacity (p < 0.05), and visual reaction time (p < 0.01) compared with placebo. These positive effects lasted for at least 3 weeks after treatment, and it was concluded that the effects of ginseng are based on clinically relevant metabolic changes

that persist for a certain period after treatment.?7! In a double-blind, placebo-controlled study with 50 ambulatory patients suffering from asthenia, depressive syndrome, or neurovegetative disorders, the effects

of 8 weeks’ treatment with 200 mg/day of a standardized ginseng extract on performance in two psychometric tests and on results from a comprehensive psychological questionnaire (Sandoz Clinical Assessment Geriatric) were studied. Significant improvement (p < 0.05 and p < 0.01) was seen in most of the parameters.?")

In a randomized double-blind study, 31 healthy male volunteers received 200 or 400 mg ginseng extract per day for 8 weeks. Ginseng had no effect on oxygen consumption, respiratory exchange ratio, minute ventilation, blood lactic acid concentration, heart rate, and

perceived exertion.) In another randomized double-blind study, 19 healthy female volunteers received daily 200mg ginseng extract or placebo for 8 weeks. It had no effect on maximal work performance and resting, exercise, recovery oxygen uptake, respiratory exchange ratio, minute

ventilation,

heart rate, and blood

lactic acid

levels.?>! In a double-blind, placebo-controlled, crossover study in 8 healthy volunteers (mean age 25yr) who regularly practised physical activities, 30 days of daily oral treatment with 400mg of a standardized ginseng extract did not improve performance at supramaximal exercise (125% of the maximum aerobic power on bicycle ergometer), nor did it influence blood lactate or blood testosterone.8° In a study on blood oxygenation status of 8 male and 2 female middle aged subjects (average 50 yr old), a significant (p < 0.05) increase of resting arterial pO» was found after 4 weeks’ oral treatment with 200mg standardized ginseng root extract per day. The resting arterial pO, was increased by 4.5 mmHg. In synergy with oxygen treatment, the increase was 10.1 mmHg. Venous pO was decreased (4.3 mmHg).87] The effects of 400 mg/day of a ginseng extract on a variety of cognitive functions were compared with placebo in a double-blind, randomized study in which 112 healthy volunteers older than 40 yr (55 on ginseng, 57 on placebo) were treated for 8-9 weeks. The ginseng

—

Ginseng, Asian (Panax ginseng)

273

group showed a tendency to have faster simple reactions and significantly better abstract thinking than the controls. However, there was no significant difference between the two groups in concentration,

memory, or subjective experience.!**!

A study investigated whether acute administration of standardized ginseng extract had any consistent effect on mood and four aspects of cognitive performance (quality of memory, speed of memory, quality of attention, and speed of attention) that can be derived by factor analysis of the Cognitive Drug Research computerized assessment battery. The study followed a placebo-controlled, double-blind, balanced crossover design. Twenty healthy young adult volunteers

and

received 200, 400, and 600mg of the extract, a matching placebo, in counterbalanced order,

with a 7day wash-out period between treatments. Following a baseline cognitive assessment, further test sessions took place 1, 2.5, 4, and 6hr after the day’s

treatment. The most striking result was a significant improvement in “quality of memory’’ and the associated “‘secondary memory”’ factor at all time points following 400mg of ginseng. Both the 200 and 600 mg doses were associated with a significant decrement of the “speed of attention’’ factor at later testing times only. Subjective ratings of alertness were also reduced 6 hr following the two lowest doses.**! The effects of a standardized ginseng extract on psychological mood states, and the perceptual response to submaximal and maximal exercise stress were examined in a study with 19 young adult females who received either 200 mg/day of a standardized ginseng root extract (n = 10) or placebo (n = 9). The results did not support claims of the efficacy of ginseng to alter psychological function characteristics at rest and during exercise stress"! The effects of a standardized ginseng extract (300mg/day) on healthy, untrained male students and on healthy male students who received regular bicycle ergometer training were compared with placebo in an 8 week, randomized, double-blind study (n = 41). Ginseng administration at the prescribed dose exhibited training-like effects on VO2 max as well as anaerobic power and leg muscle strength. But no synergistic effect on these fitness variables occurred when both ginseng administration and exercise training were combined.4° The effect of acute administration of standardized ginseng extract was investigated on mood and four aspects of cognitive performance mentioned preciously derived from factor analysis of the cognitive drug research computerized test battery. Following a double-blind,

placebo-controlled,

balanced, crossover

design, 30 healthy young adult volunteers received 400 mg of ginseng, and a matching inert placebo, in a counterbalanced

order, with a 7day wash-out period

between treatments. Following baseline evaluation of cognitive performance and mood measures, participants’ cognitive performance and mood were assessed again 90min after drug ingestion.,In line with previous research, a fractionation of the effect of ginseng administration was observed. Ginseng significantly improved speed of attention, indicating a beneficial effect on participants’ ability to allocate attentional processes to a particular task. However, no significant effect was observed on any other aspect of cognitive performance. In addition, participants’ self-reported mood measures did not differ significantly across treatments. It is interesting to note that previous research demonstrated no improvement on attentional processes, but significant improvements on quality of memory following administration of 400mg of ginseng when participants were tested 1, 2.5, 4, and 6hr postingestion.'*! It may be the case that ginseng may offer performance at varying time points. This may be due to different chemical constituents of ginseng displaying several pharmacokinetic properties and psychopharmacological actions.'47!

Immunomodulation

The effects of ginseng root extract (200 mg orally/day) on immune parameters were studied in an 8 week three leg trial involving 60 healthy volunteers of both sexes aged between 18 and S0yr. Study medication was either a standardized ginseng extract or a nonstandardized aqueous ginseng extract or placebo. The statistically significant differences from baseline that have been observed are listed below. The standardized extract led to an increase in the following: chemotaxis of circulating polymorphonuclear leukocytes (p < 0.05 at week 4and p < 0.001 at week 8), phagocytosis index and phagocytosis fraction (p < 0.001 at weeks 4 and 8), total lymphocytes (T3) (p < 0.05 at week 4 and p < 0.001 at week 8), T-helper (T4) subset (p < 0.05 at week 4 and p < 0.001 at week 8), helper/suppressor (T4/T8) ratio (p < 0.05 at weeks 4 and 8), induction of blastogenesis in circulating lymphocytes (p < 0.05 at weeks 4 and 8 after induction by cocanavalin A and pokeweed mitogen, p < 0.001 at weeks 4 and 8 after induction by lipopolysaccharide) and natural killer cell activity (p < 0.05 at week 4 and p < 0.001 at week 8). With the aqueous extract, a rise was observed in the following: chemotaxis of circulating polymorphonuclear leukocytes (p < 0.05 at week’ 4 and 8), phagocytosis index and phagocytosis fraction (p < 0.05 at week 8), total (T3) lymphocytes (p < 0.05 at week 4 and p < 0.001 at week 8), T-helper (T4) subset (p < 0.05 at week 8), induction of blastogenesis in circulating lymphocytes (p < 0.05 at week 8 after induction by cocanavalin

Ginseng, Asian (Panax ginseng)

274

A and pokeweed mitogen), and natural killer cell activity (p < 0.05 at week 8). With the placebo, only an enhancement in natural killer cell activity was statistically significant (p < 0.05) after 8 weeks. It was concluded that ginseng extracts act as an immunostimulant in humans, and that the standardized extract

was more active than the aqueous one 41) Healthy volunteers (n = 227) were enrolled in a placebodouble-blind, randomized, multicenter, controlled clinical trial to investigate potential effects of a standardized ginseng extract on resistance against influenza and the common cold. Study duration was 12 weeks and the study medication was either 200mg standardized ginseng extract (n = 114) or placebo (n = 113) per day. All participants received an antiinfluenza polyvalent vaccine at week 4. Results from examinations

at weeks

4, 8, and

12 showed

highly

significant differences (p < 0.0001) between ginseng extract and placebo with regard to the frequency of influenza or colds between weeks 4 and 12 (15 cases in the verum group vs. 42 cases in the placebo group). Antibody titers at week 8 were also much higher after verum (272 units vs. 171 units after placebo) as well as natural killer cell activity which was almost twice as high

in the verum group compared to the placebo group.*7! A controlled single-blind study was performed to investigate the effects of standardized ginseng root extract (200mg/day) in 40 patients suffering from chronic bronchitis. It was shown that the extract significantly (p < 0.001) improves alveolar macrophage activity compared to baseline.!*"! The effects of a standardized ginseng root extract (200 mg orally per day for 3 mo) were studied in a pilot trial involving 15 patients with severe chronic respiratory diseases. Respiratory parameters, such as vital capacity, expiratory volume and flow, ventilation volume, as well as walking distance, were examined.

The results led to the conclusion that the extract improves pulmonary function and oxygenation capacity, which seems to be the reason for improved walking capacity.™4) A study in two equal groups of 10 young healthy males was undertaken to investigate the effects of 8 weeks’ administration of a standardized ginseng extract (300mg/day) in comparison with the effects of placebo. It was concluded that ginseng caused no significant changes in peripheral blood leukocytes and lymphocyte subsets.!**!

INDICATIONS Uses Supported by Clinical Data

Radix ginseng is used as a preventive and restorative agent for boosting mental and physical capacities,

immunity against infections, and in subjects experienc-

ing debility fatigue, tiredness, and loss of concentra-

tion, and during pregnancy.>**

Uses Described in Pharmacopoeias and in Traditional Systems of Medicine Radix ginseng is also used in the treatment of impotence, prevention of hepatotoxicity, and gastrointestinal disorders such as gastritis and ulcer.

Uses Described in Folk Medicine, but not

Supported by Experimental or Clinical Data Treatment of liver diseases, coughs, fever, tuberculosis, rheumatism, vomiting during pregnancy, hypothermia,

and dyspnea."!

POSOLOGY Adult daily dose: 0.5—2.0 g dried root; doses of equiva-

lent preparations should be calculated accordingly.>**!

CONTRAINDICATIONS None have been reported.**

INTERACTIONS AND SIDE EFFECTS Data from clinical trials suggest that the incidence of adverse events with P. ginseng preparations is similar to that with placebo. The most commonly experienced adverse effects are headache, sleep and gastrointestinal disorders. The possibility of more serious side effects is indicated in isolated case reports and data from spontaneous reporting schemes. However, causality is often difficult to determine from the evidence provided. Combination products containing ginseng as one of several constituents have been associated with serious adverse events and even fatalities. Interpretation of these cases is difficult as ingredients other than P. ginseng may have caused the problems. Possible drug interactions have been reported between P. ginseng and warfarin, phenelzine, and alcohol. Collectively, these data suggest that P. ginseng monopreparations are rarely associated with adverse events or drug interactions. The ones that are documented are usually mild and transient. Combined preparations are more often associated with such events, but causal

attribution is usually not possible.”

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275

A study in humans has shown that P. ginseng extract after oral administration for 14 days does not

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GA '¢ advocated the use of glucosamine as part of a defined therapeutic approach to osteoarthritis that in addition to the supplements glucosamine and chondroitin, recommends regular exercise, healthy diet, and weight control, “traditional medications’’ as indicated, and a positive attitude. Osteoarthritis has been described as “the coming epidemic of arthritis.”’"'”! This article estimates the prevalence of osteoarthritis in the United States to be 20

million

in 2002,

and,

assuming

current

demo-

graphics, it will climb to 40 million by 2020. Patients with osteoarthritis frequently seek medical care for improvement in their symptoms. The Arthritis Foundation estimates that there are over 7 million physician visits annually for osteoarthritis. A recent study addressing primary care utilization found that osteoarthritis patient visits accounted for more than one-half of general medical visits that involved rheumatologic complaints."®) Currently, recommended medical therapy includes patient education in joint protection, weight reduction, physical therapy, and analgesia most often with acetaminophen.!!41?-71] Often-times, these recommendations fail to meet the patient’s expectations, and miscreates a frustrating gap between hopes and reasonably attainable results. In this setting, patients are turning to complementary and /or alternative therapies in an effort to obtain an added measure of improvement. Likewise, physicians are frustrated by the lack of evidence-based information to establish a foundation for the rational use of these therapies. Often, studies attempting to demonstrate the efficacy have been hampered by serious flaws. of CAM Publication bias in the medical literature, even of trials

that have been poorly developed and performed, is toward reports of positive results. Consequently, due to a lack of scientifically credible information, both patients and health care practitioners are often

unable to develop rational therapeutic strategies that include CAM. In an effort to encourage rigorously designed scientific trials that address

CAM

efficacy, the National

Institutes of Health (NIH) established the Office of Alternative Medicine and, subsequently, the National

Center for Complementary and Alternative Medicine (NCCAM). The stated mission of NCCAM is to “support rigorous research on complementary and alternative

medicine,

to train

researchers

in CAM,

and to disseminate information to the public and professionals on which CAM modalities work, which do not, and why.’’??! When the Office of Alternative Medicine was established in 1992, its budget was $ 2 million. The 2003 budget for NCCAM was $ 113.4 million.??! In this complex medical/political milieu, the use of glucosamine in the treatment of osteoarthritis has become very popular over the past several years. The Nutrition Business Journal estimates that the United States consumes over 3000t of glucosamine annually worth almost $ 800 million in consumer spending.?4)

GLUCOSAMINE

PREPARATIONS

Glucosamine is prepared commercially by acid hydrolysis of chitin [poly-B-(1 — 4)-N-acetyl-p-glucosamine], which is a major component of the shells of Crustacea such as crabs and shrimps. Because it is derived from shellfish hydrolyzates, persons with shellfish allergies should probably avoid exposure, or use with caution. Along with cellulose, chitin is the most widely prevalent natural biopolymer. In shellfish, chitin is clustered with proteins and calcium carbonate. The purification of chitin and its subsequent hydrolysis yields glucosamine. As a weak organic base, glucosamine can be transformed into either a hydrochloride or a sulfate salt form. Commercially available forms of glucosamine include: 1) glucosamine sulfate; 2) cocrystals and coprecipitates of glucosamine sulfate with potassium or sodium chloride; 3) glucosamine hydrochloride; and 4) physical mixtures of glucosamine hydrochloride and potassium or sodium sulfate. Glucosamine is available in highly purified final forms. Details of the various preparations are summarized below.

Glucosamine Sulfate

The “pure’’ hygroscopic, subsequently of the amino perties, this

sulfate salt of glucosamine is intensely and due to the resultant hydration and low pH, there is potential for oxidation group (Fig. 3). Because of these proformulation must be preserved with a

Glucosamine

282 HOCH

Ho

G

\

“ON

HO Fig. 3

Chemical structure of glucosamine sulfate.

desiccant under extremely controlled conditions and would be prohibitively expensive. For these reasons, commercial manufacture of “‘pure glucosamine sulfate,’’ and development of a clinical application of this formulation is not feasible.

Glucosamine Bonded with Sulfate (Not as a Salt) There are other substances available that are sometimes termed “glucosamine sulfate.’’ These compounds are

not

the

sulfate

salts

of glucosamine.

Rather,

they are composed of glucosamine with sulfate groups covalently bonded to the hexosamine at different sites. Examples are p-glucosamine 2,3-disulfate, p-glucosamine 2,6-disulfate, p-glucosamine 3,6-disulfate, and p-glucosamine-6-sulfate. Their structures are available through the International Union of Pure and Applied Chemistry (IUPAC). These molecules are not a component of the so-called “‘stabilized glucosamine sulfate’’ (which is a salt form of glucosamine discussed later), nor are they available in oral dosage forms. One example is illustrated in Fig. 4.

Cocrystals and Coprecipitates of Glucosamine Because glucosamine hydrochloride was readily available but could not be patented, efforts were directed toward the use of glucosamine sulfate for commercial purposes. However, due to the issues described above, commercial development for mass distribution could not be accomplished. To overcome these obstacles, a process was developed and patented that yielded

SO,

Ita:

HO

Ne

ee

Co

+, SO,

-2

Na* cr

ome 2

iy

S42 aay

OH

OH

OH

Fig. 5 Chemical structure of glucosamine sulfate/sodium chloride coprecipitate.

glucosamine sulfate in a cocrystallized matrix with sodium chloride (Fig. 5). This method “stabilized’’ the glucosamine sulfate, in that addition of sodium chloride led to a reduction in the hygroscopic properties of the compound and made it possible to produce oral dosage forms. This product has since been used in commercially sponsored clinical trials of glucosamine in osteoarthritis. : Subsequent to the award of patent protection for the production of this “stabilized glucosamine sulfate,’’ there has been commercial promotion alleging that this preparation is therapeutically superior to others, though no clinical studies have been conducted to prove it. In this regard, it is important to recognize that since the biological acid in the stomach is HCl, all dietary glucosamine (independent of the salt form ingested) likely enters the small intestine for absorption as glucosamine HCl. It is also interesting to note that all of the published pharmacokinetic studies on glucosamine in humans have been conducted using $C _radiolabeled glucosamine hydrochloride mixed

with unlabeled “‘stabilized glucosamine sulfate.”’U!-!) At least one other glucosamine stabilization method has been patented. Similar to the process described above, this technique utilizes lyophilization (freezedrying) to coprecipitate glucosamine sulfate with potassium chloride. This method has also been patented, and the resultant product is commercially available in the United States. In both instances, glucosamine hydrochloride (see below) is the glucosamine substrate that is either cocrystallized or coprecipitated to produce the final “‘stabilized glucosamine sulfate’ salt.

Glucosamine Hydrochloride

HOCH

HO

ou

s z, z7

NH»

Fig. 4 Glucosamine-6-sulfate (not a salt covalently bonded to structure).

form:

sulfate

Glucosamine hydrochloride is a much more stable salt form of glucosamine than glucosamine sulfate and is produced from chitin in an acid extraction using hydrochloric acid (Fig. 6). It is available as an extremely (>99%) pure compound that is very stable and has a long shelf life. The material can be certified by the Food and Drug Administration (FDA) as compliant with current Good Manufacturing Practices (cGMP) and can be produced to strict pharmacologic standards.

Glucosamine

Fig. 6

283

the actual level of glucosamine can range from 895 to 1245 mg/day. Most often, salt forms of pure substances are prepared to improve solubility characteristics. Hydrochloride salts are among the most commorily used forms of salts of weak organic bases, because chloride is readily available, is found naturally in the human body, and produces salts with good stability characteristics. An additional advantage is that on a moleculeper-molecule basis, these salts are smaller than those

Chemical structure of glucosamine hydrochloride.

made

Physical Mixtures

with

ions such

as citrate, lactate, and

sulfate

(e.g., the molecular weight of HCl is 36, whereas that of H2SO, is 101). This is important when considering whether a dose is referenced to the amount of active parent drug or to the quantity of the salt form as seen in Table 1. Finally, therapeutic drug monitoring in patients receiving a salt form of a drug is conducted using blood concentration of the parent drug, not the

Because of the patent issues described above, some products marketed as “glucosamine sulfate’ are simply a physical combination of glucosamine hydrochloride and a sulfate salt such as potassium sulfate. The rationale for using this combination is the possibility that both glucosamine and ionic sulfate may be therapeutic, or that ionic sulfate may promote glucosamine absorption in the gut. However, it must be recognized that when the sulfate dissociates from the glucosamine in the gastrointestinal (GI) tract, highly charged anion(s) are generated that do not readily cross GI tract membranes and potentially result in an osmotic diarrhea. In large doses, this is the basis of the mechanism of action for cathartic laxatives that contain sulfate salts. Normal levels of sulfate in the blood (about 0.3 mM) are critical for many cellular functions, including the synthesis of GAGs. However, the pharmacokinetics of dietary sulfate and its potential effect on cartilage metabolism are unknown, though some authors have suggested a role for sulfates in osteoarthritis.2°7°

salt. In the instance

of glucosamine,

the salt form

dissociates when it dissolves in the GI tract. Hence, in performing pharmacokinetics of glucosamine HCl (or H2SO,), only the glucosamine moiety is measured. In the case of glucosamine hydrochloride, glucosamine

sulfate,

and

any

of its stabilized

forms,

the

dissolution of these molecules will also involve dissociation of the salt. There has been no published evidence,

nor

have

we

observed

any

difference

in

the rate of dissolution of any of the glucosamine containing preparations.

CLINICAL TRIALS USING GLUCOSAMINE IN OSTEOARTHRITIS Many of the controlled clinical trials with glucosamine in osteoarthritis patients have been of marginal quality

Comparison of Salt Forms

due to insufficient sample size, lack of statistical rigor, potential for sponsor bias, inadequate concealment,

As detailed in the subsections above, there are practical

varies due to the size of the associated salt form. Thus,

and lack of intention-to-treat principles. One systematic review of published randomized trials whose aim was to determine the effectiveness of glucosamine in osteoarthritis’! identified six studies found to be acceptable for systematic quality assessment.?*>") The authors found that each one of these studies

as can be seen, in the usual daily dose of 1500 mg/day,

demonstrated

therapeutic considerations inherent in the physical characteristics of the various glucosamine preparations. These are summarized in Table 1. The actual quantity of glucosamine found in the preparations

Table 1

a positive effect, and the pooled effect

Quantities of glucosamine present in different preparations*

Comparative attributes

Glucosamine HCl

Purity (as the salt form) (%)

Glucosamine SO,-2NaCl

Glucosamine SO,-2KCI

79.5 (20.5% NaCl)

75 (25% KCl)

Weight percentage as glucosamine

62.7

5)

Dose (mg) to yield 1500 mg of glucosamine

2392

2521

940.5

892.5

Glucosamine content (mg) per 1500 mg of substance

1246.5

Does not include comparison of physical mixtures of glucosamine hydrochloride and potassium sulfate because these mixtures can be prepared in varying concentrations.

Glucosamine

284

size was deemed to be moderate. Subsequent to this review, three independently funded studies have been

published.44°! In general, experiments with larger

sample sizes and those without industry support tended to have smaller effect sizes. Two other glucosamine clinical trials merit specific comment. Both of these recently published studies present data from patients who received long term glucosamine therapy with the primary objective being to evaluate progressive loss of joint space in the knee and thus assess the potential for disease modification using standard, serially obtained, antero-posterior, weight-bearing knee X-rays as the outcome measure. Other outcomes were also addressed aimed at evaluating improvement in joint pain over the duration of the trial. Both studies were industry supported.

The first study®7! evaluated 212 patients followed for three years on 1500 mg glucosamine per day versus placebo. It assessed change in medial compartment joint space width as determined on standing, weightbearing antero-posterior knee radiographs as the primary outcome. Symptomatic outcomes were assessed using the WOMAC instrument. The authors reported that the patients taking glucosamine experienced no loss in joint space, while those on placebo continued to show progressive cartilage loss. Glucosaminetreated subjects also experienced improved symptoms in total WOMAC index based on intent-to-treat statistical principles. In

the

second

study

evaluated,2*!

202

patients

received 1500mg glucosamine per day or placebo. Once again, radiographic medial joint space narrowing as described in the study above was the primary outcome measure. Symptomatic evaluation was measured using both the WOMAC and Lequesne instruments. The researchers found that patients taking glucosamine showed no progression of medial joint space narrowing, while the placebo-treated subjects experienced progressive joint space narrowing. The study also reported a completer’s analysis that demonstrated significant improvement in symptoms based on both the above-mentioned indices. At least two major concerns have been raised regarding the validity of the selected radiographic outcome measure in these studies. The first is that because of anatomic positioning in the extended AP view of the knee, the joint space width does not actually measure articular cartilage only, but others as well such as the meniscus and status of the collateral ligaments and therefore may not indicate true joint space width. Utilization of standardized radiographic protocols'”! or the development and validation of other quantitative measures of articular cartilage will be necessary to demonstrate whether these agents are potentially disease modifying. Secondly, positioning of the joint for

radiography in terms of extension may be influenced by the amount of joint pain at the time the film was taken.'“°! Thus, patients with less painful knees may have had less guarding and therefore more extension that could give the appearance of wider joint space width. In any event, the possibility of disease modification by glucosamine as determined by altering radiographic evidence of progressive joint space narrowing is an intriguing and important question that warrants further study. A seemingly overlooked, yet remarkable, finding in both of these trials was the improvement that was seen in joint pain over the years of study follow-up. Sustained lessening in pain of the degree and duration suggested by these trials has never been reported before for any agent in the management of osteoarthritis. This is certainly a puzzling information regarding glucosamine efficacy in a controlled setting.

SAFETY The safety profile of glucosamine in the published studies described earlier is uniformly favorable and comparable to placebo. A few minor adverse events have been reported, including GI complaints such as heartburn, diarrhea, constipation, epigastric pain, and

nausea.“'] One concern about the use of glucosamine is its potential to cause or worsen diabetes. In animal models, increased glucosamine levels in cells have been

associated with insulin resistance (a major factor in the genesis of Type 2 diabetes mellitus) and alterations in insulin production.474! Whether the doses commonly used in humans are sufficient to cause significant alterations in glucose homeostasis is not clear at this time. A recent study by Scroggie, Albright, and Harris,4*! however found that glucosamine treatment

of known diabetics did not change either their diabetes management or their diabetes control as assessed by levels of hemoglobin Alc.

RECOMMENDATIONS Glucosamine is a natural aminosaccharide present in the exoskeletons of arthropods and is obtained from chitin by acid decomposition. It does not exist as a natural biosynthetic product in cells. Instead, it is generated as glucosamine-6-phosphate by the reaction of fructose-6-phosphate and glutamine (see Fig. 2 pathway for detail). Glucosamine-6-phosphate precedes in the biosynthesis of the GAG component of proteoglycans such as cartilage aggrecan. Loss of cartilage aggrecan due to excessive proteolysis is part of the clinical syndrome identified as osteoarthritis. Dietary glucosamine may slacken, stall, or even counter this

Glucosamine

285

degenerative process. While there have been clinical studies suggesting these effects, meticulous and objective experiments are required to validate them. Even more importantly, if glucosamine has a beneficial effect

Largo, R.; Alvarez-Soria, M.A.; Diez-Ortego, L.; Calvo, E.; Sanchez-Pernaute, O.; Egido, J. et al.

Glucosamine inhibits IL-lbeta-induced NFkappaB activation in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2003, J/ (4), 290-298.

on osteoarthritis, more basic studies will be necessary

to determine its pharmacokinetic profile, establish what the biological mechanism of its effects are (which cells and metabolic pathways are affected), and how such effects could be maximized. Finally, if glucosamine is proven to be effective in the treatment of OA, a feasible plan to regulate its manufacture and distribution will be required. This will assure the patient of a safe and uncontaminated product.

ACKNOWLEDGMENTS

Ma, L.; Rudert, W.A.; Harnaha, J.; Wright, M.;

Machen, J.; Lakomy, R. et al. Immunosuppressive effects of glucosamine. J. Biol. Chem. 2002, 277 (42), 39,343-39,349.

10.

his

Ph.D., Anna Plaas, Ph.D., and Jamie Barnhill, Ph.D.,

assistance

in

preparing

this

Setnikar, I.; Giacchetti, C.; Zanolo, G. Pharmaco-

kinetics of glucosamine in the dog and in man. Arzneimittelforschung 1986, 36 (4), 729-735.

The authors gratefully acknowledge John D. Sandy, for their valuable manuscript.

Setnikar, I.; Giachetti, C.; Zanolo, G. Absorption,

distribution and excretion of radioactivity after a single intravenous or oral administration of [14C] glucosamine to the rat. Pharmatherapeutica 1984, 3 (8), 538-550.

12)

13s

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effect

of glucosamine

36.

eM

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3h:

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in outpatients

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tion of glucosamine sulfate compared to ibuprofen for the treatment of temporomandibular

a

B.P. Knee pain reduces joint space width in conventional standing anteroposterior radiographs

C.; Paget-

Cartilage

osteoarthritis:

Buckland-Wright, J.C.; Wolfe, F.; Ward, R.J.; Flowers, N.; Hayne, C. Substantial superiority

(MTP),

44.

Osteoarthritis

knee

26 (12), 2664-2674.

Dellio, S.D. Effect of glucosamine hydrochloride in the treatment of pain of osteoarthritis of the knee. J. Rheumatol. 1999, 26 (11), 2423-2430.

arthritis of the knee. 1994, 2 (1), 51-59. 34.

R.; Wein,

clinical trial. Lancet 2001, 357 (9252), 251-256. _ Pavelka, K.; Gatterova, J.; Olejarova, M.; Machacek, S.; Giacovelli, G.; Rovati, L.C.

of semiflexed (MTP) views in knee osteoarthritis: a comparative radiographic study, without fluoroscopy, of standing extended, semiflexed

gonarthrosis. Clin. Ther. 1981, 3 (5), 336-343. Pujalte, J.M.; Llavore, E.P.; Ylescupidez, F.R.

Double-blind clinical evaluation of oral glucosamine sulphate in the basic treatment of osteoarthrosis. Curr. Med. Res. Opin. 1980, 7 (2), 110-114.

double-

Reginster, J.Y.; Deroisy, R.; Rovati, L.C.; Lee, R.L.; Lejeune, E.; Bruyere, O. et al. Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled

placebo-controlled, double-blind study. Intern. Med. 2002, /62 (18), 2113-2123.

Vajaradul, Y. Double-blind clinical evaluation of

intra-articular

randomized,

Rindone, J.P.; Hiller, D.; Collacott, E.; Nordhaugen, N.; Arriola, G. Randomized, controlled trial of

of

Reichelt, A.; Forster, K.K.; Fischer, M.; Rovati,

L.C.; Setnikar, I. Efficacy and safety of intramuscular glucosamine sulfate in osteoarthritis of the knee. A randomised, placebo-controlled, doubleblind study. Arzneimittelforschung 1994, 44 (1), 75-80. Rovati, L. The clinical profile of glucosamine sulfate as a selective symptom modifying drug in osteoarthritis: current data and perspectives. Osteoarthritis Cartilage 1997, 5 (Suppl. A), 72.

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Glucosamine sulfate use and delay of progression

treatment of osteoarthritis: a systematic quality assessment and meta-analysis. J. Am. Med. Assoc. 2000, 283 (11), 1469-1475. 28.

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glucosamine for treating osteoarthritis of the knee. West. J. Med. 2000, 772 (2), 91-94.

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Metabolism 2001, 50 (7), 767-770. McAlindon, T.E.; LaValley, M.P.; Gulin, J.P.; Felson, D.T. Glucosamine and chondroitin for

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Hughes,

blind, placebo-controlled trial of glucosamine sulphate as an analgesic in osteoarthritis of the knee. Rheumatology (Oxford) 2002, 4/ (3), 279-284.

Hoffer, L.J.; Kaplan, L.N.; Hamadeh, M.J.; Grigoriu, A.C.; Baron, M. Sulfate could mediate

the therapeutic

2g

joint osteoarthritis: a randomized double blind controlled 3month clinical trial. J. Rheumatol. 2001, 28 (6), 1347-1355.

Hochberg, M.C.; Altman, R.D.; Brandt, K.D.; Clark, B.M.; Dieppe, P.A.; Griffin, M.R. et al.

Heyneman, C.A.; Rhodes, R.S. Glucosamine for osteoarthritis: cure or conundrum? Ann. Pharmacother. 1998, 32 (5), 602-603. McClain, D.A.; Crook, E.D. Hexosamines and insulin resistance. Diabetes 1996, 45 (8),

1003-1009. Tang, J.; Neidigh, J.L.; Cooksey, R.C.; McClain, D.A. Transgenic mice with increased hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance. Diabetes 2000, 49 (9), 1492-1499. Rossetti,

L.

Perspective:

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nutrient sensing. Endocrinology 2000, 141 (6), 1922-1925. Scroggie, D.A.; Albright, A.; Harris, M.D. The effect of glucosamine-chondroitin supplementation on glycosylated hemoglobin levels in patients with type 2 diabetes mellitus: a placebocontrolled,

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Intern.

Med.

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2003,

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(13),

-

Glutamine Steve F. Abcouwer University of New Mexico School of Medicine, Albuquerque, New Mexico, U.S.A.

INTRODUCTION Glutamine is the most abundant amino acid in the body, due to its relatively high concentration in the blood and comparatively large stores of free glutamine in muscle. It is found in all proteins, and thus any protein source provides glutamine in the diet. The free form is also found in meat, milk, fruits, and vegetables. However, glutamine is not an essential amino acid, since it can be readily produced from glutamate in all tissues, with muscle tissue being the primary source of glutamine in the blood. Although there is no dietary reference

intake

(for

under

normal

conditions,

glutamine

is not

a necessary

dietary

constituent),

group differentiates glutamine from the closely related amino acid glutamate (L-glutamic acid, Glu, E). The interconversion of glutamine and glutamate by addition and removal of this amide group makes glutamine a convenient nitrogen shuttle and nitrogen donor in many synthetic biochemical reactions. In this way, glutamine can also serve as a source of intracellular glutamate. Because glutamate is a critical intracellular anion,

this function

is vital for the control

of cell

volume. Through glutamate, glutamine carbons can also enter the tricarboxylic acid cycle (TCA cycle). Thus, glutamine can serve as an important source of reducing equivalents (e.g., NAD(P)H, FADH>2) and, ultimately, cellular energy.

during critical illness, severe trauma, intestinal disease,

starvation, total parenteral nutrition (intravenous feeding), wasting (excessive loss of lean body mass), and extreme endurance exercise, the body’s need and consumption of glutamine can exceed the ability of tissues to produce this amino acid. Under such stress conditions, dietary glutamine is beneficial. Hence, glutamine is referred to as a “conditionally essential’ amino acid. It is becoming one of the most popular and profitable nutritional supplements due to claims that consumption can boost immune function and increase muscle mass and volume. However, while glutamine is nontoxic and probably harmless, the benefits of consuming dietary supplements containing this amino acid have not been proven.

BIOCHEMISTRY AND FUNCTIONS Biological Synthesis and Utilization Glutamine is formed directly from glutamate by the addition of ammonia in an ATP-requiring reaction catalyzed by the enzyme glutamine synthetase (GS, E.C. 6.3.1.2). This enzyme is found in the cell cytoplasm. Its function is solely to form glutamine at the expense of cellular glutamate and energy. The amino acid may

ree 5 CF

NAME AND GENERAL DESCRIPTION Amino

Registry Glutamine (1-glutamine, Gln, Q, CAS number 56-85-9) is a nonessential, neutral, polar amino acid, one of the 20 common

in proteins. Its molecular

molecular formula is C;H,)N2O3. Unlike most amino

acids, glutamine contains two nitrogen molecules: one is part of the “alpha amino’’ group, and the other is part of an amide or “amido’’ group of the amino acid side chain (Fig. 1). The addition of the amide

Steve F. Abcouwer,

CH

Ph.D., is Assistant Professor at the Depart-

ment of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico, U.S.A. 20022066 Encyclopedia of Dietary Supplements DOT: 10.1081 /E-EDS-1

Copyright © 2005 by Marcel Dekker. All riglits reserved.

ci

ee

nites

Cr

O

yaan

ig

:

:

|

amino acids found

weight is 146.15, and its

O

|

WY 6a

iG

Amide

group

Fig. 1 The structure of glutamine. Two representations of glutamine: On the left is the structural formula, with the amino nitrogen and amide nitrogen side groups indicated. On the right is a conformational formula. In solution at neutral pH, both the amino and carboxylic acid groups of glutamine are charged. 287

Glutamine

288

be formed because it is needed for synthetic reactions, ammonia detoxification, or for export to other tissues.

Glutamine is readily converted to glutamate by several amidotransferase enzymes and glutaminase enzymes (GA, E.C. 3.5.1.2). The former enzymes transfer the amide nitrogen of glutamine to other molecules in the course of biosynthesis. In this way, glutamine is necessary for the production of other amino acids, purine and pyrimidine bases, amino sugars, and several coenzymes.''! Glutaminase produces glutamate and ammonia. Two GA genes exist: One is expressed exclusively in the liver and is therefore referred to as hepatic glutaminase (hGA), whereas the other is ubiquitously expressed and is referred to as kidney glutaminase (kGA).”! The kidney GA gene gives rise to several isoforms due to alternative splicing of the transcript.) The functional significance of these GA isoforms is not fully known. However, the expression of one isoform known

as GAC

is controlled by acidity, which

has important implications for the control of chronic metabolic acidosis.“

skeleton of glutamate enters the TCA cycle. In this way, glutamate carbons are utilized for anaplerosis (as carbon donor to replenish the tricarboxylic acid cycle) and are oxidized to COz making glutamine an important source of cellular energy."*! Oxidation of glutamine carbons to CO and glutamine to pyruvate (a process referred to as “glutaminolysis’’ in analogy to glycolysis) produces reducing equivalents in the forms of NADH, NADPH, and FADH). These reducing equivalents are utilized for ATP synthesis by oxiand synthetic reactions, dative phosphorylation, 2).04) (Fig. stress oxidative cellular protection against

PHYSIOLOGY Cellular Functions

i

In 1955, Harry Eagle pioneered the growth of mammalian cells in culture. In the course of developing culture media for these cells, he tested the requirements for numerous

salts,

vitamins,

minerals,

carbohydrates,

and amino acids."'*! The scientist discovered that glutaMetabolic Functions

mine was necessary to support the growth and viability of cell in culture, and at concentrations

The normal plasma concentration of glutamine is relatively high, whereas the plasma concentration of glutamate is quite low.:*! Cells also exhibit large capacities for the import of glutamine, much larger than that for glutamate.!” Once inside the cell, glutamine is readily converted into glutamate by the action of amidotransferase and glutaminase enzymes. In fact, in most tissues (with the notable exception of muscle), the intracellular concentration of glutamine is much lower than that of glutamate!°*! Thus, glutamine represents the primary source of intracellular glutamate (for review see Ref.”!), Glutamate is an important intracellular anion, playing a vital role in maintenance of cell osmolarity and, thus, cell volume." Glutamate formed from glutamine is itself indispensable for many cellular processes (for review, see Refs.7!), For example, transamination reactions utilizing the amino nitrogen of glutamate convert certain keto acids to amino acids. In this way, glutamate is central to the cell’s amino acid economy. The anion also serves as a precursor for proline synthesis. It supports the synthesis of the tripeptide molecule glutathione, the cell’s major store of reducing equivalents."") Glutamate does this directly by serving as a substrate for glutathione synthesis, and indirectly by providing a means for the cell to import cysteine, another substrate for glutathione synthesis."7! Glutamate is oxidatively deaminated by the enzyme glutamate dehydrogenase (GDH) to form a-ketoglutarate,

with the concurrent reduction of NAD* (or NADP*) to NADH (or NADPH). As «-ketoglutarate, the carbon

greater than

that of any other amino acid.''*! Eagle and colleagues subsequently determined that both protein synthesis and nucleic acid synthesis were dependent on glutamine") Now it is known that nearly all mammalian cell cultures benefit from the addition of glutamine to their media. Thus, cell culture media is

almost always supplemented with concentrations of glutamine that are an order of magnitude greater than those of other amino acids. However, during all this time, the exact nature of this dependence on glutamine has not been clarified. Perhaps this is because the metabolic functions of glutamine and glutamate are so varied. Indeed, a supply of glutamine is needed to support numerous cellular processes.

Support of Cell Proliferation The need for glutamine is particularly acute for proliferative cells. In the adult, cell proliferation is most active in the intestine, immune system, and during wound healing. Cells within the intestinal epithelium constantly divide to cope with cell loss and renewal. Replacement of damaged epithelial cells through a process of crypt cell proliferation and differentiation along the crypt—villus axis seems to be supported by glutamine."'*! In culture, intestinal epithelial cells are avid glutamine consumers, and their growth is glutamine dependent." Cell growth and replacement also characterize the immune system. In response to immune challenge, immune cells of

599

Glutamine

Nucleotides Nicatinamide

Amino-sugars Asparagine

Glutathione

Alanine

Carbamoyl

Proline

Aspartate

phosphate

Arginine

able

_—y

Serine

NADH

— >» FADH2 /

coo

!

Cc

mA Nn,

NH

|

CH,

Glutamine

’

— > Pyruvate

TAs

| CH,

it

Gs \

Aspartate

>

Glucose

Go|

NH; GA

=>

-NH,

COO aN

CH, |

i

-NH,

ATs

Cesta

CH,

|

Cc mA bG 7

Glutamate

NH, : GDH

S NH

;

Cc Goes

a-Ketoglutarate

Fig. 2 Metabolic functions of glutamine. Once inside a cell, glutamine is readily converted to glutamate by glutaminase enzymes (GA) that produce free ammonia, and by several amidotransferase enzymes (ATs) which utilize the amide nitrogen of glutamine to transfer ammonia to other molecules in synthetic reactions (a partial list is shown). Glutamine is formed from glutamate by glutamine synthetase (GS), utilizing ammonia and ATP (not shown). The relatively high intracellular concentration of glutamate makes it an important osmolyte for the control of cell volume. Glutamate is also utilized for synthesis of glutathione (a tripeptide containing glutamate), proline, and arginine. It is oxidatively deaminated to form a-ketoglutarate and ammonia in a reversible reaction catalyzed by glutamate dehydrogenase (GDH). The forward reaction utilizes NAD(P)* and H,O (not shown). In the reverse reaction, NAD(P)H is oxidized and HO is formed. Transaminase enzymes (TAs) transfer the ammonia from the amino group of glutamate to keto acids, forming new amino acids. For example, alanine is formed from pyruvate and aspartate from oxaloacetate in this manner. Other intermediate reactions in the synthesis of amino acids are catalyzed by aminotransferase enzymes utilizing glutamate, for example, in the formation of 3-phosphoserine from 3-hydroxypyruvate during serine synthesis. As a-ketoglutarate, the carbon skeleton of glutamine enters the tricarboxylic acid cycle (TCA cycle). In this way, these carbons are oxidized to CO>, forming reducing equivalents in the forms of NADH and FADH)j that are utilized for ATP production. From TCA cycle intermediates, the carbon skeleton of glutamine can provide substrates for formation of many metabolic intermediates, including aspartate, pyruvate, and glucose.

both T and B lineage followed by programmed the infection has abided. exhibit marked glutamine tion and proliferation.?°!

undergo clonal expansion cell death (apoptosis) when Immune cells of all types dependence for both activaGlutamine also inhibits cell

death caused by several cellular stresses, and thus has

been referred to as an “apoptosis suppressor.’’!'4]

SYSTEMIC METABOLISM Glutamine Cycling in Brain and Liver Glutamine synthetase occurs in all tissues and is especially abundant in brain and liver. In the brain, GS activity is vital for the conversion of glutamate (one of the most important neurotransmitter molecules) to glutamine by astrocytes.”"! This serves to prevent the accumulation of glutamate, which is toxic at

high levels, and to detoxify ammonia. Glutamine is then transferred from the astrocytes to neurons, which convert it back to glutamate to be released at synapses in response to stimuli (for review, see Ref.) In the liver, another glutamine-glutamate cycle operates.”*! Blood entering the liver from the gut via the portal vein carries waste nitrogen that must be disposed of by conversion to urea. Much of this nitrogen is carried by glutamine. Periportal hepatocytes extract glutamine and convert it to glutamate, producing ammonia that then enters the urea cycle. In addition, the alpha amino nitrogen of glutamine is converted to ammonia by oxidative deamination of glutamate. The alpha amino group of glutamate can also be transferred to oxaloacetate to form aspartate that can enter the urea cycle. Thus, as the blood moves down the liver sinusoids, it is stripped of glutamine. A high level of GS in the liver is concentrated in the pericentral and perivenous hepatocytes.“

Glutamine

290

In the perivenous section of the sinusoid, excess ammonia is scavenged and incorporated into glutamine In this way,

by GS.

ammonia

and

is removed

the

in the outgoing venous

concentration of glutamine blood is elevated.

Glutamine Production by Muscle and Lung The muscle, lung, and adipose tissue are major sources of glutamine in circulation, and these tissues increase

glutamine production during catabolic states. The expression of the GS gene in these tissues is increased in response to stress hormones, principally glucocorticoids.?°! The human GS gene has not been characterized. However, the rat GS gene includes two regions containing glucocorticoid response elements (GRE), which are responsible for increased transcription in response to glucocorticoid hormones.?°! This phenomenon of increased gene expression is particularly evident in muscle and lung tissues.”°! Thus, in response to stress hormones, muscle and lung tissues produce increased amounts of GS mRNA that is translated into GS protein. In

addition,

the

ultimate

accumulation

of

In

times

of stress,

glucocorticoid

hormones

increase GS transcription, and the resulting elevated level of GS mRNA leads to increased GS protein production. Nevertheless, GS protein will not appreciably accumulate unless there is a need for increased glutamine production, as signaled by intracellular glutamine depletion.?” Importantly, stress hormones also signal changes that lead to increased glutamine export from cells.”®! Thus, as these hormones signal for increased GS

expression,

the

intracellular

glutamine

nia,

GDH

catalyzes

the

oxidative

deamination

of

glutamate to form «-ketoglutarate and ammonia. The ammonia formed from these two reactions binds hydrogen ions to form ammonium ions that are eliminated in the urine along with acid anions. During shock, starvation, uncontrolled type-I diabetes, and severe diarrhea, metabolic acidosis can occur due to the increased production of ketoacids (e.g., acetoacetate and B-hydroxybutarate) or the loss of bicarbonate ions. To dispose of these extra acid anions, the kidney greatly increases its utilization of glutamine, thereby producing ammonia. This is accomplished by increasing the expression of GA and GDH within the kidney tubules. The expression of GA (the GAC isoform) and GDH in response to acidosis is controlled by a unique mechanism that involves stabilization of these mRNA by zeta-crystallin in response to acidic pH.47°! The consequence of greater utilization of glutamine by the kidney during acidosis is that the demand for systemic glutamine synthesis is correspondingly increased (Fig. 3).

GS

protein is regulated by a unique feedback mechanism that responds to the need for glutamine synthesis. Namely, the degradation rate of the GS protein is increased by glutamine.”” Thus, when glutamine is abundant, GS protein is rapidly degraded. When intracellular glutamine is depleted, GS protein degradation slows and the GS level increases. In this way, the amount of GS protein is indexed to the need for glutamine.

enzymes glutaminase and GDH, using glutamine as the source.?7! Once GA forms glutamate and ammo-

stores

become depleted. This leads to a synergistic mechanism by which GS protein is produced at an accelerated rate and degraded at a reduced rate. This results in a robust increase in GS activity, until such time that the production of glutamine is sufficient to match the rate of its export.

Control of Acidosis

Glutamine utilization by the kidney is essential for controlling the amount of acid in the blood. This is accomplished by ammonia formation by the kidney

ROLE

IN CATABOLIC

DISEASE

Catabolic States

Catabolic states describe any metabolic situation in which fat and lean body masses are utilized faster than they are restored. However, the term catabolic state is

usually used to describe a pathological state where fat and lean body mass (primarily muscle) is utilized in response to hormonal signals and/or increased metabolic demands that are not being met by nutrient uptake. Severe trauma, burn, infection, starvation, or

chronic diseases such as HIV/AIDS and cancer cachexia may cause it. Increased protein turnover during these states produces greater amounts of waste nitrogen and thus requires increased nitrogen shuttling by glutamine.”"! These states are also associated with ketosis causing metabolic acidosis. Glutamine demand by the kidney is increased to counter this acidosis. In addition,

massive

immune

activation

and expansion

may also cause an increased glutamine demand as immune cells consume more of this amino acid. If infection occurs, the liver increases its consumption of glutamine and other amino acids to support acute phase protein synthesis: If sustained, catabolic states can lead to severe depletion of lean body mass, thereby diminishing the ability of muscle tissue to produce glutamine and satisfy this increased demand, and ultimately leading to impaired immunity, poor wound healing, and loss of intestinal barrier function.22!

Glutamine

291

Adipose

Enteral Glutamine |

= growth Energy Glucose

Blood : =

=N Aspartate Glucose Acute phase proteins

eh Glucose

Parenteral Glutamine

a growth Energy Immunoglobulins

Fig. 3. Systemic glutamine metabolism. Free glutamine in circulation is derived mainly from muscle, and secondarily from lung and adipose tissue. Glutamine is also obtained from the liver and brain (not shown). Blood glutamine can also be provided directly by parenteral nutrition. Glutamine from the diet and in circulation is extracted by the intestine and is used to support cell growth, energy production, and acts as a substrate for gluconeogenesis. The amino acid extracted by the liver is a major carrier of nitrogen that enters the urea cycle. Aspartate formed from glutamine can also enter the urea cycle, carrying both nitrogen and carbon. Glutamine is also a major substrate for liver gluconeogenesis. In times of infection, it aids acute phase protein synthesis by the liver by supporting amino acid synthesis. The kidney uses glutamine as a source of ammonia to neutralize hydrogen ions and eliminate acid anions, and thus control acidosis. Glutamine also serves as a source of energy and as a gluconeogenic precursor for the kidney. Immune cells, both gut-associated and in circulation, consume glutamine to sustain cell growth and provide cellular energy. It also supports the synthesis of immunoglobulins and other proteins by immune cells. (View this art in color at www.dekker.com.)

alanine as nitrogen carriers.?*! Thus, lean body mass is converted into energy, and a mixture of amino acids is released that is dominated by glutamine and alanine.?°! The lung also increases production of glutamine during catabolic states.°”| Adipose tissue has also been implicated as a producer of glutamine.?*! However, the source of nitrogen to support the production of glutamine by these tissues has not been established. Glutamine may be produced from glutamate and ammonia extracted from blood.®*! It is possible that branched-chain amino acids produced in the liver could be extracted by these tissues and used to support glutamine synthesis.2>! Glutamine is an ideal nitrogen shuttle because it can be readily formed from intracellular glutamate, and because each molecule carries two ammonia equivalents. Alanine is ideal as a nitrogen shuttle because it can be readily formed from pyruvate by a single transamination reaction, and as it can then serve as a ready source of energy and glucose when it is converted back to pyruvate. Thus, both glutamine and alanine carry nitrogen and serve as energy sources for visceral tissues. In addition, both serve as major gluconeogenic precursors for the liver, kidney, and intestine.2?-401 Thus, in catabolic states, muscle protein breakdown

and conversion of unknown substrates by the lung produce glutamine that is released by these tissues. The kidney, intestine, immune system, and healing tissues utilize this glutamine. The amino acid also serves as a major precursor for glucose formation. Nitrogen carried by glutamine is disposed of as urea produced in the liver and as ammonia produced primarily in the kidney. If a catabolic state persists, loss of lean body mass diminishes the ability of muscle to produce glutamine and maintain interorgan glutamine flux.

NUTRITIONAL SUPPLEMENTATION Total Parenteral Nutrition (TPN)

Interorgan Transport In severe During catabolic states, stress hormones trigger an increased rate of muscle protein degradation, while decreasing muscle protein synthesis. 7) Mhass free amino acids are produced. The carbon skeletons of branched-chain amino acids are preferentially utilized for oxidative energy production by the muscle, sparing glucose for use by other tissues.24! The muscle releases other amino acids produced from protein degradation, which are then utilized in other tissues, especially those

catabolic

states or situations

where

oral

nourishment cannot be tolerated, intravenous feeding, referred to as TPN, is used. Until recently, TPN formu-

lations did not include glutamine. This was in part due to the fact that glutamine is unstable in solution; it slowly decomposes to form ammonia and pyrrolidonecarboxylic acid." Lack of enteral feeding during TPN

leads to intestinal atrophy.“*) Because glutamine is

in the intestine, kidney, liver, and immune system. In the muscle, the amino groups from oxidation of

such an important substrate for the intestine, it was reasoned that its inclusion in TPN solutions would alleviate intestinal atrophy. Numerous studies using animal models have confirmed this assumption (for

branched-chain amino acids ultimately become waste nitrogen in the form of ammonia. In order to dispose of this nitrogen, muscle tissue utilizes glutamine and

have demonstrated significant benefits in inclusion of glutamine in TPN solutions (for review, see Ref. a):

review, see Ref.!**!). In addition, many human trials

Glutamine

292

Including glutamine-containing dipeptides that are cleaved in the circulation to produce free glutamine solved the problem of glutamine instability.4>! However, the inclusion of up to 25g/day glutamine in TPN solution has very little effect on glutamine concentrations in blood and muscle.'**! In contrast to TPN, the benefits of enteral glutamine supplementation are still being debated.” In fact, several efforts to reverse wasting and cachexia by glutamine feeding have not been successful.48-47! On the other hand, two recent studies°°*!! have shown that

a combination of an oral glutamine, arginine, and the leucine metabolite, B-hydroxy-B-methylbutyrate, was able to inhibit lean muscle loss during HIV/AIDS wasting and cancer cachexia . This has led to a clinical trial of this nutrient combination entitled “Adjuvant Nutrition for Critically Ill Trauma Patients’’ being sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Support of Intestinal Renewal and Function

Glutamine’s ability to support intestinal renewal and function has made it a prime candidate for nutritional support of patients with intestinal diseases, including short-bowel syndrome and inflammatory bowel disease, such as Crohn’s disease (for review, see Ref.7}). However, most studies showing the benefits of enteral glutamine feeding on intestinal repair and function have been conducted using rats and pigs. Although glutamine is now being routinely incorporated into treatments that include various growth factors and nutrients, there are few clinical data to confirm that it has beneficial effects in humans.°?*4) Glutamine feeding is also believed to improve intestinal mucositis caused by chemotherapy or radiation treatment.°°* Results of studies with animals have suggested that it can accomplish this. However, there are not sufficient controlled clinical data on humans to reach a conclusion.>7>*! A recent phase III, randomized, doubleblind clinical study conducted by the North Central Cancer Treatment Group found that glutamine feeding had no effect on acute diarrhea in patients receiving pelvic radiation therapy.!©!

decrease

morbidity

and

reduce

hospital

costs

(for

review, see Ref.°4), However, subsequent clinical trials

have not demonstrated appreciable benefits of glutamine in this patient group. A trial of glutamine in TPN for very low-birth-weight infants found that glutamine reduced the time until these babies could tolerate full enteral feeding (13 vs. 21 days).°] However, glutamine did not reduce the incidence of sepsis

or age at discharge. A clinical trial of glutamine feeding for extremely low-birth-weight babies found that glutamine fed infants did not exhibit greater tolerance of enteral feeding, suffer less necrotizing enterocolitis, or exhibit greater weight gain?! Another study found that enterally fed glutamine was completely metabolized in the gut of preterm infants and had no effect on whole-body protein and nitrogen kinetics.!4) Nevertheless, a clinical trial examining the benefits of

nutritional support including glutamine is now being conducted by the National Center for Research Resources entitled ““Gluconeogenesis in Very Low Birth Weight Infants who Are Receiving Nutrition by Intravenous Infusion.”’

Sickle Cell Disease

It has been suggested that glutamine feeding may alleviate the anemia associated with sickle cell disease. In 1975, a study found that incubation of sickle cells in high concentrations of homoserine, asparagine, and glutamine reduced the sickling of the red blood

cells.'°°! A subsequent study discounted the effects of these amino acids when it was found that they did not restore the deformability of sickle cells and did not raise the minimum gelling concentration of deoxyhemoglobin §, in spite of noticeable morphologi-

cal effects on the cells.°°! On the other hand, recent studies performed at the UCLA School of Medicine found that oral glutamine does improve the redox

state of sickle cells, indicated by increased NADH/

(NADH + NAD‘) ratio.!©” A clinical trial entitled “~-Glutamine Therapy for Sickle now being sponsored by the FDA Products Development.

Cell Anemia’’ is Office of Orphan

Very-Low-Birth Weight Infants

Athletic Performance

Another group of patients who may benefit from glutamine feeding is very low-birth-weight infants. These babies are born with underdeveloped alimentary systems that make them unable to tolerate oral feedings and render them prone to necrotizing colitis and susceptible to sepsis due to poor barrier function of the intestinal epithelium. Several studies have shown that glutamine feeding of premature infants can

Glutamine has recently become one of the most popular dietary supplements. Its use to boost athletic performance and promote muscle gain is based on three observations: 1) Glutamine concentration in the plasma is decreased following extreme endurance exercise, such as marathon

Enhancement

running'®). 2) Muscle

break-

down leads to release of large amounts of glutamine from the muscle®*!, and 3) Glutamine, in its role as

Glutamine

293

the primary source of glutamate, is vital for the maintenance of cell volume.""”! It is rational to believe that consumption of glutamine would prevent depletion of plasma glutamine, even boosting its concentration

in the blood. However, studies in animals and humans have shown that enteral consumption of large amounts of glutamine causes only slight and transient increases

in blood glutamine levels.! If the muscle were no

longer required to supply glutamine, then increasing glutamine supply might be expected to deter muscle protein breakdown. Although this is seemingly logical, there is no scientific evidence that glutamine supplementation, either by enteral or parenteral routes, can decrease muscle glutamine release or increase muscle glutamine import. Both muscle protein breakdown

and glutamine release are hormonally controlled.?*!

Thus, the balance between anabolic and catabolic hormones is more likely to influence net buildup or loss of lean body mass. Furthermore, increasing plasma glutamine concentration would increase muscle cell glutamate concentration, osmolarity, and cell volume only if muscle glutamine uptake is appreciably increased for sustained periods. There is no evidence to suggest that this can be accomplished by oral glutamine consumption. Though only short-term studies have been performed so far, they do not support the hypothesis that oral glutamine supplementation improves athletic performance.!®"””!

supplementation

as an immune

modulator,

and

the

mechanism by which oral glutamine consumption may support immune function have yet to be determined.

CONCLUSIONS In conclusion, glutamine is a nitrogen donor in metabolic reactions and the main interorgan nitrogen shuttle. As a source of glutamate, it is essential for maintenance of cellular volume, amino acid economy,

glutathione, energy, and reducing equivalents. Glutamine is a conditionally essential amino acid, and sey-

eral limited studies have suggested that metabolic support of catabolic patients with glutamine may

improve their condition and speed their recovery.!””! Glutamine, together with other nutrients, may also benefit those with intestinal deficiencies or sickle cell disease. Dietary supplementation is claimed to increase buildup, and mass muscle athletic performance, improve immune function. However, controlled clinical studies have not yet substantiated these claims.

Further, several recent clinical studies have not confirmed that the benefits of glutamine feeding observed in animals can be translated to humans.

REFERENCES Immune System Enhancement

1.

Because immune cells are dependent upon glutamine, this amino acid has been touted as an “immune booster.’’ Even juice vendors offer immune booster additions that include glutamine. Several animal studies have found beneficial effects of glutamine feeding on measures of immune function, especially mucosal immune function.!"! Because glutamine feeding has minor effects on plasma glutamine concentration, this effect is probably not due to delivery to immune cells in the circulatory system. Oral glutamine may directly affect the proliferation and development of gutassociated lymphoid tissue (GALT). These immune cells mature and expand their numbers while residing in the intestine. They are later associated with mucosal

2.

3.

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18 (5), 367-370.

glutamine,

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mine supplementation combined with resistance training in young adults. Eur. J. Appl. Physiol. 2001, 86 (2), 142-149. Kudsk, K.A. Effect of route and type of nutrition on intestine-derived inflammatory responses. Am. J. Surg. 2003, 785 (1), 16-21. Kudsk, K.A.; Wu, Y.; Fukatsu, K.; Zarzaur, B.L.; Johnson, C.D.; Wang, R.; Hanna, M.K.

Glutamine-enriched total parenteral nutrition maintains intestinal interleukin-4 and mucosal immunoglobulin a levels. J. Parenter Enteral Nutr. 2000, 24 (5), 270-274 (discussion 274-275). Garrel, D. The effect of supplemental enteral glutamine on plasma levels, gut function, and outcome in severe burns. J. Parenter Enteral Nutr. 2004, 28 (2), 123. Andrews,

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Melis, G.C.; ter Wengel, N.; Boelens, P.G.; van

Leeuwen, P.A. Glutamine: recent developments in research on the clinical significance of glutamine. Curr. Opin. Clin. Nutr. Metab. Care 2004, 7 (1), 59-70.

of sickling in erythro-

cytes by amino acids. Blood 1975, 45 (1), 45-48.

66.

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Shirahama, K.; Kubota, S.; Yang, J.T. Do amino

acids reverse the sickling of erythrocytes containing hemoglobin S? Hemoglobin 1980, 4 (2), 149-155.

FURTHER

Nuihara, Y.; Zerez, C.R.; Akiyama, D.S.; Tanaka,

For a comprehensive guide to glutamine and sources of information, see ‘Glutamine—A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References, ICON Health Publications,

K.R. Oral L-glutamine therapy for sickle cell anemia: I. Subjective clinical improvement and favorable change in red cell and redox potential. Am. J. Hematol. 1998, 58 (2), 117-121. Castell, L. Glutamine supplementation in vitro and in vivo, in exercise and in immunodepression. Sports Med. 2003, 33 (5), 323-345.

READINGS

March 2004; ISBN: 0597844399. For an update on U.S. government sponsored clinical trials of glutamine, see http://www.clinicaltrials.

gov/ct/search?term=Glutamine.

|

Goldenseal (Hydrastis canadensis) Dennis J. McKenna

Gregory A. Plotnikoff Center for Spirituality and Healing, Academic Health Center, University of Minnesota, Minneapolis, Minnesota,

U.S.A.

INTRODUCTION

are metabolized by P450 3A4. Reported adverse reactions to goldenseal or berberine are rare however.”!

Goldenseal (Hydrastis canadensis L.) is a plant native to North America and is used in its herbal traditions. A principal active ingredient, the alkaloid berberine, is

shared with several medicinal plants used in traditional Asian medicines. Traditional uses include soothing irritated skin and mucous

membranes,

easing dyspepsia,

and reducing debility. Preclinical studies suggest clinically relevant activity for cancer, cardiac diseases, and gastrointestinal and infectious diseases among others. There are no published clinical trials of goldenseal, and most of the available preclinical and clinical data are on the alkaloids berberine and B-hydrastine. Accordingly, much of the information summarized in this entry applies to berberine, and only indirectly to goldenseal, under the assumption that extracts of the

plant containing berberine or B-hydrastine will display activities similar to those of the alkaloids. A few clinical trials of berberine support use for cardiac arrhythmias, congestive heart failure, diarrhea, and protozoal

infection. Berberine has poor oral absorption, but human pharmacokinetic studies have not been published.

Injected,

inhaled,

or skin-absorbed

berberine

affects cytochrome P450 metabolism and may displace albumin-bound bilirubin and pharmaceuticals. In a study of 21 commercial ethanolic herbal extracts potentially inhibitory to cytochrome P450 3A4 (CYP3A4), goldenseal displayed the most pronounced activity, at a concentration of 0.03% of the full strength

preparation."] Thus, there is a significant potential for goldenseal extracts to elicit herb/drug or herb/herb interactions in patients concomitantly taking pharmaceutical medications or other herbal supplements that

Dennis J. McKenna, Ph.D., is Senior Lecturer and Research Associate at the Center for Spirituality and Healing, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, U.S.A. Gregory A. Plotnikoff, M.D., M.T.S., is currently a Visiting Research Fellow at the Department of Oriental Medicine, Keio University School of Medicine, Tokyo, Japan.

Encyclopedia of Dietary Supplements DOK: 10.1081 /E-EDS-120022109 Copyright © 2005 by Marcel Dekker. All rights reserved.

CLASSIFICATION AND NOMENCLATURE Scientific name: Hydrastis canadensis L. Family: Ranunculaceae Common

names: Goldenseal, yellow root, turmeric

root, eye root, Indian dye, yellow puccoon, ground raspberry

H. canadensis (Fig. 1) is a perennial herbaceous plant found

in rich, shady woods

and moist meadows

in

eastern North America, especially in Ohio, northern Kentucky, Indiana, and Virginia, whereas in Canada,

it is restricted to southwestern Ontario.! The name “‘goldenseal’’ comes from the yellow scars left on the rhizome by the stem that bursts forth every spring; these scars look like the imprint of an old-fashioned letter seal. Hydrastis is a Greek word meaning “to accomplish with water.’’?-4) Goldenseal grows to about 30cm in height with a simple, hairy stem, usually bearing a single-lobed basal leaf and two-lobed cauline leaves near the top. The flower

is terminal,

solitary,

and

erect,

with

small

greenish-white sepals and no petals, and blooms in May and June. The fruit is an oblong, compound, orange-red berry containing two black seeds in each carpel. The medicinal rhizome is horizontal, irregularly knotted,

bears

numerous

long slender

roots,

and is

bright yellow with an acrid smell.[*-° Populations of goldenseal in the wild have been greatly diminished in recent years due to overcollection and habitat loss, which has placed this plant on the endangered species list. Due to concerns about overharvesting and increasing market demand, it is now commercially cultivated across the country, especially in the Blue Ridge Mountains. Recently, other species of plants purported to be H. canadensis have been sold as the bulk dried herb on the U.S. wholesale market. This substitution is due to the high market price goldenseal now commands, and the shortage of cultivated supply. Care must be taken in ascertaining

accurate identification of the dried material.”

297

Goldenseal (Hydrastis canadensis)

298

Goldenseal

Fig. 1 Goldenseal (Hydrastis canadensis L.): whole plant, flower, and rhizome. (View this art in color

Hydrastis canadensis

at www.dekker.com. )

HISTORY AND TRADITIONAL USES Goldenseal has been used as both a dye and a medicine in North America. The root of goldenseal supplied Native Americans with a brilliant yellow dye for coloring their clothing and weapons, as well as for painting their skin.“! Goldenseal’s ability to soothe irritated mucous membranes led to its topical and oral use for numerous uncomfortable conditions. Native Americans taught the first European settlers to use goldenseal root to treat skin diseases, ulcers, gonorrhea, and arrow wounds. The Iroquois employed goldenseal for heart troubles,

The

Cherokee

utilized

fevers, and tuberculosis.!®!

it for

cancer

and

general

debility.”! Both tribes used the plant for dyspepsia, appetite improvement, and inflammatory dermatoses. Folk use expanded later to include treatments of sore eyes, hepatitis, and menstrual difficulties. Goldenseal became known as one of the most powerful North American medicinal plants, and was included in the U.S. Pharmacopoeia from 1831 to 1936, and then in the National Formulary until

1960.“ It is now commonly used in the United States as a treatment for canker sores, and sore mouths and throats. Many herbal practitioners advise that topical use, such as gargling with a solution of goldenseal for sore throat, is more effective than similar

amounts taken orally in capsules."°! Today, many North American herbal practitioners consider it to be indispensable for its many purported medicinal effects: digestive, antibiotic, immunostimulatory, antispasmodic, sedative, hypotensive, uterotonic, cholerectic, carminative, antifungal, and antimicrobial.!"!" No

clinical trials have been published to date. Goldenseal’s medicinal effects are primarily attributed to the alkaloid berberine, on which there are the

most preclinical data.''*] Berberine is also found in barberry or Oregon grape root (Mahonia aquifolium Nutt.), another traditional Native American herbal medicine

used for similar symptoms, !"7] Likewise,

in

traditional Asian medicines, berberine-containing species are used for similar indications as in North America. For example, in traditional Chinese medicine, three species of Coptis are used for problems affecting the cardiovascular and gastrointestinal systems.''?) They are widely used in China today for treatment

of congestive heart failure.!'*! Kampo,

the

Japanese herbal medicine tradition, incorporates the berberine-containing Chinese cork tree (Phellodendron amurense Rupr.) as a cooling agent for hot illnesses including irritated skin and membranes.!!?! Ayurveda, a traditional Indian system of herbal medicine, utilizes Berberis aristata for intestinal infec-

tions. Vietnamese

traditional

herbal

medicine

uses

Goldenseal (Hydrastis canadensis)

299

B. asiatica Griff. for dyspepsia, dysentery, eye inflammation, and toothache."">! Some studies have commenced on the other abundant alkaloid, B-hydrastine.”! This is a central nervous system (CNS) stimulant and has direct myocardial and intestinal smooth-muscle-depressant effects.!7!7 The British Herbal Compendium" states that the activity of goldenseal is mainly due to B-hydrastine, which is vasoconstrictive, and active on the nervous, reproductive, respiratory, and cardiac systems. Both berberine

heen

O

Berberine

CH30

ZA

N

OCH3

and §-hydrastine are choleretic, spasmolytic, sedative, and antibacterial; canadine is a stimulant to uterine muscle.!!7]

CHEMISTRY Alkaloids

The primary active constituents of goldenseal are the alkaloids B-hydrastine (1.5-4%) and_ berberine (0.5-6%). The plant contains lesser amounts of the alkaloids canadine (tetrahydroberberine), berberastine, hydrastindine, isohydrastindine, (S)-corypalmine, (S)isocorypalmine, and 1-«-hydrastine (Fig. 2).!'*)

Other Constituents Other constituents include meconin, chlorogenic acid, lipids, resin, starch, sugars, and a small amount of volatile oil.!'7!

Fig. 2 Structures of berberine and B-hydrastine, the major alkaloids of Hydrastis canadensis (goldenseal).

Cardiovascular and Circulatory Functions (In Vitro, Organ Isolates) ; Yao et al.?° reported that 5-hydroxytryptamine-induced Palmery,

Formulation and Analysis High performance liquid chromatography (HPLC) analysis of commercial goldenseal products demonstrates wide variation in berberine, f-hydrastine, alkaloid ratio, and total alkaloid content.

Berberine

content ranged from 0.82% to 5.86%, while that of

B-Hydrastine between 0% and 2.93%."'8] analysis guidelines have been published.!'?!

HPLC

PRECLINICAL STUDIES ON GOLDENSEAL AND BERBERINE As noted above, there have been no clinical and few

Cometa,

berberine could relax muscle contractions.

and Leone?!) have shown

that an

extract of goldenseal exhibits an inhibitory action on adrenaline-induced contractions in rat thoracic aorta in vitro. The constituents responsible were identified as the alkaloids canadaline, berberine, and canadine.

Including the inactive alkaloid B-hydrastine, the alkaloid mixture showed an ICs of 2.86 x 10~’M. Therefore, Palmery, Cometa, and Leone!!! concluded that

the mixture of active alkaloids, producing a greater adrenolytic action than any one alone, acted synergistically. Moreover, acting in a dose-dependent manner, the total extract of the roots and rhizomes was able to inhibit contractions induced by higher doses of adrenaline, whereas

the individual

alkaloids

did not.

The

authors concluded that these alkaloids appear to account for the vasoconstrictive activity of goldenseal that has led to its popular use.

preclinical investigations of goldenseal itself, but a rather large number of studies have investigated the activities of its major alkaloid, berberine. In this entry,

Immune Functions (In Vivo, Male Rats)

we havé reviewed what little preclinical data have been published on goldenseal itself, and have summarized the preclinical and clinical data on berberine.

Rehman et al.?! examined the effects of continuous treatment with a goldenseal root extract on antigen-

Goldenseal (Hydrastis canadensis)

300

specific immunity in male rats over a 6-week treatment period (6.6g in glycerin solvent/L drinking water) compared to glycerin-only treated rats. No significant difference was found in the consumption levels of treated and nontreated controls. Rehman and colleagues recorded changes in immunoglobulins G and M (IgG and IgM), finding no significant difference in IgG levels during the first 3 weeks and a trend toward lower levels in the last 3 weeks of treatment compared to the controls that reached significance on day 42 only. IgM levels became significantly higher in the goldenseal group on day 4 and continued to remain so on days 11 and 15. The effect amounted to an accelerated antibody response, which permitted a more rapid increase in levels of IgM, or an enhancement of “the acute primary IgM response.’’ The authors commented that further studies of immunomodulatory medicinal plants should take the matter of time dependence into consideration to pinpoint times of maximal effects.

Respiratory and Pulmonary Functions (In Vitro, Organ Isolates) A relaxing effect was shown from an ethanolic extract of goldenseal roots in carbachol-precontracted guinea pig trachea.?°! Complete relaxation of carbacholprecontracted isolated guinea pig trachea was obtained from a total extract of the roots in a cumulative dose of 5 mg/ml. Further studies have shown that the constituents responsible are the alkaloids canadaline, canadine, berberine, and hydrastine (ECso 2.4, 11.9, 34.2,

72.8 g/ml, respectively). Although as yet unclear, the activity appears to involve interactions of the alkaloids with adenosine and adrenergic receptors.°7

PRECLINICAL STUDIES ON BERBERINE Cancer Antiproliferative activity

Antimicrobial and Antifungal Activity (In Vitro) Scazzocchio et al.?! evaluated a “‘total’’ standardized extract of goldenseal for relative killing time in a low-density inoculum. The undiluted extract showed

the

most

activity,

and

there

was

corre-

spondingly weaker activity at two lower dilutions. The standardized extract and four derivative alkaloids were least effective against Candida albicans and Escherichia coli. Compared with the alkaloids tested, however,

the undiluted

standardized

extract

In vitro antiproliferation of 6 types of esophageal cancer lines was found from coculturing the cells with berberine (IDso 0.11-0.90 pg/ml).?*! Consecutive intraperitoneal (i.p.) dosing inhibited ascites tumor proliferation in Swiss albino mice and resulted in a 32% increase in life span compared to controls.?*! Chemopreventive activity In vitro, berberine inhibited carcinogenicity of arylamine and its main metabolizing enzyme, N-acetyl-

showed the most potent activity, killing the fungus at 15sec vs. 1-2hr for the alkaloids (canadaline, canadine, berberine, and -hydrastine). Undiluted, berberine was the most potent alkaloid against C. albicans (killing time, 1 hr at 3.0mg/ml), equivalent to the standardized extract at a 50% dilution. Canadaline killed C. albicans at over 2hr. The results suggested that isoquinoline alkaloids with an open C ring, such as canadaline, appear to show greater antimicrobial activity. In most of the

transferase, in human bladder tumor cell colon tumor cells?" and leukemia cells.2°

micro-organisms

Chemotherapy adjunct activity

tested, canadine also showed more

potency than berberine. Goldenseal is a very weak antibiotic against common bacteria such as the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus and Streptococcus pyogenes. For the latter, a weak pathogen, the mean inhibitory concentration of goldenseal was 4000 times higher than that of penicil-

lin.'**! Goldenseal’s value and potential value lie elsewhere. For example, it demonstrates relevant activity against the oral pathogens Streptococcus mutans and Fusobacterium nucleatum.?*!

lines,°®

Berberine displayed dose-dependent in vivo activity against carcinogenesis induced by 20-methylcholanthrene or N-nitrosodiethylamine (NDEA) in mice and

rats.7]

.

Berberine also suppressed tumor induction by proinflammatory tumor promoters teleocidin and 12-O-tetradecanoylphorbol-13-acetate (TPA).@3!

Berberine displayed a synergistic effect with radiation treatment and cyclophosphamide in Swiss albino mice implanted with Dalton’s lymphoma ascites tumor cells.!77!

Cytotoxicity In vitro cytotoxic activity of berberine has been demonstrated in a wide variety of tumor cells including uterine, ovary, and larynx carcinomas,!*“! gliomas,?>! leukemias,°° and hepatomas.?7]

Goldenseal (Hydrastis canadensis)

301

Cardiovascular and Circulatory Functions Antiarrhythmic effects Berberine was shown by Huang et al.?*! to inhibit experimental ventricular arrhythmias induced by

was shown to inhibit ion transport. Based on studies using a human model of intestinal ion transport, the antisecretory activity of berberine appears to be due to a direct action on epithelial cells, possibly through the blockade of potassium channels.'*>!

aconitine, ouabain, and barium chloride in rats by 62%.

Hepatic functions

Cardiotonicity; cardioprotection

Berberine has shown significant pre- and posttreatment hepatoprotective effects in rat models of acetaminophen-induced hepatotoxicity as measured by reduction of serum alkaline phosphatases and serum transaminases. Although the alkaloid does display some ameliorative effects as a pretreatment in CCl, induced models of hepatoxicity, it did not have comparable posttreatment activity. In both instances, berberine was more effective in the animals exposed to acetaminophen than those exposed to CCl,."¢

Berberine significantly reduced creatine phosphokinase release during the reoxygenation period, and ultrastructural damage was reduced.'°”!

Congestive heart failure Berberine and its derivatives have positive inotropic, negative chronotropic, antiarrhythmic, and vasodilator properties, each of which can be beneficial in congestive heart failure.) For rats with verapamil-induced cardiac failure, pretreatment with berberine resulted in significantly less severe cardiac failure compared with untreated controls.4"] In a rat model of cardiac hypertrophy, berberine administered for 8 weeks, beginning 4 weeks after aortic banding, resulted in significant reductions in

whole

ventricular rats./47]

heart,

left

ventricular

size compared

weight,

and

Endocrine and Hormonal

Functions

Adrenal functions

Berberine has exhibited a-adrenergic activities in isolated animal organs.2°*7!

antagonist

left

with control aorta-banded

Hypertension

Immunology:

Berberine alkaloids have displayed potent macrophageactivating activity, in turn inducing cytostatic activity against

Berberine is reported to have an antihypertensive effect at low concentrations (

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Green Tea Polyphenols Shengmin Sang Joshua D. Lambert Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy,

Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A.

Chi-Tang Ho Cook College, Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A.

Chung S. Yang Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, U.S.A.

INTRODUCTION

BACKGROUND

Tea (Camellia sinensis, Theaceae) is second only to water in worldwide popularity as a beverage. The three major types of tea, green, oolong, and black, differ in terms of the manufacture and chemical composition.

Tea is one of the most widely consumed beverages in the world; it has been used for medicinal purposes in China and Japan for thousands of years. More than 300 different varieties of tea are produced from the leaves of C. sinensis by various manufacturing processes. Generally, tea is divided into three types: green (nonfermented), oolong (semifermented), and black (fermented). Green tea and oolong tea are more popular in China, Japan, Korea, and some African

There are numerous studies in humans, animal models,

and cell lines to suggest potential health benefits from the consumption of tea, including prevention of cancer and heart disease. Many of the health benefits have been attributed to the polyphenolic components: Epigallocatechin-3-gallate (EGCG) and the related catechins have been the most widely studied in terms of disease prevention and treatment. The present entry summarizes the data concerning the preventive effects of the green tea polyphenols on heart diseases, diabetes,

neurodegenerative

disorders,

and

cancer.

Greater attention is given to cancer prevention as this area has been most widely studied. Importance is also accorded to the bioavailability and biotransformation of the catechins. Such factors are likely to influence the potential health benefits of tea consumption, and a complete understanding of them will aid in better assessing the beverage’s disease preventive activity.

countries, whereas black tea is preferred in India and

the Western countries. Experimental and epidemiological studies have linked the consumption of tea to reduced risk of cardiovascular diseases and cancer."! These effects have been attributed to its polyphenol compounds. Catechins are the most abundant polyphenols in green tea. A typical cup of brewed green tea contains, by dry weight, 30-40% catechins including EGCG, epigallocatechin (EGC), epicatechin-3gallate (ECG), and epicatechin (EC) (Fig. 1). EGCG is the most abundant

catechin in green, oolong, and

black teas. Green and oolong teas typically contain 30-130mg of EGCG per cup (237ml), whereas

black tea may have up to 70mg of EGCG per cup.”!

Shengmin Sang, Ph.D., is Assistant Research Professor, Dept. of Chemical Biology, at the Susan Lehman Cullman Laboratory for Cancer

Research,

Ernest

Mario

School

of Pharmacy,

Rutgers

University, Piscataway, New Jersey, U.S.A. Joshua D. Lambert, Ph.D., is Research Associate, Dept. of Chemical Biology, at the Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey, U.S.A. Chi-Tang Ho, Ph.D., is Professor II, Dept. of Food Science, Cook

College, Rutgers University, Piscataway, New Jersey, U.S.A. Chung S. Yang, Ph.D., Professor II and Chair, Dept. of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey, U.S.A. Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022069 Copyright © 2005 by Marcel Dekker. All rights reserved.

The main pigments in black tea are theaflavins (Fig. 1) and thearubigins, which are formed by the oxidation and polymerization of catechins during fermentation. The resulting brewed black tea contains 3-10%

catechins,

2—6%

theaflavins,

and

more

than

20% thearubigins.”! Green tea and its constituents have been extensively studied both in vitro and in animal models of carcinogenesis.'"! Whereas these compounds have been shown to be efficacious in a number of animal models of carcinogenesis, the epidemiological evidence regarding the effects of tea consumption on cancer risk in humans is conflicting. Likewise, the primary cancer preventive 327

Green Tea Polyphenols

328

HO

O

a

R

“VOR,

OH OH Epicatechin: R,=R,=H

Theaflavin: R,=R,=H

Epigallocatechin: R,=H; R,=OH

Theaflavin 3-gallate: R,=Galloyl; R,=H

Epicatechin 3-gallate: R,=Galloyl: R,=H Epigallocatechin 3-gallate: R,=Galloyl: R,=OH

Theaflavin 3’-gallate: R,;=H: R,=Galloyl Theaflavin 3,3’-digallate: R,=R,=Galloyl

mechanisms of tea in animal models remain unclear. Although many of the beneficial effects have been attributed to the strong antioxidative activity of the polyphenols, this mechanism has not been firmly established in

Fig. 1 Structures of the major tea polyphenols. (Used with permission from Dr. Chung S. Yang.) E

contrast, 8 weeks of treatment with green tea and black tea had no effect on the incidence of atherosclerotic lesions, low-density lipoprotein (LDL) oxidation, and lipid peroxidation in New Zealand white rabbits.!1"]

animals or humans.! In the present entry, we discuss the disease preventive activities of green tea and tea polyphenols, potential mechanisms by these activities are realized, and the current knowledge of the bioavailability and biotransformation of these compounds.

CARDIOVASCULAR

DISEASE

Animal Studies

Both animal and human studies have suggested a cardioprotective effect for tea.'*! Treatment of nephrectomized rats with 0.25% green tea extract for 4 weeks resulted in attenuation of left ventricular hypertrophy and hypertension.'”! Studies with isolated myocytes showed that green tea extract inhibited ouabaininduced reactive oxygen species (ROS) production and cell proliferation. Both green tea and black tea (1-2% in the diet) have been shown to reduce serum cholesterol levels in hypercholesterolemic rats at least in part by inhibiting the absorption of cholesterol from the gut."! Vinson and colleagues have demonstrated that 1.25% green or black tea (as the drinking fluid) reduced cholesterol (20-29% decrease), triglycerides (20-32% decrease), and lipid peroxides (27-49% decrease) in cholesterol fed hamsters after 15 days of treatment.'©! Treatment of CS7bL/6J apolipoprotein (apo) E-deficient mice with 0.08% green tea extract for 14weeks reduced the number of atheromatous areas in the aorta by 23% and aortic cholesterol and triglyceride levels by 27% and 50%, respectively."°! By

Human

Studies

Studies in humans have yielded mixed results. A Phase II randomized controlled study has shown that consumption of 4 cups/day of green tea but not black tea results in a 31% decrease in the urinary concentrations of 8-hydroxydeoxyguanosine, a marker of oxidative stress, in smokers.!!7! Hirano et al. have reported

that intake of green tea was inversely associated with incidence of myocardial infarction (1-3 cups/day reduced prevalence by 35%), but had no effect on the incidence of coronary artery disease.""*! Another study of 512 subjects in Japan suggested that drinking 2-3 or 4 cups/day was inversely associated with the development of coronary atherosclerosis with odds ratios of 0.5 and 0.4, respectively."4!

NEURODEGENERATIVE

DISORDERS

Green tea and its components, especially EGCG, have been shown in some experiments to inhibit the development of Parkinson’s disease. In a case-control study in China, consumption of 3 or more cups of tea per day was shown to reduce the risk of developing this disease by 28%.''*) Laboratory trials with mice further support a potential protective effect for green tea. Choi et al. reported that oral administration of green tea and EGCG attenuated the development of

Green Tea Polyphenols

329

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced Parkinson’s disease in mice.!'®! EGCG prevented the loss of tyrosine hydroxylase positive cells in the substantia nigrum and decreased the expression of neuronal nitric oxide synthetase (nNOS).!'* Potential mechanisms for the antiparkinsonian effects of EGCG include inhibition of catechol-O-methyltransferase (COMT) and of 6-hydroxydopamine-induced neuronal cell death,!'7'8!

are effective in reducing the incidence and multiplicity of UVB-induced skin tumors, orally administered decaffeinated teas are much less effective. Addition of caffeine restores the activity of the decaffeinated teas.?° A recent study has shown that topical application of caffeine or EGCG to SKH-1 hairless mice that have been pretreated twice weekly with UVB for 20 weeks decreases the multiplicity of skin tumors by 44-72% or 55-66%, respectively. In addition, both

compounds have increased the apoptotic index of the tumors by 56-92% as measured by immunohisto-

DIABETES

chemistry for caspase-3 positive cells.?”!

Obesity and diabetes have become widespread health

problems and contribute to increased risk of other diseases,

including

heart

diseases,

and cancer.

sumption of 1.5L of oolong tea for 30 days has been shown to decrease the plasma glucose levels by 30% in individuals with Type I diabetes.!'*! Likewise, green tea, but not an equivalent amount of caffeine, was shown to increase the 24h energy expenditure and fat oxidation in healthy human volunteers.?°! Sabu, Smitha, and Kuttan have demonstrated that green tea polyphenols [500 mg/kg, intragastric (i.g.)] increased glucose tolerance in normal rats, and daily administration of 50-100 mg/kg, i.g. for 15 days reduced the plasma glucose levels by 29-44% in alloxan-treated rats.?!! In vitro, EGCG inhibited inter-

leukin-1B and interferon-y-induced cytotoxicity in insulinoma cells.?7! The author suggested that this effect may be beneficial in preventing islet cell death in Type I diabetes as these mediators are important in the pathology of that disease. CANCER Animal Studies

Green tea has shown cancer chemopreventive activity against ultraviolet-light, chemical-induced, and genetic models of carcinogenesis. The organ sites include the lung, skin, oral cavity, pancreas, bladder, small

esophagus, stomach, intestine, colon, and

Epidemiological Studies

Con-

liver, pros-

tate.!'?341 Chung et al. have demonstrated that 2% green tea inhibits NNK-induced lung tumors per mouse by ~45%. The corresponding amount of EGCG or caffeine inhibits lung tumor multiplicity by 28% or 15%, respectively.?>! In the inhibition of intestinal tumorigenesis in Apc™" mouse model, EGCG is active, but caffeine is not (unpublished data from our laboratory). It appears that the major active constituent of green tea is EGCG in this model. In addition to polyphenols, caffeine has also been shown to be an important cancer preventive component of tea. For example, Huang et al. have reported that whereas orally administered green tea and black tea

Although data from animal models suggest that tea and its components cancer preventive agents, they are inconclusive. These studies have reviewed elsewhere.!'**7°! Because

of carcinogenesis may be efficacious epidemiologically been extensively of direct contact

between tea constituents and the gastrointestinal tract,

these organs are most likely to be affected by tea. Prevention of gastric cancer by tea has been suggested by at least six case-control studies. In addition, a nested case-control study of gastric cancer in men of the Shanghai cohort in China with greater than 4 yr follow-up has found an inverse correlation between urinary tea polyphenol positivity and risk of gastric

cancer (OR = 0.52; 95% CI = 0.28-0.97).2° Conversely, analysis from two population-based prospective studies in Japan found no association between tea consumption and the risk of gastric cancer over 9- and 7-yr periods.8!! Similarly, whereas Su and Arab have reported that tea consumption by men and women in the United States was significantly inversely associated with the risk of colon cancer,?7! a prospective cohort study by The Netherlands Cohort Study on Diet and Cancer found no association between black tea consumption and risk of colorectal, stomach, lung, or breast cancer

in men or women.'*! Such inconsistent results may be due to a number of confounding factors, including diet, smoking status, age, alcohol consumption, problems quantifying tea consumption, and interindividual differences in cancer susceptibility and in the biotransformation of tea constituents. Mechanisms

of Cancer Prevention

Numerous potential mechanisms have been proposed for the cancer preventive activity of tea and its constituents based on studies with cancer cell lines. In vitro,

tea polyphenols, especially EGCG, have been shown to cause growth inhibition and apoptosis in a number of human

tumor

cell lines including

melanoma,

breast

Green Tea Polyphenols

330

cancer, lung cancer, leukemia, and colon cancer.34371

The relative importance of any of these mechanisms in vivo remains to be determined. In general, biologically

important activities that can be modulated by low concentration of an agent are likely to be more relevant in vivo. One problem faced by most studies is the relatively high concentrations of tea compounds used in in vitro studies. These levels often far exceed those found in animal plasma or tissue following tea consumption.

Antioxidant/pro-oxidant activity

containing

EGCG, as well as other tea polyphenols, has been shown to have strong antioxidant activity in vitro by scavenging reactive oxygen and nitrogen species and chelating redox-active transition metal ions. They have also been suggested to function indirectly as antioxidants through inhibition of the redox-sensitive transcription factors, nuclear factor-kB (NF«B) and activator protein-1 (AP-1), inhibition of “‘pro-oxidant’’ enzymes, such as inducible nitric oxide synthase, lipoxygenases, cyclo-oxygenases and xanthine oxidase, and induction of Phase II and antioxidant enzymes, such as glutathione S-transferases and superoxide dismutases.4*! Several human intervention studies with green and black teas demonstrate a significant increase in plasma antioxidant capacity in humans ~1 hr after consumption of moderate amounts (1-6 cups/day).°?! However,

some

of the effects

tumorigenesis. This enhanced activity can result from activation of one or more mitogen-activated protein kinase (MAPK) pathway.'7! EGCG (5-20M) has been shown to prevent 12-tetradecanoylphorbol-13acetate (TPA) and epidermal growth factor (EGF)induced transformation of JB6 mouse epidermal cells in a dose-dependent manner.'?! This inhibition is shown to correlate with decreased jun N-terminal kinase (JNK) activation leading to inhibition of AP-1 activity. Topical application of EGCG to B6D2 trans- | genic mice, which carry a luciferase reporter gene

are

small,

and

some

studies do not demonstrate such an effect. The results now available are insufficient to draw a firm conclusion,

and additional human intervention studies with more specific markers are needed. In contrast to the potential antioxidative activity of tea polyphenols, recent experiments have suggested that the cell-killing activity of these compounds, at least in vitro, may be related to their pro-oxidant activity. For example, we have shown that EGCG-induced apoptosis in H661 lung cancer cells and Ras-transformed human bronchial cells is completely or partially blocked by

the AP-1

binding

sequence,

results

in a

60% inhibition of UVB-induced transcription of the luciferase reporter gene.!*7! NF«B activation by both lipopolysaccharide and tumor necrosis factor « (TNFa) is restrained by EGCG-mediated inhibition of inhibitor «B (IxB) phosphorylation and degradation. In cultured intestinal epithelial cells, EGCG

is found

to be the most

potent inhibitor of IxB kinase activity among green | tea catechins, with an ICs o of approximately 18 uM."*7! Since NF«B is known to be antiapoptotic, its prevention by tea polyphenols is expected to increase apoptosis. We have demonstrated that EGCG and theaflavin digallate (TFdiG) affect different steps in the Ras— MAP kinase signaling pathway (Fig. 2).'“*! Treatment of 30.7b Ras

12, Ras-transformed

mouse

epidermal

cells, with 20uM of either compound results in decreased levels of phosphorylated Erk1/2 and

MEK1/2.

EGCG

inhibits

the association

between

Raf-1 (an upstream protein kinase) and MEK1. TFdiG and EGCG can also directly prevent the kinase activity of this protein by competing with Elk-1 for access to

the inclusion of catalase in the medium.°7*°! Catalase,

however, does not affect the growth inhibitory activity of EGCG. In the presence of HT29 cells in McCoy’s 5A medium, treatment with 501M EGCG results in the production of up to 23 uM H>0>.4" Both of these

observations suggest a role for H,O> in some of the observed activities of EGCG in vitro. It is not known whether this mechanism is relevant in vivo.

Inhibition of protein kinases, transcription factors, and growth factor signaling Enhanced activities of the transcription factors, AP-1 and NF«B, have been implicated as key events in some

carcinogenesis pathways, including UVB-induced skin

Fig. 2. Inhibitory mechanism of EGCG and TFdiG on the Ras-MAP kinase pathway (Ref.'*)), (Used with permission from Dr. Chung S. Yang.)

Green Tea Polyphenols

331

the active site. TFdiG, but not EGCG, enhances the degradation of Raf-1/44! Similarly, treatment of Ras-

Other potential mechanisms

transformed human bronchial cells with EGCG and TFdiG has been shown to restrain c-jun and Erk1/2 phosphorylation as well as the phosphorylation of

EGCG has also been shown to inhibit many enzyme activities that may contribute to the prevention of carcinogenesis (reviewed in Ref."°!). These » include the inhibition of: topoisomerase I activity in several human colon carcinoma cell lines (by 3-17uM EGCG), chymotryptic activity of the 20S proteasome

EIK-1

and

MEK1/2,

which

are

downstream

and

upstream of the MAP kinase cascade, respectively.4°! Overexpression of growth factor and its receptors such as EGF, platelet-derived growth factor (PDGF), and others can result in enhanced proliferation by cancer cells.'*°! Selective blockade of growth factor receptor-mediated signal transduction, either by competition with ligand or inhibition of kinase activity, has been shown to inhibit cell growth and induce apoptosis. Using in vitro kinase assays, Liang et al. have demonstrated that EGCG potently and selectively inhibits the kinase activity of EGF-R, PDGFR, and fibroblast growth factor (FGF)-R with ICs of 1-2uM."*! In A431 human epidermoid carcinoma cells, pretreatment with 5uM EGCG completely abolishes ligand-induced autophosphorylation. The fact that preincubation is required to demonstrate this effect suggests that the auto-oxidation of EGCG may denature the EGF-R, resulting in lower ligand binding and autophosphorylation.

in leukemic,

breast cancer,

and

prostate

Tea catechins have been shown to undergo extensive biotransformation and to have low bioavailability

(Fig. 3).!'4°] Whereas cell-line studies typically require OCH3

OH

OH OV

HO

COMT

ONG

HO.

Oo= droxypheny ) ydroxyphenyl -(3',4'-Dihy 5-(3',4'-Dih

'

Shape

-g-valerolactone (M6)

EC

OH

‘OH

LON

“4

OH

ww

r

—$—$——_—_ Oo."

fs

SAME SAH

a

\

OH

3'-MeEC

OCH3

OH

‘|

HO

OH

OES

oO

:

OH

OH

ee icroflora

he

ae

a

ay

OH

cell

BIOTRANSFORMATION AND BIOAVAILABILITY OF TEA POLYPHENOLS

OH

OH

cancer

lines (1-10 uM EGCG), and matrix metalloproteinases (MMPs) 2 and 9 (9-13 uM EGCG). It is evident that different pathways may be involved in the protective effects that tea polyphenols exhibit. The relative importance of each of these potential mechanisms in vivo depends on whether effective tissue concentrations of the tea polyphenols can be achieved. In most cases, this remains to be determined.

Ou

tom

SAH

SAM

‘OH

ORs

HO

COMT

OH | 4-MeEGC

ae '

5-(3',4',5'-Trihydroxyphenyl)

-g-valerolactone (M4)

UGT/SULT

Catechin and MeCatechin

Conjugation

Conjugates OH igh

oe ORR

ee

Be Sater.

m

SAM

5-(3',5 Hoasi ipiiewil

~

SAH

OH

.

COMT 3

j

"0

OH

OH

O

-g-valerolactone (M6’)

EGCG 0H

On

OCH3

:

4',4"-DiMeEGCG

OH

(Re f.4¢l) (Used with permission from Dr. Chung Fig. 3. Potential Phase II biotransformation pathways for the tea catechins S. Yang.)

Green Tea Polyphenols

332

concentrations of compounds in the 5S—100 uM range, such levels are not generally observed systemically. The low bioavailability of the tea polyphenols is likely due to their relatively high molecular weight and the large number of hydroxyl groups, which not only serve as functional handles for Phase II enzymes but may also reduce the absorption of the compounds from the intes-

tinal lumen.” Correlating mechanistic data in vitro with effects in vivo should be done with careful consideration of the poor bioavailability of the tea polyphenols.

intravenous

and

In humans,

EGC

has been detected mainly as the

glucuronidated form (57-71%) or sulfated form (23-36%) with only a small amount present as the free form (3-13%).°!! Methylation of EGC also occurs in humans leading to the formation of 4/-O-methyl EGC, which is present mainly as the glucuronide or sulfate conjugate.'"! In contrast, the sulfated form of EC is more abundant (66%) than the glucuronidated form (33%),?"! whereas EGCG is present mainly in the free form in plasma.! Recent work in our laboratory has shown that EGCG is time- and concentrationdependently sulfated by human, mouse, and rat liver cytosol.>3! The rat has the greatest activity, followed by the mouse and the human. Further studies are required to determine the structure of these sulfated metabolites. In addition to these conjugation reactions, the tea catechins undergo metabolism in the gut to form the ring-fission products 5-(3',4’,5'-trinydroxyphenyl)y-valerolactone (M4), 5-(3’,4’-dihydroxyphenyl)-yvalerolactone (M6) and _ 5-(3',5'-dihydroxypheny]l)y-valerolactone (M6’) (Fig. 3).°4°°! We have found that these ring-fission products are present in human urine (4-8M) and plasma (0.1-0.2,.M) approximately 13hr after oral ingestion of 20mg/kg decaffeinated

green

tea.>4] M4,

M6,

and

M6’

retain

the

polyphenolic character of the parent compound, and have the addition of a potentially, biologically active valerolactone structure. Pharmacokinetics

The pharmacokinetic parameters of the tea catechins have been thoroughly determined in the rat by both

The

and 41 min, respectively. The absolute bioavailability of EGCG, EGC, and EC following i.g. administration of decaffeinated

green

tea is 0.1%,

14%,

and

31%,

respectively.°° In another study, the EGCG levels in the tissues and blood correspond to 0.0003-0.45% of the ingested dose, further demonstrating the poor

bioavailability of EGCG in the rat.” Studies with bile :

administration

The catechins are subject to extensive biotransformation including methylation, glucuronidation, sulfation, and ring-fission metabolism (Fig. 3). Methylated forms, have been observed in the rat with 4’,4’’-di-Omethyl-EGCG being the major metabolite detected in the bile of the rat following oral EGCG administration.48°" Rat liver cytosol shows higher COMT activity toward EGCG and EGC than do human or mouse liver cytosol.

of administration.

kinetics of i.v. EGCG, EGC, and EC fit to a two-compartment model with elimination half-lives of 212, 45,

duct-cannulated

Enzymology of Biotransformation

ig. routes

rats

have

of 100mg

shown

EGCG,

that 3.28%

after

oral

of the dose

is recovered in the bile as EGCG (2.65%) and methylated metabolites (0.63%).'*8! With the exception

of 4”-O-methyl-EGCG and 4',4”-di-O-methyl-EGCG, which were present as the sulfated form, the other metabolites and EGCG are present largely (>58%) as the glucuronidated form.

Studies of PH]-EGCG in both the rat and the mouse have shown that following a single i.g. dose, . radioactivity is found throughout the body.©**) After 24h, 10% of the initial dose (radioactivity) is in the blood with 1% found in the prostate, heart, lung, liver, kidney, and other tissues. Excretion in the feces is the

major route of elimination with 25-30% of the total radioactivity excreted after 24h. In the rat, i-v. administration of [>H]-EGCG resulted in 77% of the dose being excreted in the bile and only 2% in the urine,“°! Treatment of rats with a green tea polyphenol preparation (0.6% w/v) in the drinking fluid has been shown to result in increasing plasma levels over a 14-day period with levels of EGC and EC being higher than those of EGCG.&'! Plasma levels then decrease over the subsequent 14 days suggesting an adaptive effect. EGCG levels have been found to be highest in the rat esophagus, intestine, and colon, which have direct contact with tea catechins, whereas

they are lower in the bladder, kidney, colon, lung, and prostate. When the same polyphenol preparation is given to mice, the levels in the plasma, lung, and liver are much higher than in rats. These levels appear to peak on day 4 and then decrease to less than 20% of the peak values in days 8-10.64 In mice, the absolute bioavailability of EGCG

has

been found to be higher than that in rats (26.5% vs. 0.1%). Concentrations of EGCG in the small intestine and colon are 45 and 7.9 nmol/g following i.g. administration of 75mg/kg EGCG. The levels in other tissues are less than 0.1 nmol/g. Following i.v. administration of EGCG, levels are highest in the liver (3.6nmol/g), lung (2:7nmol/g), and small intestine (2.4nmol/g). While greater than 50% of plasma EGCG is found as the glucuronide, EGCG is found mainly as the free form in the tissues.!©7! Several studies of the systemic bioavailability of orally administered green tea and catechins in human

Green Tea Polyphenols

333

volunteers have been conducted. Most recently, we have shown that oral administration of 20mg green tea solids/kg body weight results in Cmax in the plasma for EGC, EC, and EGCG of 728.8, 427.6, and 170.1 nM, respectively.!*! T,,., has been found to range from 1.3 to 1.6hr with tyoB of 3.4, 1.7, and 2hr for EGCG, EGC, and EC, respectively. Plasma EC and EGC

are present mainly in the conjugated

well-designed intervention and prospective epidemiological studies.

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F.L. Synthesis of (—)-[4-3H]epigallocatechin gallate and its metabolic fate in rats after intrave-

61.

YangweC.S.) Chenwele, LeeaMoe Balentine, D.; Kuo, M.C.; Schantz, S.P. Blood and urine levels

tion to mice. J. Nutr. 2003, 733 (12), 4172-4177. Lee, M.J.; Lambert, J.D.; Prabhu, S.; Meng, X.; . Lu, “Hy; |Maliakal;. Ps" Hos Giise Vanes Gs.

Delivery of tea polyphenols to the oral cavity by green tea leaves and black tea extract. Cancer Epidemiol. Biomark. Prev. 2004, /3 (1), 132-137.

67.

Lee, M.J.; Lambert, J.D.; Prabhu, S.; Meng, X.;

LujoH:; ‘Maliakal,’ PgeHo) |CA.arYang) Gs: Delivery of tea polyphenols to the oral cavity by green tea leaves and black tea extract. Cancer Epi. Biomark Prev. 2004, 73 (1), 132-137.

Hawthorn (Crataegus) Werner R. Busse Wiltrud Juretzek

Egon Koch Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany

INTRODUCTION

Small false fruits (berries) are formed, and are red, black, or yellow and mealy.

Hawthorn is one of the most popular herbal medicines for the heart worldwide. In Europe, particularly in Germany, Austria, and Switzerland, hawthorn preparations are eligible for marketing authorization as drugs for the treatment of mild forms of heart insufficiency. In the United States, hawthorn products are regulated as dietary supplements. Numerous preparations are available, including teas, homeopathic preparations, and tinctures as well as simple and standardized extracts. Different starting materials are used. Extracts

are prepared from leaves, flowers, fruits, or

leaves and flowers. A substantial number of hawthorn products in the United States consist of comminuted hawthorn leaves, flowers, or fruits. Over the last decade, the pharmacodynamic

action

of standardized extracts from leaves and flowers as well as different fractions thereof has been extensively evaluated in in vitro studies and animal experiments. Similarly, the clinical efficacy of hawthorn extracts has been assessed in more than a dozen double-blind,

placebo-controlled clinical trials. The results of these studies are reviewed in this entry.

GENERAL

DESCRIPTION

Plant Description

Starting Material The starting material consists of collected wild plant parts. Leaves and flowers

Hawthorn leaves and flowers consist of the whole or cut, dried, flower-bearing branches of C. monogyna Jacq. (Lindm.), C. laevigata (Poir.) DC. (C. oxyacanthoides Thuill.), or their hybrids or, more rarely, other European Crataegus species, including C. pentagyna Waldst. et Kit ex Willd., C. nigra Waldst. et Kit, and C. azarolus L. The preparation should contain not less than 1.5% flavonoids, calculated as hyperoside (C2;H290}2; M, 464.4), with reference to the dried substance (European Pharmacopoeia; EP). The United States Pharmacopoeia (USP) only recognizes the first two species. According to the USP, the preparation should contain not less than 0.6% C-glycosylated flavones, expressed as vitexin (Co,H290}2), and not less than 0.45% O-glycosylated flavones, expressed as hyperoside (C2,H290j9), calculated on a dry basis (USP-NF).

Fruits (berries)

Hawthorn species (Crataegus L.; family Rosaceae) grow as shrubs or trees with hard wood and generally thorny twigs throughout the temperate zones of the world. Leaves are more or less lobed, with margins typically slightly serrated. Flowers are arranged in clusters, and are mostly white and sometimes red.

Hawthorn berries consist of the dried false fruits of C. monogyna Jacq. (Lindm.) or C. laevigata (Poir.) DC., or their hybrids, or a mixture of these (EP). They should contain not less than 1.0% procyanidins, calculated as cyanidin chloride (C,2H;;ClO¢; M, 322.7) with reference to the dried product (EP). Constituents of the Starting Material

Werner

R.

Busse,

Ph.D.,

is Director,

International

Division,

Leaves and flowers

Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany. Wiltrud Juretzek, Ph.D., is at Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany.

The major constituents are flavonoids (up to 2%) such

Egon Koch, Ph.D., is Head of the Department of Pharmacology at Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany.

and vitexin as well as procyanidins

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022112 Copyright © 2005 by Marcel Dekker. All rights reserved.

as

vitexin-2’’-O-a-L-rhamnoside,

hyperoside,

rutin,

formed

by the 337

Hawthorn (Crataegus)

338

condensation of catechin and/or epicatechin with varying degrees of polymerization (Fig. 1). The most important are oligomeric procyanidins (OPCs) containing 2-8 monomeric units, e.g., the dimeric procyanidin By (Fig. 2). The content of OPCs is approximately 3% {4 Further constituents are triterpenoid acids (approximately 0.6%), e.g., ursolic, oleanolic, and crataegolic

acid, and phenol carboxylic acids such as chlorogenic and caffeic acid, as well as various amines."

Fruits (berries) Fig. 2

The fruits contain relatively low levels of flavonoids. The procyanidins contained in the fruits reportedly have a higher degree of polymerization than those in the leaves and flowers. Total procyanidins amount up to 3% of which about 1.9% is OPCs. Triterpenoid acids are also present in the fruit (approximately 0.45%).

Procyanidin Bp.

Preparations Hawthorn

extracts from

are characterized

leaves,

flowers,

and

fruits

by different quantitative flavonoid

patterns." Leaves and flowers

Extracts are produced from the herbal product by a suitable procedure using either water or a hydroalcoholic solvent equivalent in strength to a minimum of 45% ethanol. Aqueous extracts contain a minimum of 2.5% flavonoids and hydroalcoholic extracts a minimum of 6.0% flavonoids expressed as hyperoside (dried extracts) (EP). Standardized dry extracts are adjusted to 18.75% olligomeric procyanidins (WS® 1442; extraction solvent 45% ethanol) or 2.2% flavonoids (LI 132; extraction solvent 70% methanol), with a ratio of starting material to genuine extract (DER) of 47:1. The daily recommended dose is currently set at 160-900 mg in 2 or 3 divided doses.!'~*!

Fruits (berries)

HO

~~

“OH

OH

Cc OH HO

re) ee : OH

“OH

Extracts are produced either from dried fruits, in compliance with the European Pharmacopoeia using alcohol (25-60%, v/v), or from fresh fruits. To date, no official monograph is available advocating the use of preparations from hawthorn fruits. Water extracts, water—alcohol extracts, wine infusions, and fresh juice

from hawthorn fruits have been utilized traditionally to strengthen and invigorate heart and circulatory function.

OH

Fig. 1 (A) Vitexin [B-p-glucopyranosyl-5,7-dihydroxy-2(4-hydroxyphenyl)-4H-1-benzopyran-4-one]; (B) hyperoside [2-(3,4-dihydroxyphenyl)-3-(B-p-galactopyranosyloxy)-5,7-dihydroxy-4H-1-benzopyran-4-one]; (C) .-epicatechin [QRcis)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran3,5,7-triol].

ACTION AND PHARMACOLOGY Pharmacological investigations with Crataegus preparations have been reported in a great number of publications. Unfortunately, many of these studies have been performed with insufficiently characterized

Hawthorn (Crataegus)

339

extracts: Information on plant species, plant parts, and solvent and production conditions, for example, is not

provided. Moreover, the applied pharmacological models and experimental details are frequently ill defined. Thus, formation of a clear judgment on the pharmacological activities of many of these products is not possible. Almost all clinical studies reported until now have been performed with two different extracts, prepared from leaves and flowers of selected Crataegus species with either 70% methanol (LI 132) or 45% ethanol (WS® 1442).* The present entry will mainly review the pharmacological actions of these well-defined preparations.

Positive Inotropic Action

At concentrations between 30 and 180yg/ml, LI 132 was found to raise the contraction amplitude of isolated cardiomyocytes of rats by up to 53%!” and to improve oxygen utilization in comparison to fadrenergic agonists or the cardiac glycoside ouabain. It is supposed that the inotropic action of LI 132 may be due to enhanced intracellular Ca** sensitivity. An increase of contraction amplitude was also observed in electrically stimulated canine papillary muscles." In isolated, electrically stimulated left ventricular muscle strips of human failing myocardium, WS 1442 significantly augmented force of contraction by about 30% (50ug/ml) and improved the frequency-dependent force generation.”! In normal human myocardial

tissue, WS

1442 raised the Ca** gradient as well as

the force generation and from cell membranes. As the activity of adenylate mechanism of WS 1442 the cAMP-independent cardiac

glycosides.

displaced bound *H-ouabain the extract did not influence cyclase, the pharmacological is suggested to be similar to positive inotropic action of

However,

this

conclusion

is

weakened by the fact that an extract fraction enriched for water-soluble low molecular weight constituents displaced *H-ouabain but did not elicit any inotropic effect. Likewise, a significant dose-dependent effect of WS 1442 on the shortening of isolated and electrically stimulated myocytes isolated from right atria and left ventricles of failing human hearts has been reported,!'° Using an isolated guinea pig heart preparation, two independent research groups"'!!7] observed a maximal increase of contraction force between about 10 and 20% at concentrations of 10 pg/ml LI 132.

Increase of Coronary Flow and Vasorelaxing Effects An increase of coronary flow has repeatedly been reported after perfusion of isolated hearts with medium containing ill-defined hawthorn extracts. These earlier observations were confirmed by a comprehensive study investigating the influence of LI 132 on different functional parameters in isolated guinea pig hearts. At a concentration of 3yg/ml, LI 132 maximally enhanced coronary flow by 64% A similar effect was brought about

by amrinone

and milrinone,

while epinephrine

had only a marginal effect, and digoxin concentrationdependently reduced coronary perfusion!" Since the nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine and the soluble guanylyl cyclase inhibitor ODQ completely abolished the increase in coronary flow induced by WS 1442 in the isolated rat heart, it has been concluded that Crataegus extracts increase the endothelial release

of NO."'*! Endothelium-dependent NO-mediated relaxation induced by an extract from hawthorn fruits has also

been observed in rat mesenteric arteries''*! and by another extract and a procyanidin-enriched fraction in

the rat aorta.!'°! Siegel et al. proposed that dilatation of normal and arteriosclerotic human coronary arteries mediated by LI 132 could be due to the opening of K* channels, an action compatible with enhanced NO synthesis. Therefore, relaxation of the noradrenalinprecontracted rat aorta has been proposed as a bioassay to investigate the pharmacological equivalence of different hawthorn extracts.!'* In unanesthetized dogs, the effect of oral (p.o.) treatment with WS 1442 on local blood flow in the myocardium of the left ventricle was measured by means of chronically implanted heat-conduction probes. WS 1442 led to a dose-dependent temporary rise in blood flow, and repeated application caused a sustained increase of basal blood flow.!!7! In a pilot study, the effect of LI 132 on the microcir-

culation in the mesenteric vessels of rats was compared with those of B-acetyldigoxin by intravital microscopy. Compared with digitalis, the Crataegus extract improved the erythrocyte flow rate in all investigated vessel types, and reduced both leukocyte adhesion to the endothelium and leukocyte diapedesis.""*!

Antiarrhythmic Effects Evidence for an antiarrhythmic potential of LI 132 was provided by Poepping et al.,!71 who observed a prolongation of the refractory period in isolated rat cardiac myocytes. Similarly, in isolated guinea pig hearts, an increase in left ventricular pressure and coronary flow was obtained, while at the same time, the duration of the

*WS® 1442 is a registered trademark.

refractory period was prolonged."""! This combination

Hawthorn (Crataegus)

340

of effects was unique among inotropic drugs, as epinephrine, amrinone, milrinone, and digoxin shortened the effective refractory period in a concentrationdependent manner. In guinea pig papillary muscles, LI 132 was observed to significantly increase action potential duration and time required for recovery of the maximum upstroke velocity of the action potential. These effects indicate class III and class I antiarrhythmic

effects, respectively.!'*! Using the patch-clamp tech-

nique, the researchers who conducted this experiment subsequently obtained evidence that the prolongation of the action potential duration in isolated guinea pig ventricular myocytes is due to a weak blockade of both the delayed and the inward rectifier potassium current."'7] These investigators also attempted to get information on the mechanism responsible for the positive inotropic action of LI 132. As no influence on the L-type calcium current was detected, an inhibition of phosphodiesterase or a B-sympathomimetic action, which had previously been proposed to account for the cardiotonic action of hawthorn extracts, can be excluded. In vivo, antiarrhythmic effects of Crataegus extract WS 1442 were investigated in a rat model of ischemia/reperfusion-induced arrhythmia. Oral treatment for 7days (100 mg/kg/day) effectively protected animals from reperfusion-induced arrhythmias, mortality, and hypotensive crisis following 7 min of occlusion of the left coronary artery. Treatment with the extract, however, did not modify the elevated plasma creatine

kinase concentrations during reperfusion.!"7! The influence of intake of a diet containing 2% LI 132 for 3mo on the incidence of reperfusion arrhythmias was also studied ex vivo in isolated rat hearts after global no-flow ischemia. Depending on the duration of ischemia, the average prevalence of malignant arrhythmias was significantly reduced by up to 83% in hearts of treated animals.?°! However, in a recent publication, no protection against reperfusion-induced arrhythmias in isolated hearts was reported after 8 weeks’

treatment (0.5 g/kg/day) of Wistar rats with LI 132.7!) Cardioprotective Effects Besides protecting against arrhythmias, hawthorn extracts have also been shown to prevent the leakage of intracellular enzymes upon ischemic injury. For this investigation, male Wistar rats were fed for 3 mo with a diet containing 2% Crataegus extract LI 132. As an index of myocardial cell damage, the concentration of lactate dehydrogenase (LDH) was determined in the perfusate of the isolated heart. LDH activity increased slightly during occlusion of the left coronary artery and was elevated dramatically after reperfusion.

However,

in treated animals,

pressed significantly

(control:

LDH

release was

sup-

3795 + 512mU/min;

LI 132: 1777 + 452mU/min).”7!

Fractionation of WS 1442 established that its cardioprotective effect is almost exclusively due to its standardized content of 18.75% OPC. A subfraction of WS 1442 enriched for OPCs was found to exert potent antioxidative action and to inhibit the enzymatic activity of neutrophil elastase.) Since restoration of blood flow into a previously ischemic tissue is , associated with the formation of oxygen free radicals as well as the accumulation and activation of leukocytes, it has been suggested that these activities may contribute to protection against reperfusion injury. Since ischemia lasting for more than 20-30 min causes irreversible tissue damage and cell death, Veveris, Koch,

and Chatterjee'4! now evaluated whether treatment of rats with WS 1442 also improves cardiac function and prevents myocardial infarction during prolonged ischemia and reperfusion lasting for 240 and 15 min, respec-

tively. Oral administration of WS 1442 (10 100mg/kg/day) for 7days before ligation of the coronary artery dose-dependently suppressed decrease of the pressure rate product. Treatment attenuated the elevation of the ST segment in ECG,

diminished

or left the also the

the incidence of ventricular fibrilla-

tions, and reduced the mortality rate. Furthermore, the area of myocardial infarction within the ischemic zone was significantly smaller in treated rats when compared with controls. It is suggested that these pharmacological effects are accounted for by the combined antioxidative,

leukocyte elastase-inhibiting, and endothelial synthesis-enhancing properties of WS 1442.

NO

Other Pharmacological Activities Koch and Chatterjee!*! investigated the effects of WS 1442 in a rat model of endotoxin shock, in which the

observed cardiovascular pathologies are suggested to be mediated by an excessive formation of oxygenderived free radicals and enhanced production of NO by inducible NO synthase. Oral treatment (100 mg/kg) lhr before injection of endotoxin significantly inhibited the endotoxin-induced deterioration of cardiac output and prevented an increase in peripheral resistance. As WS 1442 had no effect on heart rate and mean arterial blood pressure, the increased cardiac output in treated animals appears to be due to improved ventricular diastolic filling and/or enhanced myocardial contractility. The beneficial effects of WS 1442 in endotoxin shock may also be related to its

positive effects on endothelial NO

synthesis and/or

its antioxidative properties. Platelet-derived growth factor (PDGF) has been reported to play an important role in the pathogenesis

.

Hawthorn (Crataegus)

341

of atherosclerosis as well as restenosis after angioplasty. Since polyphenols have been reported to inhibit tyrosine phosphorylation of the PDGF receptor beta (PDGFR-B) and Crataegus extracts are rich in these constituents,

the effect of WS

1442 on the

PDGF signal transduction pathway and neointimal formation were investigated in a rat balloon angioplasty model. WS 1442 concentration-dependently inhibited phosphorylation of the human PDGFR-B (ICs 1.4ug/ml). In PDGF-stimulated NIH3T3 fibroblasts, auto phosphorylation of PDGFR-f and DNA synthesis were suppressed half-maximally at concentrations of 31 and 3.1 g/ml, respectively. Oral treatment (300mg/kg) of rats from day 2 before to day 13 after carotid artery balloon angioplasty significantly reduced neointimal formation and increased the luminal area in parallel. These results provide evidence that WS 1442 may also have therapeutic potential for the prevention of restenosis in humans.?° Hypocholesterolemic activity of different hawthorn extracts has been reported by two research teams. In one study, rats fed with an atherogenic diet were treated for 6 weeks with a tincture prepared from the fruits of C. oxyacantha. In the treated animals, the total as well

as the low-density-,

very-low-density-,

and high-density-lipoprotein (LDL-, VLDL-, and HDL)-cholesterol concentrations were significantly reduced. Detailed investigations revealed that application of the tincture enhanced cholesterol uptake into the liver by an elevated expression of LDL receptors. However, accumulation of cholesterol in the liver was

prevented by suppression of cholesterol biosynthesis as well as increased cholesterol degradation.” The same authors reported that under identical experimental conditions, the tincture reduced lipid peroxidation and precluded atherosclerotic changes in the aorta of treated rats. In addition, decrease of the glutathione and a-tocopherol content of the liver, aorta, and heart was inhibited.?*! The total serum cholesterol and triacylglycerol concentrations were also decreased in hamsters that were fed for 4weeks with a hypercholesterolemic diet containing an ethanolic extract from the fruits of C. pinnatifida (0.5%). Treatment with the extract led to greater excretion of both neutral and acidic sterols. Enzymatic tests indicated that the hypocholesterolemic activity may be mediated by upregulation of hepatic cholesterol-7x-hydroxylase and :cholesterol downregulation of intestinal acyl-CoA

acyltransferase.” Other pharmacological effects reported for different hawthorn extracts include stimulation of superoxide dismutase activity in erythrocytes of treated mice,®°! inhibition of thromboxane A» (TXAz2) synthesis and a stimulation of prostaglandin I, (PGI) production,°"! and inhibition of angiotensin converting enzyme.7!

Pharmacokinetics

The absorption and distribution of '*C-labeled catechins tri- and higher polymeric procyanidins, and OPC total fraction have been determined in mice after oral administration. Total radioactivity was measured in blood and different organs without determination of individual metabolites. As early as lhr after oral administration, absorption of radioactivity could be detected for all labeled substances. The absorption rate for the OPC total fraction was about 31%, and those

for individual substances ranged from 16% to 40%. The accumulation of radioactivity was higher after repeated oral administration than after a single dose.23}

TOXICOLOGY Doses of rats and toxicity. values of and rats,

up to 3000 mg/kg of WS 1442 were given to mice by the oral route without any sign of Following intraperitoneal injection, LDs9 1170 and 750 mg/kg were calculated in mice respectively. No abnormalities in the general

state of health as well as clinical, chemical, hemato-

logical, gross morphological, and histological findings were observed after oral treatment of rats and dogs

at doses of 30, 90, or 300mg/kg/day for 26 weeks. Similarly, in a battery of genotoxicity assays, no evidence of mutagenic or clastogenic action was obtained.°“! While reports on carcinogenicity studies with Crataegus

extracts

are not available, animal

as

well as clinical and postmarketing surveillance studies do not indicate any carcinogenic potential. Oral application of WS 1442 at doses up to 1600 mg/kg body weight to rats and rabbits did not induce teratogenic effects. Furthermore, this extract affected neither the peri- and postnatal development nor the fertility of treated male and female rats and their Fl descendants (Schlegelmilch, personal communication).

INDICATIONS AND USAGE Hawthorn extracts from leaves and flowers are recommended as an oral treatment option for chronic heart failure (CHF),>! e.g., declining cardiac performance corresponding to functional stage II of the New York Heart Association (NYHA)."5661 Stage II NYHA heart failure is characterized by freedom from symptoms at rest and a slight limitation of physical activity; ordinary physical activity results in fatigue, palpitations, dyspnea, or anginal pain. The majority of clinical studies have been performed with standardized

Hawthorn (Crataegus)

342

hydroalcoholic extracts of hawthorn leaves and flowers

(extract designations WS 1442, LI 132) (Table 1). In mainly placebo-controlled, double-blind studies, a statistically significant reduction in subjective discomfort and an improvement in cardiac performance due to an increase in left ventricular ejection fraction (LVEF), more efficient cardiac work (reduction in pressure-rate product), and an increase in physical stress tolerance (increase in exercise tolerance, elevation of anaerobic threshold) have been demonstrated in CHF NYHA II patients treated with hawthorn

the standardized extract of leaves and flowers (WS 1442, LI 132), the daily doses ranged from 160 to 900 mg extract. In the CHF NYHA III study, daily doses of 900 and 1800 mg WS 1442 were used.

CONTRAINDICATIONS for the use of hawthorn

Contraindications

extracts

have not been reported.!>-*!

extracts.2>¢1

In a placebo-controlled, double-blind study of 40 patients with CHF NYHA II resulting from coronary heart disease, an increase in LVEF of approximately 1.5%, measured during bicycle ergometric loading, was found with WS 1442 (daily dose 480mg p.o.; 4weeks), whereas the LVEF in the placebo group dropped by around 0.2% (p = 0.0002). At rest, the LVEF increased by about 2.5% with WS 1442 and decreased by about 0.3% with placebo (p = 0.0001).°4 In another placebo-controlled, double-blind study of 136 patients with CHF NYHA II, WS 1442 (daily dose 160mg; 8 weeks) led to a reduction in the pressure-rate products (difference 50W load vs. resting) of approximately 6.2, whereas no effect was observed with placebo (p = 0.018).%! In a double-blind, comparative study of 132 patients

with CHF NYHA II, LI 132 (daily dose 900 mg; 7 weeks) was shown to be as effective as the angiotensin-I converting enzyme (ACE) inhibitor captopril (daily dose 37.5mg; 7 weeks). The work tolerance determined during bicycle exercise increased in the Crataegus group from 83 to 97W, whereas in the captopril group it increased from 83 to 99 W; in both groups, the frequency and severity of the symptoms decreased by about 50%." A recent placebo-controlled, double-blind study of 209 patients with CHF stage NYHA III investigated the efficacy of WS 1442 (daily dose 1800 or 900mg; 16 weeks) as an add-on therapy to the basic treatment with a diuretic (daily dose 50mg triamterene/25 mg hydrochlorothiazide). After therapy with 1800mg WS 1442, the maximal tolerated workload during bicycle exercise showed a statistically significant increase in comparison to both placebo (p = 0.013) and 900mg WS 1442 (p = 0.01). Typical heart failure symptoms as rated by the patients were reduced to a greater extent by WS 1442 than by placebo (1800mg: p = 0.004:

900mg: p = 0.04).

Table 1 provides an overview of 13 clinical trials on the efficacy of Crataegus extracts with a total of 1080 patients, of whom 871 were suffering from CHF NYHA II and 209 from CHF NYHA III. Duration of treatment lasted between 4 and 16weeks (mean 7.9 weeks). In CHF NYHA II studies performed with

PRECAUTIONS

AND ADVERSE

REACTIONS

Precautions

A physician must be consulted in cases where symptoms continue unchanged for longer than 6 weeks or when fluid accumulates in the legs. Medical intervention is absolutely necessary when pain occurs in the | region of the heart, spreading out to the arms, upper abdomen,

or the area around the neck, or in cases of

dyspnea.!!°.6 Adverse Effects

Adverse

effects

of

Crataegus

extracts

are

not

known."'>*l In a 16-week placebo-controlled, double-

blind study of 209 patients with CHF NYHA III (daily dose 900 or 1800mg WS 1442 as add-on therapy to pre-existing diuretic treatment), no adverse effects related to the extract were observed, even with the higher daily dose of 1800mg. The tolerability of the treatment was rated best in the 1800mg WS 1442 group by both patients and investigators. The incidence of adverse events was also lowest in this group, particularly with respect to dizziness and vertigo. Of the placebo patients, 10% complained of dizziness or vertigo, whereas only 4.3% of the patients treated with 900mg WS 1442 and 1.4% of those treated with

1800 mg WS 1442 had these complaints.“®!

Drug Interactions No interactions with other drugs are known to date. A randomized, crossover trial with 8 healthy volunteers was performed to evaluate the effect of WS 1442 on digoxin pharmacokinetic parameters. Subjects were randomized into one of two groups: digoxin 0.25mg/day alone for 10days or digoxin 0.25mg/day with 900mg WS 1442/day for 21 days. There were no statistically significant differences in any measured pharmacokinetic parameters. This suggests that WS 1442 and digoxin, in the doses and dosage forms studied, may be coadministered safely,44

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344 Hawthorn (Crataegus)

Hawthorn (Crataegus)

In

345

a placebo-controlled,

patients with CHF

NYHA

double-blind

study

of

III, no interactions were

seen between WS 1442 (daily dose 900 or 1800 mg) and a diuretic used as basic medication (combination

Table 3. Compendial status of hawthorn European

Hawthorn leaf and flower,

Pharmacopoeia

2003 Hawthorn leaf and flower

triamterene/hydrochlorothiazide).”!

dry extract, 2003 Hawthorn fruits, 2003

British Pharmacopoeia

OVERDOSAGE Symptoms of overdosage have not been reported.

Hawthorn fruits, 1998

U.S. Pharmacopoeia—

Hawthorn leaf with flower,

National Formulary

2003 Powdered hawthorn leaf with flower, 2003

CONCLUSIONS Extracts from different parts of Crataegus species are used commonly throughout the world as_ herbal remedies for the treatment of mild forms of heart insufficiency. The majority of pharmacological, toxicological, and clinical studies have been performed with standardized hydroalcoholic extracts from leaves and flowers (WS 1442, LI 132). These extracts have been demonstrated to possess cardiotonic as well as cardioprotective activities. Clinical trials, many of them conducted in accordance with the highest standards of good clinical practice, have confirmed that these extracts can be used as an alternative in early CHF and as an adjunct in the therapy of later stages of chronic heart failure. The evidence for the efficacy and safety of other hawthorn preparations needs to be evaluated on a case-by-case basis. Promising results from pharmacological studies on vasoprotective effects of hawthorn extracts warrant further clinical trials in the prophylaxis and treatment of other cardiovascular diseases.

Table 2

American Herbal

Hawthorn leaf with flower,

Pharmacopoeia

1999

ESCOP monograph

Hawthorn leaf and flower, 1999

WHO

Folium cum Flore Crataegi, 2001

COMPENDIAL/REGULATORY

Listed in Australian Register of Therapeutic Goods

Austria

Authorized as prescription (Rx) or over-the-counter (OTC) drug

Belgium

Authorized as OTC drug

Canada

Available without restriction; not suitable for self-medication of cardiac diseases

Denmark

Authorized as herbal medicinal product

France

Authorized as traditional herbal medicine

Germany

Authorized as OTC drug

Poland

Authorized as OTC drug

Switzerland

Authorized as OTC drug

United Kingdom

Not included in general sales list Regulated as dietary supplement

United States

STATUS

The regulatory and compendial status of hawthorn are summarized in Tables 2 and 3.

REFERENCES 1.

2.

Regulatory status of hawthorn

Australia

monograph

Crataegi cum Flore (Hawthorn Leaf and Flower); ESCOP Monographs on the Medicinal Uses of Plant Drugs; European Scientific Cooperative on Phytotherapy, October 1999, Fascicule 6, 1-11 (ISBN 1-9019604-06-X). Upton, R. Hawthorn Leaf with Flower—Crataegus spp. Analytical, Quality Control, and Therapeutic Monograph, American Herbal Pharmacopoeia and Therapeutic Compendium (Series 1), 1999; 1-29.

3.

Blumenthal, M. The ABC Clinical Guide to Herbs, American Botanical Council: Austin, TX,

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6.

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Hawthorn leaf with flower. In The Complete German Commission E Monographs; Blumenthal, M.., Busse, W., Goldberg, A., Gruenwald, J., Hall, T., Riggins, C.W., Rister, R.S., Eds.; Integrative Medicine Communications: Boston, 1998; 142-144. Poepping, S.; Rose, H.; Ionescu, L,; Fischer, Y.; Kammermeier, H. Effect of a hawthorn extract

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rat cardiomyocytes. Arzneimittel-Forschung Drug Res. 1995, 45, 1157-1161. Siegel, G.; Casper, U.; Schnalke,

19.

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Molecular physiological effector mechanisms of hawthorn extract in cardiac papillary muscle and coronary vascular smooth muscle. Phytother. Res. 1996, 10, S195-S198. Schwinger, R.H.G.; Pietsch, M.; Frank, K.; Brixius, K. Crataegus special extract ws® 1442 increases force of contraction in human myocardium cAMP-independently. J. Cardiovasc.

20.

1B

Joseph,

G.; Zhao,

gisches

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zu

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W.

ZAM

Amrinon,

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im

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Klaus,

24.

W.

Crataegus extract prolongs action potential duration in guinea-pig papillary muscle. Phytomedicine 1996, 3, 257-261.

13.

14.

Koch, E.; Chatterjee, S.S. Crataegus extract WS®1442 enhances coronary flow in the isolated rat heart by endothelial release of nitric oxide. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2000, 361 (Suppl.), R48. Chen, Z.Y.; Zhang, Z.S.; Kwan, K.Y.; Zhu, M.;

25:

26.

15.

extracts in rat

mesenteric artery. Life Sci. 1998, 63, 1983-1991. Kim, S.H.; Kang, K.W.; Kim, K.W.; Kim, N.D.

Procyanidins in Crataegus extract evoke endothelium-dependent vasorelaxation in rat aorta. Life Sci. 2000, 67, 121-131.

16.

2

Vierling, W.; Brand, N.; Gaedcke, F.; Sensch, K.H.; Schneider, E.; Scholz, M. Investigation of

medicine 2003, /0, 8-16. Mavers, W.H.; Hensel, H.

Verdnderungen

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Ernst,

F.D.; Reuther,

G.; Walper,

A. Haimod-

ynamische und hamorheologische Wirkungen von Crataegus-Extrakt. Muench. Med. Wochenschr. 1994, 7/136, 57-60.

Al Makdessi, S.; Sweidan, H.; Miiller, S.; Jacob, R.

Myocardial protection by pretreatment with Crataegus oxyacantha. Arzneimittel- Forschung/ Drug Res. 1996, 46, 25-27. Chatterjee, S.S.; Koch, E.; Jaggy, H.; Krzeminski, T. In-vitro- und insvivo Uniemuchurican zur kardioprotektiven Wirkung von oligomeren — Procyanidinen in einem Crataegus-Extrakt aus Blattern mit Bliiten. Arzneimittel-Forschung Drug Res. 1997, 47, 821-825. Veveris, M.; Koch, E.; Chatterjee, S.S. Crataegus

special extract WS® 1442 improves cardiac function and reduces infarct size in a rat model of prolonged coronary ischemia and reperfusion. Life Sci. 2004, 74, 1945-1955. Koch, E.; Chatterjee, S.S. Crataegus extract WS® 1442 compensates the decrease of cardiac output in endotoxemic rats. Eur. Cytokine Network 2000, 71, 527 Koch, E.; Spérl-Aich, G.; Zirrgiebel, U.; Trotzke,

Rajendran,

an

S.; Deepalakshmi,

P.D.; Parasakthy,

atherogenic

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29.

Niranjali, D.S. Protective effect of tincture of Crataegus on oxidative stress in experimental atherosclerosis in rats. J. Clin. Biochem. Nutr. 1996, 20, 211-223. Zhang, Z.; Ho, W.K.K.; Huang, Y.; Chen, Z.Y. Hypocholesterolemic activity of hawthorn fruit is mediated by regulation of cholesterol-7ahydroxylase and acyl CoA : cholesterol acyltransferase. Food Res. Int. 2002, 35, 885-891.

der

lokalen Myokarddurchblutung nach oraler Gabe eines Crataegusextraktes bei nichtnarkotisierten Hunden. Arzneimittel-Forschung/Drug Res. 1974, 24, 783-785.

Rothfug, M.A.; Pascht, U.; Kissling, G. Effect of

K.; Devaraj, H.; Devaraj, S.N. Effect of tincture of Crataegus on the LDL-receptor activity of hepatic plasma membrane of rats fed

the pharmaceutical and pharmacological equivalence of different hawthorn extracts. Phyto-

17.

Cardiol.

F. Inhibition of PDGF receptor-beta signal transduction and intimal hyperplasia in a rat carotid artery balloon angioplasty model by oral treatment with the standardized Crataegus extract WS® 1442. Naunyn-Schmiedeberg’s Arch. Phar macol. 2004, 369, R96.

Ho, W.K.K.; Huang, Y. Endothelium-dependent

relaxation induced by hawthorn

Basic Res.

long-term application of Crataegus oxyacantha on ischemia and reperfusion induced arrhythmias in rats. Arzneimittel-Forschung/Drug Res. 2001, 51, 24-28.

Pharmakolo-

Crataegus-Extrakt

Epinephrin,

Pharm. Pharmacol. Lett. 1993, 3, 45-48. Al Makdessi, S.; Sweidan, H.; Dietz, K.; Jacob, R. Protective effect of Crataegus oxyacantha against reperfusion arrhythmias after global no-flow

ischemia in the rat heart. 1999, 94, 71-77.

Pharmacol. 2000, 35, 700-707. Schmidt-Schweda, S.; Burstin, J.V.; Mollmann, H.;

Wollner, S.; Holubarsch, Ch. Der positiv inotrope Effekt des Crataegus Spezialextrakts WS® 1442 in isolierten Myozyten aus menschlichem Vorhofund Ventrikelmyokard wird vorwiegend durch oligomere Procyanidine vermittelt. Z. Kardiol. 2000, 98 (Suppl.), 164

Krzeminski, T.; Chatterjee, S.S. Ischemia and beneficial arrhythmias: induced reperfusion L. oxyacantha effects of an extract of Crataegus

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Effect of extracts of some medicinal plants on superoxide dismutase activity in mice. Planta Med. 1987, 53, 309-310.

a

Vibes,

J.; Lasserre,

B.; Gleye,

J. Effect

ities of cardiac tissue. Med.

Vertraeglichkeit von Crataegus-Extrakt WS® 1442 bei herzinsuffizienten Patienten mit einges-

of a

methanolic extract from Crataegus oxyacantha blossoms on TXA2 and PGI2 synthesising activ-

38.

Sci. Res. 1993, 21,

Lacaille-Dubois,

M.A.; Franck, U.; Wagner,

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Search for potential angiotensin converting enzyme (ACE)-inhibitors from plants. Phytomedicine 2001, 8/, 47-52.

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ah

University: Marburg, 1983 (dissertation). Schlegelmilch, R.; Heywood, R. Toxicity of Crataegus (hawthorn) extract (WS® 1442). J. Am. Coll. Toxicol. 1994, 73, 103-111. Schmidt,

analysis of randomized

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K.; Ernst, E. Hawthorn

extract for treating chronic heart failure: meta-

36.

39.

Hecker-Niediek, A.E. Untersuchungen zur Biogenese, Markierung und Pharmakokinetik der Procyanidine aus Crataegus-Spezies. Marburg

Pittler, M.H.;

chraenkter linksventrikulaerer Funktion., Perfusion 2001, 74, 212-217. Weikl, A.; Assmus, K.D.; Neukum-Schmidt, A.; Schmitz, J+ Zapie, G: Noli, \H.5= Sicerist, 1)

Crataegus-Spezialextrakt WS® 1442. Objektiver

435-436.

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Eichstaedt, H.; Stoerk, T.; Moeckel, M.; Danne, O.; Funk, P.; Koehler, S. Wirksamkeit und

trials. Am. J. Med. 2003,

114, 665-674. Loew, D. Phytotherapy in heart failure. Phytomedicine 1997, 4, 276-271.

41.

Wirksamkeitsnachweis bei Patienten mit Herzinsuffizienz (NYHA II). Fortschr. Med. 1996, //4, 291-296. Tauchert, M.; Ploch, M.; Huebner, W.-D. Wirksamkeit des Weissdorn-Extraktes LI 132 im Vergleich mit Captopril. Muench. Med. Wochenschr. 1994, J (Suppl.), 27-33. Tauchert, M. Efficacy and safety of Crataegus extract WS® 1442 in comparison with placebo in patients with chronic stable New York Heart Association class-III heart failure. Am. Heart J. 2002, 143, 910-915. Tankanow, R.; Tamer, H.R.; Streetman, D.S.; Smith, S.G.; Welton, J.L.; Annesley, T.; Aaronson, K.D.;

Bleske,

B.E.

Interaction

study

between

digoxin and a preparation of hawthorn (Crataegus oxyacantha). J. Clin. Pharmacol. 2003, 43, 637-642.

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5-Hydroxytryptophan Pedro Del Corral Karel Pacak Clinical Neuroendocrinology Unit, Pediatric Reproductive Endocrinology Branch,

National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A.

INTRODUCTION 5-Hydroxytryptophan precursor

cerebellar

(5-HTP)

is

for the neurotransmitter,

the

immediate

serotonin.

It is

available for purchase over the counter in food supplement stores and via the internet. Because 5-HTP crosses the blood-brain barrier and readily converts to serotonin, it is thought that 5-HTP may be taken orally to boost serotonin levels. Hence, the precursor

may be used to modulate physiological processes mediated by serotonin (see Fig. 1). S-HTP is produced commercially by extraction from the seeds of Grif fonia simplicifolia, an African plant, and as of the Fall of 2003, it was sold in the United States for ~US$ 14-16 for 45-60 capsules of 50mg. The entry will review the literature on 5-HTP; particular interest will be on studies performed on humans using 5-HTP for therapeutic purposes. Trials using animal models will be referenced when no available information was found in human studies. This article gives an overview of pharmacokinetics and metabolism of 5-HTP; then it covers several clini-

cal areas in which 5-HTP has been suggested to be at least partially therapeutic. First, we discuss studies on depression, from the early studies of the 1960s, which

provided the first indications that 5-HTP could have an antidepressant property to a meta-analysis published in 2002. The use of combination/augmentation therapy (5-HTP + antidepressants) will also be

ataxias.

Fourth,

we

assess

the side effect

profile of 5-HTP, its frequency, severity, and duration, including the infrequent but potentially lethal condition, eosinophilia-myalgia syndrome. The entry ends with a summary and recommendations on the use of 5-HTP.

PHARMACOKINETICS

AND METABOLISM

5-Hydroxytryptophan is the immediate precursor for 5-hydroxytryptamine (serotonin) synthesis (see Fig. 2). Serotonin does not cross the blood-brain barrier. Therefore,

the neuronal

availability

of serotonin

is

highly dependent on tryptophan uptake, which uses a transporter to cross the blood-brain barrier to get intracellular access before it is metabolized by tryptophan hydroxylase, the rate limiting step to produce serotonin from 5-HTP. Importantly, this is a saturable process in which tryptophan competes with other amino acids, suggesting that their elevated levels could inhibit/diminish tryptophan transport rates into the neuron. Conversely, it may be taken as a dietary supplement, which does not require a transporter system, and crosses the blood-brain barrier. Studies in rodents indicate that ~7% of '*C labeled 5-HTP from the arterial circulation is extracted by the brain.!!! Following 5-HTP administration, a dose-dependent serotonin release that could last for more than 2hr

addressed. Second, we study the utilization of 5-HTP

for its purported anorectic effect and potential application in obese individuals. Third, we examine the use of 5-HTP in several neurological disorders including different kinds of headaches, and its controversial use in +7 5-Hydroxytryptophan

Pedro Del Corral, Ph.D., M.D., is at the Clinical Neuroendocri-

Branch, Endocrinology nology Unit, Pediatric Reproductive National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A.

Karel Pacak, M.D.,-D.Sc., is at the Clinical Neuroendocrinology Unit, Pediatric Reproductive Endocrinology Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022065 Copyright © 2005 by Marcel Dekker. All rights reserved.

Satiety

Zin

+ 5-Hydroxytryptamine

/ Mood

Fig. 1 Possible medicine.

therapeutic

\

Fibromyalgia

Headaches

uses

of 5-HTP

in clinical

349

5-Hydroxytryptophan

350

There are few studies on the effects of S-HTP on obesity, and they suggest an anorectic effect of 5-HTP.U I

NH NH

Aromatic Amino acid

For

decarboxylase

HO

NH,

HO

NH, 4~—~

OH

5-Hydroxytryptophan

5-Hydroxytryptamine

Fig. 2 Chemical structure of 5-hydroxytryptophan conversion to 5-hydroxytryptamine.

and

after administration has been reported.”*! Subsequent to oral intake of 5-HTP, the peak plasma concentration is observed at 2—3hr, and the bioavailability is ~50-70%.4°! The former may be substantially increased by pretreatment with carbidopa, a peripheral aromatic amino acid decarboxylase inhibitor. For example, a single dose of 5-HTP with carbidopa resulted in a 15-fold higher plasma peaks than did

100 mg without carbidopa,?” suggesting that if carbidopa is coadministered with 5-HTP, smaller doses of the later may be used. Additionally, subject-to-subject variability in S-HTP plasma half-life ranges from 24 to Thr after a single dose of 200mg. Little is known about intracerebral 5-HTP and the effects of carbidopa on brain concentration of 5-HTP in humans. The development of positron emission tomography (PET) and new tracers has emerged as the dominant methodology to assess neurotransmission in humans. In the early 1990s, Agren et al." at Uppsala University, Sweden, were the first to use PET and demonstrate ''C-labeled 5-HTP uptake across the blood-brain barrier in eight healthy volunteers, with most

of the tracer

accumulating

in the

striatum and in the prefrontal cortex. The authors suggested that after intravenous injection, ''C-5-HTP crosses through the blood-brain barrier by a simple diffusion mechanism prior to subsequent decarboxylation to '!C-serotonin. In a subsequent study, the same investigators”! showed that administration of the peripheral .L-aromatic amino acid decarboxylase (AADC) inhibitor benzeride is an efficient method to raise levels of intracerebral 5-HTP uptake.

instance,

a 5-week,

double

blind,

crossover

study, without any dietary modification, found that oral administration of 300mg of 5-HTP before meals (i.e., 900 mg/day) led to mild decrease of food intake and weight loss in obese subjects.!11) A second study!!7} from the same group followed obese patients for 6weeks while treated with placebo or the same 5HTP dose + no dietary restriction, followed by an additional 6 weeks in which the patients continued placebo or 5-HTP treatment plus dietary restriction diet of 1200 cal. The cohert receiving 5S-HTP during the first 6 weeks experience a small but significant weight loss compared with the group receiving placebo. However, during the second 6 weeks, the patients on 5-HTP experienced further weight loss compared with first 6 weeks and the placebo group plus dietary restriction. Food records indicated that subjects on 5-HTP reduced their carbohydrate intake by about 50% | by the end of the first 6weeks, and a questionnaire revealed that early satiety was reported by 100% and 90% of the patients receiving 5-HTP during the first 6 weeks and the second 6 weeks, respectively. Nausea was reported by 80% of the treatment group during the first 6 weeks and 20% during the second 6 weeks. The authors argued that the episodic nausea was unlikely to have been a major contributor to weight loss since it declined substantially over time as the weight reduction was enhanced. The authors suggested that optimal adherence to dietary prescription observed in these patients receiving 5-HTP resulted in early satiety and reduced carbohydrate intake’?! In a third study,"*! this group extended their findings to noninsulin dependent overweight diabetics. The authors postulated that low brain serotonin levels may contribute to excess energy intakes of diabetic patients. Importantly, this study was only 2 weeks long and based on their previous studies, one might wonder whether there would have been further benefits with an additional

4-6 weeks.

Thus,

it remains to be established whether longer periods of time would lead to greater weight loss and declines in glycosylated hemoglobin.

CLINICAL USE OF 5-HTP

Depression

Obesity

The hypothesis that S-HTP was involved in the pathogenesis of depression was based on observations of lowered concentrations of 5-hydroxyindoleacetic acid (S-HIAA, a metabolite of serotonin) in cerebrospinal fluid of depressed patients. Therefore, it seemed reasonable to consider that exogenous administration of S-HTP could increase endogenous brain serotonin synthesis, presumably boosting synaptic serotonin

It has been long known that brain serotonin systems contribute to the modulation of food intake and

satiety.!"°! An increase of intrasynaptic serotonin tends to reduce food consumption. Thus, one might consider that individuals taking 5-HTP might experience increase satiety and weight loss over a period of time.

5-Hydroxytryptophan

activity.!4! Its use in depression has been studied for at least 40 yr.'>'° Initial success was reported in treating some depressed patients with small amounts of intravenous 5-HTP combined with a monoamine inhibitor. However, in a subsequent study, these results were not replicated. Preliminary data collected during the 1960s and 1970s showed some evidence suggesting a possible antidepressant effect. There were a few well-conducted studies in the following years. Van Praag et al.!'7! published the results of the first double blind placebo controlled study of 5-HTP in depressed patients. Three out of five patients responded compared to zero out of five receiving placebo. In an open label study, Sano!!! administered 5-HTP (50-300 mg) to 107 depressed individuals and found 70% response rate, suggesting beneficial effects in the treatment group. Other studies (see Ref.''*! for review) were plagued by methodological issues, such as low number of patients, lack of adequate controls, including placebo treatment, and coadministration of other antidepressant agents, such as monoamine oxidase inhibitors. During the 1980s, Van Praag and de Haan!'”! reported that 200 mg of oral 5-HTP given with 150 mg of carbidopa was more effective than placebo in preventing relapses in formerly depressed unipolar and bipolar patients during a 12-mo period. Specifically, during placebo treatment, 17/20 patients experienced relapse compared with 6/20 taking S-HTP. The authors concluded that 5-HTP plus a peripheral decarboxylase inhibitor may be an effective prophylactic agent to prevent recurrent depression in unipolar and bipolar patients. Van Hiele?! performed a clinical study of S-HTP (50-600 mg dosing, mean was 200mg) plus 150mg of carbidopa in 99 depressed patients who had previously failed to respond to psychotherapy, tricyclics, lithium, and electroconvulsive therapy. During the experimental period, many of the patients continued treatment with tricyclics, lithium, or other neuroleptics. Although the author reported that 51% of the patients underwent partial or complete recovery, it is not possible to know whether the recovery from depression was due to the experimental treatment or to the combination with the agents the patients were already taking before the study. Interestingly, the author stated that some of the responders had been followed on 5-HTP and peripheral decarboxylase inhibitor for up to 3 yr. In a recent review of the literature, most studies of the 1980s were short in duration, had small sample sizes, and used a wide range of oral doses from 200 to 3000 mg/ day.?") Despite these limitations, a careful evaluation of the literature reviewed by several investigators suggests a 25-50% efficacy of 5-HTP in alleviating depression. 1471771 The treatment response does not appear to be dose (100-300mg/day) dependent, and there is no evidence that the use of decarboxylase

351

inhibitor increased the efficacy of 5-HTP.2°??) A recent meta-analysis reviewed the literature on 5-HTP and depression from 1966 to 2000.°*! The authors found that 5-HTP was more effective than placebo at alleviating depression. In this review, a large body of evidence was subjected to very basic criteria for assessing reliability and validity, and was found to be largely of insufficient quality to inform clinical practice. The small size of the studies and the large number of inadmissible, poorly executed trials cast doubt on the result from potential publication bias, and suggest that they are insufficiently evaluated to assess their effectiveness. The authors suggested that well-designed studies are required before the true efficacy of 5-HTP is known. Less is known about the pathophysiological mechanisms by which serotonin synthesis is reduced in depression. Several investigators have examined whether brain 5-HTP uptake in depression is different from that in healthy controls.”! The PET study showed that S-HTP uptake across the blood-brain barrier was about 30% lower in depressed patients compared with that in healthy volunteers. This study described lower 5-HTP uptake in depressed patients, but the nature of this anomaly remains elusive. Although one might consider 5-HTP to mediate its effects through serotonin, there is evidence hinting to the possible stimulation of dopaminergic activity. For instance, Takahashi, Kondo, and Kato”*! administered 300 mg of oral 5-HTP daily for 2weeks to 24 depressed patients,

and

CSF

5-HIAA

and

HVA

levels

were

measured. The authors reported significant increases in CSF 5-HIAA in responders and nonresponders, whereas rise in HVA was seen only in responders (30% of the patients). This suggested that 5-HTP dosing may alter serotonergic and dopaminergic turnover, and that only those patients who experience increases in both CSF metabolites could expect antidepressant effects from 5-HTP. This is consistent with the earlier observations that dopaminergic and noradrenergic neurons possess transport sites for 5-HTP.! The efficacy of 5-HTP to treat depression has been found to be similar to that of imipramine and clomipramine.?!*7] The therapeutic effects of the tricyclic antidepressant clomipramine and of the monoamine oxidase nialamide are potentiated by 5-HTP in combination with carbidopa.?!?7! It is unknown whether this potentiating effect is observed with other antidepressants.) It has been reported“ that patients resistant to fluvoxamine treatment, a serotonin reuptake inhibitor, fail to respond to subsequent 5S-HTP. This should not have been an unexpected finding since both agents target the same neurotransmitter. Perhaps the next step would be to crossover patients responsive to serotonin

reuptake

inhibitors

to 5-HTP,

a positive

response would extend the findings of Nolen et al,261

5-Hydroxytryptophan

352

and would suggest that serotonin sensitive depressed patients could be tried on 5-HTP.

headaches in children. One study”?! has reported 70% effectiveness compared with a 10% placebo response in children and adolescents with recurrent headaches and parasomnias.

Headaches

The hypothesis that serotonin might be involved in the mechanisms of chronic primary headaches was set forward in the late 1950s.°” By the 1970s, a study using 200mg/day of oral 5-HTP for 2mo in 20 patients reported improved migraine headaches comparable to the therapeutic effect of methysergide, an ergot alkaloid.?*! A double blind trial on a group of 80 patients suffering from common or classic migraine were pretreated with placebo for 30 days and then randomized to groups receiving 1.4mg/day of pizotifen (a serotonin antagonist and antihistamine) or 5-HTP (400 mg/day), respectively, for 60 days. The authors assessed headache severity by a 4-degree analog scale. The 5-HTP treatment group showed a significant improvement in the most severe headache subgroups (i.e., degrees 3 and 4) compared with placebo. The prophylactic effects were similar to those of pizotifen.?*! In a subsequent study,?"! the same group of researchers set to identify and describe responsive subgroups to 5-HTP treatment in 100 patients within similar categories of primary headaches. The subjects ingested 100mg of 5-HTP at meal times for 4mo. Responders were defined as exhibiting a 60% or greater reduction

in the Pain Total Index (a composite of pain severity and pain duration). The study found 74% of the patients improved with treatment, and the effect became noticeable by the second month. Personal history of depression, age of onset less than 20 yr, and previous positive response to pizotifen were identified as having significant prevalence in the responder group. Additionally, the responders were more likely to present with throbbing and anterior pain, whereas the nonresponders presented with generalized and posterior headaches. [31,32] Other investigators could not reproduce the therapeutic effects of 5-HTP to the same extent. For example, in one study,?" there was 48% response rate in patients,

which

was

not

statistically

significant,

probably because of the relatively large and prolonged placebo effect in the patients with chronic primary headaches. This small discrepancy might be explained by the shorter duration (2 vs. 4mo) of the latter study compared to the former, and by the fact that 82% of the patient population had very frequent, severe, and long lasting headaches refractory to previous prophylactic agents. A recent parallel, randomized, double blind study found 5-HTP and placebo equally effective in patients treated for chronic tension headaches during an 8 week period.&7! Less is known about the effects of 5-HTP on the management of

Sleep Aid In adults, there is an association between sleep and the serotonergic system. In the 1960s, there were reports

on the effect of 5-HTP intravenously on sleep. One study was a case report of a single individual in whom progressive increases in the dosages of intravenous 5HTP from 50 to 150mg augmented the percentage of the night spent in REM sleep from 22% to 30%.24) Another trial administered 40mg of intravenous 5HTP and reported a shortened period from sleep onset to the first REM period in one of six patients.°! Yet another examined the effects of 600 and 200mg of 5-HTP administered orally to healthy volunteers during a five night period, while electroencephalograms were performed throughout the night. Increases in REM sleep ranged from 5% to 53% for both patients receiving the 200 and 600mg dose.@*! During the last three decades, little new information has been reported

in humans. Animal models have shown that the destruction of the raphe nuclei or the administration of the serotonin synthesis inhibitor p-chlorophenylalanine induces insomnia that is selectively antagonized/reversed by S-HTP.?” Future studies in humans should explore the possible sleep-inducing effect of 5-HTP, and if so, the dose-response.

Fibromyalgia This syndrome is characterized by chronic aching of skeletal muscles, multiple tender joints, fatigue, morning stiffness, and disturbed sleep. There are also reports of reduced blood serotonin levels in this patient popu-

lation.©*! In a 30-day double blind trial, Caruso et al.29! found 100mg of oral 5-HTP three times a day more effective than placebo in patients with fibromyalgia. In a subsequent study,"! the authors were interested in examining the efficacy and tolerability of long term orally administered 5-HTP at the same dose. Good clinical improvement was found in 50% of the patients as early as the 15th day and retained to up to the 90th day of treatment; it was well tolerated and its side effects were mild and transient. Neurological Disorders

Theré is some evidence that 5-HTP at doses of about 10 mg/kg/day for 4mo can improve postural equilibrium, dysarthria in patients with various inherited

5-Hydroxytryptophan

353

and acquired cerebellar ataxias, and particularly in those with lesions located precisely in the anterior lobe vermis.*'! Improvements in coordination have been reported in patients with Friedreich’s ataxia; however, the effect is only partial and not clinically major./7! A recent report"?! described a group of five boys between ages | and Syr presenting with floppiness in infancy followed by motor delay, hypotonic-ataxic syndrome, learning disability, and short attention span. All patients had 51-65% reduction of CSF 5-HIAA compared with age-matched median values, as well as decrease in urinary 5-HIAA excretion. The levels of tryptophan in CSF and serum were normal. Urine and CSF serotonergic metabolites were unaltered by tryptophan loading, but normalized following 5-HTP treatment. The authors stated that this new neurodevelopmental syndrome responsive to treatment with 5-HTP and carbidopa might result from an overall reduced capacity of serotonin production due to tyrosine hydroxylase gene regulatory defect/ inactivation or selective loss of serotonergic neurons.

ADVERSE SIDE EFFECTS The most common

side effects are nausea, vomiting,

fatigue/sleepiness. Adverse events appear to be dose related to some degree. Studies using higher doses of 5-HTP are more likely to see side effects.?!! Coadministration of L-aromatic amino acid decarboxylase inhibitors prevents the conversion of 5-HTP to serotonin in the periphery, which in turn increases the plasma concentration of 5-HTP, associating it to a higher incidence of nausea.!7! In 1994, the dietary supplement industry was deregulated by passage of the Dietary Supplement and Health Education Act. As a consequence, the purity, safety, and efficacy of dietary supplements are not evaluated by the U.S. Food and Drug Administration. The potential dangers of ingesting dietary supplements were demonstrated in 1989 with the outbreak of eosinophilia-myalgia syndrome, which affected more than 1500 people and caused about 30 deaths.) Eosinophilia-myalgia like syndrome has _ been

described in individuals taking 5-HTP."*! The compound thought to cause this syndrome was structurally characterized as a 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-B-carboline.!**! The quantity of contaminant per dosage of 5-HTP varies between preparations, and a threshold to prevent eosinophilia-myalgia syndrome may not be established. It is conceivable that high doses of 5-HTP could increase serotonin levels excessively, and theoretically it could lead to serotonin syndrome, which is characterized by altered mental status,

autonomic dysfunction, and neuromuscular abnormalities. To our knowledge, there are no published cases of

serotonin

syndrome

linked to 5-HTP

consumption.

Administration of 5-HTP to individuals without known affective disorders may cause mood elevation,

and at times mania.?!! CONCLUSIONS The

serotonin

precursor,

5-HTP,

is commercially

available over the counter in food supplement stores and via the internet. It is sold in the United States for ~US$ 15-20 for 45-60 capsules of 50mg. Following oral administration of 5-HTP, plasma concentration peaks are observed at 2-3hr and can be raised by prior treatment with an L-aromatic amino acid decarboxylase inhibitors. Brain PET scans indicate most 5-HTP accumulates in the striatum and in the prefrontal cortex. Individuals taking the supplement might experience increased satiety and weight loss over a period of time. The literature suggests that 200300 mg of 5-HTP three times a day with meals exerts an anorectic effect, which leads to weight loss in obese individuals, even without any dietary modification. 5-Hydroxytryptophan seems to preferentially reduce carbohydrate intake by as much as 50% in overweight/ obese and noninsulin dependent overweight diabetics. There is reasonable evidence to suggest that gradual individually titrated dose of 50-300 mg/day taken with meals may reverse/attenuate depression in about 50% of patients. The treatment response does not appear to be dose dependent,

and there is no evidence that

the use of decarboxylase inhibitor increases the efficacy of 5-HTP.

Of note,

it is acknowledged

that

most

studies were plagued by methodological issues, and further studies are necessary before appropriate clinical recommendations can be established. Moderate-to-severe primary chronic headaches may be alleviated in patients receiving oral 5-HTP at 100mg three times a day, particularly in patients presenting with anterior throbbing pain. Children may also respond well to treatment. The therapeutic effect takes several months before a clear response is observed. 5-Hydroxytryptophan at 100 mg three times a day may also be useful in about 50% of patients with fibromyalgia. Other possible therapeutic effects of 5HTP on inducing sleep, treating selected forms of ataxias, and treating inborn errors of serotonin metabolism

have not been well studied. More studies are warranted before

any

recommendation

can

be made.

Nausea,

vomiting, fatigue/sleepiness are the most common side effects of 5-HTP and appear to be somewhat dose related. The most serious side effect, albeit uncommon,

is eosinophilia-myalgia syndrome. Its incidence is associated to the presence of a contaminant acquired during the commercial synthesis of 5-HTP. Therefore, it is recommended that individuals choosing to take

5-Hydroxytryptophan

354

5-HTP

cognitive/motor

changes,

and

make

arrangements

with their primary care physicians to follow eosinophil counts regularly.

COMPENDIAL/REGULATORY

5-hydroxytryptophan in human brain studied by positron emission tomography. Psychiatr. Res. Neuroimaging 1992, 45, 215-225.

of myalgias or

be alert to the development

10.

bi

Int. J. Eat. Disord. 1981, /, 2-15. Ceci, F.; Cangiano, C.; Cairella, M.; Cascino, A.; Del Ben, M.; Muscaritoli, M.; Sibilia, L.; ,

Rossi-Fanelli, F. The effects of oral 5-hydroxytryptophan on feeding behavior in obese adults

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of Public Health, Loma Linda University, Loma Linda, California, U.S.A.

| M.W. 270

O

Fig. 1 Chemical formulas and molecular weights of the soybean isoflavone aglycones. 363

Isoflavones

364 R3

R4

RS5

Chemical Name

M.W.

Genistein, 7-O-B-D-glucopyranoside

432

(Genistin) 6"-O-Acetylgenistin

474

ee OH

: Pia

a ee COCHS

OH

H

COCH2COOH

H

H

H

H

COCHS3

6"-O-Acetyldaidzin

H

H

COCH2COOH

6"-O-Malonyldaidzin

H

Glycitein, 7-O-B-D-glucopyranoside (Glycitin)

446

4H

6"-O-Malonylgenistin

518

4',7-Dihydroxyisoflavone, 7-0-B-D-

416

glucopyranoside (Daidzin)

H

OCH3

|

458

5024

H

OCH3

COCH3

6"-O-Acetylglycitin

488

H

OCH3_|

COCH2;,COOH

6"-O-Malonylglycitin

532

O

|

Q

O

HO Ry

O

Ray

Fig. 2 Chemical formula and molecular weights of the soybean isoflavone glycosides.

KO

traditional soyfood provides approximately 20-35 mg of isoflavones. Isolated soy proteins vary in isoflavone content (range 0.5—2.0mg/g), although the average is about 1mg/g. As a result of processing losses, the isoflavone content of alcohol-washed soy protein concentrates,

the most

common

—

type of concentrate,

is

only 5—20% that of the water-washed concentrates. The United States Department of Agriculture, in conjunction with Iowa State University, has recently

established an online database (http://www.nal.usda. gov/fnic/foodcomp/Data/isoflav/isoflav.html) of the isoflavone content of foods. In addition to soyfoods and supplements,

isofla-

vones are used as food fortificants, being added to both

soy and non-soy products. One brand of isoflavones, Novasoy®, which is produced by the Archer Daniels Midland Company, has achieved GRAS. status

through self-affirmation and review by an outside panel of experts. It can be added to adult single meal replacements, and health beverages and bars. Novasoy is an example of a supplement that is derived from soybean molasses (distilled ethanol extract from soy flakes) and that reflects the isoflavone profile of soyfoods (Fig. 3). In contrast, a second source of isoflavone supplements, which is commonly referred to as soygerm, is derived from the hypocotyl portion of the soybean and in comparison to soyfoods is very low in genistein and high in glycitein.“ As is the case with many types of supplements, a recent analysis found that there is frequently a discrepancy between the isoflavone content and the amount listed on the product label, as only about half of the

supplements

analyzed were found to contain within

Soygerm

*)

~—

Food

Es | oaccein | aa

Glycitein

Fig. 3 Relative isoflavone profiles of different sources of isoflavones.

Isoflavones

365

20% of the stated amount.! Most supplements contain the amount of isoflavones found in | serving of a traditional soyfood.

ASIAN ISOFLAVONE INTAKE Older values reported in the literature for Asian isoflavone intake are exaggerations. There are quite good data on Asian soy consumption, especially for Japan. In fact, the Ministry of Health and Welfare in Japan has conducted a national household survey (NHHS) of food intake since 1946. The NHHS was conducted on a quarterly basis between 1946 and 1963 and has been done on an annual basis thereafter. It includes randomly selected prefectures comprising 10,00020,000 subjects of all ages. Substantial data now indicate that Japanese adult intake is approximately 50mg/day.”*! Intake in China, including Hong Kong, is generally similar to or lower than that in Japan. For example, a Chinese prospective study published in 2003 that included nearly 65,000 women from Shanghai found daily median soy protein intake to be only 7.36 g/day. Smaller studies conducted in Shanghai reported both lower (3.5g soy protein/day)!'" and higher (~11g soy protein/day)""7! intakes. The findings from three studies from Hong Kong are quite variable; daily soy protein intakes for women were reported to be 2.5 g,"7)

4.9¢"I and 7.9¢."°] Traditional soyfoods have an isoflavone (mg): protein (g) ratio of approximately 3.5:1. Thus, it is clear that mean isoflavone intake is 100 mg

isoflavones /day.'>)

Most of the isoflavones absorbed are excreted from

the body within 24hr after a single ingestion. Studies - Suggest that } as does estrogen, but unlike estrogen, isoflavones do not stimulate endometrial cell proliferation.?* As evidence of the difference between isoflavones and estrogen is an Australian study that found that in postmenopausal women, isoflavone-rich soy protein did not affect any of the estrogen-regulated proteins examined.?7] Even categorizing isoflavones as SERMs does not fully describe their likely physiological effects, however, as isoflavones, especially genistein, affect signal transduction pathways by inhibiting the activity of many enzymes (e.g., tyrosine protein kinase, mitogen activated kinase, DNA topoisomerase, etc.) and regulating cellular factors that control the growth and differentiation of cells.?*! Also, isoflavones demonstrate antioxi-

dant activity in vitro?! and in humans,°°! although the effects in humans have been very inconsistent. Arguably, the nonhormonal properties of isoflavones are responsible for more of the interest in these compounds than the hormonal effects. This is especially true with regard to genistein and anticancer effects.°®! The nonhormonal properties also account for why isoflavones are being studied in connection with such diseases as malaria, diabetes, cystic fibrosis,

and alcoholism. Not surprisingly, given the disappointing results of the Women’s Health Initiative (WHI), there has been particular interest in isoflavones functioning as possible alternatives to conventional hormone replacement therapy (HRT).°?! Finally, it is important to recognize that isoflavones differ markedly from estrogen in many respects; recent research shows that isoflavones affect the expression of many genes differently from estrogen.24! Consequently, it is not prudent to draw conclusions about their health effects—good or bad—on the basis of what estrogen is thought to do.

CHRONIC DISEASE PREVENTION AND TREATMENT Cancer

Genistein inhibits the growth of a wide range of both hormone-dependent and-independent cancer cells in vitro.?*] The in vitro concentrations required to inhibit cancer growth are typically much higher than serum isoflavone levels, but animal data suggest that the in vitro anticancer effects are relevant.?*! In fact,

it appears that the in vitro studies underestimate the in vivo anticancer activity of isoflavones since, as cited below, many studies have found that tumor inhibition

occurs in response to the ingestion of amounts of isoflavones that produce serum levels that are much

lower than the concentration needed to inhibit the growth of cancer cells in vitro. Also, Dalu et al. found that genistein affected signal transduction pathways in the prostate of rats even though genistein prostate concentrations only reached the low nanomolar range.P4l In

rodents,

isoflavone

administration

has

been

shown to inhibit the growth of several types of cancers,

including bladder,?”! skin,®* prostate,??! and mammary™“°! tumors. However, most focus has been on the latter two, in part because of the low rates of these cancers in soy-consuming countries.“"] Recently, an international group of experts suggested that isoflavones inhibit the progression of latent prostate cancer to the more advanced stages of this disease.“ Research in animals generally shows that isoflavone-rich soy protein and isolated isoflavones inhibit prostate tumors induced by several different methods, and several epidemiologic studies lend support to the animal experiments, although the design of these epidemiologic studies limits the value of the findings.) In one, consuming tofu at least five times a week was associated with a 65% reduction in risk (p = 0.054) among Japanese Hawaiians.*! In another, consuming soy milk more than once a day was associated with a 70% reduced risk among Seventh-day Adventists.!°! These

intake

levels are quite modest.

However,

the

design of both these prospective studies limits the value of their findings. Most notably, not only did few men actually develop prostate cancer, but importantly, neither study actually presented total soy intake. The only epidemiologic study that did found soy intake to be protective against prostate cancer.'*¢ In this study, risk was reduced by approximately 50% in Chinese men in the fourth quartile of soy intake. The mean intake for the fourth quartile was not reported, but the cutoff for total soyfoods was a rather modest >111.8g/day. The authors reported the intake cutoffs for the fourth quartile for genistein and daidzein as >62.0 and >36.3 mg/day, respectively. In contrast to the data on prostate cancer and ‘soy, the epidemiologic data generally do not show that adult soy intake reduces postmenopausal breast cancer risk, although there are many possible mechanisms by which it can do so. For example, soy increases the length of the menstrual cycle, which is associated with

protection against breast cancer.47!

A very intriguing hypothesis is that exposure to isoflavones when young reduces breast cancer risk markedly later in life.“*! In support of this hypothesis is a large Chinese case-control study that found that women who consumed soy during their teenage years were about half as likely to develop breast cancer as adults as were Chinese women who consumed little soy during this period.*! Exposure to genistein causes breast tissue differentiation and reduces the number of terminal end buds, the anatomical structure within the

Isoflavones

367

rodent mammary gland that is the likely site of tumor development.'*8°°! There is evidence that these kinds of changes are also important in humans,.?!

Coronary Heart Disease In 1999, the U.S. Food and Drug Administration (FDA) approved a health claim for the cholesterollowering effects of soy protein.?! The FDA does not require that soy protein contain isoflavones to qualify for the health claim. Data do not indicate that isoflavones by themselves lower cholesterol, although they may slightly raise high-density-lipoprotein (HDL)cholesterol levels.*! In any event, there are intriguing, but still speculative, data suggesting that independent of effects on cholesterol, isoflavones reduce coronary

heart disease risk through multiple mechanisms. For example, some data indicate that isoflavones inhibit low-density-lipoprotein (LDL)-cholesterol oxidation,°"! enhance systematic arterial compliance?>! and flow mediated dilation“! reduce platelet aggregation,>>! and inhibit smooth muscle cell proliferation.” ‘| These effects, if confirmed, will likely prove to be of

greater clinical value than the cholesterol-lowering effects of soy protein. In support of the possible coronary benefits of isoflavones is a recent prospective study involving nearly 65,000 women

from Shanghai, which found that those

in the highest quartile of soy protein intake were 86% less likely to have a nonfatal heart attack in comparison to women who consumed relatively little soy protein." These results suggest that soy has effects on heart disease risk far beyond the modest cholesterol-lowering properties of soy protein.

Osteoporosis

Conventional HRT is no longer being recommended for long-term use because results from the WHI showed that the harm of HRT outweighed the benefits.°7]

However,

this

trial

also

found

that

HRT

reduces bone loss and fracture risk in postmenopausal women.?! Isoflavones have been posited to reduce bone resorption in perimenopausal and postmenopausal women in a manner similar to that of estrogen. While

still speculative, there is in vitro, animal, and

clinical support for this hypothesis and for the potential for isoflavones to not only inhibit bone resorption but stimulate bone formation.°”°*! For example, Italian researchers found that over the course of one year, daily consumption of 54mg genistein increased bone mineral density at both the spine and hip to a similar extent as conventional HRT.&?! More recently, ina l-yr study, Chen et al.8*] reported that in comparison

to placebo, isoflavone supplements (80 mg/day) reduced bone loss at the hip. In contrast to these favorable findings, a few studies have failed to show that isoflavones or isoflavonerich soy protein affect bone mineral density. Also of possible relevance is a 3-trial involving 500 women, which found that the synthetic isoflavone ipriflavone failed to favorably affect bone mineral density (BMD). Furthermore, most of the isoflavone trials have involved relatively few subjects and have been relatively short (! The complete analysis, including the separation of 12 kava pyrones, can be accomplished in under 20 min using the appropriate combination of solvent, column, and column temperature.

PRECLINICAL STUDIES While the intoxicating, sedative qualities of kava extracts were obvious from Forester’s description of kava’s traditional use, it was not until the early 1900s that serious investigations into the pharmacological properties were made.!'*!"! These early studies clearly demonstrated that the active components of kava extracts existed in the lipophilic portions. Although aqueous extracts had similar activity, they were much less potent. Because the traditional method of preparing kava involved extensive chewing of the root, Schiibel and many others postulated that enzymatic activity in the saliva was required to increase the potency of the extracts. However, this has since been proven incorrect, since the chewing action merely creates an emulsion that aids in the extraction of the lipophilic components. Van Veen was able to demonstrate that both the lipophilic extract and the purified form of dihydrokavain caused pigeons and monkeys to

become sleepy.!'” At high doses, the animals lost control of their limb and quickly entered into long periods of sleep. These results, in addition to loss of righting reflex, were confirmed by Meyer et al., following oral administration of dihydromethysticin and dihydrokawain to mice, rats, rabbits, and cats.!!®! As a result

of the continued study, Meyer found that both kava extracts and individual pure chemical constituents produced centrally acting smooth muscle relaxation and antagonized strychnine-induced convulsions."*! Interestingly, none of the individual kava pyrones exhibited an equivalent potency as that induced by extracts, causing a number of investigators to propose a synergistic action of the natural combination of compounds.

The increasing interest in the use of kava not only for its well-documented

effects as a sleeping aid, but

also for its anxiolytic qualities, has prompted numerous investigations into its effectiveness as a treatment for anxiety disorders and mild forms of depression. Neuropharmacological trials of the binding of individual kava pyrones to gamma-amino butyric acid (GABA) and benzodiazepine receptors in isolated rat and mouse synaptosomal membranes found only weak binding to GABA, receptors and no significant binding to GABAg or benzodiazepine receptors.?" The weak receptor binding of the individual kava pyrones and kava extract did not correlate with the observed centrally acting pharmacological properties of kava in animal and human studies. Although this study is widely cited as providing the molecular level mechanism of action of kava pyrones, it does in fact conclude the opposite. Other experiments suggest the mechanism of action to involve sodium- and calcium-gated channels along with inhibition of noradrenalin uptake.?!?7] Another evaluation indicates that central nervous system receptor binding of indicated components other than kava pyrones may be responsible for the anxiolytic activity in kava extracts. Leaf and stem extracts contained components with potent binding to GABA,g, dopamine D3, opioid, and histamine receptors, which were unrelated to the concentration

of kava pyrones.7?) A number of animal studies have been carried out to examine the metabolism of kava pyrones. In rats, metabolism was extensive and consisted primarily of hydroxylation of the benzene ring and hydrolysis of the pyrone ring.?*! Analysis of human urine following ingestion of a traditionally prepared kava beverage detected the known kava pyrones along with a complex mixture of hydroxylated metabolites.?>! Other metabolism included the demethylation of the methoxy substituent on the pyrone ring, as well as reduction of the 3,4-double bond of the pyrone ring. Although kava had not previously been shown to be hepatotoxic, recent reports of liver failure associated with consumption of kava-containing products have prompted new studies related to its metabolism. Since one well-known pathway by which natural products and drugs can cause hepatotoxicity is through the inhibition or modulation of cytochrome P450 (Cyp450) enzymes, the ability of individual kava pyrones to inhibit specific Cyp450 enzymes was investigated. Although kavain was associated with little or no inhibition,

methysticin,

dihydromethysticin,

and des-

methoxy-yangonin were all found to cause significant inhibition of the isozymes Cyp2C9, Cyp2Cl9, and Cyp3A4.°*l More importantly, this study showed that methysticin and dihydromethysticin form irreversible metabolic complexes following incubation with these Cyp450 isozymes. Since the affected enzyme systems

Kava (Piper methysticum)

377

are responsible for the metabolism of more than 90% of all drugs, there is a significant risk of adverse reaction associated with kava consumption and conventional drug therapy. This would not be unexpected since drug/natural product combinations have been associated with adverse reactions involving a number of natural products, such as grapefruit juice and St. John’s wort. The delayed onset and low incidence of hepatotoxicity associated with kava products suggested an idiosyncratic reaction similar to those observed for some drug products. In many cases, these reactions are often associated with bioactivation to reactive electrophilic metabolites. Further study into the chemical reactivity of the principal chemical constituents of kava extracts was conducted to evaluate their ability to generate reactive metabolites in the presence of human liver microsomes and hepatocytes. The results of these investigations showed significant and extensive formation of glutathione reactive metabolites with several of the kava pyrones.?7! Characterization of the glutathione conjugates pointed to a mechanism involving the generation of ortho-quinones as the reactive intermediates. Although it is often difficult to link reactive metabolite formation to a specific toxicity, identification of the reactive metabolites has provided important mechanistic insights for future toxicological studies. For example, should toxicity be mediated through this mechanism, rats could not be used since they do not have the ability to metabolize methysticin and dihydromethysticin to the same reactive metabolite observed in humans (Fig. 4). Since cases of hepatitis similar to those reported for kava extracts have often been associated with the

(Fig. 5) was isolated from the outer stem peelings of a kava cultivar from Papua New Guinea known as Isa. Peelings from 10 other cultivars, including another variety from Papua New Guinea, were also included in the analysis, but they did not have detectable levels of

the epoxide. This new chemical compound was found to be toxic in in vitro cell cultures of hepatocytes. Since stem peelings are not used traditionally, and have only been recently used to fill the enormous demand for kava, the authors speculate that this alkaloid may be responsible for some of the observed hepatotoxicity. Some support for this hypothesis may be found in the data generated from the Port Import/Export Reporting Service (PIERS), which records more than 100tn of kava root peelings being imported to the United States prior to the year 2001, presumably for use in kava preparations. While the toxicity of the pipermethystine epoxide remains to be proven, it would not explain the hepatotoxicity observed with a patient taking only kavain, implying that there may be multiple pathways of hepatotoxicity.

CLINICAL STUDIES Although there are a number of potential medicinal uses for kava root extracts, the principal purpose is for the relaxing, anxiolytic effects. Its pharmacological properties have led to a continuous, worldwide increase in use as an over-the-counter dietary supple-

ment or a medicinal herbal preparation. The German Commission E monographs permit daily doses of 60-120 mg of total kava pyrones for the treatment of

on the liver,

anxiety, stress, and restlessness. While the mechanism

numerous studies are underway to evaluate other chemical constituents present for the same activity. One such investigation into direct acting hepatotoxic contaminants present in kava extracts has identified an epoxide form of pipermethystine.”*! The epoxide

of action may not be completely understood, a recent review of clinical trials conducted on the effectiveness of kava for treating depression and anxiety disorders clearly suggests efficacy in comparison to placebo

direct action of a chemical contaminant

treatment.”

HO OCH; Humans

HO

| Oo

=

OCH;

oO

|

O O

Rats

O bos

OCH, OH

O

| Oo

Fig. 4 Schematic representation of the difference in primary metabolism of dihydromethysticin and methysticin in rats and humans. Demethylenation of methysticin and dihydromethysticin can lead to the formation of a reactive ortho-quinone species.

Kava (Piper methysticum)

378

OAc

ela

NO

Oo Pipermethystine

oO

OAc

OCS

Oo

Npe oO

3a ,40.-epoxy-5ppipermethystine

Fig. 5 Chemical structures of possible contaminants of kava extracts, pipermethystine and the in vitro hepatotoxic epoxide form of pipermethystine.

The review included meta-analysis of data collected from seven double-blinded randomized clinical trials. One of the most recent clinical trials included in the meta-analysis was that conducted by Volz and

Kieser.°! It included 101 patients and employed a carefully formulated dose of kava equivalent to 210mg of kava pyrones per day. Using the Hamilton Anxiety Scale as a measure of efficacy, the study found a significant improvement over the control group following 8 weeks of treatment. Although adverse events were reported, they were confined to relatively minor complaints including headache, restlessness, and drowsiness. Kava has been used, and even abused for its intoxi-

cating properties, for centuries with little but isolated or circumstantial reference to human toxicity. Prior to numerous recent reports of hepatotoxicity, reported human toxicity was limited to a scaly dermopathy observed with chronic, high consumption rates. This condition is believed to result from inhibition of cholesterol metabolism.8"! Clinical observations on an Australian Aboriginal community described greatly increased levels of gamma-glutamyl transferase, along with decreased levels of bilirubin, plasma protein, and urea in all frequent kava users.) Although many of the individuals in this study consumed very high levels of kava, with most exceeding 100g/week and some exceeding 400 g/week, no reports of hepatotoxicity were observed. A number of other epidemiological investigations have also been conducted on the carcinogenicity of kava extracts. However, no study has shown any causal link with cancer. - The toxicity of kava extracts to humans is a matter of fierce debate. Prior to 2000, kava was among the top

10 best selling botanical supplements in the United States, and worldwide millions of doses were consumed annually. However, based on a number of reported cases of severe hepatotoxicity, some involving liver transplant, sales have been banned in a number of countries including Germany, Switzerland, and United

Kingdom. In a summary of 29 cases of kava-implicated hepatotoxicity reported in Germany between 1990 and 2002,*! nine people developed fulminant liver failure,

and eight of them received liver transplants. There seemed to be no correlation between illness and a particular brand of kava. Additionally, hepatitis was reported in patients consuming kava extracts prepared with acetone and alcohol, implying that the method of extraction had little to do with the associated toxicity. It is also noteworthy to mention that many of the individuals exceeded the German Commission E recommended daily dose of 120 mg. So far, a definitive

for kava-induced

mechanism

hepatotoxicity has not been identified, but it could include both idiosyncratic and immunoallergenic mechanisms. The most likely mechanism of toxicity could be related to individual metabolic idiosyncratic reaction, since the affected individuals generally demonstrated a long latency period and rarely showed evidence of autoimmune response. A recent study of the metabolic profiles of kava-sensitive individuals showed that subjects with Cyp2D6 deficiency seemed to be at risk for developing kava-related hepatotoxi- _ city.4! Although those with this deficiency are known to be sensitive to a broad range of drugs, there is insufficient evidence to come to this conclusion. Another traditional use has been for the relief of pain

and

inflammation

associated

with

arthritis,

chronic gout, menstruation, and toothache. Extracts have been shown to produce both short acting locai anesthetic action, as well as longer lasting pain relief. The former effect has been likened to that produced by lidocaine,°*! and has not been attributed to the indi-

vidual kava pyrones but to other components present in the extract. The individual kava pyrones have been found to produce analgesic action similar to aspirin. An investigation into the mechanism of the antiinflammatory response to the kava pyrones found that all six of the primary kava pyrones, in addition to flavokawain, had inhibitory effects on cyclo-oxygenase

(COX) enzymes COX-1 and COX-2.°*! Since the mechanism of action of nonsteroidal anti-inflammatory drugs, like ibuprofen, is attributed to their ability to inhibit the COX enzymes, the finding that kava pyrones, in particular dihydrokawain and yangonin, also perform a similar function is a likely explanation for their activity. Kava has a long history of use as a treatment for urinary tract infections and gonorrhea. It was widely believed that drinking the extract would not only relieve the symptoms associated with gonorrhea, but ultimately result in a cure."! Some supporting scientific evidence for this traditional use is found with the observed broad spectrum of antimicrobial and antifungal effects of kava extracts.0”] Finally, kava extracts have also long been used as a natural remedy for the relief of symptoms associated with

menopause.

Indeed,

extract for the treatment

clinical

evaluation

of menopausal

of the

symptoms

Kava (Piper methysticum)

379

does conclude that some efficacy is observed.°*! One study evaluated the combined effectiveness of hormone replacement therapy along with a daily dose of 100 mg kava extract containing 55% kavain.©*! Results of this trial found considerable relief of psychological symptoms associated with menopause when kava was combined with traditional hormone replacement therapy.

Kubatova,

A.;

CONCLUSIONS 1 Kava extracts have a well-documented, effective pharmacological action in inducing sleep and reducing mild cases of anxiety. They are also used extensively as a and numerous

13:

14.

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2»

Parmar, V.S.; Jain; S.C.; Bisht, K.S.; Jain, R.; Taneja, P.; Jha, A.; Tyagi, O.D.; Prasad, A.K.;

3.

Wengel, J.; Olsen, C.E.; Boll, P.M. Phytochemistry of the genus Piper. Phytochemistry 1997, 64, 597-673. Borsche, W.; Blount, B.K. Research on the composition of the roots of kava. XIII. Report on a new compound with experimental part (German). Chem. Ber. 1933, 66, 803-806.

4.

Macierewicz, stance

Z. Synthesis

of yangonin.

of the mother

Roczniki

Chem.

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Dharmaratne,

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Lebot, V.; Levesque, J. The origin and distribution of kava (Piper methysticum Forst f., Piperaceae):; a phytochemical approach (National Tropical Botanical Garden, Hawaii). Allertonia 1989, 5, 223-380. Lebot, V.; Levesque, J. Evidence for conspecificity of Piper methysticum Forst f. and Piper wichmannii

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optimization in the development of a highperformance liquid chromatographic method for the analysis of kava pyrones in Piper methysticum preparations. J. Chromatogr. A 2002, 948, 51-63. Schubel, K. The chemistry and pharmacology of kawa-kawa (Piper methysticum, Rauschpfeffer) (German). Naunyn-Schmiedeberg’s Arch. Exp. Pathol. Pharmakol. 1924, /02, 250-282. Van Veen, A.G. On the intoxicating substance from kawakawa of wati-plant (Piper methysticum) (Dutch). Geneeskd. Tijdschr. Ned. 1938, 78. Meyer, H.J.; Oberdorf, A.; Seifan, E. Investiga-

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pharmacol. Bull. 1967, 4, 10-11. Davies, L.P.; Drew, C.A.; Duffield, P.; Johnston,

G.A.; Jamieson, D.D. Kava pyrones and resin:

chemistry 2002, 59, 429-433.

6.

P.; Lebot, V. Identification

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1950, 24,

I.A. Kavalactones from Piper methysticum, and their 13C NMR spectroscopic analyses. Phyto-

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YS.

1.

S.B.

determining kavalactone content and chemotype in kava (Piper methysticum Forst f.). Biochem.

kava bars are

present in Hawaii and elsewhere in the South Pacific and United States. Despite the implied, broadly accepted safety of consuming kava extracts, there appears to be a sufficient amount of evidence to support a significant risk of severe hepatotoxicity when consuming these products. Since the time required to solve the mechanism of toxicity and conduct appropriate animal studies is likely to take many years, extreme caution should be urged when consuming kava-containing products.

Hawthorne,

A.A. High performance liquid chromatography of kava lactones from Piper methysticum. J. Chromatogr.

for alcohol,

D.J.;

root. J. Chromatogr. A 2001, 923, 187-194. Smith, R.M.; Thakrar, H.; Arowolo, T.A.; Safi,

10.

substitute

Miller,

Comparison of subcritical water and organic solvents for extracting kava lactones from kava

studies on GABAA, GABAB

and benzodiazepine

binding sites in rodent brain. Pharmacol. Toxicol. 1992, 71, 120-126.

2

Magura, E.I.; Kopanitsa, M.V.; Gleitz, J.; Peters, T.; Krishtal, O.A. Kava extract ingredi-

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Seitz, U.; Schule, A.; Gleitz, J. [3H]-Monoamine

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Scheline,

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some human urinary metabolites of the intoxicating beverage kava. J. Chromatogr. 1989, 475, 273-281.

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Volz, H.P.; Kieser,

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R.B. Identification of novel electrophilic metabolites of Piper methysticum Forst. (kava). Chem.

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Lactobacilli and Bifidobacteria Linda C. Duffy Infectious Diseases Division, University of Buffalo—State University of New York, Women and Children’s Health Research Foundation, Women and Children’s Hospital/Kaleida Health, Buffalo, New York, U.S.A.

Stephen Sporn Springfield, Missouri, U.S.A,

Elizabeth Griffiths Haiping Qiao Pearay Ogra Infectious Diseases Division, University of Buffalo—State University of New York, Women and Children’s Health Research Foundation, Women and Children’s Hospital/Kaleida Health, Buffalo, New York, U.S.A.

INTRODUCTION

GENERAL DESCRIPTION

Lactic acid bacteria are found naturally in the human and animal gastrointestinal tract, mouth, and vagina. Lactobacillus acidophilus is the most well studied of the lactic acid bacteria, which are important in the fermentation of milk products, fruits, and vegetables. Lactobacilli and a related genus comprising bifidobacteria produce organic compounds (lactic and/or acetic acid) that increase acidity (pH 4—S) of the intestine and vagina, and favorably alter the balance of intestinal microflora.") Typical lactobacilli (e.g., L. acidophilus

Lactobacilli and bifidobacteria are considered commensal “‘beneficial’’ bacteria that have been shown in extensive studies to inhibit growth of harmful pathogens, reduce toxin production, promote good digestion by increasing bioavailability of some nutrients, boost innate

immune

gastrointestinal

function,

and

and

vaginal

increase

infections

resistance

to

(Fig. 2). In

addition, selected strains have been shown to reduce

serum cholesterol levels. Less evidence regarding their anticarcinogenic effects.

is available

and L. casei) ferment lactic acid, whereas bifidobac-

teria are considered heterofermenters of acetic and lactic acid. Each genus and species of lactic acid bacteria are generally rod-shaped, Gram-positive prokaryotes, but may differ considerably in their pleomorphic and anaerobic properties (Fig. 1).

Linda C. Duffy, Ph.D., is Professor of Pediatrics and Epidemiology/Biostatistics at Women and Children’s Hospital, University of Buffalo, Buffalo, New York, U.S.A.

Stephen

Sporn, N.D., is Doctor

of Naturopathy,

Springfield,

Missouri, U.S.A.

Elizabeth Griffiths, Ph.D., is Research Associate at the Department of Pediatrics, Infectious Diseases Division, UB—SUNY, Women and Children’s Health Research Foundation, Women and

Children’s Hospital/Kaleida Health, Buffalo, New York, U.S.A. Haiping Qiao, M.D., is Research Associate at the Department of Pediatrics, Infectious Diseases Division, UB—SUNY,

Women

and

Children’s Health Research Foundation, Women and Children’s Hospital/Kaleida Health, Buffalo, New York, U.S.A. Pearay Ogra, M.D., is Professor at the Department of Pediatrics, Infectious

Health

Diseases

Research

Division,

UB—SUNY,

Foundation,

Women

Women

and

and Children’s

Children’s

Hospital/

Kaleida Health, Buffalo, New York, U.S.A.

Encylopedia of Dietary Supplements DOL: 10.1081 /E-EDS-120022911

Copyright © 2005 by Marcel Dekker. All rights reserved.

Fig. 1 Lactobacillus casei are Gram-positive, facultatively anaerobic, nonmotile and nonspore-forming, rod-shaped [cell size range = (0.7 — 1.1) x (2.0 — 4.0)mm] members of the industrially important lactic acid bacteria. (Photo courtesy of Jeff Broadbent, Utah State University.) (View this art in color at www.dekker.com. ) 381

Lactobacilli and Bifidobacteria

382

Lactobacillus and Bifidobacterium

Lactic and acetic acid

ingested strains

Lowers Luminal pH Increases Luminal pH

Enhances

Integrity of mucosal barrier

Breakdown gut mucosal

barrier

Potential pathogenic Microorganisms (PPMs)

NH4 Glutamic acid

Bifidobacterium

Fig. 2. The relationship between the ingestion of lactic acid bacteria and protective effects against overgrowth of potentially pathogenic micro-organisms.

Glutamine

colony counts

(intestine/stools)

BIOAVAILABILITY Autochthonous L. acidophilus and _ bifidobacteria strains in humans may remain stable lifelong. With lactobacilli particularly, some succession of strains may be caused by transient species derived from food or

from the oral cavity.?! Multiple combinations of lactobacilli and bifidobacteria abound in the intestinal tracts of breast-feeding infants, and persist in substantially lower logarithmic quantities in adults to promote gastrointestinal health. They are greatly influenced by food and alcohol consumption

habits, antibiotic use,

and host physiology.?! L. acidophilus, and only a few strains of bifidobacteria, grow well in the vagina and urethra."! While multiple strains of commensal bacteria coexist in large quantities in the ileum and large bowel, lactic acid bacteria use lactose as their main source of carbon to produce energy. L. acidophilus is a rich source of lactase, the enzyme needed to digest milk sugar. Fructo-oligosaccharides (FOS) are naturally occurring carbohydrates that cannot be digested or absorbed by humans. They support the growth of bifidobacteria in the lower colon, where the bifidobacteria thrive on vegetable fiber, and complex sugars, that are included in garlic, onion, artichoke, asparagus, and chicory root.) By matching host carbohydrate synthesis with the capacity of these commensal bacteria to produce glycosidases, oligosaccharide outer chains in mammalian glycans may help prime colonization of the developing intestine.

Proprietary and Probiotic Strains

The adaptive tolerance of commensal bacteria to live in host gastrointestinal niches provides the rationale for giving biologically active supplements (probiotics) as prophylactic and therapeutic agents. Probiotic bacteria are generally, though not exclusively, lactic acid

{

bacteria and include single or combined strains of L. acidophilus, L. casei (rhamnosis), L. bulgaricus, L. plantarum, L. salivarius, L. reuteri, L. brevis, L. paracasei, L. johnsonii, L. lactis, L. fermentum, L. gasseri,

Bifidobacterium lactis,

B.

breve,

bifidum,

B. longum,

B. adolescentis,

B. infantis,

and

B.

Streptocotcus

thermophilus. Proprietary strains are generally recognized as safe (GRAS) and are used in the production of yogurt, various fermented milk products, and dietary supplements. The claimed health benefits of probiotic bacteria include: increased nutritional value (improved digestion and increased absorption of vitamins and minerals), promotion of intestinal lactose digestion, positive influence on intestinal and urogenital flora (antibiotics and radiation-induced colitis, yeast infections and vaginitis in women), prevention and reduction of intestinal tract infections (bacteria or virus induced, Candida enteritis, and Helicobacter pylori), regulation of gut motility (constipation and irritable bowel syndrome), decreased incidence and duration of diarrhea (antibiotic associated, Clostridium difficile, travelers, rotavirus), maintenance of gut mucosal integrity, improvement of immune system function,

prevention

of colon

cancer,

reduction

of

catabolic products eliminated by the kidney and liver, prevention of osteoporosis, better development (growth), anticarcinogenic, antimutagenic, and antiallergic activities (Fig. 3).2:" Lactobacilli and_ selected bifidobacteria strains have been consumed in fermented food products for hundreds of years without serious side effects. Single and combined cultures are increasingly used as probiotics (live microbial supplements) in pharmaceuticals and as dietary supplements. Enhanced viability and _strainspecific effects have produced variable results, and are the subject of on-going studies in the emerging field of probiotic biology.! Probiotic strains of L. acidophilus have been the most studied, although an increasing number of probiotic bifidobacteria strains have demonstrated beneficial effects in animal models.’*! Probiotic

_

Lactobacilli and Bifidobacteria

Harmful/Pathogenic

383 Effects

Health Promoting Effects

| Pseud-Aeruginosa

Production of Toxin

\ aa Staphylococci

‘ Clostridia

(Systemic Effects)

Inhibition of harmful bacteria

Aid in digestion/ absorption of food

Stimulation of immune Eubacteria

Bifidobacteria Bacteroides

response; Anti-tumor properties; Cholesterol reduction

Synthesis of Vitamins

= Number/g of stool (log 10 scale)

strains require additional safety and efficacy testing. Yet evidence-based results suggest that they may enhance production of antibacterial products and reduce pathogen attachment to epithelia, which can cause intestinal inflammation, toxin production, and clinical disease. Among the most promising strain-specific effects are their potential roles as adjuvants in stimulating immunoprotective responses.”! Probiotics represent an important,

yet unproven,

antimicrobials, to which may develop resistance.

alternative

to conventional

pathogenic

micro-organisms

ACTION AND PHARMACOLOGY Although best known for their ability to reimplant and balance

the intestinal microflora,

lactic acid bacteria

also possess antibacterial activity (bacteriocins). Numerous species, including L. acidophilus and bifidobacteria, are involved in the production of short-chain

fatty acids and amino acids (arginine, cysteine, and glutamine). L. acidophilus and selected bifidobacteria strains produce

lactase, an enzyme

important

Fig. 3 Dichotomy of microflora based on potentially toxic or beneficial properties. (From Ref; adapted from Ref.”!.)

play an important role. Among the possible methods of probiotic action is the promotion or maintenance of the gut defense barrier, and this may occur through both immunologic and nonimmunologic pathways." When pathogenic micro-organisms, antibiotics, chemicals, radiation therapy, or even dietary antigens perturb either the intestinal epithelium or the normal microflora, causing inflammation and increased intestinal permeability, the host’s gut defense can be compromised, and this leads to disease. Lactic acid-producing strains can stabilize the intestinal barrier by virtue of their ability to survive and colonize the intestinal tract. The ability of lactic acid bacteria to adhere to the intestinal epithelium may provide the niche from which they compete successfully with pathogenic bacteria in preventing the latter’s attachment to and penetration of the mucosa.|*! Adherence to intestinal cells may be nonspecific in nature or may involve interaction with specific receptors. Once the strains succeed in colonizing and multiplying, even if transiently within the gut milieu, they may also

in the Luminal pathogens

digestion of milk, and both are involved in vitamin K and vitamin B synthesis (niacin, folic acid, biotin, vita-

min B6, and pyridoxine). Colonization by indigenous microflora induces expression of fucosylated glycoconjugates on the host intestinal epithelium. Resistance to colonization by enteric pathogens interacting with gut commensal flora prevents large inoculums of pathogens that lead to pathogenesis and clinical symptoms

indigenous microflora

Fucosylated GYCOCON ARR.

(Fig. 4).°!

Mechanisms

of Host Defense

Various mechanisms of action for commensal strains have been postulated to explain the reported beneficial effects following their consumption. An intact and functional gut mucosal surface is key to the maintenance of intestinal health, and the intestinal microflora

Fig. 4 Crosstalk between intestinal bacteria and the host epithelium. (From Ref.!'°l. adapted from Schauer, D.B. Paving the way for pathogens. Curr. Biol. 1997, 7 (2), R75-77.)

Lactobacilli and Bifidobacteria

384

influence composition of the microflora by the secretion of bacteriocins (antimicrobial substances that may kill or inactivate certain disease causing bacteria). They may also provide stability to the gut intestinal barrier by means of immunomodulatory activities especially stimulation of specific secretory immunoglobulin A (IgA) production, resulting in immune exclusion or immune elimination of pathogenic invaders. Both in vitro and in vivo studies have measured the effect of lactic acid strains on cytokine production and the role played in the inflammatory and immune

biofilm type plaques may attenuate inflammatory cellular responses. Nutrient exchange in the gut could occur through biofilms, although biofilm plaque has only been reliably reported in the oral cavity. Addi-

responses to various antigens. The results, which show

Implications for Vaccine Development

stimulation of certain cytokines and downregulation of others, suggest that one benefit of oral probiotic therapy may be in controlling the balance between proinflammatory and anti-inflammatory cytokines, and this, in itself, may be influenced by the immunologic state of the host, e.g., healthy vs. allergic.!'"! In addition, there is evidence that lactic acid bacterial enzymes can modify foreign dietary antigens to render them less immunogenic, which could have clinical applications in the treatment of allergic infants.!"*! Recent evidence suggests that stimulation of specific and nonspecific immunity may be important mechanisms by which lactic acid strains can impart protective immunity. The underlying mechanisms of immunoregulation are not well understood, but specific cell wall components may act as adjuvants and increase humoral immune responses. Commensal strains tested in animal models and in a variety of small controlled human trials have generally been shown to be safely tolerated and effective in mediating acute diarrhea and gastrointestinal inflammation. Survival under conditions of gastric acidity and the ability to adhere to intestinal cells have been substantiated in vitro and in vivo for L. acidophilus and other probiotic strains.""7! The combined findings to date indicate that the mechanisms by which lactic acid bacteria modulate host immunity are complex. Much less is known about the safety and efficacy of other commensal, probiotic

strains including L. acidophilus Gilliland," which may have the potential to reduce serum cholesterol. In vivo observations in the oral cavity strongly support the notion that the growth of natural flora in Table 1

tionally, there have been a number of interesting obser-

vations made production of in its inhibition duction and in

recently with L. acidophilus in the mucous by stimulating mucous genes of apoptosis induced by cytokine proits interaction with toll-like receptors.

Nonpathogenic bacteria may directly influence the intestinal epithelium of the host to limit immune activation. One aspect of probiotic modulation of the immune response is through its effects on cytokine production. Cytokines and their regulation of the immune system have been studied intensively in the last several years in cell lines and primary cells of both rodents and humans. Several experiments have shown — that cytokine production can be favorably altered by probiotic use (Table 1)."*! There is mounting evidence that commensals acquired postnatally are essential for the development of tolerance to luminal antigens.''*! Immune responses are attenuated if there has been prior exposure to gut antigens. This observation suggests an element of tolerance induced by the mucosal immune system. In healthy hosts, the mucosal immune system must be efficient in downregulating overactive responses to dietary antigens and commensal flora, yet differentiate and mount effective protective responses to pathogenic organisms. The search for suitable carrier organisms for the development of live oral vaccines has been concentrated on attenuated mucosal pathogens. The use of commensal bacterial strains and encoded genes might allow

better

control,

prevent

reversion

to virulence

and thus, optimization of expression systems to be used in live oral vaccines. Work with probiotic carrier strains performed in mouse infection models and optimized model antigens will help to reveal the efficacy and safety of this approach. While genetic manipulation of probiotic strains might also lead to

Probiotic effects on cytokine production

Probacteria

Species

Assessment

Lactobacillus casei, oral (dry)

Human

Serum IFNy

Lactobacillus GG, oral (live)

Human

TNF« in patients with food allergy

Lactobacillus, Bifidobacterium, and streptococcus (several strains), oral (live)

Rodent

Mitogen-induced IL-6, IL-12, IFNy, and TNF« production by intestinal lymphoid cells

IFN, interferon; TNF, tumor necrosis factor; IL, interleukin; NO, nitric oxide.

(From Ref.'°),)

Effect

Increased

Decreased fecal TNFa

:

Enhanced IL-6 and IL-12 (L. casei and L. acidophilus). Enhanced IFNy and NO (L. acidophilus)

Lactobacilli and Bifidobacteria

385

promising results, human immunodeficient states might introduce limitations to vaccination strategies with live carrier vaccines.!!7]

PREPARATION Commercially available fermented foods, although of high nutritional value and shown to have benefit in countering antibiotic-induced and traveler’s diarrhea, can be unreliable sources of delivering optimal quantities of these commensal bacteria, since the acid and peroxide production may kill the strains themselves, if concentrations of these natural byproducts become excessive. A variety of alternative delivery systems and biotherapeutic approaches are underway, including expression of heterologous proteins of lactobacilli

and bifidobacteria strains."®! Antigen delivery systems in development require more precise identification of mechanisms of action including liposomal and microparticle components and biofilm plaques, immune adjuvancy, and the potential for use of these commensal organisms as live attenuated vectors. Live vectors are highly immunogenic in themselves, and there may be potential danger for their use in immunocompromised individuals. Lactobacilli and bifidobacteria can be found in natural health foods and fermented dairy products (sweet acidophilus milk and yogurt). Supplementary sources can also be taken in powder, liquid extract, capsule, and tablet form. Tablet manufacture

generates heat,

which can reduce the number of viable organisms. Some manufacturers have incorporated a low-temperature tableting process to increase viability. Most strains in tablet form should be freeze-dried, packaged,

and stored in opaque, glass, and moisture-proof containers in the refrigerator. Another technique to enhance product viability includes “‘flushing’’ the container with nitrogen gas. . Studies with unheated and heated homogenates indicate that candidate strains of lactobacilli and bifidobacteria possess a heat-stable antiproliferative component(s). The potential for generating microbiologically nonviable, yet immunologically active, dietary supplements that are easier to store also warrants further investigation.7! Fig. 5 depicts a typical manufacturing process for both sweet and fermented acidophilus milk.?°! The production of kefir and yogurt also follows, a similar production pattern.

dietary supplement should be of human origin, be nonpathogenic and nontoxic, contain a large number of viable cells, be capable of surviving and metabolizing in the gut, remain viable during storage and use, remain active in the presence of antibiotics, be antagonistic to pathogens, and, most important, exert beneficial effects

on

the host.

In addition,

the probiotic

ideally should be readily available through regular retail suppliers and be affordable to the consumer. Probiotic strains of bacteria consumed as dietary supplements (or in dairy products) are measured by the amount of viable colony forming units (CFUs) per dosage. Dosage regimens may differ from individual to individual. It is debatable how often or whether probiotics should be ingested regularly in order to enhance persistent health effects. Generally, it is suggested to gradually increase the dosage over time. When the primary reason for supplementation is to aid digestion, lactobacilli and/or bifidobacteria tablets should be consumed with meals. If, however, the primary goal is to increase the population of these organisms

in the lower

intestinal

tract, then dosing

in-between meals with a full glass of water is recommended. A combination of both dosing approaches could also be used to maximize the potential benefits of supplementation. Many practitioners suggest “dosing away’’ from antibiotic regimens to decrease risk of potential side effects. Daily recommended usage varies, with general consumption levels indicated at between | and 10 billion bacteria daily; more may cause gastrointestinal irritation. One to 2 billion CFUs per day of L. acidophilus is considered to be the minimal amount necessary for the healthy maintenance of intestinal ecology. It is primarily available in 4701000 mg capsules that contain 2.5-10 billion live bacteria. Bifidobacteria supplements are less available in health food stores. Limited research has recommended bifidobacteria consumption at 8g per day. However, some studies indicate that 4 g per day appears sufficient to increase bifidobacteria levels in the gastrointestinal tract. Alternative recommendations to regular use suggest probiotic levels have strain-specific effects and should be used only for 1-2 weeks a year. Since oral

antibiotics kill normal populations of microbial flora, consuming an acidophilus preparation for up to a month beyond the antibiotic treatment period is recommended as safe. Studies are also underway to determine whether combinations of lactobacilli and bifidobacteria reimplant better or have enhanced antibacterial or immunoprotective effects.?4

INDICATIONS AND USAGE Labeling

It is possible to manipulate the composition of the gastrointestinal tract microflora in adults through dietary supplementation with probiotics. An effective probiotic

Several reports of misleading labeling of dietary supplements have been published. Labeling has been

Lactobacilli and Bifidobacteria

386

Acidophilus milk Fermented

Intensive heat treatment of milk (2 x 90°C for 20 min, or 1 x 140°C for 2 s)

1 Cooling to 37°C

a Inoculation with 5% Lactobacillus acidophilus culture

J Incubation overnight at 37°C (final lactic acid concentration 0.6—0.7%)

1 Cooling to 5°C

Cooling to 5°C

1 Addition of 1 x 10'° Lactobacillus acidophilus cells/mL

1 Bottling

1

+

Storage at 5°C

Storage at 5°C

criticized for overstating the level of viable bacteria, for inaccurately indicating the species of probiotic bacteria present, and for the presence of species of bacteria not listed on the label (e.g., Enterococcus spp.).°7! In commercial yogurt, the number of L. acidophilus cells does not currently appear on the labels because their concentrations cannot be accurately measured in the presence of other cultures, particularly L. bulgaricus and S. thermophilus,

recommendations for specific probiotic strains.?7) Dose—Range Effects general,

urgently needed to establish optimum dose schedules in target groups. There are several reports of disease prevention or enhancement of immune function resulting from the oral administration of probiotics such as Lactobacillus and Bifidobacterium species.°*! When ingested in large enough quantities (1 x 10'°/L) and for long enough periods (>2 weeks), these bacteria can be incorporated into the resident gut flora.

which are the bacteria that convert

milk into yogurt. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results using these methods indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains (L. acidophilus and L. casei). Accurate labeling is critical to standardizing and establishing uses and

In

Fig.5 The manufacturing process for acidophilus milk, both sweet and fermented. (From Ref.”°!; reproduced with permission © American Society for Clinical Nutrition.)

Bottling

in the

United

States,

food

products

containing probiotic bacteria make no mention of the numbers of bacteria present in the product per serving. Most list bacterial genera and species added as live cultures, but not levels. California and Oregon are unique in that they legislate a minimum requirement for acidophilus-containing fluid milk products (10°/ml). In the United States, yogurt is not required to contain any viable cultures. Dose-range escalation studies are

ADVERSE EFFECTS Lactobacilli and bifidobacteria are extremely rare causes of infection in humans. In the general literature, they have been estimated to represent only 0.05-0.4% of infective endocarditis and bacteremia cases.?°! This lack of pathogenicity extends across all age groups and to immunocompromised individuals. Most of the rare cases of infection with lactobacilli occur in patients with predisposing conditions; lactobacillemia is a frequent marker of a serious or fatal underlying disease.”°! Recent evidence confirms that the increasing consumption of probiotic lactobacilli and bifidobacteria has not led to an increase in opportunistic infection in consumers. In addition, while immunocompromised patients are at a higher risk for opportunistic infections than the general population, consumption of probiotic products containing lactobacilli or bifidobacteria has not been shown to increase the risk of infections due to these organisms in such

individuals.”

Lactobacilli and Bifidobacteria

387

Side effects from consumption of probiotic strains that may indicate allergic reactions include: breathing problems

or tightness in throat or chest, chest pain,

skin hives, rash, itchy, or swollen skin. Other possible adverse

events

may

include

nausea,

diarrhea

(loose

stools), burping, hiccups, or gas.

DRUG INTERACTIONS/SAFETY Certain medications may theoretically interact with lactobacilli and bifidobacteria. Gut bacteria may be depleted by use of the contraceptive pill, antibiotics, and diets high in sugar and refined carbohydrates. Lactobacilli and bifidobacteria should be considered safe; however, some restrictions need to be placed on

probiotic formulations. Bacteria used should not contain genes encoding transmissible resistance to antibiotics. They should not produce biogenic amines (e.g., histamine, tyrosine, or phenylethylamine), and should not degrade mucin. The assessment of clinical value should probably include an absence of potential to deconjugate bile salts, induce platelet aggregation, and bind to fibrinogen and fibronectine.?7!

to increase host resistance to gastrointestinal and vaginal infections, effectiveness in treating antibioticinduced diarrhea, reduction of serum cholesterol and

blood pressure, treatment of allergy and inflammatory skin conditions, stimulation of phagocytosis, antitumor effects, and adjuvant properties for passive immunity and new generation vaccine delivery. Confirmation of clinical results for most candidate strains of lactobacilli and bifidobacteria is still lacking, with the exception of acute diarrhea or antibiotic-induced diarthea (Table 2).!'7) Genetically engineered commensals may, in the future, be used as platforms for delivering drugs, antimicrobials, and live vaccines to defined host

sites. Product development in the field of probiotic biology requires interdisciplinary expertise from epidemiologists, microbiologists, gastroenterologists, immunologists, nutritionists, computational scientists, and food technologists. Comparative studies with germfree, knockout,

and conventional

animals, given advances

in new molecular biotechnology, will also help to establish a more accurate concept of host—microbial relationships,

in attempts

to standardize

what

con-

stitutes a safe and efficacious, probiotic dose of these organisms.

Compendial/Regulatory Status

CONCLUSIONS Research Summary A large body of research over the past 100 yr demonstrates the health benefits of fermenting lactobacilli and bifidobacteria in the gastrointestinal tracts of breast-feeding infants and adults. Tannock’s”®! recent compendium of probiotic species of lactobacilli and bifidobacteria summarizes the evidence for their ability

Choice of strain selection with proven safety and probiotic efficacy, appropriate inoculum, incubation conditions, microbial interactions, viability and delivery systems are imminent in establishing health claims and promoting product manufacture of these prophylactic and therapeutic agents. For successful transfer of candidate strains of lactic and acetic acid-producing bacteria from the laboratory scale to industrial production, graphical optimization methods relying on

Table 2 Important studies for the safety assessment of probiotic lactic acid bacteria and other bacteria Safety factor to be assessed -

Type of property studied

Intrinsic properties of lactic acid bacteria

Adhesion factors, antibiotic resistance, existence of plasmids and

plasmid transfer potential, harmful enzyme profile

Metabolic products

Concentrations, safety, and other effects

Toxicity

Acute and subacute effects of ingestion of large amounts of

Mucosal effects

Adhesion, invasion potential, intestinal mucus degradation, infectivity in immunocompromised animals (e.g., following lethal irradiation)

tested bacteria

Dose-response effects

Dose-response studies by oral administration in volunteers

Clinical assessment

Potential for side-effects, careful evaluation in health volunteers and

disease-specific studies

Epidemiological studies (From Ref.""7!.)

Surveillance of large populations following introduction of new strains and products

Lactobacilli and Bifidobacteria

388 h. [29]

statistical standards are essential in process researc Continuing challenges include conclusive evidence for probiotic-mediated responses to support purported health claims. To fully explore immunomodulatory mechanisms, further studies on the relative immunogenicity of strain-specific effects and “broken immune tolerance’’ in inflammatory bowel disorders are critically needed. Live oral vaccines and use of probiotic adjuvants offer several benefits over conventional vaccine delivery and represent a top priority in modern vaccinology.

10.

Lu, L.; Walker, W.A. Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium. Am. J. Clin. Nutr. 2001, 73 (6), 1124S-1130S.

We

IPs Kankaanpaa, Y.; Sutas, E.; Isolauri, on effects : Probiotics S. Salminen, Arvilommi, H.;

immunity. Am. J. Clin. Nutr. 2001, 73 (2 Suppl.), 444S-450S. (Dx,

Salminen,

S.; Isolauri, E.; Salminen,

E. Clinical

uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges. Antonie van Leeuwenhoek 347-358.

|e

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The Lactic Acid Bacteria: The Lactic Acid Bacteria in Health and Disease; Wood, B.J.B., Ed.; Elsevier Science Pub. Ltd.: Essex, England,

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intestine: composition

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Lactobacillus_

Microbiol.

1985,

Erickson, K.L.; Hubbard, N.E. Probiotic immunomodulation in health and disease. J. Nutr. 2000,

Ingredients

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Duffy, L.C.; Zielezny, M.A.; Riepenhoff-Talty, M.; Dryja, D.; Sayahtaheri-Altaie, S.; Griffiths, E.; Ruffin, D.; Barrett, H.; Rossman, J.; Ogra,

P.L. Effectiveness of Bifidobacterium bifidum in mediating the clinical course of murine rotavirus diarrhea. Pediatr. Res. 1994, 35 (6), 690-695. Gill, H.S. Stimulation of the immune

system by

lactic cultures. Int. Dairy J. 1998, 8, 535-544.

I.B.; Schmidt,

M.A.

Bacterial inter-

Kullen, M.J.; Klaenhammer, T. Genetic modification of intestinal lactobacilli and bifidobacteria. In Probiotics: A Critical Review; Tannock, G., Ed.; Scientific Press) Wymondham, UK, 1996; 65-83. :

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Kaila, M.; Isolauri, E.; Saxelin, M.; Arvilommi, H.; Vesikari, T. Viable versus inactivated lactobacillus strain GG in acute rotavirus diarrhoea. Arch. Dis. Child. 1995, 72 (1), 51-53.

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Heller, K.J. Probiotic bacteria in fermented foods: product characteristics and __ starter organisms. Am. J. Clin. Nutr. 2001, 73 (2S), 3748-3798.

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Marteau, P.; Salminen, S. Safety of probiotics. In

J. Nutr. Biochem. 1998, 9 (12), 668-675.

rhesus rotavirus-challenged BALB/c mice treated with bifidobacteria and prebiotic supplements. Pediatr. Res. 2002, 5/ (6), 750-755.

Autenrieth,

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Walker, W.A.; Duffy, L.C. Diet and bacterial colonization: role of probiotics and prebiotics. Qiao, H.; Duffy, L.C.; Griffiths, E.; Dryja, D.; Leavens, A.; Rossman, J.; Rich, G.; RiepenhoffTalty, M.; Locniskar, M. Immune responses in

Moreau, M.C.; Corthier, G. Effect of the gastro-

play at intestinal mucosal surfaces: implications for vaccine development. Trends Microbiol. 2000, 8 (10), 457-464.

Fuller, R. Probiotic foods: current use and future

Food

Nelson,

of cholesterol

intestinal microflora on induction and maintenance of oral tolerance to ovalbumin in C3H/HeJ mice. Infect. Immun. 1988, 56 (10), 2766-2768.

1995,

Stark, P.L.; Lee, A. The microbial ecology of the large bowel of breast-fed and formula-fed infants during the first year of life. J. Med. Microbiol. 1982, 15 (2), 189-203. Int.

S.E.;

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130 (2S), 403S—409S.

Gibson, G.R.; Roberfroid, M.B. Dietary modulation of the human colonic microbiota: introdu-

developments. 23-26.

Gilliland,

acidophilus. Appl. 49 (2), 377-381.

Reuter, G. The lactobacillus and bifidobacterium

and succession. Curr. 2001, 2 (2), 43-53.

Conway, P.L.; Gorbach, S.L.; Goldin, B.R. Survival of lactic acid bacteria in the human

Assimilation

1992; Vol. 1, 69-114.

microflora of the human

1996, 70,

stomach and adhesion to intestinal cells. Sci. 1987, 70 (1), 1-12.

Mitsuoka, T. The human gastrointestinal tract. In

'

Probiotics, Other Nutritional Factors and Intestinal Microflora. Nestle Nutrition Workshop Series, Hanson, L., Yolken, R., Eds.; Lippincott Raven Pub.: Philadelphia, PA, 1999; Vol. 42, 259-269.

Lactobacilli and Bifidobacteria

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Sanders, M.E. Considerations for use of probiotic bacteria to modulate human health. J. Nutr. 2000, 130 (2S), 384S-390S.

PE

Hammes, W.P. Safety assessment of lactic acid bacteria and probiotics. Monatsschr. Kinderheilkunde 1998, 746 (8S), S31-S38.

23,

Yeung, P.S.; Sanders, M.E.; Kitts, C.L.; Cano, R.;

28.

Tannock,

Tong, P.S. Species-specific identification of commercial probiotic strains. J. Dairy Sci. 2002, 85 (5), 1039-1051.

24.

Gill, H.S.; Rutherfurd, K.J.; Cross, M.L.; Gopal,

P.K. Enhancement of immunity in the elderly by dietary supplementation with the probiotic Bifidobacterium lactis HNO19. Am. J. Clin. Nutr. 2001, 74 (6), 833-839.

25.

Gasser, F. Safety of lactic-acid bacteria and their occurrence in human. clinical infections. L’Inst. Pasteur 1994, 92, 45-67.

26.

S.P.; Hammes, W.P.; Holzapfel, P.; Schrezenmeir, J.; Vaara,

Valtonen,

V. Safety of probiotics that contain Clin.

Infect.

Probiotics:

A Critical

Review;

29,

Report Food of

of

a

Consumption Chemicals;

WHO/FSF/FOS Organization.

FAO/WHO and

February

97.5,

Consultation.

Exposure

1998,

Assessment

10-14,

1997,

World

Health

FURTHER READINGS

Bull.

Borriello, Marteau,

lactobacilli or bifidobacteria. 2003, 36 (6), 775-780.

G.W.

Horizon Scientific Press: Wymondham, UK, 1996.

W.; M.;

Dis.

http: //www.academyhealth.com/abbif.html. http: //www.flora-source-probiotics.com. http: //www.horizonpress.com/hsp/abs/abspro.html. http: //www.pubmedcentral.nih.gov. http: //www/usda.gov

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are now under investigation.?”! The concentration is approximately |1mM in the human fovea (the conephotoreceptor-dense area of the central macula that processes fine pattern information). Retinal concentrations may be 1000 times higher than those in serum and other tissue (Table 1). The macula also contains the highest proportion of lutein and zeaxanthin as total carotenoids. Within the macula, the cellular distribution of lutein is believed to be highest in photoreceptor axons and interneurons of the inner plexiform layer.'7*! In eccentric retinal areas, lutein is believed to be present mainly in the rod photoreceptor outer segments.?°?"! The distribution of total and individual macular xanthophylls varies with retinal eccentricity.!”*!°! The average mass of lutein and zeaxanthin per unit retinal area was 1.33 and 0.81 ng/mm/ at the foveal center and at an eccentricity of 1.6-2.5mm, respectively. The lutein: zeaxanthin ratio at 0-S° was 1.0:1.6 in one study. At 5-19°, the ratio was

was 2.0:1.0. Research groups examining the distribution of macular xanthophylls have observed similar patterns, with relatively more zeaxanthin in the fovea.”! The distribution of xanthophylls may have implications for light processing and tissue protection, as subtle differences in the structure and physical form of lutein and zeaxanthin lead to positional differences in phospholipid membranes. Snodderly and others have examined the distribution of macular xanthophylls in nonhuman primates.7%393!] Fig. 3 shows the macaque retina in cross-section and the distribution of macular pigment within layers, relative to the fovea.

Plasma, Serum, Cell, and Macular Concentrations

Lutein and zeaxanthin are among the six carotenoids that contribute 60-70% of total plasma carotenoids

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Lutein

414

under fasting conditions. These xanthophylls are commonly reported as a single value, since their elution

profiles are similar; when grouped as such, they follow lycopene to represent the second highest concentration of carotenoids within total plasma composition. The plasma lutein : zeaxanthin ratio is 4 or 5:1. Landrum and Bone!” present the current knowledge on serum response to lutein and zeaxanthin: Serum lutein and zeaxanthin levels of people consuming common Western diets are substantially lower than those attainable with supplementation; higher serum concentrations of lutein and zeaxanthin metabolites are found with supplementation; meso-zeaxanthin may be produced from lutein; within the central macula, meso-zeaxanthin attains highest concentrations and represents approximately half of the zeaxanthin present in this region. In experimental populations fed a low-carotenoid diet, plasma carotenoid levels decreased at a rate proportional to the concentration of nutrient in the diet for 2-4weeks.

Thereafter, the rate declined slowly to

a plateau. Initial half-lives of lutein/zeaxanthin were 12 days in a 30-day feeding study and 19days in a 64-day study.“! The mean plasma depletion half-life of lutein/zeaxanthin is estimated to be between 33 and 61 days from a metabolic ward study on a 13-week vitamin C-free diet providing 0.4mg carotenoids/ day."*] Serum concentrations were approximately 45-50% of baseline in 68- and 90-day feeding studies using low-carotenoid diets.°?77) Stable isotopes have been used to characterize xanthophyll plasma response across a 528-hr period. 'C-Labeled lutein was fed as part of a low-carotenoid meal and showed a rapid serum response, with a unimodal distribution peaking at 16hr.°* Fig. 4 shows

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the results from this study. It is important to note that the 3-mg (5.3-umol) lutein dose was dissolved in higholeic safflower oil and represented a physiological level attainable through diet. A monophasic 16-hr peak was also reported in a feeding study of an ethyl acetate form of lutein in tocopherol-free corn oil (0.5 pmol/kg body weight, 3.84mmol/L) with samples taken across an 840-hr period.?*! A number of intervention studies with lutein supplements and/or feeding regimens have been developed to characterize plasma and serum lutein responsiveness. While ability to compare across studies is constrained by differences in study design, implementation, and analysis, a number of consistencies emerge in the results: The serum or plasma response to nearly equal intakes of lutein was approximately two times higher in the groups receiving lutein in supplement form; and diets with 79-3) Similarities in the structural characteristics of carotenoids cause difficulty when trying to adequately identify individual carotenoids using only fixed wavelength or retention time data. The use of photodiode array detection, allowing for the collection of spectral data across a wide range of wavelengths, has improved our ability to more accurately characterize individual carotenoids. Measurements of retention time, peak resolution, and spectral data for individual absorbing species, and the use of authentic standards for comparison of UV/VIS spectra and retention times are required.)

Ss

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of selected lycopene

Lycopene

424

Our ability to detect very low levels of carotenoids in biological samples has been somewhat limited by methodology and detection that does not adequately quantify carotenoid concentrations. The development of a C9 reversed-phase gradient HPLC method coupled with a coulometric electrochemical (EC) array detector provides a much lower detection limit and a unique opportunity to quantify low levels of carotenoids in tissue samples and in plasma chylomicron fractions.?*! The improved sensitivity of this HPLCEC method (1-10 fmol) also allows for detection and quantification of compounds from extremely small sample sizes. Mass spectrometric and tandem mass spectrometric analyses, which provide molecular weight and characteristic fragmentation patterns, provide additional information that increases our confidence in the identification of various carotenoids.7! Both electron impact and fast atom bombardment have also been used in mass spectrometric analysis of carotenoids.2+7*! Identification and structure elucidation of isomeric carotenoids have been facilitated with the aid of high-resolution nuclear magnetic resonance (NMR) spectroscopy. Hengartner, Bernhard, and Meyer?” reported the use of H- and C-NMR,

and UV/VIS,

mass, and infrared (IR) spectroscopy to fully characterize 15 (E/Z)-isomeric forms of lycopene. In addition, the use of HPLC-MS with atmospheric pressure chemical ionization allows lutein stereoisomers to be distinguished from zeaxanthin stereoisomers during one chromatographic run with detection in the picogram range. In contrast, HPLC-NMR coupling provides unequivocal identification of each stereoisomer with a concentration in the nanogram range.*! The rapid improvement in analytical technology will significantly impact future investigations designed to elucidate the biological impact of lycopene and its isomers on tissues and organs. Investigators ranging from epidemiologists and clinical scientists to those involved in animal studies will be able to more precisely quantitate lycopene and its isomers in extremely small biological samples.

STABILITY AND ISOMERIZATION Consumers use the intensity of the red color as an index of quality for tomato products. Therefore, reducing the loss of lycopene throughout the production process and during storage has always been an important issue for the food processing industry. Exposure to thermal treatments during food-processing operations causes well-documented changes in the physiochemical stability of carotenoids. Boskovic!*”! and Cano et al,'4° observed that processing and extended storage of dehydrated tomato products resulted in a loss of

all-trans lycopene content by up to 20%. Food-processing techniques, such as canning and freezing, led to a significant reduction in lycopene and total carotenoid content in papaya slices. In contrast, many studies have found that hydrocarbon carotenoids such as lycopene, o-carotene, and B-carotene in processed fruits and vegetables are fairly heat resistant.414! According to Khachik et al.,4!! most of these carotenoids remain stable after bench-top food preparation. Additionally, no major changes in phytofluene, phytoene, and C- , carotene were observed during the processing of tomatoes.“*! Saini and Singh'*! reported that thermal processing had no effect on the lycopene content in juices made from several high-yield tomato hybrids. Zanori et al.'4°! reported that despite the oxidative and thermal severity of the drying process, reflected in the 5-hydroxymethyl-2-furfural and ascorbic acid values, lycopene displayed high stability during drying of tomato halves. Additionally, Nguyen and Schwartz!!7} reported that processing does not have a significant effect on the stability of lycopene, independent of product type, moisture content, container type, tomato variety, and severity of heat treatments. Although lycopene may be fairly stable during standard food-processing procedures, less is known about the impact of heat on isomerization. Studies have shown that heating tomato juice and bench-top preparation of a spaghetti sauce from canned tomatoes increases cis isomer concentrations.?*** In contrast, Khachik et al.7) observed that common heat treatments during food preparation, such as microwaving, steaming, boiling, and stewing, did not significantly change the distribution of carotenoids in tomatoes and green vegetables. Other studies have also reported low levels of lycopene cis isomers in thermally processed tomato products.!'*?7] Nguyen, Francis, and Schwartx*! reported that during typical cooking of tomatoes, factors such as genotypic differences in the overall carotenoid

composition,

the presence

of oil,

and physical changes to tomato tissues did not influence the thermal isomerization of all-trans lycopene, all-trans 6-carotene, all-trans y-carotene, or prolycopene. Observations in our laboratory indicate that lycopene is quite stable in its native matrix within tomato tissue. However, extensive physical and thermal processing that disrupts cell wall constituents can expose lycopene to degradative reactions. In addition, food products containing lipids or supplements formulated with oil will partially solubilize lycopene, leading to isomerization reactions. Further heating such as cooking of foods in the presence of oils and fats will facilitate solubilization,

isomerization,

and oxidation

reactions. Difference in formulation and the extent of processing treatments may account for discrepancies in literature reports on the stability and isomerization susceptibility of lycopene. In addition, once extracted

Lycopene

425

into organic solvents, lycopene is very labile, and analysis should take place immediately without exposure to heat, light, or oxygen. Additional information needs to be gathered on the thermal behavior of lycopene before definitive answers can be offered regarding its physical state and stability during processing and cooking. Nevertheless, it is evident that the pigment is more stable in native tomato fruit matrices than in isolated or purified form due to the protective effects of cellular

constituents such as water.!*!

BIOAVAILABILITY Phytochemicals present in tomatoes and tomato-based products must be readily bioavailable for absorption to mediate their hypothesized beneficial health effects. Bioavailability is defined as the fraction of an ingested nutrient that is accessible to the body through absorption for use in normal physiological functions and for metabolic processes.°°! Carotenoids are strongly bound to intracellular macromolecules in many foods,

and absorption may therefore be limited unless they are released from the food matrix.°" Differences in the bioavailability of lycopene may account, in part, for the relatively poor correlation between blood lycopene concentrations and estimated dietary intake. Carotenoid absorption involves micelle formation, intestinal uptake, and chylomicron transfer via the lymphatics to the bloodstream (Fig. 3).531 Little is known about how lycopene in chylomicrons is subsequently accumulated by the liver and other tissues, repackaged in lipoproteins, and returned to the circulation. Lycopene is carried in the plasma entirely by lipoproteins, and no other lycopene-specific binding

or carrier proteins have been identified thus ieee

B-C in food matrix

Details of how hepatocytes, the initial source of circulating lipoproteins, transfer lycopene into specific secreted lipoproteins, and how this process may be regulated are unclear. However, it is likely that dietary and pharmacologic agents that influence lipoprotein metabolism will impact circulating lycopene concentrations also. It is hypothesized that highly lipophilic carotenoids, such as lycopene, are present within the hydrophobic core of the lipoprotein particle. Several studies have evaluated the bioavailability of the lipophilic carotenoids found in tomatoes and tomato-based products. Heating tomato juice was shown to improve the uptake of lycopene in humans.°! These observations seem to be the result of thermal weakening and disruption of lycopene—protein complexes, rupturing of cell walls, and/or dispersion of crystalline carotenoid aggregates. Likewise, various food-processing operations such as chopping and pureeing, which result in a reduction in physical size of the food particle, will also enhance lycopene bioavailability.°°°" Lycopene bioavailability was studied after a single dose of fresh tomatoes or tomato paste by measuring concentrations of lycopene in the chylomicron fraction of the systemic circulation.” Each source of lycopene (23mg) was consumed with 15g corn oil. Tomato paste was found to yield a 2.5fold greater total all-trans lycopene peak concentration and a 3.8-fold greater postprandial response area under the curve than fresh tomatoes. When compared with fresh tomatoes, tomato paste resulted in a significantly higher area under the curve for cis-lycopene isomers.

In addition, van

het Hof et al.58! studied the

effect of mechanical homogenization and heating on the bioavailability of multiple carotenoids from canned tomatoes. Interestingly, homogenization enhanced triglyceride-rich layer (TRL) and plasma lycopene response (TRL fraction: 31% and 62% higher for

Abbreviations B-C: B-carotene CM: chylomicrons RE: retinyl esters CCE: carotenoid cleavage enzyme

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(gut)

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te

cc

Retinol

Intestinal mucosa

cell

Fig. 3. Intestinal absorption of carotenoids. (From Ref. el.)

Lycopene

426

mildly and severely homogenized tomatoes, respectively, vs. no homogenization; plasma: 16% and 21% higher, respectively; P < 0.05). Additional heating also increased lycopene responses in TRL (P = 0.14) and plasma (P = 0.17). Interestingly, similar effects were found for B-carotene. These observations support the conclusion that food processing, homogenization, and cooking enhance lycopene bioavailability. When compared to these food matrix effects, microencapsu-

continuous dietary intake and that mobilization of alltrans lycopene from liver or other tissues, or reconversion of cis isomers to trans, cannot maintain the cis: trans ratio. In addition, it is plausible to hypothesize

that there is a biological preference for certain lycopene isomers to be cleared from serum, distributed to tissues,

or participate in reactions that cause degradation. Although data are still limited, it is apparent that carotenoids are not uniformly and equally dispersed

lation and solubilization in oleoresin may provide a

in human tissues.'*°°?-7!! The tissue-specific carotenoid —

more bioavailable form of most carotenoids. However,

patterns reported thus far suggest a process whereby certain carotenoids may exert unique biologic effects in one tissue but not in another (Table 2). Presently, there is no evidence for a specific receptor or enzymatic process that mediates lycopene uptake by the tissue. We must therefore assume that uptake in the tissue is related to lipoprotein metabolism. Lycopene has been shown to exist in over 15 different geometrical configurations in human prostate tissue, where the cis isomer content is even greater (at 80-90%) than that observed in serum.*”! The chemical’ and physiological processes that account for the high proportion of cis isomers in tissue remain speculative. An intriguing hypothesis is that isomerization reflects the participation of lycopene in antioxidant reactions within the prostate. Isomerization changes the structure of lycopene in a fashion that could alter its intracellular distribution within organelles and membrane structures that in turn could influence biological processes. These are hypotheses that will need additional investigation.

in studies measuring the plasma response to lycopene intake from supplement sources vs. processed tomatobased foods, lycopene bioavailability does not appear to be statistically different." Digestive processes will also certainly influence carotenoid bioavailability. Several factors affect initial carotenoid release from the physical food matrix and transfer and distribution into lipid droplets within the stomach and proximal duodenum." Pancreatic lipases and bile salts act upon the carotenoid-containing lipid droplets entering the duodenum and form multilamellar lipid vesicles containing the carote-

noids.©*! The transfer of lycopene, like that of other carotenoids, from the micelle into the mucosal appears to occur via passive diffusion.!©7°!

cells

Perhaps of major importance, dietary lipids appear to serve a critical role in the dissolution and subsequent absorption of very hydrophobic carotenoids (e.g., lycopene). Factors such as structural features, dietary fat content, fatty acid patterns, fiber, and other food com-

ponents may influence the carotenoid content of micelles and subsequent mucosal transfer.*! In addition, drugs that interfere with cholesterol absorption!

ROLE IN CHRONIC DISEASE

and nonabsorbable fat analogs, such as sucrose poly-

esters"! have been reported to reduce carotenoid absorption.

PLASMA AND TISSUE DISTRIBUTION Serum lycopene levels can range from 50 to 900 nmol/L. It is well known that between 10 and 20 cis isomer peaks are typically observed in human blood and together account for the majority of lycopene in serum.*”-°°) Interestingly, we observed that the ratio of cis: trans lycopene isomers changes in those on a lycopene-free diet. According to Allen et al.,!°” plasma lycopene isomer concentrations exhibit a 61:39 ratio for cis:trans at the start of a lycopene-free diet, whereas after 2 weeks, the ratio shifts to 70:30, which

was highly significant. In a study by Hadley, Schwartz, and Clinton,'**! the percentage of cis: trans lycopene isomers increased from 58:42 to 62:38 following a l-week washout phase. Studies suggest that the alltrans lycopene content of serum is maintained through

Multiple mechanisms of action for lycopene’s purported beneficial health effects have been proposed. As reviewed by Heber and Lu,'”” the pigment has been shown to display antioxidant, antiproliferative, and prodifferentiation activities. In addition, other potential methods such as stimulation of xenobiotic metabolism, modulation of cyclo-oxygenase pathways, and inhibition of cholesterogenesis and/or inflammation are discussed. Although these may participate collectively to impart lycopene’s health benefits, much of the focus has been on its antioxidant activity. It has been widely postulated that oxidation may contribute to the damage of cellular DNA,

proteins,

and lipids that initiate or enhance the progression of cancer. Recent research has focused on the role of reactive oxygen species (ROS) or free radicals that are produced from exogenous and endogenous factors. Mammals have developed multiple defenses against reactive oxygen. Nutritional substances such as vitamin

E, vitamin

C, selenium,

and carotenoids!®!

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Lycopene

Table 2

427

Lycopene concentrations in human tissue Concentration (nmol/g wet wt.) a

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0.78

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0.31

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0.62

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2.45

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0.22

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0.28

0.25

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0.42

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ei

ee

’Khachik et al.!77), “Rao, Fleshner, and Agarwal.!4! “Freeman et al.!7>). *Kucuk et al.!7°,

‘Clinton et al.471.

Clinton. [4

systems, such as antioxidant enzymes (glutathione peroxidase, catalase, CuZn- and Mn-superoxide dismutase) and antioxidant quenchers (ceruloplasmin,

Lycopene may also interact with ROS such as hydrogen peroxide and nitrogen dioxide.®?**!

transferrin, ferritin, Cd/Hg/Zn/Cu: metallothioneins), which participate in the free radical defense system and provide protection against oxidative damage. Many of the proposed biological effects and health benefits of tomatoes and tomato-based products are hypothesized to be associated with the ability of certain phytochemicals such as lycopene to enhance the endogenous defense system by protecting against in vivo oxidative damage. The ability of lycopene to act as an antioxidant and scavenger of free radicals is considered by most investigators as the most likely mechanism that could account for the hypothesized beneficial effects on human health.'”?*"! As a result of having an extensive chromophore system of conjugated carbon-carbon double bonds, lycopene can accept energy from various electronically excited species. This characteristic

gives lycopene the ability to quench singlet oxygen'*®! formed by energy transfer from a metastable excited photosensitizer.” Singlet oxygen (10>) is a very reactive high-energy and short-lived oxygen species produced in biologic systems that can react with biomolecules.

CANCER In recent decades, we have seen an accumulated body of evidence strongly supporting the conclusion that diets rich in fruits and vegetables are associated with a lower risk of many malignancies. A comprehensive review of the epidemiological evidence regarding lycopene-containing tomatoes and tomato-based products and cancer risk was published by Giovannucci.! Nearly 80% of the 72 studies reported in the review revealed evidence of a protective association between consumption of tomatoes, tomato-based products, or carotenoids provided by these foods and the risk of cancer at several sites. In more than 60% of these trials,

the inverse associations were statistically significant. The observed inverse relationship was strongest for lung, stomach, and prostate cancer and was supportive for cervical, breast, oral cavity, pancreatic, colorectal,

and

esophageal

cancer.

In addition,

epidemiologic

investigations of colon,**! upper aerodigestive,®” prostate®8°"l

and

lung cancer”!

further

support

the

Lycopene

428

concept that lycopene-containing tomato products have cancer-preventive properties. Prostate cancer is the most common noncutaneous malignancy in American men, and is the second leading cause of cancer-related deaths.°7! Although an association with prostate cancer has not been as strong in comparison with other malignancies, epidemiologic studies have shown that an increase in lycopene consumption as well as serum lycopene concentrations is inversely correlated with the risk for prostate cancer. In 1991, Le Marchand et al.4! studied a multiethnic Hawaiian cohort and found no association between estimated lycopene intake and prostate cancer risk.

conducted

by Nomura

Japanese-American

men

et al.8! in a cohort

examined

from

of 6860

1971

to

1975, however, showed no association between several

sive ongoing epidemiologic studies in adult men, Giovannucci et al.?°! investigated the relationship between the risk of prostate cancer and estimated

plasma micronutrients and carotenoids, and prostate cancer risk. A nested case-control investigation was undertaken, which involved the analysis of carotenoids in blood samples from men enrolled in the Physicians’ Health Study (a randomized, placebo-controlled trial of aspirin and f-carotene). In this study, subjects in the highest quintile (>580.1 ng/ml) of serum lycopene levels had a significantly lower risk of prostate — cancer compared with those in the lowest quintile (6.4mg lycopene/day) was compared with the lowest quartile (Contents per 10g dried maca tuber! “Ref! Number of fatty acids: 20. Three major acids are given. Values are in percentage of total fatty acid.

b) glucosinolates; c) alkaloids; and d) macamides. In

The glucosinolates are a class of secondary metabolites found in 15 botanical families of dicotyledonous plants, notably including the Brassicaceae. Over 100 have been reported to date from plant sources. Glucosinolates are present at approximately 1% in fresh L. meyenii root, but no novel ones have been reported so far in maca. The presence of two main glucosinolates, glucotropeolin (3) and m-methoxybenzylglucosinolate (4), have been reported from maca,!'*!*'7] and their combined presence in L. meyenii may be used as a chemotaxonomic

marker, since the combination

of 3 and 4 does not occur in other members of the Brassicaceae.!!*! Glucosinolates and their derived products have received increasing attention due to their biological activities; examination of glucosinolate degradation products in the hexane extract has revealed the presence of benzyl isothiocyanate (5) and its m-methoxy derivative (6),''”! the former reported to be present in the range of 0.1-0.15% in standardized maca product.) Several maca products derived from processed hypocotyls of L. peruvianum and other organs have been assessed by high performance liquid chromatography (HPLC) for glucosinolate content. The most abundant glucosinolates were found to be 3 and 4 in fresh and dry hypocotyls and leaves. The richest sources of glucosinolates are seeds, fresh hypocotyls, and sprouts, in that order. Maca seeds and sprouts differ in profile from hypocotyls and leaves due to the presence of several modified benzylglucosinolates, including 5-methylsulfinylpentylglucosinolate (7), indolyl-3-methylglucosinolate (8), pent-4-enylglucosinolate, 4-methoxyindolyl-3-methylglucosinolate, glucolepigramin, and 4-hydroxybenzylglucosinolate, while the liquor and tonic contain sinigrin (9) 0141

Alkaloids Qualitative detection of alkaloid like compounds in L. meyenii was first reported by Dini et al?! and a further detailed chemical analysis of the tubers by

Muhammad et al.!!7! reported the benzylated deriva-

addition, the presence of malic acid and its benzoate

tive of 1,2-dihydro-N-hydroxypyridine, named macari-

ester (2),!'4! as well as five sterols’! and catechins, is also reported (Table 2 and Fig. 2).

dine (10). From

the methanol

extract

of the tuber,

(1R,35S)-1-methyltetrahydro-B-carboline-3-carboxylic

Maca (Lepidium meyenii)

438

Table

2 Compounds isolated from and identified in maca

Macamides

Feature

Constituents

Class

Reference

N-Benzyloctanamide (macamide A)

n

[11]

N-Benzyl-16-hydroxy-9-oxo-10£,12E, 14E-octadecatrienamide

n

{11]

n

[11]

n

[12]

(macamide B) N-Benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide (macamide C) N-Benzyl-5-oxo0-6E,8E-octadecadienamide (15)

Organic acids

N-Benzylhexadecanamide (16)

[12]

Caprylic acid, capric acid, lauric acid, myristic acid,

(Deeley)

palmitic acid, palmitolic acid, stearic acid, oleic acid,

linoleic acid, linolenic acid 5-Oxo-6E,8E-octadecadienoic acid (1)

Alkaloids

n

[17]

Malic acid benzoate (2)

[17]

Macaridine (10)

‘

(1R,3S)-1-Methyltetrahydro-B-carboline-3-carboxylic acid (11) 1,3-Dibenzyl-4,5-dimethylimidazolium chloride (lepidiline A) (13) 1,3-Dibenzyl-2,4,5-trimethylimidazolium chloride (lepidiline B) (14) Glucosinolates

Isothiocyanates

Sterols

Essential oil

Others

[12]

Malic acid n

[12]

n

[18]

[17] n

:

[18]

Benzylglucosinolate (glucotropeolin) (3) m-Methoxybenzylglucosinolate (4) p-Methoxybenzylglucosinolate

[14]

5-Methylsulfinylpentylglucosinolate (glucoalyssin) (7)

[14]

p-Hydroxybenzylglucosinolate (glucosinalbin) m-Hydroxybenzylglucosinolate (tentative identification) Pent-4-enylglucosinolate (glucobrassicanapin) Indolyl-3-methylglucosinolate (glucobrassicin) (8) 4-Methoxyindolyl-3-methylglucosinolate (4-methoxyglucobrassicin)

[14]

[14, 16, 17] [14, 17]

—

[14]

[14] [14] [14]

Sinigrin (9)

[14]

Benzyl isothiocyanate (5) m-Methoxybenzy] isothiocyanate (6)

[LSsul7 [17]

Sitosterol

[9]

Campesterol

[9]

Ergosterol

(9]

Brassicasterol A’*?-Ergostadienol

[9]

Phenylacetonitrile (85.9%), benzaldehyde (3.1%), 3-methoxyphenylacetonitrile (2.1%), and others

[15]

[9]

Uridine (12)

17]

Catechins

[32]

Polysaccharide ae a

a

eee

[11]

n = New compound.

acid (11), and uridine (12) and its benzoyl derivative have been isolated.!!7] Recently, two new 1,3-dibenzylimidazolium chloride derivatives have been isolated from root extracts and identified as lepidiline A (13)

and lepidiline B (14)!'®! (Fig. 2).

Macamides

Maca contains novel polyunsaturated acids and their amucdes?: called macaene and macamide by Zheng et al.) Prom purified standardized products of

Maca (Lepidium meyenii)

439

fe)

e OOOGORY

fe)

A iF

ae 7%.

fe)

1

OH 2

5

Ph

MeO

.

Ny

fe)

We

NO Ns

A

6

H

j

i

Ss

oS ICHs

ie HO3;80_.

HO3SO = Oo @ no ;

7

Zz HAD

Fig. 2 Selected structures of compounds isolated from maca. (Continued next page)

maca,

three

new

macamides,

N-benzyloctamide,

N -benzyl-16-hydroxy-9-oxo-10EF,12E,14E-octadecatrienamide, and octadecatrienamide,

by HPLC!" macamide

and

VN-benzyl-9,16-dioxo-10£,12E,14Ehave been isolated and identified

In addition, macaene

have

17 other been

analog

reported,

of but

their chemical identity has not been disclosed. Two additional alkamides, N-benzyl-5-oxo-6E,8E-octadeca-

dienamide

(15) and

N-benzylhexadecanamide

(16),

have been isolated from tubers.!!7!

BIOLOGICAL STUDIES The following sections include discussions of the results of both preclinical (in vitro and/or animal) and clinical (human patients) studies. While these studies suggest potential beneficial effects of maca, demonstration of efficacy in humans requires the

conduct of clinical trials utilizing randomized, double blind, placebo controlled protocols.

Fertility-Enhancing and Aphrodisiac Activities The aphrodisiac activities of maca have been reported by several research groups. Oral administration of the purified lipid extract decreased the latent period of erection (LPE) in male rats with erectile dysfunction, as well as enhanced the sexual function of mice and rats by increasing the number of complete intromissions and the number of sperm-positive females in normal mice.!''! Gonzales et al.!!?-7° reported that maca does not affect serum reproductive hormone levels in adult men, but rather improves sperm motil-

ity and sperm production in a dose-dependent manner.

In

a _ similar

protocol,

Gonzales

et

al.Pu

demonstrated the improvement of sexual desire after 8 weeks of treatment. Maca demonstrated an effect

Maca (Lepidium meyenii)

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Magnesium Robert K. Rude University of Southern California, Los Angeles, California, U.S.A.

INTRODUCTION Magnesium (Mg) is an essential nutrient and is vital for numerous biological processes. This entry reviews the biochemistry, physiology, and homeostasis of Mg. Dietary intake and requirements as well as current recommendations are discussed. Causes of and risk factors for the element’s deficiency are reviewed along with the clinical manifestations of moderate-to-severe depletion. Possible complications of deficiency such as hypertension,

cardiovascular

disease,

and

osteo-

porosis when dietary Mg intake falls below the recommended daily allowance (RDA) are discussed.

Magnesium is the fourth most abundant cation in the body and the second most prevalent intracellular cation. Most intracellular Mg binds to ribosomes, membranes, and other marcomolecules in the cytosol and nucleus."! The nutrient provides specific structure and catalytic activity for enzymes.

Enzyme Interactions Magnesium is involved in more than 300 vital bolic reactions.”! Essential for many enzymatic tions, magnesium has two general interactions: binds to substrate, thereby forming a complex the

enzyme

interacts,

as

in the

metareaca) It with

reaction

of

kinases with MgATP; and b) It binds directly to the enzyme and alters its structure and/or serves a catalytic role. Overall, the predominant action of the nutrient is related to utilization of adenosine triphosphate (ATP), which provides high-energy phosphate and exists in all cells primarily as MgATP. Thus, it is imperative for the function of the glycolytic cycle, citric acid cycle, protein kinases, RNA and DNA polymerases, lipid metabolism, and amino acid activation,

Robert

K.

Rude,

Medicine, University California, U.S.A.

MD.

of

is Professor

Southern

at

the

California,

ger systems.

Structural Modification of Nucleic Acids and Membranes

Another important role of Mg is its ability to form complexes with nucleic acids. The negatively charged ribose phosphate structure of nucleic acids has a high affinity for Mg. The resulting stabilization of numerous ribonucleotides and deoxyribonucleotides induces important physicochemical changes, which affect DNA maintenance, duplication, and transcription.”! Magnesium, calcium (Ca), and some other cations react

BIOCHEMISTRY AND PHYSIOLOGY

which

besides playing a critical role in the cyclic adenosine monophosphate and phospholipase C second messen-

Department

Los

of

Angeles,

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022061 Copyright © 2005 by Marcel Dekker. All rights reserved.

with hydrophilic polyanionic carboxylates and phosphates of the membrane components to stabilize the membrane, thereby affecting fluidity and permeability, which influences ion channels, transporters, and signal

transducers.

lon Channels

Ion channels constitute a class of proteins in the cell membrane, which allow passage of ions into or out of cells when the channels are open. They are classified according to the type of ion they allow to pass, such as sodium (Na), potassium (K), or Ca.?! Magnesium plays an important role in the function of a number of such channels. A deficit of Mg results in cellular K depletion.“! Magnesium is necessary for the active transport of K out of cells by Na, K-ATPase. Another mechanism for the K loss is its increased efflux from cells via other Mg-sensitive K channels, as has been seen in skeletal muscle and in heart muscle. Therefore, a deficiency in the nutrient leads to diminution of intracellular K. The arrhythmogenic effect of Mg deficiency, as discussed below, may

be related to its

effect on maintenance of intracellular K. Magnesium has been called nature’s physiological Ca channel blocker."! During Mg depletion, intracellular Ca rises. This may be due to both an increase from extracellular Ca and release from intracellular Ca stores. Magnesium decreases the inward Ca flux through slow Ca channels. In addition, it decreases Ca transport out of the sarcoplasmic reticulum into the cell cytosol. 445

Magnesium

446

There is an inverse ability of inositoltriphosphate to release Ca from intracellular stores in response to changes in Mg concentrations, which would also allow greater rise in intracellular Ca during a fall in the latter.

BODY COMPOSITION AND HOMEOSTASIS Composition The distribution of Mg in various body compartments of apparently healthy adult individuals is summarized in Table 1. Approximately 50-60% is in the skeleton, of which two-thirds is within the hydration shell and one-third on the crystal surface! and may serve as a reservoir for maintaining extracellular and intracellular Mg. Except for 1% in the extracellular fluid, the rest is intracellular.

Cellular Mg Homeostasis Significant amounts of Mg are found in the nucleus, mitochondria, the endoplasmic and sarcoplasmic reticulum, and the cytoplasm." Total cell concentration ranges between 5 and 20 mM. In the cytosol, 90-95% is bound to ATP, adenosine diphosphate (ADP), citrate, proteins,

and

nucleic

acids.

The

remainder

is free,

constituting 1-5% of the total cellular Mg. The concentration of free ionized form in the cytoplasm is 0.2— 1.0mM. Concentration in the cytoplasm is maintained relatively constant due to limited permeability of the plasma membrane to Mg and to the operation of Mg transport systems. Transport into or out of cells requires the presence of carrier-mediated transport systems. Efflux of Mg from the cell is coupled to Na transport and requires extrusion of sodium by Na, K-ATPase. There is also evidence for a Na-independent efflux of Mg. Influx is linked to Na transport, but by Table 1 Distribution and concentrations of magnesium (Mg) in a healthy adult (total body: 833-1170 mmol, or 20-28 g) Percentage of total body Mg

Site

Concentration/ content

Bone

53

0.5% of bone ash

Muscle

27

9mmol/kg wet weight

Soft tissue

19

9mmol/kg wet weight

Adipose tissue

0.012

0.8 mmol/kg wet weight

Erythrocytes

0.5

1.65-2.73 mmol/L

Serum

0.3

Mononuclear Platelets Immol

0.88 + 0.06mmol/L 2.91 + 0.6 fmol/cell 2.26 + 0.29mmol/L

= 2mEq

= 24.3 mg.

a mechanism other than efflux. The molecular characteristics of Mg transport proteins have not been described.

Studies

in

prokaryotes,

however,

have

identified four separate transport proteins. The processes that maintain or modify the relationships between total and ionized internal and external Mg are incompletely understood. Its transport is influenced by hormonal and pharmacological factors. Magnesium efflux is stimulated by «- and B-agonists and permeant cAMP in heart, liver, and thymocytes. Activation of protein kinase C by diacyl glycerol or by phorbol esters stimulates influx but not efflux. Epidermal growth factor has been shown to increase transport into a vascular smooth muscle cell line. Insulin and dextrose increase Mg uptake in a number of tissues, including skeletal and cardiac muscle. The mechanism of insulin-induced Mg transport is likely due to an effect on protein kinase C. It is hypothesized that this hormonally regulated uptake system controls intracellular Mg concentration in cellular subcytoplasmic compartments. Its concentration in these compartments would then serve to regulate the activity of Mg-sensitive enzymes.

BODY HOMEOSTASIS Homeostasis of the individual with respect to a mineral depends on the amounts ingested, the efficiency of intestinal and renal absorption and excretion, and all other factors affecting them. A schema for Mg balance is given in Fig. 1.

Dietary Intake Magnesium is widely distributed in plant and animal sources but in differing concentrations. In terms of major food sources,'” vegetables, fruits, grains, and animal products account for approximately 16% each; dairy products contribute 20% in adolescents and 10% beyond the third decade. The 1994 U.S. Department of Agriculture Continuing Survey of Food Intakes by Individuals (CSFII) indicated that the mean daily intake was 323mg in males and 228mg in females, which was similar to that reported in the Third National Health and Nutrition Examination Survey (NHANES II). These values fall below the current RDA recommendation of approximately 420mg for males and 320 mg for females.'*! It has been suggested that 75% of subjects in the United States have dietary Mg intake that is below the recommended one. Intestinal Absorption

In humans, the primary sites of intestinal Mg absorption are the jejunum and ileum."! With a normal dietary

Magnesium

447

KIDNEY

INTESTINE

Fig. 1 Magnesium homeostasis in humans. A schematic representation of magnesium metabolism indicating a) its absorption from the alimentary tract, b) its distribution into bone, and c) its dependence on the kidney for excretion. Homeostasis depends upon the integrity of intestinal and renal absorptive processes.

Mg intake, 30-40% is absorbed. There exist both a passive paracellular mechanism and an active transport process for Mg absorption. The former is dependent on transcellular potential difference generated by sodium transport and accounts for about 90% of intestinal Mg absorption. A Mg-specific transport protein/channel,

which is not yet well defined, accounts

for the remainder

of absorption."°! Net absorption

increases with increasing intake. However,

fractional

Mg absorption falls. Absorption fell progressively from approximately 65% with an intake of 36mg down to 14% for 1000mg consumption.!!! No hormone or factor has been described that regulates intestinal Mg absorption, although vitamin D and its metabolites may increase this activity.” 1,25(OH)>-vitamin D enhances intestinal absorption of both Ca and Mg in normal human subjects and patients with chronic renal failure. In balance studies,

vitamin D accelerated intestinal Mg absorption ‘but much less than Ca, and mean Mg balance was not affected. In contrast to Ca, there is no significant

correlation between serum Mg absorption.

1,25(OH) -vitamin D and

but not all studies. Increased dietary Mg decreases phosphate absorption, perhaps secondary to formation of insoluble Mg phosphate. A rise in intake of dietary fiber reduces Mg utilization in humans, presumably by decreasing absorption. High dietary zinc intake lessens Mg absorption and balance, while vitamin Bg depletion was associated with negative Mg balance. The presence of excessive amounts of free fatty acids and oxalate may also impair Mg absorption.

Bioavailability of Mg Salts Many salts of Mg are available as oral dietary supplements including oxide, hydroxide, citrate, chloride, gluconate, lactate, aspartate, aspartate hydrochloride, and glycinate. The fractional absorption of a salt depends on its solubility in intestinal fluids and the amounts ingested.!'"!] Absorption of enteric-coated Mg chloride is 67% less than that of the acetate in gelatin capsules. Magnesium citrate has higher solubility than the oxide form.

In humans,

the former was

observed

to have

better absorption than the latter in one study.""”! Little difference in absorption has been demonstrated among other salts however.

Bioavailability: Influence of Other Dietary Factors Renal Mg Regulation

Absorption of ingested Mg is influenced by its dietary concentration as well as dietary components inhibiting or promoting its absorption.”! Studies in healthy indiCa intake does not significantly affect Mg absorption

The kidney is the critical organ regulating Mg homeostasis, which involves a filtration/reabsorption process.''7] About 2400mg of Mg is normally filtered daily through the glomeruli in the healthy adult. Of

or retention.

dietary

this, only about 5% is excreted in the urine. The frac-

Mg has been associated with either decreased Ca absorption or no effect. High levels of dietary phosphate are reported to impair Mg absorption in some

tional absorption of the filtered load in the various segments of the nephron is summarized in Fig. 2. Approximately 15-20% of the filtered Mg is

viduals, for the most part, indicate that increasing oral On

the other

hand,

increased

Magnesium 448

Proximal convoluted tubule

Distal convoluted tubule

Loop of Henle

5 - 10%

Thick ascending limb Cortical

15 - 20%

Collecting duct

65 - 75%

100% Mg Thin

Medullary

descending

limb

> y 3-5%

reabsorbed in the proximal convoluted tubule, presumably by a paracellular mechanism. The majority (65-75%) is reclaimed in the cortical thick ascending limb of Henle. The mechanism also appears to be paracellular transport. A recently described gene, paracellin-1, has been shown to encode for a protein thought to mediate Mg transport at this site. The distal convoluted tubule reabsorbs 5—-10% of filtered Mg via an active transcellular pathway. No hormone or factor has been described that regulates renal Mg homeostasis. Micropuncture studies in rodents show that vasopressin, glucagon, calcitonin, and parathyroid harmone (PTH), when added to segments of the cortical thick ascending limb of loop of

Henle and/or the distal convoluted tubule, significantly increased Mg absorption."’7! The physiological significance of these observations, however, is unclear.

A number of conditions affect absorption, principally in the ascending thick limb. Inhibition occurs with hypermagnesemia and hypercalcemia.''*) This is thought to occur because these cations bind to a calcilum-sensitive receptor on the basolateral aspect of these tubular cells and decrease transepithelial voltage, thereby decreasing paracellular absorption of both Mg and Ca. Decreased Mg intake in experimental animals and humans rapidly reduces Mg excretion, even before plasma Mg levels fall below the normal range, suggesting an adaption of the kidney to Mg insufficiency.

Tissue Sources

Extracellular, intracellular, and bone Mg fall during depletion. Bone may serve as an important reservoir

Fig. 2 Fractional segmental reabsorption of filtered Mg in the nephron. The percentage absorption of filtered Mg has been determined by micropuncture techniques in varas_ the ious laboratory animals Mg proceeds through the nephron. Approximately 15-20% of the Mg is reabsorbed in the proximal convoluted tubule. The major site for Mg reabsorption is the thick ascending limb of the loop of Henle, primarily in its cortical portion. Here, 65-75% of Mg leaves the lumen. In the distal convoluted tubule, 5-10% of Mg is reabsorbed. (From Cole, D.E.€.; Quamme, G.A. J. Am. Soc. Nephrol. 2000, 77, 1937-1947, with permission.)

for Mg, as human iliac crest content fell an average

of 18% with depletion.)

MAGNESIUM

REQUIREMENTS

Assessment

For healthy older children, adolescents, and adults, the

primary approach for assessing dietary Mg requirement has been the dietary balance study. For infants and young children, figures are based on estimates of intakes through milk and other foods that allow good development. Such data have been transmuted into the dietary reference intakes (DRI) in the United States.!®! The U.S. reference intakes are given in Table 2. These are estimates that meet the needs of 97-98% of healthy individuals. The balance study poses some problems. Laboratory analysis of foods revealed a Mg content 115124% greater than those calculated by tables of food composition."*) A critique of previous RDA for Mg noted that most published balance data often did not meet the criteria for acceptable methodology."'*! The balance studies were usually short term, done mostly in adolescents and younger adults, and data presented for pregnant women were less than adequate. Published information about the elderly was meager. The need was pointed out for improved definition of acceptable standards. While the database of the 1997 reference values (Table 2) has eliminated the poorer experiments, a question remains as to the accuracy of many balance studies in terms of adherence to acceptable methodology.

Magnesium

Table

449

2 Recommendations for daily intakes of

Mg (mg) 0-0.5 0.5-1.0 1-3 4-8 9-13 14-18 19-30

30 Ws) 80 130 240 410 400 420 420 420

Bl=50 51-70 >70

30 18) 80 130 240 360 310 320 320 320

Pregnancy

III.

Potassium homeostasis A. Hypokalemia 1. Renal potassium wasting 2. Decreased intracellular potassium

IV.

Cardiovascular A. Cardiac arrhythmia 1. EKG: prolonged P-R interval and Q-T interval, U waves 2. Atrial tachycardia, premature contractions, and fibrillation 3. Junctional arrhythmias 4.

CLINICAL PRESENTATION

OF DEFICIENCY

As Mg plays an essential role in a wide range of fundamental biologic reactions, it is not surprising that its deficiency may lead to serious clinical symptoms." Indications and signs of deficiency are given in Table 4. Since deficiency occurs in a number of disease states, the clinical presentation of Mg deficiency may coexist or be masked by the signs and symptoms of the primary disorder.

Moderate-to-Severe Deficiency When Mg deficiency is recognized in the clinical setting, it is usually of moderate-to-severe depletion. Biochemical, neuromuscular, and cardiac complications are the most prevalent findings in the Mg-deficient patient.

Hypocalcemia Calcium is the major regulator of PTH secretion. Magnesium modulates PTH secretion via the Ca-sensing receptor in a manner similar to Ca. Magnesium deficiency, however, perturbs Ca homeostasis.?!! When it becomes moderate to severe, symptomatic hypocalcemia develops. Impaired PTH secretion is suggested as most patients with hypocalcemia have low

»

B. C. D.

Ventricular premature contractions,

tachycardia, fibrillation 5. Sensitivity to digitalis intoxication 6. Torsades de pointes Myocardial ischemia/infarction (putative) Hypertension (putative) Atherosclerotic vascular disease (putative)

or inappropriately normal serum PTH levels. Administration of Mg will result in an immediate rise in the serum PTH. Normal or elevated serum PTH in the face of hypocalcemia in some patients suggests an endorgan resistance to PTH as well. Skeletal and renal resistance to PTH in hypocalcemic Mg-deficient patients has been described. The mechanism for impaired PTH secretion and action in Mg deficiency remains unclear, although a defect in the second messenger systems is suggested. Adenylate cyclase requires Mg for cyclic AMP generation. The PTH has also been shown to activate the phospholipase C second messenger system. Magnesium depletion could perturb this system, as a Mgdependent guanine nucleotide regulating protein is involved in activation of phospholipase C, and the nutrient has also been shown to be a noncompetitive inhibitor of IP3-induced Ca release. Magnesium is also important in vitamin D metabolism and/or action." Patients with hypocalcemia and Mg deficiency are resistant to vitamin D, la-hydroxyvitamin D, and 1,25(OH),-vitamin D. In addition,

Magnesium

452

serum concentrations of 1,25(OH)>-vitamin D are low

or low normal in most hypocalcemic Mg-deficient patients. Since PTH is a major trophic for 1,25(OH)>vitamin D formation, the low serum PTH

concentra-

tions could explain the low levels. Magnesium deficiency may directly impair the ability of the kidney to synthesize 1,25(OH),-vitamin D as the element supports the 25-hydroxy-1a-hydroxylase in vitro. Hypokalemia

A common feature of Mg depletion is hypokalemia.!?7! Experimental human Mg deficiency demonstrated a negative K balance resulting from increased urinary K loss. During depletion, there is also loss of intracellular K. Attempts to replete the K deficit with K therapy alone are not successful without simultaneous Mg therapy. The reason for this disrupted K metabolism may be related to Mg dependence of the Na, K-ATPase.

Also, intracellular Na and Ca rise, while

Mg and K fall. Magnesium also appears to be important in the regulation of K channels in cardiac cells that are characterized by inward rectification. This biochemical feature may be a contributing cause of the electrocardiographic findings and cardiac dysrhythmias discussed later. Neuromuscular manifestations

Neuromuscular hyperexcitability is a common complaint of a patient with Mg deficiency.!’*! Latent tetany, as elicited by positive Chvostek’s and Trousseau’s sign, or spontaneous carpal—pedal spasm may be present. Seizures may also occur. Although hypocalcemia contributes to the neurologic signs, deficiency without hypocalcemia has been reported to result in neuromuscular hyperexcitability. Other signs occasionally seen include vertigo, ataxia, nystagmus, and athetoid and choreiform movements. Muscular tremor, fasciculation, wasting, fatigue, and weakness

may be present. Reversible psychiatric aberrations have also been reported. There may be several mechanisms underlying these neuromuscular problems. Magnesium has been shown to stabilize the nerve axon. Lowering its serum concentration decreases the threshold of axonal stimulation and increases nerve conduction velocity. It also has been shown to influence the release of neurotransmitters, such as glutamate, at the neuromuscular junction

by competitively inhibiting the entry of Ca into the presynaptic nerve terminal. It is likely that a decrease of extracellular Mg would allow a greater influx of Ca into the presynaptic nerves and the subsequent release of a greater quantity of neurotransmitters, resulting in hyper-responsive neuromuscular activity.

Cardiovascular manifestations

Dysrhythmias. Cardiac dysrhythmias are an important consequence of Mg deficiency. Electrocardiographic abnormalities of such shortage in humans include prolonged P-R interval and Q-T interval. Intracellular K depletion and hypokalemia are complicating features and may contribute to these abnormalities. Magnesium-deficient patients with cardiac dysrhythmias have been treated successfully by administration of the nutrient.°*) Supraventricular dysrhythmias including premature atrial complexes, atrial tachycardia, atrial fibrillation, and junctional arrhythmias have been described. Ventricular premature complexes, ventricular tachycardia, and ventricular fibrillation are more serious complications. Such dysrhythmias may be resistant to usual therapy. As intracellular Mg depletion may be present despite a normal serum Mg concentration, deficiency always must be considered as a potential factor in cardiac. dysrhythmias.

Acute Myocardial Infarction. Acute myocardial infarction (AMI) is the leading cause of death in the United States. Magnesium deficiency may be a risk factor as it has been shown to play a role in systemic and coronary vascular tone, in cardiac dysrhythmias as mentioned above, and by inhibiting steps in the coagulation process and platelet aggregation. Over the past decade, debate has arisen over the clinical utility of adjunctive Mg therapy for AMI. While several small controlled trials suggested that adjunctive Mg therapy reduced mortality from AMI by 50%, three major trials define our understanding regarding Mg therapy in AMI.4! Leicester Intravenous Magnesium Intervention Trial (LIMIT-2) was the first study involving large numbers of participants. Magnesium treatment showed an approximately 25% lower mortality rate. The Fourth International Study of Infarct captopril, nitrates, and Mg on AMI. Unlike LIMIT-2, the mortality rate in the Mg-treated group was not significantly different from that in the control group. It was suggested that the ISIS-4 design masked the benefits of Mg therapy. Two major criticisms involved the timing of the therapy and the severity of patient illness. The recently published magnesium in coronaries (MAGIC) trial was designed to address the issues regarding ISIS-4 study design—namely, early intervention in higher risk patients would more likely show the benefit of Mg therapy.?*! Over a 3 yr period, 6213 participants were studied. The Mg-treated group mortality at 30 days was not significantly different from that of placebo. The overall evidence from clinical trials does not support the routine application of adjunctive Mg

therapy in patients with AMI at this time.

Magnesium

453

Chronic Latent Deficiency Although the diets ordinarily consumed by healthy Americans fall below the RDA with respect to Mg levels,'*! they do not appear to lead to symptomatic depletion of the nurtrient. A number of clinical dis-

to the finding that Mg inhibits the synthesis of thromboxane A» and 12-HETE, eicosanoids thought to be involved in platelet aggregation. It also inhibits the thrombin-induced Ca influx in platelets as well as stimulates synthesis of prostaglandin I, (PGI,), the potent antiaggregatory eicosanoid.

orders, however, have been associated with a low Mg

diet. It has been suggested that milder degrees of inadequacy may contribute to disease states such as hypertension, coronary artery disease, and osteoporosis.

Hypertension A number of studies have demonstrated an inverse relationship between dietary Mg intake and blood pressure.?*! Hypomagnesemia and /or reduction of intracellular Mg have also been inversely correlated with blood pressure. Patients with essential hypertension were found to have reduced free Mg concentrations in red blood cells (RBCs). The Mg levels were inversely related to both systolic and diastolic blood pressure. Intervention studies with Mg therapy in hypertension have led to conflicting results. Several have shown a positive blood-pressure-lowering effect of supplements, while others have not. Other dietary factors may also play a role. Recently, a diet of fruits and vegetables, which increased Mg intake from 176 to 423 mg/day (along with an increase in potassium), significantly lowered blood pressure. The addition of nonfat dairy products that increased Ca intake as well further lowered blood pressure. The mechanism by which Mg deficit may affect blood pressure is not clear but may involve decreased production of prosta-

Pre-eclampsia and eclampsia

Pre-eclampsia, which complicates | in 2000 pregnancies in developed countries, is responsible for over 50,000 maternal deaths per year. Magnesium therapy has been utilized in the management of pre-eclampsia and eclampsia for decades and contributes to the very low mortality rate in developed countries.?*! A large randomized trial (the MAGPIE trial) examined the efficacy of Mg therapy in preventing eclampsia.?*! MgsOg, showed a lower risk (0.8%), as compared with nimodipine, a specific cerebral arterial vasodilator (2.6%) for eclampsia. Documentation of deficiency in women with pre-eclampsia has been difficult to establish. No difference was found in the plasma Mg levels of women with pre-eclampsia and those of healthy pregnant women. However, women with pre-eclampsia had a decreased RBC Mg level. In another study, those with pre-eclampsia or preterm labor had no differences in ionized or total serum Mg levels. While subtle deficits in total body Mg may contribute to hypertension during pregnancy, its role may relate more to the stabilizing neuronal and vascular effects rather than to the correction of an electrolyte deficit. Magnesium therapy is clearly indicated for women with pre-eclampsia. It has been shown to decrease the incidence of eclampsia,

enhanced vasoconstrictive effect of angiotensin II and norepinephrine.

and likely to reduce overall mortality. There is also evidence of better fetal outcomes—less low birth weight— from fetal growth retardation or preterm delivery.

Atherosclerotic vascular disease

Osteoporosis

Another potential complication of Mg deficiency is the development of atheromatous disease.?7! Lipid alterations have been reported in hypomagnesemic human subjects. However, they are often complicated by factors related to underlying lipoprotein abnormalities occurring in diabetes, coronary artery disease, myocardial infarction, and other diseases. Epidemiological studies have related water hardness (Ca and Mg content) inversely to cardiovascular death rates. Mg inhibits platelet aggregation, and platelet hyperactivity is a risk factor in the development of cardiovascular diseases. Diabetic patients with Mg depletion have been shown to have increased platelet aggregation. Mg therapy in these subjects returns the response toward normal. The antiplatelet effect may be related

Dietary Mg depletion in animals has been shown to lead to a decrease in skeletal growth and increased skeletal fragility.) A decrease in osteoblastic bone formation and an increase in osteoclastic bone resorption are implicated as the cause in decreased bone mass. In humans, epidemiologic studies suggest a cor-

cyclin, increased production of thromboxane

Aj, and

relation between bone mass and dietary Mg intake.”! Few studies have been conducted assessing Mg status in patients with osteoporosis. Low serum and RBC Mg concentrations as well as high retention of parenterally administered Mg has suggested a deficit. Low skeletal Mg content has been observed in some, but not all, studies. The effect of supplements on bone mass has generally led to an increase in bone mineral density, although study design limits useful information.

Magnesium

454

Larger long-term placebo-controlled double-blind investigations are required. There are several potential mechanisms that may account for a decrease in bone mass in Mg deficiency. Magnesium is mitogenic for bone cell growth, which may directly result in a decrease in bone formation. It also affects crystal formation; a lack results in a larger, more perfect crystal, which may affect bone strength. Inadequacy results in a fall in both serum PTH and 1,25(OH),-vitamin D as discussed above. Since both hormones

are trophic for bone, impaired

secretion or skeletal resistance may result in osteoporosis. Low 1,25(OH) -vitamin D may also result in decreased intestinal Ca absorption. An increased release of inflammatory cytokines may result in activation of osteoclasts and increased bone resorption in rodents.?7!

MANAGEMENT

OF DEPLETION

Seizures, acute arrhythmias, and severe generalized spasticity require immediate intravenous infusion. One to two grams of MgSO,e7H>,O (8.2-16.4mEq Mg) may be infused over 5—10 min, followed by continuous infusion of 6g over 24hr or until the condition is controlled. Correction of electrolytes (especially K) should accompany the Mg therapy. Serum Mg and other electrolytes should be determined twice daily. Less severe manifestations (e.g., paresthesias with latent or active tetany) are likewise best treated by the intravenous route. When renal function is good, 6 g (48 mEq) of Mg sulfate may be given intravenously over 24hr. This may be continued for 3—S days until the signs and symptoms and/or electrolytes abnormalities are corrected. When this route cannot be used, intramuscular injections can be given, although these are painful. The dosages given must always exceed the daily losses as indicated by serum levels and urinary excretion. The return to the normal or slightly higher range of serum Mg levels with any of these schedules is relatively rapid. However, repletion of Mg lost from bone and other tissues requires more prolonged therapy. When intestinal absorption is normal and renal Mg wasting is present, supplements should be added to the

usual diet to tolerance (onset of diarrhea) to maintain normal serum levels. In some instances, oral supplementation may not be sufficient, and intramuscular and/or intravenous Mg may be required. Those with continuing severe Mg and K losses in the urine (as in cisplatin nephrotoxicity) may require long-term supplements by intravenous infusion via an indwelling central catheter for home administration. When depletion is modest and persistent, initial efforts should be directed to increased intake of

Mg-rich foods. When necessary and feasible, oral supplemention may be taken. Quantities of 300-600 mg may be given in divided doses 3-6 times /day.

CONCLUSIONS In conclusion, Mg is a vital nutrient necessary for essential biological processes. Despite its rigid homeostasis by the body, deficiency is not uncommon due to numerous

diseases,

disorders,

and

medications

that!

impair the normal metabolism of Mg. The relatively low dietary intake compounds this problem in many patients. Indeed, even the reduction in suggested dietary Mg may contribute to chronic disease states in otherwise healthy individuals. It is clear that education is necessary for the general population to increase awareness of the importance of Mg in maintaining health. In addition, further research efforts in both basic science and clinical science are needed to clarify the role of Mg deficiency in disease states.

REFERENCES 1.

Maguire, M.E.; Cowan, J.A. Magnesium chemistry and biochemistry. Biometals 2002, 75, 203-210.

2.

Cowan, J.A. Structural and catalytic chemistry of magnesium-dependent enzymes. Biometals 2002,

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magnesium transport and homeostasis: role of intracellular compartments. Miner. Electrolyte Metab. 1993, 19, 282-289. Pennington, J.A.T.; Young, B. Total diet study nutritional elements. J. Am. Diet. Assoc. 1991, 91, 179-183. Food and Nutrition Board, Institute of Medicine. In Dietary Reference Intakes for Calcium, Phos-

phorus, Mg, Vitamin D, and Fluoride; National Academy Press: Washington, DC, 1997. Kerstan, D.; Quamme, G.A. Physiology and pathophysiology of intestinal absorption of magnesium. In Calcium in Internal Medicine; Massry,

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Springer-Verlag London Ltd.: Surrey, U.K., 2002; 171-183. 10.

Voets, T.; Nilius, B.; Hoefs, S.; van der Kemp, A.W.C.M.; Droogmans, G.; Bindels, R.J.M.;

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Hoenderop, J.G.J. TRPM6

channel

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135

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in intestinal

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Intestinal absorption of magnesium from food and supplements. J. Clin. Invest. 1991, 88, 396-402. Quamme, G.A.; de Rouffignac, C. Epithelial magnesium transport and regulation by the kidney. Front. Biosci. 2000, 5, 694-711. Shils, M.E.; Rude, R.K. Deliberations

Endres,

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Chemistry,

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ples of Bone Biology, 2nd Ed.; Bilezikian, J.B.,

25.

26.

18.

Quamme, G.A. Renal magnesium handling: new insights in understanding old problems. Kidney Int. 1997, 52, 1180-1195. MeNair,

P.; Christensen,

M.S.; Christiansen,

C.

et al. Development of bone mineral loss in insulintreated diabetes: a 1 and 1/2 years follow-up in sixty patients. Eur. J. Clin. Invest. 1982, 72, 81-85.

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Song,

Y.;

Buring,

J.E.;

Manson,

J.E.,

et

H.; Domanski,

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controlled trial. Lancet 2002, 360,

1189-1196. Touyz, R.M. Role of magnesium in the pathogenesis of hypertension. Mol. Aspects Med. 2003, 24, 107-136. Appel, L.J.; Moore, T.J.; Obarzanek,

E. et al. A

clinical trial of the effects of dietary patterns on blood pressure. DASH Collaberative Research Group. N. Engl. J. Med. 1997, 336, 1117-1124. 27.

Maier, J.A.M. Low magnesium and atherosclero-

sis an evidenced based link. Mol. Aspects Med. 2003, 24, 137-146. 28.

Belfort, M.A.; Anthony, J.; Saade, G.R. et al. A

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comparison of magnesium sulfate and nimodipine for the prevention of eclampsia. N. Engl. J. Med. 2003, 348, 302-311. Tong, G.; Rude, R.K. The role of dietary magnesium deficiency in osteoporosis. In Nutrition Aspects of Bone Health; New, S.A., Ponjour, J.P., Eds.; The Royal Society of Chemistry:

Raisz, L., Rodan, G., Eds.; Academic Press: San Diego, 2001; 339-358.

17.

E.; Cooper,

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E.R., Eds.; W.B. Saunders: Philadelphia, PA, 2001; 795-821. Ryzen, E. Magnesium homeostasis in critically ill

Antman,

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Zehender, M.; Meinertz, T.; Faber, T., et al. Anti-

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absorption. J. Biol. Chem. 2004, 279, 19-25. Fine, K.D.; Santa Ana, C.A.; Porter, J.L. et al.

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Cowan, J.A. Introduction to the biological chemistry of magnesium. In The Biological Chemistry of

Zi

W.C., et al. Magnesium intake and risk of type 2 diabetes in men and women. Diab. Care 2004, 27, 134-140. Rude, R.K. Magnesium deficiency in parathyroid function. Bilezikian,

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Magnesium homeostasis and clinical disease of magnesium deficiency. Ann. Pharmacother 1994, 28, 220-226.

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Ed.;

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Touyz, R.M. Magnesium in clinical medicine. Front. Biosci. 2004, 9, 1278-1293. Laires, M.J.; Monteiro, C.P.; Bicho, M. Role of cellu-

lar magnesium in health and human disease. Front. Biosci. 2004, 9, 262-276. Shils, M.E. Magnesium. In Handbook of Nutritionally Essential Mineral Elements; O’ Dell, B.L., Sunde, R.A., Eds.; Marcel Dekker: New York, 1997;

117-152.

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473 Milk Thistle (Silybum marianum)

Milk Thistle (Silybum marianum)

474

lines! It may also have a chemopreventive role in tongue and bladder carcinogenesis.!°”°*! Silymarin has also been investigated as a possible adjunctive agent in mitigating some of the toxicity associated with chemotherapeutic agents. Silibinin and silychristin exerted a protective effect on monkey kidney cells and rats exposed to vincristine or cisplatin

or virus-induced hepatitis.!168?-*°! Variations in the form and dosage of silymarin make comparisons difficult. In the only double-blind placebo-controlled trial, 59 subjects were randomly assigned to Legalon or placebo.'8°! Substantial increases in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin were noted, but p values were not reported.

chemotherapy.!”©7*! In germ cell tumors, silibinin did

not interfere with the antineoplastic effects of cisplatin or ifosfamide. It potentiated the cytotoxic effect of cisplatin and doxorubicin in breast cancer and ovarian cell lines.'©:79! The flavonolignan mixture may increase the chemosensitivity of DUI45 prostate cancer cells resistant to chemotherapy.'’!! The clinical significance of these investigations in humans as well as the effective dose, timing, and duration of treatment with silymarin in humans with cancer needs further investigation.

INDICATIONS

AND USAGE

Subacute or acute liver disease

Two double-blind, randomized, controlled trials and one observational study have investigated silymarin in the treatment of patients with liver disease.43:8687] In a large observational study (n = 2637), subjects with liver disease of various etiologies and severity were supplemented for 8 weeks with a mean Legalon dose of 267 + 103.6mg. Considerable decreases in ALT, AST, and y-glutamyl transferase (GGT) and a decrease in the number of patients with hepatomegaly were noted, but p values were not reported. In the two. placebo-controlled double-blind, decreases in ALT, AST, and GGT

Liver Disorders

Although research has been conducted in humans with silymarin and a variety of diseases of the liver, its mechanisms of action are largely unknown. Some human studies suggest that silymarin may be more effective in the earlier stages of liver disease.??! This may be explained by its ability to prevent toxins from entering the hepatocyte, thereby preventing initial damage to the cell (Table 4). The studies investigating silymarin in humans with liver disease are described below.

trials, significant were observed.

Primary biliary cirrhosis In a nonrandomized pilot study (n = 27), silymarin (140 mg three times daily) was administered to patients with primary biliary cirrhosis nonresponsive to standard medical care.'**! No noteworthy changes were observed.

Cirrhosis

Lipidemia

Three double-blind, randomized, controlled trials utilizing similar dosing regimens have investigated milk

One study suggests that silymarin may be a potential therapeutic agent in the prevention of atherosclerosis.'°°! Oxidation of LOL particles plays an important role in the development of atherosclerosis. Silymarin may reduce atherosclerosis through its effects on apolipoproteins A-I and A-II. Apolipoproteins reside on the surface of HDL and activate lecithin : cholesterol acyltransferase (LCAT), thereby clearing cholesterol from extrahepatic tissues. Apolipoprotein A-I facilitates uptake of cholesterol into cells. A small trial of 12 weeks’ silymarin supplementation in 14 adults with Type-II hyperlipidemia resulted in significant reduction in apolipoprotein A-I levels, thereby showing that silymarin is not beneficial in this condition. Significant decreases in apolipoprotein A-II levels were also observed. However, the role of apolipoprotein A-II in atherosclerosis is not well defined.?" As apolipoprotein A-II is inversely associated with insulin resistance and plasma triglycerides, silymarin may be useful in both atherosclerosis and diabetes. Further study is needed.

thistle in the treatment

of alcohol-induced

cirrhosis,

The studies have reported mixed results (Table 4). Ferenci et al. followed patients with alcohol- and non-alcohol-induced cirrhosis for two years and found increased survival in patients supplemented with

420 mg/day of silymarin. However, no effects on measurements of liver function were found. A later study utilizing a similar dosing regimen and duration found that silymarin had no effect on survival.*°! In a smaller study, patients with alcohol-induced cirrhosis were supplemented with 150mg/day of silymarin. Significant increases in glutathione levels and decreased malondialdehyde levels were observed.'*"! No significant effects on measurements of liver function tests were found. Alcoholic or virus-induced hepatitis

Five studies have investigated the effect of silymarin in the treatment or prevention of progression of alcohol-

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Omega-3 Fatty Acids William S. Harris Lipid and Diabetes Research Center, Mid America Heart Institute, Saint Luke’s Hospital, Kansas City, Missouri, U.S.A.

INTRODUCTION Long-chain, highly unsaturated fatty acids (HUFA) of the omega-3 (w-3 or n-3) family include eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). EPA and DPA are 20- and 22-carbon molecules, respectively, with five double bonds A5,8,11,14,17, while DHA is a 22carbon and six double-bond molecule A5,8,11,14,17,20.

All fatty acids (FA) in the w-3 family are characterized by having their first double bond at the 3rd position counting from the terminal (@ or nth) methyl group in the molecule (Fig. 1). As with the omega-6 family, @-3 FA cannot be synthesized de novo by mammals and must be obtained from the diet.

BIOCHEMISTRY AND FUNCTIONS Synthesis of EPA and DHA EPA and DHA are natural constituents of the lipids of most marine and many freshwater animals. These FA are originally synthesized by microalgae at the base of the marine food chain.""! They move up the chain via phytoplankton, zooplankton, and small fish to larger fish and marine mammals. Biosynthesis, once assumed to be relatively straightforward, vis-a-vis moving up in chain length and desaturation in a natural progression (C18—C22; 3-6 double

bonds),

has

now

been

shown

to be more

complicated and involves two separate organelles (Fig. 1).°%! The parent -3 FA, «-linolenic acid (ALA), is elongated and desaturated (via A6 and A5 desaturases) to C24:6 in the endoplasmic reticulum (ER). This FA is then translocated to the peroxisome where it is B-oxidized to 22: 6,4! and then returned to

the ER for incorporation into glycerophospholipids for export. It has been shown that feeding pure EPA

raises DPA but not DHA levels in plasma lipids, whereas pure DHA raises EPA levels.°-”! Presumably, supplemental DHA is able to enter the peroxisome and be degraded to EPA. But the latter FA is apparently not able to enter the proper compartment in the ER in order to be elongated and desaturated to DHA.

Efficiency of Conversion of ALA to EPA and DHA In the adult human, the only known role for ALA is to serve as a precursor of DHA, but conversion is exceedingly slow. Reported rates vary from a low of far less than 1%!®?! to 3-6% in young men," 9% for young women,""! and even 15%!!°! depending on the methods used to assess conversion. One of the primary reasons why ALA is poorly converted to the longer-chain EPA and DHA is because it is mostly (about 75%) shunted to B-oxidization.!"7!

Tissue Distribution of EPA and DHA

EPA and DHA are found esterified predominantly in membrane phospholipids of tissues, circulating cells, and plasma lipoproteins.!'”'®! The distribution within plasma differs by lipid class (Fig. 2), with phospholipids carrying by far the most w-3 FA. With respect to cell membranes, the erythrocyte may be used as a model. Omega-3 FA typically constitute about 4-6% of total red blood cell (RBC) phospholipid FA in Western diet where fish intake is sparse. In Japan where EPA and DHA intake is approximately 5-10 higher, RBC EPA + DHA is approximately twice as high.'"3-'4] Certain tissues are particularly rich in DHA. Spermatozoa

contain

between

6 and

8%

DHA?!

and rod outer segments of the retinal epithelium can have up to 30% DHA."'4

Functions of EPA and DHA

William

S. Harris, Ph.D.,

is Professor

at the Department

of

Medicine, University of Missouri-Kansas City, Missouri, U.S.A.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022075

Copyright © 2005 by Marcel Dekker. All rights reserved.

In tissues undergoing physiological stresses, phospholipase A, becomes activated by a G-protein mediated pathway, liberating the HUFA at position 2 of membrane phospholipids.!'7!7! Once cleaved, these HUFA 493

Omega-3 Fatty Acids

494

@-3 family

He VV C18:3

COOH

: ER: A5 and A6 desaturase, elongase Vv

C20:5

|ER: elongase

C22:5

iER: A6 desaturase, elongase

C24:6

i Peroxisome: B-oxidation

C= N/V

C22:6

NN

COOH

Resolvins

~* 40,17S docosatrienes

ER: esterification

3-series prostaglandins Thromboxane Az Prostacyclin I, 5-series leukotrienes

are available for conversion into eicosanoids (20carbon moieties) and docosanoids (22-carbon moieties). The principal 20-carbon tri-, tetra-, and pentaenoic HUFA are dihomo-y-linolenic acid (DGLA; C20: 3 n-6), arachidonic acid (AA; C20:4 n-6) and EPA. The eicosanoids made from each substrate include a bewildering range of molecular species including prostaglandins, thromboxanes, prostacyclins, lipoxins, leukotrienes, hydroxy acids (metabolites of P-450 enzymes), etc.?°! These three substrates give rise to three classes of eicosanoids: DGLA to the 1|-series prostaglandins and the 3-series leukotrienes, AA to

the 2- and

4-series,

Fig. 1 The three principal FA in the w-3 family. ALA is derived from plant oils such as flaxseed, soybean, and canola. EPA and DHA are generally derived from marine organisms. There is limited conversion of ALA to EPA and DHA. There is retroconversion of ingested DHA to EPA, but very little DHA is produced from ingested EPA. EPA is the precursor ~ for a wide variety of eicosanoids including the 2-series prostaglandins, and the 5-series leukotrienes. ER, endoplasmic reticulum.

and

EPA

to the

3-series

and

5-series, respectively.?4 With regard to biochemical potency, the eicosanoids produced from AA are generally more potent mediators

of

inflammation,

vasoconstriction,

and

platelet aggregation than those from EPA”! or DGLA.”*! AA is the predominant HUFA in tissue membranes in Western cultures where intakes of EPA and DHA are very low. In platelets, for example, AA constitutes about 25% of total FA and DGLA about 1.5% in subjects consuming Japanese! and Western"! diets, whereas EPA can vary from , and the subsequent interaction of the free acids with ion channels.°” As noted below, the evidence for an antiarrhythmic effect of the

relatively low doses of EPA + DHA associated with reduced risk for CHD death (about 500-1000 mg/day) is relatively sparse. A pilot study in 10 patients undergoing cardiac electrophysiological testing reported beneficial effects of an infused, w-3 FA-rich emulsion on the susceptibility of the myocardium to dysrhythmias.°*! Tissue levels of EPA + DHA achieved by supplementation with 1 g/day increase from about 1.7% to 2.9% of total cardiac FA.?! Tt is unknown whether this level of tissue enrichment with EPA + DHA produces the same biophysical effects on membrane function than the higher levels produced in experimental settings in cells, animals, or humans.

FOOD SOURCES Fish are the primary source of the w-3 FA. The content per serving can vary markedly depending on the species (Table 1 A and B). Higher-fat fish such as salmon, mackerel, albacore tuna, herring, and sardines are

excellent sources of EPA and DHA, whereas low-fat fish such as bass, perch, and cod are poor ones. Com-

mercially available dietary supplements providing EPA and DHA are madé with refined fish oils obtained from the high-fat species and others such as menhaden and anchovy. Fish oils are also derived from fish liver;

495

indeed, for centuries cod liver oil was (and continues to be) the most widely utilized fish oil in the world. A variety of fortified foods have reached the market in which deodorized -3 rich oils are incorporated into breads, spreads, salad dressings, milk products, frozen desserts, etc. Efforts

to increase

the w-3 content

of

traditional foods have included feeding fish oils or flaxseed oil to pigs and chickens.“°! With the recent demonstration that transgenic mice carrying the fat-1 gene from C. elegans are able to convert w-6 FA into @-3 FA and thereby enrich mouse tissues with EPA and DHA,"*"! beef, pork, and chicken may some day be engineered so as to join fish as major sources of m-3 FA. Recognizing that fish contain @-3 FA only because their food has them, and that harvesting fish to obtain these FA cannot be sustained forever, interest has turned to micro-organisms, the ultimate sources of

these FA in the marine food chain. A number of species of algae, fungi, and bacteria have been discoyered which, under

specific environmental

conditions,

synthesize either DHA! or EPA.*! Omega-3 fatty acids derived from microbial sources are free of the “fishy’’ flavors characteristic of most fish oil supplements, and are uncontaminated by any lipid-soluble pollutants (as are the refined fish oils). Organic solvents are, however, used for the recovery of cellular lipids

and must be quantitatively removed. At this point, cost issues have largely prohibited their wide-scale use in supplements, but increased demand may bring more “single cell oils’? to the market.

SAFETY OF -3 FATTY ACIDS Omega-3 fatty acids have been a part of the human diet for thousands of years with no known toxicity. The apparent safety of these FA has been underscored by a 1997 U.S. Food and Drug Administration (FDA) ruling given in response to a request that menhaden oil (a very common fish oil) be granted Generally Recognized as Safe (GRAS) status. The FDA ruled that intakes of up to 3 g/day of marine w-3 FA should be considered GRAS for inclusion in the diet. Thus, the safety of w-3 FA up to this level of intake is not in serious question. The extensive (tablespoon amounts) use of fish liver oils (cod and shark) is ill-advised, however, owing to their high levels of fat soluble vitamins (A and D). Although safety is apparently not an issue, there are side effects of supplementation. Perhaps the most common (seen in about 50% of subjects) is a “fishy’’ belch occurring soon after the capsules are taken. This can be minimized by bedtime consumption of supplements or enteric coating. In controlled trials such as the GISSI Prevention Study (see below) in which w-3 and vitamin E capsules

Omega-3 Fatty Acids 496

Table 1A Approximate levels of EPA + DHA in fish and the amounts required to provide about 1 g/day of EPA + DHA Ounces/day EPA

Fish

+ DHA

g/3 oz serving (edible portion)

required to provide

~1gm of EPA + DHA per day

Tuna

12

Light, canned in water, drained

0.26

White, canned in water, drained

0.73

Fresh

0.24-1.28

2.5-12

Sardines

0.98-1.70

2-3

Sockeye or Pink

1.05

3

Chinook

1.48

2

Coho, farmed

1.09

3

Coho, wild

0.91

3

Atlantic, farmed

1.09-1.83

1.5-2.5

Atlantic, wild

0.9-1.56

2-3.5

0.34-1.57

2-8.5

4

Salmon

Mackerel Herring Pacific

1.81

Ire)

Atlantic

7A

2

Trout, rainbow Farmed

0.98

Wild

0.84

oils Table 1B Approximate levels of EPA + DHA in fish + EPA of g/day 1 and the amounts required to provide about DHA Fish oil capsules

o-3 Fatty acids (g)/ oil (g)

: Oil/day (g)

Cod liver oil*

0.19

5

Standard fish body oil

0.30

5

Omega-3 FA

0.50

2

0.7-0.9

1

concentrate

Highly concentrated @-3 FA

Fish intakes (oz/d) are only estimates since EPA + DHA content can vary markedly with season, diet, age, and storage/preparation methods. (Values derived from literature?” and the USDA Nutrient Data Laboratory, http://www.nalusda.gov/fnic/foodcomp/.) @This amount of cod liver oil would provide the recommended dietary allowance of vitamin A and twice that of vitamin D.

upset was reported by 7% of the former and 8% of. the latter. Finally, although refined and concentrated -3 FA products are very low in organochloride contaminants, less well-controlled preparations can contain detectable amounts.°! Nevertheless the risk— benefit ratio for these products continues to be very

favorable.44 3.5

Cod Atlantic

0.13

23

Pacific

0.24

eS

Farmed

0.15

20

Wild

0.2

15

0.42

7

POTENTIAL HEALTH BENEFITS OF OMEGA-3 FATTY ACIDS

Catfish

Flounder/Sole Oyster

Pacific

oily)

DS

Eastern

0.47

6.5

Lobster

0.07-0.41

7.5-42.5

Crab, Alaskan King

0.35

8.5

Shrimp, mixed species

0.27

11

Clam

0.24

12.5

Scallop

0.17

Wes

In early 2004, the first of a series of United States government-mandated Evidence-Based Reviews on @-3 FA and a wide variety of disease conditions were released by the Agency for Health Care and Policy Research (AHCPR), now known as the Agency for Healthcare Research and Quality. These studies examined the existing medical literature in 12 disease categories. At the time of this writing (June 2004), reports had been issued for coronary heart disease (CHD), asthma, inflammatory/autoimmune diseases, and glycemic control in diabetes. These will be summarized first.

Coronary Heart Disease were tested in a postmyocardial infarction population of 11,324 patients, less than 5% cited side effects as their

reason for discontinuing therapy. Gastrointestinal disturbances were the most common. In another study

in which 6.9g of EPA + DHA (in 10 capsules) was given for 6mo to 221 patients, there was no difference in adverse event rates between the fish oil group and a control group given corn oil placebos.4! Gastrointestinal

The first three reviews published focused on the effects of w-3 FA on cardiovascular disease (CVD): 1) clinical endpoints; 2) risk factors; and 3) possible arrhythmogenic mechanisms. The first concluded that the evidence for CVD benefit from consumption of fish or fish oil supplements was strong (http://www.ahcpr.gov/ clinic/epcsums/o3cardsum.htm). The second found a

Omega-3 Fatty Acids

497

consistent beneficial effect of EPA + DHA on serum triglyceride levels, but beyond that, the benefits of @-3 FA on CVD risk are not well explained by their effects on CV risk factors (http://www.ahcpr.gov/clinic/ o3cvrinv.htm). The third came to the conclusion that EPA and/or DHA “might’’ have antiarrhythmic effects but that the data were not conclusive (http:/ /www. ahcpr.gov/clinic/o3arrinv.htm). Some of the effects on membrane function and ion channel activity were discussed above and have recently been reviewed?” as has the evidence for effects on serum lipids.“ The strongest support for a CHD benefit from increased w-3 FA intake comes not from effects on surrogate risk markers but from studies on event rates themselves. These are summarized below and in a recent scientific statement from the American Heart Association.4*!

A third study to examine the relationship between a blood measure of w-3 FA and risk for CHD death was reported by Lemaitre et al.?! utilizing data from the Cardiovascular Health Study. These investigators found a strong, protective relationship between serum phospholipid EPA + DHA and risk for fatal ischemic heart disease. The odds ratio associated with a onestandard deviation increase in this biomarker was 0.30 (95% CI, 0.12-0.76). Four other analyses!>*! found strong inverse relationships between CHD events and other biomarkers of EPA + DHA intake (coronary artery, serum cholesteryl ester, or total plasma FA). One study reported no relationship between plasma w-3 FA and the 5-yr risk of myocar-

dial infarction.>?! Randomized

clinical trials

Epidemiological studies Data indicating that w-3 FA may protect against CHD began to accumulate in the 1970s when Danish investigators found that acute myocardial infarction rates were significantly lower in Greenland Inuits

than in age- and sex-matched Danes.4*! On further investigation, a strong link between the w-3 FA in their diets and their apparent protection from heart

attacks

began

to emerge.°!

In studies

of other

fish-eating populations such as the Japanese, w-3 FA intakes were also associated with lower rates of

CHD,&4

In three U.S. epidemiological studies, @-3 biomarkers were related to risk.©?>*! Siscovick et al.7! obtained blood samples from 80 adults experiencing primary cardiac arrest in the Seattle area and from 108 healthy matched controls. The cases did not have known CHD at the time of their events. The EPA + DHA content of RBC membranes was determined in these samples and related to risk for primary cardiac arrest. The multivariable-adjusted odds ratios for primary cardiac arrest in the highest quartile was about 10% of that in the lowest quartile (95% confidence interval (CI), 0.1-0.4). Albert et al.! reached the same conclusion using data from the Physicians’ Health Study. In this study, 14,916 healthy male physicians were screened for a wide variety of risk factors and provided baseline blood samples between 1982 and 1984. Over the next 17 years, 94 men experienced sudden cardiac death. Whole blood long-chain w-3 FA (i.e., percentage of total FA as EPA + DHA + DPA) in these cases was compared to that of 184 age- and smoking-statusmatched controls. As in the Seattle study, risk for sudden cardiac death was reduced by about 90% in those subjects with the highest blood @-3 levels compared with those with the lowest levels.

The strongest evidence for w-3 FA benefit in CHD has come from randomized, controlled clinical trials with

“hard’’ CHD endpoints such as total mortality, fatal and nonfatal stroke, and/or myocardial infarction.

The first was the Diet And Reinfarction Trial (DART) that reported a 29% reduction in all-cause mortality over a 2-yr period in male myocardial infraction survivors advised to increase their intake of oily fish. This provided an additional 500-800mg of w-3 FA per

day.'°°! The greatest benefit in DART was seen in fatal myocardial infarctions suggesting an antiarrhythmic effect. A post hoc analysis of DART in capsule users suggested that the protective effect was attributable

to w-3 fatty acids in the fish." Burr et al.!°! conducted a second study similar to DART

in patients with stable angina. In this trial,

as with the original,

a recommendation

to increase

the intake of oily fish was given. National Health Service records were followed for up to 8-yr to determine the effects of this advice on death from any cause. The authors found no benefit from fish advice, and disturbingly, in a subset randomized to fish oil capsules, a significant increase in the risk for death. There were many weaknesses of and caveats to this study (funding interruption, no confirmation of compliance past 6-mo, possible “risk compensation,”’ or avoidance of heart medicines in favor of fish oil capsules, etc.). Thus its findings, although disquieting, do not at this point outweigh the evidence for beneficial effects. The largest study to test the efficacy of m-3 FA for secondary prevention of CHD was the GISSIPrevenzione Study.'**4! Patients who had survived a heart attack (n = 11,324) were randomized to either 300 mg of vitamin E, 850mg of w-3 FA ethyl esters, both, or usual care alone. After 3.5 yr, the group given the w-3 FA alone experienced a 20% reduction in

Omega-3 Fatty Acids

498

all-cause mortality (p = 0.01), and a 45% reduction in sudden death (p < 0.05) compared to the usual care group. Vitamin E provided no additional benefit. This trial, although very large and carried out in a relatively “real-life’’ setting, did not include a placebo arm, and dropout rates were high (>25%) in both the w-3 and vitamin

E groups.

further

research

Thus,

remains

there

to determine

a need

the efficacy,

for

optimal

dose, and mechanism of action of w-3 FA supplements

and

A (IgA) nephropathy,

systemic

lupus

erythe-

matosus (SLE), the Evidence Based Review concluded,

“There appears to be no effect on most clinical out-

comes

in rheumatoid

arthritis, although tender joint

counts appear to be reduced. There are insufficient data to draw conclusions about IBD, renal disease, SLE, bone density or fractures, requirement for anti-

inflammatory or immunosuppressive drugs’’ (http: fi

www.ahcpr.gov/clinic/epcsums/o3lipidsum.htm).

for the prevention of CHD death. Blood w-3 FA as a CHD risk factor

Given the evidence summarized above, blood biomar-

kers of EPA + DHA are independently associated with increased risk of death from CHD. This suggests that they might serve as a new risk marker or factor. We have proposed that the content of (i.e., percentage of total FA as) EPA + DHA in RBC membranes (the “Omega-3 Index’’) be considered such a risk factor.°*! Based on clinical studies in our laboratory and a review of the literature, we proposed that an Omega-3 Index of 810% was associated with the greatest cardioprotection, whereas an index of less than 4% was associated with the least. Although further work will be needed to thoroughly validate and refine such a marker, the Omega-3 Index may represent a novel, physiologically relevant, easily modified, independent, and graded risk factor for death from CHD that could have significant clinical utility.

Asthma

The ratio nale for hypothesizing an effect on asthma (and other inflammatory diseases) is based on the previously discussed competition between AA- and EPA-derived eicosanoids. There has also been evidence presented pointing to beneficial effects of o-3 FA on inflammatory cytokines.?7! The Evidence-Based Review on @-3 FA and asthma concluded: “Aside from an acceptable safety profile, it is impossible to definitively conclude anything with respect to the value of using omega-3 FA supplementation in asthma...’’ (http: //www.ahcpr.gov/clinic/o3asminv.htm).

Other Conditions

The impact of o-3 FA on other conditions is the subject of ongoing evidence-based reviews. The reader is encouraged to consult the AHRQ website at

http://www.ahcpr.gov/ where future reviews will be posted.

A summary of the effects of m-3 FA in cancer,

neurological and psychological diseases, and human development will be presented.

Cancer

While there is growing evidence that m-3 FA may play a palliative role in certain cancers, the data are still largely epidemiologic or derived from animal or

cell culture studies.°°” Randomized controlled trials have, to date, focused on high-risk biomarkers for cancer, not on cancer incidence or mortality itself. The cancers that may be modified by m-3 FA include those of the prostate and digestive tract, especially the colon. A 30-yr study among 6272 Swedish men examined the relationship between fish intake and prostate cancer incidence and death.!*! They found that, compared to men consuming “moderate’’ amounts of fish, those reporting little or no fish intake

had twice the incidence and three times the death rate from prostate cancer. Similar findings were reported from a biomarker-based study from New Zealand.!*! Clinical trials examining the effects of w-3 FA supplementation on large bowel epithelial cell proliferation (a cancerous precursor) in patients at high risk for colon cancer found that proliferation was significantly

reduced.!”°! Other studies have suggested a beneficial effect of w-3 Diabetes (Lipids and Glycemic Control), Arthritis and Inflammatory Diseases This report concluded that relatively high intakes of o-3 FA (e.g., 3 g/day) had beneficial effects on serum triglycerides in patients with Type II diabetes mellitus without altering glycemic control. With regard to inflammatory conditions including rheumatoid arthritis, inflammatory bowel disease (IBD), immunoglobulin

Finally,

FA

research

on with

cancer-induced cancer

cells

cachexia.!’!;/2] in culture

has

examined potential mechanisms driving the apparent beneficial effects of o-3 FA! For example, EPA inhibited the expression of genes for matrix metalloproteinases, enzymes that digest extracellular matrix and allow tumors to grow.!”4! Using Jurkat leukemic cells as a model, others have shown that DHA induced

programmed cell death (apoptosis), whereas arachidonic and oleic acids did not.!”*! Considerably more work

Omega-3 Fatty Acids

will be needed

499

before the role of w-3 FA

prevention and treatment with CHD.

becomes

in cancer

INTAKES

In the

are

DHA is a major component of nerve tissue with the brain and retina being particularly enriched in this FA. This observation led researchers to hypothesize that DHA plays either a structural or metabolic role in neural tissue. The possibility thus exists that deficiencies in brain DHA contribute to psychological pathologies such as depression, bipolar disorder, and schizophrenia. Studies in which patients with a variety of mental illnesses were found to have below normal @-3 FA levels in circulating cells, usually erythrocytes,

have also been published.!’°-’*! Others have demoncommon

US,

there

currently

(2004)

no

specific,

federally endorsed dietary recommendations for the long-chain w-3 FA. In 2002, the National Academy’s Institute of Medicine recommended intakes of ALA of up to 1.2% of energy based on median intakes of

Neurological and psychological conditions

strated that depression is more

RECOMMENDED

as clear as it is

in persons

with reduced blood levels of w-3 FA,!’?! and crosscultural studies suggest that postpartum depression may also be associated with low w-3 levels.°°! Better evidence comes from prospective interventions in which patients with cognitive disorders were treated with w-3 FA. These have generally, but not unanimously, shown improvements in patients with bipolar disorder,®" dyspraxia,*”! schizophrenia,**! and attention—deficit—hyperactivity disorder.®7! Hostility and

aggression! and borderline personality disorder!) may even by diminished by increased w-3 FA intakes. The opportunities for fruitful research into the role of these FA in cognitive disorders are plentiful.

Human Development (Cognitive, Behavioral, and Visual)

The presence of DHA in breast milk suggests that it’ is also necessary for early development. Early evidence for this hypothesis came from animal studies where the ability to learn mazes was correlated with w-3 FA levels in tissues.®*! Human LQ. at 4yr of age has been correlated with maternal EPA + DHA intake.” Based on the various studies that have examined the need for w-3 FA in early development, the current consensus is that formulas intended for term infants need only contain ALA (because the synthetic systems for conversion to DHA are reasonably well developed at birth), but those meant for preterm infants should have preformed DHA (since metabolic conversion from shorter-chain precursors is not sufficiently developed in these infants).%*! While there are theoretical reasons why pregnant women and’ nursing mothers should increase their intake of DHA, convincing evidence demonstrating clear benefit to their children is presently lacking.

healthy individuals in the United States.®*! Although the report noted that up to 10% of the ALA target could be supplied as EPA and DHA (about 300 mg), it did not make a specific recommendation per se for these FA. A numer of expert panels from around the world have recommended intakes of 200-800 mg/day of EPA + DHA.° 4] The American Heart Association*! currently recommends that for patients with known CHD, an intake of about 1g of EPA + DHA appears to be prudent, and for those without known CHD, two (preferably oily) fish meals per week. This would translate into an intake of about 500 mg/day of EPA + DHA, an amount that has been associated with the lowest rates of death from CHD observed in major epidemiological trials conducted in the United States.67?°-°8l

CONCLUSIONS There is tantalizing evidence suggesting that increased intakes of w-3 FA may contribute to the prevention of various diseases, most notably CVD. Although dietary sources should continue to be the first line approach to increasing intakes of w-3 FA, growing concerns about the safety of the fish supply will undoubtedly lead many to consider using supplements that carry a lower risk for contamination. Since an apparent cardioprotective intake is about 0.5—1 g/day, and as most capsules contain at least 300mg of EPA + DHA per l-g capsule, only 2-3 capsules may be needed to confer benefit. There is no evidence to date that such an intake would

have any adverse health effects, or

interact negatively with any known drugs. Consequently, interest in and utilization of @-3 FA for health promotion is likely to continue to grow.

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Omega-6 Fatty Acids William L. Smith University of Michigan Medical School, Ann Arbor, Michigan, U.S.A.

INTRODUCTION The term “essential fatty acids’? (EFAs) applies to those fatty acids that are necessary for human health and required in the diet. There are two different types of EFA deficiencies characterized by a lack of either omega-6 (w6) fatty acids or w3 fatty acids. Both w3 and w6 EFAs originate from plants and photosynthetic algae, and thus, are found throughout the food chain. The two best known 6 EFAs are linoleic acid (LA; 9Z, 12Z-octadecadienoic acid) and arachidonic acid (AA; 5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid). The most common @3 EFAs are a-linolenic acid (ALA; 9Z, 12Z, 15Z-octadecatrienoic acid), eicosapentaenoic acid (EPA; 5Z, 8Z, 11Z, 14Z, 17Z-eicosapentaenoic acid), and docosahexaenoic acid (DHA; 4Z, 7Z, 10Z,

13Z, 16Z, 19Z-docosahexaenoic acid). The average intake of m6 EFAs in the United States is about 7 energy”% (en%) with a ratio of almost 8:1 @6 EFA/w3 3 EFA. Linoleic acid and AA serve as precursors for the formation by cells of oxygenated fatty acid products called eicosanoids. At least some of the requirements for @6 EFA can be accounted for by eicosanoids. Those derived from AA promote inflammatory responses and thrombosis. There are arguments that significant decreases in inflammatory and cardiovascular diseases

would occur were the ratio of the intake of m6 EFA/w3 3 EFA reduced to less than 4.0, which would decrease the formation of eicosanoids.

STRUCTURES, BIOSYNTHESIS, AND DIETARY SOURCES OF EFAs Fig. 1 shows the structures of some common saturated and monounsaturated fatty acids as well as the w3 and 6 polyunsaturated fatty acids (PUFAs). Fatty acids containing two or three double bonds and 18 carbons are commonly referred to as PUFAs—examples are

William L. Smith, Ph.D., is Professor and Chair in the Depart-

ment of Biological Chemistry, University School, Ann Arbor, Michigan, U.S.A.

of Michigan

Medical

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022076

Copyright © 2005 by Marcel Dekker. All rights reserved.

LA and ALA. Those having three or more double bonds and 20 or more carbon atoms are called highly unsaturated fatty acids (HUFAs)—examples are AA,

EPA, and DHA!!!

Saturated fatty acids like stearic acid (Fig. 1) can be formed through the combined actions of acetyl CoA carboxylase and the fatty acid synthetase multienzyme complex by sequential addition of two carbons to the carboxyl end of the growing fatty acid carbon chain.!! In other words, C-1 and C-2 of stearic acid are the last

two atoms added during biosynthesis. Monounsaturated fatty acids such as oleic acid can be formed from saturated fatty acids by oxidation involving molecular oxygen and a desaturase;!*©! in the case of oleic acid, this is the A9 desaturase. The PUFAs can be synthesized from monounsaturated fatty acids like oleic acid by the actions of other desaturases acting at different positions along the carbon chain to form methyleneinterrupted cis double bonds (-CH=CH-CH-CH= CH-) characteristic of biological systems. The HUFAs can be formed by two carbon chain elongation of PUFAs, followed typically by another desaturation step.4°! The formation of AA from LA is illustrated in Fig. 1, which shows the A6 desaturase, two carbon chain elongation, and A5 desaturase steps.

Animals can form saturated and monounsaturated fatty acids but do not have Al2 or A15 desaturases. Hence,

they are

unable

to introduce

double

bonds

nearer than nine carbons from the methyl end of the chain (e.g., oleic acid in Fig. 1).*°! The EFAs of the 3 and w6 families have double bonds located three and

six carbons

from

the methyl

end of the chain,

respectively (Fig. 1). Plants have variable amounts of Al2 and A15 desaturases and thus are able to synthesize the 18 carbon fatty acids LA and ALA that have

double bonds at these positions.! The LA and ALA come from plants. Diatoms, which are the photosynthetic algae of phytoplankton, some fungi and moss, and the worm C. elegans can form LA and ALA and can also elongate and desaturate PUFAs such as ALA to form HUFAs.”! For example, in diatoms, up to 30% of the fatty acid pool can comprise EPA. Finally, it should be noted again that while species containing Al2 and A15 desaturases can form 3 fatty acids from 6 fatty acids, animals are not able 505

Omega-6 Fatty Acids 506 Saturated and Monounsaturated Stearic Acid; 18:0

pp

PR

RON

PU

OH O Oleic Acid; 18:1 09

ee

i

OH

O ey

ys po

ere

Linoleic Acid (LA), 18:2 6

iia

y

o6

ae OH

A6 desaturase

sanctions meer

y-Linolenic Acid (GLA);

A,

18:3 06

2C Elongation

y

ae

SVEN

20:3 06

ae

A5 desaturase

ee ——

Arachidonic wie

OG O

ar PN

Ne aera

ORR

Acid (AA);

zi

b 3

f ae Be

Dihomo-y-linolenic Acid;

of

Be weetNE

peel

a-Linolenic Acid (ALA); 18:3 03

OH

a

Eicosapentaenoic Acid

(EPA); 20:5 w3

O OH

ai

bia

Fear Sezai

aN

Docosahexaenoic Acid

(DHA); 22:6 03

Fig. 1 The structures and biosynthetic inter-relationships of saturated and monounsaturated fatty acids and the most important @6 and 3 fatty acids.

to perform either of these conversions. Hence, they need both w3 and 6 fatty acids in their diets. A major source of 3 and 6 fatty acids for humans is plant-based cooking oils, which are primarily mixtures of triglycerides of different compositions. The relative proportions of saturated fatty acids, monounsaturated fatty acids (primarily oleic acid), LA, and ALA vary widely in oils from plants (and animal sources) (Fig. 2).'*! Safflower, sunflower, and corn oils contain the highest percentages of LA. Olive oil is

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

particularly high in oleic acid, while the highest percentage of ALA is in Flaxseed oil. Corn oil is a major cooking oil in the United States. Meats consumed in the United States except fish contain relatively high proportions of the m6 fatty acids LA plus AA, and low proportions of the 3 fatty acids ALA, EPA, and DHA. This is primarily because animals are farm raised on diets containing corn and soybeans; a part of the LA that is consumed is converted to AA (Fig. 1). Freshwater and saltwater fish

4 % Monounsaturate O% n-3 ALA

B% n-6 LA % Saturate

3 g $ g E $ 5 3 3 2 é E 5 3 3 e 6 5.6.6 8 S$ 8. = dae sO E 5 iS F = 3

re

weg

”

8

8

m

0

Fig. 2 The percentages of saturated fatty acids (blue), monounsaturated fatty acids (aqua), LA (n-6, red), and ALA (3, yellow) comprising the triglycerides (oil fraction) of various seeds, lard,

and beef. (View this art in color at www.dekker.com. )

Omega-6 Fatty Acids

507

contain relatively high levels of the 20 and 22 carbon 3 fatty acids EPA and DHA and low levels of 6 fatty acids. Farm-raised fish contain comparatively higher levels of 6 fatty acids than wild fish, again because of the former’s diets. The National of Institutes of Health maintains a web page that provides many details about the relative proportions of various fatty acids in all types of foods (http://

efaeducation.nih.gov/sig/ods.html). PATHOLOGIES ASSOCIATED WITH EFA DEFICIENCIES Studies in the late 1920s and early 1930s showed that rats fed chow diets that had been extracted with organic solvents to eliminate fats exhibited a syndrome manifested grossly by: a) reproductive difficulties, most obviously with parturition; b) scaly skin, which was associated with excessive water loss; and c) a general failure to grow and thrive.”! This syndrome is called EFA deficiency. Although most studies on EFA deficiencies have been performed with rodents, there is also evidence that it can afflict humans who are treated in hospitals, nursing homes, or maintained on artificial milk diets that lack EFAs." The gross abnormalities of EFA deficiency in rodents can be overcome by including an 6 fatty acid such as LA or AA in the diet. Either LA?!" or AAU is effective. The w3 fatty acid ALA is less effective”! or ineffective.""! It has been recognized over the last 20 yr that there is a distinct m3 fatty acid deficiency that manifests itself in the form of various neurological deficits and most likely results from a reduction in DHA in various neurons.''?! This inadequacy cannot be overcome with 6 fatty acids.'!7! @3 fatty acids as a subject is treated elsewhere in this encyclopedia. As noted above (Fig. 1), AA can be synthesized from LA, and it is generally believed that AA is the key 6 EFA.""'*! Certainly, in the case of problems with parturition seen in EFA deficiency, it is an AA metabolite,

a prostanoid,

which

is involved.

Indeed,

the descriptions of the difficulties in parturition seen in EFA-deficient rats''"! and in cyclo-oxygenase-1 knockout mice!’*! are strikingly similar. As discussed in more detail below, prostaglandin endoperoxide H synthases (known generically as cyclo-oxygenases) catalyze the committed step in the conversion of AA to oxygenated metabolites called prostanoids.""*! The growth retardation and failure-to-thrive abnormalities characteristic of EFA deficiency may also be dependent on AA. It has been proposed that the latter abnormalities result from a lack of oxygenated meta-

bolites

of AA,

which

hypothalamic/pituitary

are involved

in regulating

hormone _ secretions.!'°'”!

Early studies involving ALA

feeding indicated that

ALA, presumably because it can be converted to EPA and its oxygenated metabolites, can support body weight growth.”! Interestingly, the scaly skin syndrome of EFA deficiency can be ameliorated by LA and does not require AA or its oxygenated metabolites. Felinae have only low levels of the A6 desaturase involved in the first step in converting LA to longer chain, more HUFAs (Fig. 1), but dietary LA will resolve the scaly skin problems of EFA-deficient cats."'*] The LA (or AA) may function in skin to form O-acylated sphingolipids such as 1-O-linoleoyl-ceramide!!”! or hydroxy fatty acids such as 13-hydroxy-9Z,13E-octadecadienoic acid (13HODE).”°! However, related ceramide or hydroxyl fatty acid metabolites of AA (instead of LA) may also function to relieve the scaly skin problem, which is the reason why AA can resolve this abnormality as well as LA.

METABOLISM

OF EFAs TO COMPLEX LIPIDS

Distribution of m6 EFAs in Different

Types of Lipids The AA and LA are found in cells as a free fatty acid and in many different forms in which the carboxylate group is modified but the carbon chain remains unaltered (Fig. 3). These include: a) arachidonyl- and linoleoyl CoAs (i.e., thioesters); b) arachidonyl and linoleoyl esters in which the acyl chain is esterified at the sn2 position of glycerophospholipids (i.e., phosphatidylcholine, phosphatidylserine, phosphatidylinositol, or phosphatidylethanolamine) that comprise cell membranes; c) phospholipid degradation products in which arachidonyl and linoleoyl chains are esterified at the sn2 position of metabolic or signaling intermediates such as: i) Phosphatidic acid formed from phosphatidyl inositol derivatives by the action of phospholipase D; i1) 1,2-Diacylglycerol formed by the action of phospholipase C; iii) 2-Arachidonyl glycerol formed by sequential actions of phospholipase C and diglyceride lipase; and iv) perhaps 2-acyl-glycerophospholipids formed via phospholipase A1; d) as arachidonyl ethanolamide (anandamide); and e) 1-O-acyl sphingolipids.

6

EFAs in Membrane Phospholipids

In humans of normal weight, the largest fraction of the total EFA pool is found in membrane glycerophospholipids. The major membrane glycerophospholipids in eukaryotes are phosphatidylethanolamine and phosphatidylcholine with smaller amounts of phosphatidylserine and phosphatidylinositol (Fig. 3).7122] All of

Omega-6 Fatty Acids

508 Diet

i R1-C-OH LA

Various de novo Glycerophospholipid

Biosynthetic Pathways |

R1-C-SCoA

1,2-diacyl-glycerophospholipid (newly synthesized and lacking arachidonyl or linoleoy! groups

i RC-0:CH2

i

i

serine, inositol, or ethanolamine)

|

ou |

R2-C-SCoA

= Arachidony!| CoA

H2-C-O-CH2(CH2),CH3

O

1-O-alkyl-glycerophospholipid

Hl

H2-C-O-C-CH2(CH2),CH3

is

__Linoleoy!

Desaturation, Elongation

at the sn2 position: X = choline,

CH2-0-P-O-X

iPLA2

COA-SH Coenzyme A

0

es: H2-C-O-C-CH2(CH2),CH3

3

+

ns

from ether lipid biosynthetic

HOCH2

O

1-acyl-glycerophospholipid

Cpote'nto®

(lysophospholipid)

:

pathway (Y = choline or

ethanolamine)

Il

CH2-0-P-O-X

oH ak

i

\R1-C-SCoA or R2-C-SCoA

CoA-independent acyl transferase

ll

H2-C-O-C-CH2(CH2),CH3 Il

R1,R2-C-O-CH2

4-acyl-2-arachidonyl (or linoleoyl) glycerophospholipid

H2-C-O-CH2(CH2),CH3

0

CH2-0-P-O-X

pay

picerActions tor ree nage or AA (e.g. on ion channels)

1-O-alkyl-2-arachidonyl (or

R1.R2-C-OCH2__

ay

linoleoyl)-glycerophospholipid

r

CH2-0-P-0-Y OH

LA oUaA Eicosanoid Synthesis

Lipoxygenase Pathways Cyclooxygenase Pathway (prostaglandins, thromboxane) (leukotrienes, related mono-, di- and tri-hydroxy fatty acids)

me

Cytochrome P450 Pathways (mono-. di- and epoxy-fatty acids)

Actions on Various Eicosanoid Receptors

Fig. 3 Major pathways involved in the metabolism of LA and AA into various phospholipids and eicosanoids. The snl, 2, and 3 positions of the glycerol backbone are denoted on the structure shown on the top left. (View this art in color at www. dekker.com. )

these four classes appear in the form of diacyl derivatives with acyl groups esterified to the snl and sn2 positions of the glycerol backbone. Ethanolamine- and choline-containing phospholipids also occur in abundance as ether lipids having 1-O-alkyl or 1-O-alkenyl groups and acyl groups at the sn2 position (Fig. 3).?7)

The 1,2-diacyl glycerophospholipids contain saturated and monounsaturated fatty acids at the snl position and monounsaturated fatty acids, PUFAs, and HUFAs esterified at the sn2 position.” Similarly, the ether lipids have a saturated or monounsaturated chain at the 1-position and monounsaturated fatty

Omega-6 Fatty Acids

509

acids, PUFAs, and HUFAs at the sn2 position.22) In

diacyl glycerophospholipids, about 60% of the fatty acids at the snl position are saturated fatty acids and 40’ are monounsaturated fatty acids; the sn2 position contains about 15% monounsaturated fatty acids and 85% either as PUFAs [mainly LA (18:2 6)] or HUFAs [mainly AA (20:4 6) with some w3 fatty acids mostly EPA and DHA].”" Curiously, phosphatidylethanolamine generally contains 3-4 times more AA than LA, while phosphatidylcholine has 3-4 times more LA than AA. Glycerophospholipids having AA at the sn2 position most frequently have stearic acid (or an 18 carbon, ether-linked chain) at the snl position; those with LA at the sn2 position most commonly have palmitic acid, a 16 carbon saturated fatty acid (or a C-16 ether-linked chain) at the snl position. Phosphatidylinositol and its more highly phosphorylated forms such as phosphatidylinositol-4,5-bis phosphate that are precursors of important intracellular signaling molecules contain almost exclusively stearic acid and AA at the sn] and sn2 positions, respectively. Finally, it should be noted that different tissues and organs have different phospholipid compositions. The biological rationale for these various differences in tissue phospholipid compositions and in fatty acid compositions among different phospholipids is not known.

Biosynthesis of Membrane Phospholipids Containing m6 EFAs Free fatty acids are found in plasma bound to serum albumin, whereas lipoproteins serve to transport triglycerides, cholesterol esters, and phospholipids. These acids can enter cells by simple diffusion or be generated by hydrolysis of triglycerides or phospholipids associated with lipoproteins that have been taken up by cells. There are no known specificities for their uptake. However, once inside the cell, HUFAs

such as AA are

preferential substrates for long chain acyl CoA ligase, which has a relatively low Ky, toward AA; this serves to sequester free HUFAs in cells,[1 Fatty acyl chains can be introduced into the sn2 position of 1,2-diacyl-glycerophospholipids at one of two different steps in metabolism (Fig. 3). One is during de novo glycerophospholipid biosynthesis from glycerophosphate when an acyl group from an acyl CoA becomes esterified at the sn2 position of l-acylglycerophosphate. The second is during phospholipid “retailoring’’ when a newly formed glycerophospholipid such as phosphatidylethanolamine is hydrolyzed by Ca**-independent phospholipase A, (iPLA2) to yield 1-acyl-glycero-3-phosphorylethanolamine, which, in turn, is‘acylated in a transferase reaction in which an acyl CoA is the acyl donor. It is thought that the bulk of the PUFAs and» HUFAs are introduced into

phospholipids during “retailoring’’ (Fig. 3).!74 Finally, an «6 fatty acyl chain esterified to the sn2 position of a diacyl glycerophospholipid can be transferred to 1-O-alkyl-glycerol-3-phosphate or 1-O-alkyl-glycero-3phosphorylcholine (or -phosphorylethanolamine) by transesterification via a CoA independent transacylase.?7! This is an important process because hydrolysis of ether lipids such as 1-O-alkyl-2-arachidonylphosphatidylcholine by a phospholipase A, can provide the AA used for the synthesis of eicosanoids. At the same time, it generates 1-O-alkyl-glycero-3-phosphorylcholine, which is the immediate precursor for the acetyl transferase that generates the hormone, platelet-activat(1-O-alkyl-2-acetyl-phosphatidylcholine; ing factor Fig. 3). It should be noted that the transacylation is relatively specific for AA, although LA is also shown as being transferred in Fig. 3.!°77!

TRANSFORMATION OF 6 EFAs INTO EICOSANOIDS Free AA can be transformed to a group of compounds collectively known as eicosanoids: prostanoids, leukotrienes (LTs), and related hydroxyl fatty acids and epoxides .(Figs. 3-6).!'°! The LA can also be converted to some oxygenated products, which although containing only 18 carbons, are also generally referred to as eicosanoids.

Biosynthesis and Actions of Prostaglandins

The structures and biosynthetic inter-relationships of the most important prostanoids are shown in Fig. 4. PG is the abbreviation for prostaglandin, and TX for thromboxane. Letters after PG denote the nature and location of the oxygen-containing substituents present in the cyclopentane ring. Prostanoids with a subscript 2’? are formed from AA. The PGs are synthesized and released rapidly by cells in response to certain hormones and physical stimuli.’°! The pathway for stimulus-induced prostanoid formation involves three stages (Fig. 4): a) mobilization of free AA from membrane phospholipids through the action of cytosolic (c) PLA»; b) conversion of AA to prostaglandin endoperoxide Hz (PGH2) by a prostaglandin endoperoxide H synthase (PGHS; also known as cyclo-oxygenase or COX); and c) isomerization of PGH; to one of the major prostanoids by a specific synthase (e.g., TXA synthase). Frequently, specific prostanoids are formed by specific cells types; for example, TXA, is composed almost exclusively by platelets,

whereas

endothelial cells.

PGI,

is mainly

made

of vascular

Omega-6 Fatty Acids

510 ee

ee ee

(= PO

OL |

PGH SYNTHASE

oA es.

= ea

AA

en Cyclooxygenase

eH

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nee

sa Gene age o-f\

HO

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OH

ae

PGE2

H

OH There are two isoforms of PGHSs known as PGHS1 and -2 or COX-1 and -2.”*! The enzymes are encoded by separate genes. In general, PGHS-1 is expressed constitutively, whereas PGHS-2 is induced in response to growth factors, cytokines, and inflammatory stimuli. Both enzymes are inhibited by nonsteroidal antiinflammatory drugs such as aspirin, ibuprofen, and naprosyn; COX-2 specific inhibitors (e.g., celecoxib and rofecoxib) are available commercially. It should be noted that in addition to being able to use AA as its substrate, PGHS-2 can convert 2-arachidonoylglycerol to 2-PGH)>-glycerol efficiently, and this intermediate can be converted to 2-prostanoyl-glycerol derivatives (with the exception of TXA;).74] The importance of these 2-prostanoyl-glycerol derivatives in the actions of PGHS-2 is unknown. There are specific G protein-linked receptors for each of the known prostanoids.”°! These receptors interact with downstream effectors such as adenylate cyclase and phospholipase C to modulate the formation of second messengers such as cAMP, Ca**, and diglyceride. There may also be specific receptors for at least some of the 2-prostanoyl-glycerol derivatives.

PGI2

Fig. 4 Pathway for the biosynthesis of various prostaglandins (PGs) from AA. PGH)> is converted to PGE», PGF>,, PGI, and TXA> by specific synthases.

Prostaglandins are made by many cells and tissues, hence it has been difficult to study their biology. However, the recent availability of specific knockout mice for most of the various biosynthetic enzymes and prostanoid receptors, specific enzyme inhibitors, and specific receptor agonists and antagonists has been facilitating these studies.°**! There are some situations in which the actions of prostanoids are understood reasonably well. For example, TXA, formed by platelets is involved in thrombosis. Platelets utilize PGHS-1 to produce PGH)>, which is then converted to TXA>. Low doses of aspirin are used to block platelet PGHS-1 (and thus prevent PGH)> formation) and ameliorate thrombotic conditions.?7! PGHS-2 is the relevant enzyme involved in producing prostanoids that are involved in many inflammatory

conditions,

and

accordingly,

COX-2

inhibitors are marketed to alleviate inflammation and associated pain. There also seems to be a link between COX-2 levels in the colon and colon carcinogenesis.7*! It should also be pointed out that AA esterified at the sn2 position of any phospholipids can be oxidized nonenzymatically to yield a complex racemic mixture

Omega-6 Fatty Acids

511

eee

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of

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of esterified “isoprostanes,’’ which are then mobilized

presumably through the actions of a phospholipase A>.°! Isoprostanes are formed in abundance, particularly under conditions where tissue free radical

5 Pathway for the biosynthesis

leukotrienes.

LTB,

is formed

from LTA, by LTA, hydrolase. LTC, is formed from LTA, by a glutathione-S-transferase called LTC, synthase.

damage occurs. Some of them have potent biological activities.P°!

Biosynthesis of LTs and Related Hydroxy Fatty Acids from 6 EFAs 0

—

ae

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formed

through the

The biosynthetic pathway for the formation of LTs is shown in Fig. 5.!'°! Like prostanoids, LTs are formed in response to cellular stimuli which mobilize free AA from phospholipids by activating cPLA>. The LTs are produced from AA by the action of 5-lipo-oxygenase (5-LO), which both forms 5-hydroperoxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid (S-HpETE) and then dehydrates this product to form LTA,4. LTA, hydrolase hydrolyzes LTA, to produce LTBy, while LTA, synthase catalyzes the addition of glutathione to C-6 of LTA, to create the peptidoleukotriene called LTC,. The cellular sites of LTB, and LTC, are limited and specific. Human neutrophils produce LTB, while mast cells and eosinophils form LTC4. During the search for pharmacological antagonists of LT biosynthesis, a protein called 5-lipo-oxygenaseactivating protein (FLAP) was discovered. This protein is important for the efficient production of

Omega-6 Fatty Acids

512

LTs by cells, but it is not clear how it functions. It may serve as a protein that transfers AA to 5-Lo.">! LTB, is a potent chemotactic and chemokinetic agent for the human polymorphonuclear leukocyte. LTC, constricts bronchial smooth muscle and mediates leakage of vascular fluid in the process of edema. The biological activities of both LTB, and LTC, are mediated by specific G-protein coupled receptors.!1°31) LTC, receptor antagonists (e.g., montelukast, pranlukast, and zafirlukast) and a 5-LO inhibitor zileuton are commercially available to treat asthma. There are lipo-oxygenases other than 5-LO, including 8-, 12- and 15-lipo-oxygenases, which introduce oxygen at different positions in the AA chain.27!

Certain of these lipo-oxygenases (e.g., 15-LO) will utilize LA as a substrate such as in the formation of 13-HODE, which, as noted above, may be able to ameliorate the scaly skin of EFA-deficient animals.?°!

The enzymic specificities of various lipo-oxygenases toward C-18 and C-20 fatty acids have been studied in some detail.!!37*! A methylene-interrupted cis double bond system is required in all cases. Most lipo-oxygenases utilize AA in preference to LA. When this aspect was examined, it was found that most lipo-oxygenases preferentially utilize m6 vs. m3 fatty acids and do not function with w9 fatty acids. This is most notable in the case of 5-lipo-oxygenase and LT formation. Also of importance is the finding that LTs derived from , EPA have about one-tenth the biological activity of those formed from AA_!°77) The substrate specificities of PGHS-1 and -2 have

been examined in detail.??4°) AA is the most efficient substrate for both PGHS-1 and -2. EPA is a particularly poor one for PGHS-1 and DHA is not a one. Indeed, EPA and DHA essentially serve as inhibi-

tors of the oxygenation of AA by PGHS-1.4°*!] EPA is a much

P450 Hydroxylase Pathways That Lead to Oxygenated Products of AA The AA can be hydroxylated by specific cytochrome P450 isoforms leading to epoxyeicosatrienoic acids

(EETs; Fig. 6)."°7"! These can be hydrolyzed to yield dihydroxy acids. Arachidoric acid can also be oxidized at its w-1 or @ position to produce 19- and 20-HETE, respectively. Relatively little is known about the physiology or overall biological importance of the P450 hydroxylation products, but some of these products have potent vasoactivity in the renal and other vascular beds.

BIOLOGICAL ACTIONS OF FREE AA In addition to being able to function in an oxygenated or otherwise chemically modified form, AA may exert some actions as a free acid. It will activate NADPH oxidase,*) serve as an activating ligand for peroxisomal proliferator-activated receptors (w-hydroxylated epoxy fatty acids and certain prostanoids may also activate

PPARs)**! activate ion channels such as a two-pore domain K* channel,'°*! and stimulate apoptosis.°7! BIOCHEMICAL SELECTIVITY FOR AA AND LA

Identifying the biochemical basis as to why 6 or 3 methylene-interrupted cis double bond systems perform their critical biology is of obvious importance. Selectivity or specificity is often claimed for various biological actions of PUFAs, but careful examination of the data frequently reveals that the specificity is modest (e.g., 1.5-3-fold) and thus unlikely to have significant biological consequences.

better

substrate

for PGHS-2,

and

DHA

can also be oxygenated by PGHS-2. Moreover, neither _ of these fatty acids is an effective inhibitor of AA oxygenation by PGHS-2. LA can be converted to 9or 13-HODE by PGHS-1 and -2 at about one-fourth the efficiency of AA. The degree to which oxygenation of LA by PGHS occurs and has biological importance is still not known, although 13-HODE formed via a 15-

LO"?! has been implicated as an effector of cell growth. The efficacy of prostanoids formed from 6 vs. 3 fatty acids (i.e., AA vs. EPA) is an understudied area. The information available indicates that products derived from AA are about 10 times more active on prostanoid receptors than those from EPA.'*3] cPLA; is the only phospholipase that exhibits significant specificity toward the nature of the acyl chain at the sn2 position of phospholipids. It has a marked preference for AA over LA or oleic acid. Surprisingly, the enzyme also appears to discriminate against phosphatidylethanolamine species with DHA at the sn2 position.*! It has also been reported that DHA and AA are released from phospholipids by two separate

isoforms of PLA,."**!

Another enzyme showing some specificity toward AA-containing phospholipids is the CoA independent transacylase involved in forming 1-O-alkyl-2-acylglycerophosphate from 1-O-alkyl-glycerol-3-phosphate

and a diacyl phospholipid donor.??!

Finally, it should be noted that the K* channel TRESK is relatively specifically inhibited by AA vs. HUFAs, PUFAs, and saturated fatty acids. Halfmaximal inhibition occurs with 5-10 pM AA.&4

WHY PLANTS HAVE LA AND ALA Why these products are made by plants and a few fungi is neglected in the discussion of EFAs. The question

Omega-6 Fatty Acids

513

has been addressed with the model plant Arabidopsis that is able to form both LA and ALA because it has both Al2 and A15 desaturases. LA is found at high concentrations in chloroplast membranes and is essential for photosynthesis.* »-Linolenic acid seems not to be critical for photosynthesis but is essential for the synthesis of plant hormones such as jasmonic acid.47!

ACKNOWLEDGMENTS William

E. M. Lands from College Park, Maryland,

and John Ohlrogge from Michigan State University provided important guidance in the preparation of this entry.

ARTICLE OF FURTHER INTEREST HEALTH CONSEQUENCES OF EXCESS DIETARY o-6 FATTY ACIDS

Omega-3 Fatty Acids, p. 493.

Although an absence of LA or AA in the diet can be

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*! with highest activities found when lapachol and its 0-alkyl and 9-acetyl derivatives or B-lapachone were used. Molluscicidal activity was also elicited using other naphthaquinones.'°°!

Cancer chemopreventive action (Mice) 1,4-Furanonaphthaquinones with an OH group on the dihydrofuran ring, such as avicequinone-A and avicenol-A, showed the highest bioreactivities in the EBV

early activation model and in a chemoprovocative tumor-inducing mouse model.°7!

Neurodegenerative effects Toxicity in vivo (rodents, dogs, monkeys) Lapachol and other naphthaquinones modulate the tau aggregation of proteins, and thus are assumed to effect the treatment or prophylaxis of neurodegenerative

diseases

and/or

clinical

dementias,

such

as

Alzheimer’s disease.!*”! Snakebites

T. rosea bark has demonstrated 100% neutralization of the minimum hemorrhagic dose of Bothrops atrox venom in vitro. This correlates with traditional healers’ statements from northwestern Colombia that T. rosea

has antihemorrhagic properties.°°! In Vivo Studies Anticancer activity (rodents) Crude extracts and lapachol were tested in implanted rodents against Walker 256 carcinosarcoma. Lapachol showed highly significant antitumor activity especially when administered orally, with relatively little effect on host body weight.©"! In this model, a 92% reduction in tumor growth occurred, and lapachol’s tetra-acetylglucoside derivative increased lifespan by 80% in mice

with lymphocytic leukemia P-388.!'*! Treatment with B-lapachol following exposure to irradiation optimized the effects of delayed tumor growth in mice with RKO (colorectal cancer cells) induced tumors.©?! In human ovarian and prostate tumor prexenografted mouse models, a synergistic cytotoxic effect was demonstrated using taxol and B-lapachone.!*?!

Antischistosomiasis activity (mice) Oral administration of lapachol protected mice from topical infection with Schistosoma mansoni cercariae

Lapachol from the bark of T. ochracea was tested during rat fetogenesis from days 17 to 20 of pregnancy. While lapachol was not toxic to rat mothers, it was

fetotoxic, leading to intrauterine growth retardation of pups compared to untreated controls (P < 0.01). There was also significant weight reduction (P < 0.01) in lungs, livers, and kidneys of treated pups. Putative effects in women cannot be ignored.>%°?! In oral toxicity tests using lapachol, rodents, dogs, and monkeys developed moderate to severe anemia during the first 2weeks of treatment, but recovery was evident at 4weeks of treatment. Monkeys receiving up to 0.25 g/kg/day completed the treatment and recovery periods, with those receiving higher doses developing infrequent emesis, anorexia, pallor of mucous membranes, and periods of diarrhea. Dogs and rats were able to tolerate much larger doses of lapachol than either monkeys or mice. A gender difference was also evident in mice: males tolerated less lapachol than females.'®!

CLINICAL TRIALS Anticancer Efficacy Promising anticancer reports in humans were widely disseminated in South America in the popular press and the medical literature in the 1960s") and later, and these elicited the U.S. National Cancer Institute’s (NCI) attention.) However, according to NCI evaluations’ they did not fulfill the criteria for further development in that studies with the Tabebuia constituent lapachol were disappointing, and clinically it induced nausea, vomiting, and prolonged prothrombin times due to its anti-vitamin K action.'*! Because lapachol targets vitamin K dependent reactions, such as the

Pau d’Arco or Lapacho (Tabebuia)

532

reversible activation of ligand for the axl receptor tyrosine kinase, this compound, like warfarin, may have value in cases where axl is overexpressed. Consequently, lapachol may have value in the treatment of small cell carcinoma, metastatic colon cancer, and

adenocarcinomas of the colon.!! To overcome its insolubility, use of B-lapachone as a second anticancer agent alone or in combination with radiation therapy requires the development of pharmaceutically acceptable and solubilizing carrier molecules. '°?! Anecdotal reports of lapachol’s antitumor effects continue

to accumulate,

possibly because

ACKNOWLEDGMENTS We appreciate the personal communications from Dr. Lucia Lohmann, Brazilian researcher of the Bignoniaceae, at the Missouri Botanical Garden, St. Louis,

Missouri, and thank Gayle Engels of the American Botanical Council, Austin, Texas, and Natasha Y. Hall of the American Herbal Products Association, Silver

Spring, Maryland, for documents on pau d’arco.

additional

early studies indicated that lapachol had a selective affinity for tumor cells that impeded their respiration, resulting in the evolution of free oxygen radicals and destruction of nucleic acid. In addition to lapachol, other naphthaquinones and bioreactive compounds in these extracts may be contributing to the positive clinical effects being elicited.?*!

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Grape seed proanthocyanidins fed to mice or rats, however, were not effective in curtailing chemically induced mammary tumorigenesis.774] Several foods also contain them; however, there is a paucity of observations on their effect on carcinogenic processes. Although black and green teas have been extensively investigated for their anticancer activity, the latter contain only limited proanthocyanidins,"*! while the former have substantial concentrations of derived tannins (theaflavins, thearubigins, etc.), which are a heterogeneous mixture of oxidation products of monomeric flavonoids, and structurally different from

proanthocyanidins.?7!

Proanthocyanidins

558

Relative to oxidation of LDL, many in vitro studies

Mechanisms of action

have been conducted with isolated LDL particles, che-

Investigators have suggested several mechanisms of action for proanthocyanidins toward inhibition of carcinogenic processes. Foremost among these are their antioxidant or ROS scavenging activities,'*! based primarily on findings from cell culture and ex vivo experiments. Studies with laboratory animals indicated that GSPE provided protection against chemically induced hepato- and nephrotoxicity, increased bcl-X, expression in liver tissue,

and

antagonized

both

necrotic

and apoptotic liver cell death.”*! Further attempts to elucidate the antiapoptotic and antinecrotic mechanism suggested that cytochrome P450 2E1 activity, responsible for the metabolism of drugs and chemicals, was substantially decreased by GSPE feeding in livers

of mice”®! but not in the same organ of rats.?3! Also, cytochrome P450 1A and glutathione-S-transferase activities (carcinogen metabolism) were unaltered by a similar feeding regimen in rats.

Atherosclerosis Atherosclerosis is an inflammatory disease process.”?! One of the first events in this process is the development of endothelial dysfunction, presumably caused by such phenomena as hypertension, diabetes, oxidatively modified low-density lipoproteins (LDL), and elevated levels of homoserine. The injured endothelium becomes more permeable and attracts monocytes and other inflammatory and immune cells by secreting specific cytokines and growth factors. At the same time, upregulation of a series of adhesion molecules increases

the adhesiveness

of endothelium,

platelets,

and leukocytes. All of these events promote the formation of atherosclerotic plaques, which results in constriction of the blood vessel. The injured endothelium, in turn, may

also have decreased

ability to

dilate as a result of diminished capacity to produce nitric oxide (NO). Proanthocyanidins and/or their metabolic products may ameliorate several of the steps in this complex process. Inhibition of liposome and LDL oxidation Experiments with synthetic liposomes and proanthocyanidins isolated from cocoa revealed that oxidation was

effectively

inhibited

by flavan-3-ol

monomers,

and proanthocyanidin dimers and trimers.°" Blucidation of the antioxidant mechanism suggested that proanthocyanidins interacted with the phospholipid “head’’ groups of the liposome, which restricted both access of oxidants to the membrane surface and movement of oxidants through the internal hydrophobic region of the membrane.?!!

mical oxidants, and various individual isolated procyanidins or natural products rich in proanthocyanidins.°7! It is difficult to interpret results with polymeric proanthocyanidins in terms of biological activity, because studies to date suggest that oligomers greater than Dp2 are only minimally absorbed in human beings. Nonetheless, experiments with a cellular system (endothelial cell-mediated LDL oxidation), which may have relevance to in vivo conditions, showed a preference of antioxidants for monomeric catechin and dimers rather than higher polymers of procyanidin.&?! A highly controlled, double-blind randomized crossover human study demonstrated that proanthocyanidin-containing cocoa powder and chocolate were effective in decreasing LDL oxidation susceptibility, and slightly increasing total serum antioxidant capacity as well as high-density lipoprotein (HDL)cholesterol levels.84! The diets of this study were based on an average American diet and identical in content except for daily addition of 16g dark chocolate and 22g of cocoa powder to the test diet. Similar results of cocoa ingestion on LDL oxidation susceptibility were observed in additional studies for which dietary control was less rigorous.>! Pycnogenol (150 mg/day) fed to healthy individuals for 6weeks did not alter LDL oxidizability, but reduced LDL-cholesterol and increased HDL-cholesterol levels in the plasma of two-thirds of the subjects.°° However, the same extract (360 mg/day) given to patients with chronic venous insufficiency decreased total blood

cholesterol

and

LDL-cholesterol

values,

but did not alter HDL-cholesterol levels.2” Addition of GSPE to the diet for 8 weeks to hypercholesterolemic subjects substantially reduced the level of antibodies to oxidized LDL (measure of oxidized LDL), compared to results of the placebo control group.&*! Inhibition of inflammatory response

As noted above, atherosclerosis is an inflammatory response of the intima of the vessel wall that involves such immune cells as macrophages and T-lymphocytes.P?] A cascade of biochemical events, beginning with metabolism of arachidonic acid to such cytokines as leukotriene and prostacyclin, occurs prior to the recruitment of immune cells to the injured endothelium. Alteration of any of the steps in this cascade that changes the favorable ratio and levels of these cytokines may reduce the severity of the resulting atherosclerotic event. Short-term (6hr) in vivo experiments with human subjects fed high-proanthocyanidin-containing chocolate resulted in increased plasma levels of prostacyclin, decreased concentrations of leukotrienes, and

~

Proanthocyanidins

559

a decreased leukotriene /prostacyclin ratio, a measure

of the proinflammatory /anti-inflammatory eicosanoid balance.*! Longer-term studies (4 and 6weeks) with subjects consuming a combination of cocoa powder and dark chocolate daily, in addition to an average American

diet or a low-flavonoid diet, failed to alter

the urinary excretion of F> isoprostane, or thromboxane B> (stable metabolite of proaggregatory thromboxane production), 6-keto-prostaglandin F,, (prostacyclin stable metabolite), or their ratio.2+40! Consumption of

purple grape juice, but not several other juices or coffee devoid of proanthocyanidins, significantly increased 6-keto-prostagladin F,, at 2hr postconsumption.44 Pycnogenol (200mg/day) consumption reduced thromboxane B, levels in smokers compared to those in

nonsmokers,

but

did

not

alter

levels

in

non-

smokers.'*7] Intake of G. biloba extract (120 mg/day, 3mo) by healthy subjects reduced excretion of both 11-dehydrothromboxane By, and prostacyclin, but had no effect in Type 2 diabetics, which suggested a differential modulation of cyclo-oxygenases depending on their cellular location (platelets or endothelial cells).[7! Endothelial dysfunction causes increased expression of cellular adhesion molecules (CAMs), e.g., ICAM-1 (intracellular CAM), VCAM-1 (vascular CAM), and E-selectin, which mediate the recruitment of monocytes and their subsequent differentiation into phagocytic macrophages.7! Many in vitro studies have

been conducted employing cultured and isolated cells, treated with several sources of proanthocyanidins, both purified and products, as affectors of CAM metabolism. The results of these experiments are difficult to relate to in vivo conditions because of the mixture or size of the proanthocyanidins that was tested. However, an in vitro study with peripheral blood mononuclear cells (PBMCs), isolated from human subjects who had low production of transforming growth factor beta-1 (TGFB-1), showed that its production was greatly stimulated by dimeric proanthocyanidins isolated from cocoa.“! In contrast, TGFB-1 secretion from high-producing PBMCs at baseline was inhibited by the same dimers. Notwithstanding these observations, a 6-week study with human subjects who received a combination of dark chocolate and cocoa powder (~650mg procyanidins per day) in addition to a low-flavonoid diet failed to alter the response of several markers of inflammation, including interleukin-18, interleukin-6, TNFa-1, C-reactive protein,

and P-selectin.*!

vascular

endothelium

that

produces

adequate

NO

and prostacyclin, both inhibitors of platelet adhesion,

does not attract platelets. However, atherosclerotic events, including damage to the endothelium and plaque deposits, may result in recruitmedt and adhesion of platelets at the site of injury. In the case of a highly stenosed region of a coronary or cerebral artery, aggregated platelets may cause cessation of blood flow, resulting in a myocardial infarct or transient ischemic attack. Therefore, knowledge of components of foods and dietary supplements that downregulate platelet activity may reduce the risk of stroke and heart attacks. Both short-term (2-6hr) studies and a long-term (28 day) experiment with human subjects demonstrated that consumption of proanthocyanidin-rich cocoa beverage lowered P-selectin expression and _ platelet ageregation.“*8! The effects observed were qualitatively similar to those of aspirin, but less profound.!47! Other food sources of proanthocyanidins such as purple grape juice, and combined extracts of grape seeds and grape skins, but not citrus juices, were also active in the reduction of platelet aggregation when administered to dogs, monkeys, or human beings.6-49]

Extract of G. biloba (120mg/day for 3mo) fed to healthy volunteers modulated collagen-, but not platelet activating factor (PAF)-mediated platelet aggregation.“*] However, giving the same extract to subjects with Type 2 diabetes mellitus decreased platelet aggregation stimulated by both systems. An in vivo model based on cyclic flow reductions caused by platelet aggregation in the partially occluded circumflex coronary artery of anesthetized dogs has been employed to test platelet activity and platelet— vessel wall interactions.?7! Several dietary sources of proanthocyanidins (e.g., red wine and purple grape juice) were active in preventing thrombus formation in this model. A similar model, based on experimental venous thrombosis in spontaneously normolipidemic rats

fed

a cholesterol-rich

diet, demonstrated

that

alcohol-free red wine added to the diet reversed the prothrombotic effect of the hyperlipidemic factors.F°! Animal models and human studies

Several animal models have been developed to study environmental effects on the progression of atherosclerosis. Golden Syrian hamsters, when fed diets of high cholesterol and coconut oil for 10 weeks, have a lipid profile similar to hypercholesterolemic human beings. This treatment also results in the formation

Decreased platelet aggregation

of foam

Platelets are small cells of the blood system that are normally recruited to participate in the healing of capillary walls when damage occurs.27! Healthy

has been used as a biomarker of the early stages of atherosclerosis (atherosclerotic index).*! Addition of GSPE to a hypercholesterolemic hamster diet (50 or 100mg/kg body weight) resulted in a substantial

cells on

aorta

walls,

the extent

of which

Proanthocyanidins

560

and significant reduction of the atherosclerotic index. In addition, total plasma cholesterol and triglyceride levels were also significantly reduced in the GSPE-fed animals. New Zealand white rabbits fed hypercholesterolemic diets respond with high total plasma cholesterol levels (400+ mg/dl) and the formation of Sudanpositive stained lesions (fatty streaks) on the walls of their aorta." The extent of the Sudanophilic lesions of the aortic arch of these animals also serves as a biomarker of atherosclerosis potential. Addition of GSPE (Leucoselect-Phytosome) to hypercholesterolemic diets of a group of rabbits reduced aortic arch lesions to nearly control levels (3%), whereas atherosclerotic diets alone resulted in lesions that covered 18% of the vessel wall. Studies with proanthocyanidin-rich cranberry juice powder fed to familial hypercholesterolemic pigs significantly lowered total plasma cholesterol and LDL, and slightly raised HDL.°”! However, the same powder fed to normocholesterolemic pigs did not alter levels of circulating cholesterol fractions. Grape seed proanthocyanidins fed to rats, along with high-cholesterol diets, also reduced serum cholesterol levels compared

to non-proanthocyanidin-fed controls. Studies with human beings who consumed a combination of cocoa powder and dark chocolate for relatively long periods (4 and 6 weeks) revealed that this only slightly, but significantly, increased HDL levels in one experiment, but did not remarkably alter plasma cholesterol, triglyceride, or other lipoprotein concentrations.°4*°! In contrast, cinnamon, which contains a

series of unique trimeric and tetrameric procyanidins with A-type linkages,! significantly decreased plasma levels of triglycerides and total and LDL

cholesterol,

when administered (1-6 g/day) just for 20 days.©?! NO-dependent vasodilation

beverage demonstrated a significant increase in FMD response (4 hr postconsumption) relative to response at baseline and after consumption of low-proanthocyanidin cocoa drink.“! In addition, plasma NO concentra-

tions paralleled FMD response, supporting the concept that improved endothelial function, in part, may be due to a favorable modulation of NO bioavailability. Employing pulsatile artery dilation in a fingertip, healthy subjects who consumed proanthocyanidin-rich cocoa for 4 days had consistent and striking peripheral vasodilation compared to baseline.*! Consumption of test cocoa on day 5 gave an additional significant acute vasodilation response, whereas infusion of N°-nitro-L-

arginine methylester (L-NAME), an NOS inhibitor, completely reversed the cocoa-induced vasodilation. A surgical in situ system was employed to assess the response of cerebral microcirculation of rats to the intake of an extract of G. biloba leaves (EGb 761).P4 Normotensive and spontaneously hypertensive (SHR) rats were fed EGb

761 for 9 days, after which several

parameters of cerebral circulation were compared with those of control animals. The most significant and dramatic changes observed in EGb 761-fed SHR rats were a blood pressure decrease of nearly 30%, and increased numbers of open capillaries and circulating endothelial cells, compared to SHR controls. Only minor changes were observed (increased cerebral blood flow velocity) in EGb 761-treated normotensive animals. In vitro studies demonstrated that red wine and pycnogenol, both rich in proanthocyanidins, but not white wine, improved vasodilation and simultaneously increased endothelial NO production.?7! Characterization of proanthocyanidin fractions isolated from red wine showed that vasodilation activity was greatest in the presence of low-molecular-weight oligomers (Dp2-3), whereas higher polymers were inactive.©7! Examination of the mechanism of increased NO production with rat aorta ring strips and G. biloba extract .

suggested inhibition of Ca** influx through Ca?* chanImpairment of vasodilation is characteristic of atherosclerosis and related circulatory diseases.47! Endothelium-dependent vasodilation is mediated through the

nels, thereby activating NO release.&/! Vasoconstriction

release of NO by endothelial cells. The enzyme, nitric

oxide synthase (NOS), uses L-arginine and oxygen as substrates to produce NO, which diffuses to smooth muscle cells to stimulate vasorelaxation. Two noninvasive in situ systems have been developed to test the efficacy of various dietary components, drugs, and environmental conditions on vasodilation. A study showed that patients with coronary artery disease had improved flow-mediated vasodilation of the brachial artery (FMD) when purple grape juice was consumed compared to beverages that did not contain proanthocyanidins.©7) A similar experiment with subjects who had at least one cardiovascular risk factor and who consumed a high-proanthocyanidin cocoa

Angiotensin II is a vasoconstrictor that is produced in the pulmonary capillaries by angiotensin converting enzyme (ACE) and can be involved in the development of hypertension and atherosclerosis.°7! Several proanthocyanidins and preparations containing them inhibited ACE activity in both in vitro and in vivo experiments. These included pycnogenol, proanthocyanidins isolated: from red grapes, extracts of Erythroxylum laurifolium (endemic species on Reunion Island in the Indian Ocean), and fruits of Cupressus sempervirens L. (Italian cypress). These observations suggest another role for proanthocyanidins in the amelioration of adverse vascular phenomena.

Proanthocyanidins

561

Reperfusion Induced ischemia-reperfusion studies in hearts isolated from laboratory animals simulate myocardial infarction and recovery in human beings. This model permits investigation of various dietary interventions, and other

environmental and circulatory alterations on the recovery of hearts postischemia. Hearts from GSPE-, red wine-,

or

red wine

proanthocyanidin-fed

rats

were

more resistant to ischemia—reperfusion injury than those from control animals.°*! Blood flow parameters were

improved,

whereas

infarct

size,

formation

of

hydroxyl radicals, and malondialdehyde levels of heart perfusate were all modulated as a result of feeding animals proanthocyanidins or proanthocyanidincontaining ingredients. These same dietary treatments also reduced the levels of proapoptotic factors JNK and c-Jun, as well as the proportion of apoptotic cardiomyocytes.

Other Metabolic Alterations Bacterial antiadhesion

Anecdotal observations and recent critical evaluation of scientific literature indicate that consumption of cranberry or its products is effective in the prevention of urinary tract infections.°%°?! Although the therapeutic effect was long thought to be increased urinary acidity due to hippuric acid excretion, it is now attributed to a family of unique proanthocyanidins and/or their metabolites,!°!! which have been charac-

terized as containing a high proportion of A-type linkages.'©7! In the case of urinary tract infection, the primary effect is inhibition of cellular adherence of P-type (mannose-resistant) uropathogenic strains of Escherichia coli! In addition, evidence has been presented for similar responses with Helicobacter pylori to gastric epithelial cells'4! and a host of organ!°! isms commonly found in the oral cavity.

Diabetes, and glucose and insulin metabolism Impaired glucose uptake and insulin resistance are subtle but common metabolic alterations that may be general etiologies to several age-related disorders and chronic

diseases.'*!

Thus,

identification

of dietary

components and natural products that have the potential to maintain these metabolisms throughout life has a highly favorable risk/benefit ratio. Several foods, botanical materials, and synthetic preparations, such as tea, several spices, and GSPE, have been found to

be effective.®***! Relative to proanthocyanidins,

an

extract of cinnamon, which contained a series of two

trimers and a tetramer of flavan-3-ols, each with an

A-type linkage,! was effective in significantly reducing fasting blood glucose in a group of Type 2 diabetics.*! Although elucidation of mechanism of action suggested that cinnamon extracts altered the activity of several phosphorylation /phosphatase reactions of insulin receptors in isolated rat epididymal adipocytes, !©?'”"! it is uncertain as to which proanthocyanidins were absorbed and the precise mechanism in vivo. Cataract formation and occurrence of retinopathy are two complications of Type 2 diabetes. Cocoaderived proanthocyanidins fed to diabetes-induced (streptozotocin-treated) rats almost completely inhibited cataract

formation.'”")

In control

animals,

lens

opacity was first detected at Sweeks after start of diabetes induction, and was prevalent in a majority of lenses at 10 weeks of treatment. Although lenses of proanthocyanidin-fed animals revealed some focal hyperplasia of the epithelium and liquefaction of cortical fibers at 10 weeks of dietary treatment, opacity of lens was undetectable. A review of five clinical trials involving nearly 1300 patients, in which pycnogenol was tested for treatment and prevention of retinopathy, unequivocally showed diminished progression of the disease and partial recovery of visual acuity in subjects ingesting the supplement.'”7! Pycnogenol treatment effectively improved capillary resistance, reduced blood leakages into the retina,

and

was

as efficacious

as the drug calcium

dobesilate. Immune function

Response of the immune system is one of the first lines of defense of the body to a host of environmental challenges. Many dietary components and drugs stimulate this system to an elevated level of preparedness. Besides those components of the immune system associated with atherosclerosis, the effect of proanthocyanidins

has

also

been

tested,

in vitro, in several

cell culture and isolated human cell systems. Unfortunately, these studies have employed isolated proanthocyanidin fractions or preparations for which confirmation of absorption has not been demonstrated. Thus, the applicability of the results to in vivo conditions is in question.

DIETARY SOURCES AND INTAKE Dietary Sources

Foods A wide variety of analytical procedures have been employed for the bulk measurement of proanthocyanidins.”*] Although individual dimers and trimers have

Proanthocyanidins

562

and herbals are less precise than those for foods, as these dietary components have not been subjected to the same rigorous sampling and analysis programs. In G. biloba, a fraction was isolated, which contained

been traditionally quantified with reversed-phase highperformance liquid chromatography (HPLC) systems, only recently have normal-phase HPLC techniques been coupled with sophisticated detection instrumentation for the quantification of individual proanthocyanidin oligomers (Dp < 12) as well as bulk measurement of higher molecular weight components.!’*-74 Employing these normal-phase HPLC procedures, a large number of food samples, selected on the basis of market share and demographics within the United States, were analyzed for proanthocyanidin content.!77] These data and others have been combined into a database of values for foods available online from the USDA Nutrient Data Laboratory (http://www. nal.usda.gov/fnic/foodcomp). Data for the proanthocyanidin content of selected foods are tabulated in Table 1. The information for red grapes reported in this table are for seedless “‘eating’’ grapes, whereas cultivars of red wine grapes and their wines

monomeric

have higher proanthocyanidin contents.!”*”! This is reflected in the data for several red wines common in Spain, which contained dimers through polymers Dp13 and represented 77-84% of total flavanols.!”) In general, a large number of vegetables, many spices, and some fruits, particularly citrus, had undetectable levels

of proanthocyanidins.'”” Fifty-six different kinds of common Spanish foods have been analyzed for flavanols, including dimers and trimers, but not higher oligomers.®°! Results indicated that procyanidin B2 was the most abundant dimer or trimer, and flavanols were at very low levels or undetected in most vegetables.

Supplements Qualitative and quantitative data on the proanthocyanidin content of dietary supplements, botanicals,

Table 1

and polymeric flavonoids, and accounted

for 24-36% of the mass depending on the method of extraction.!*"] This fraction contains many flavonol glycosides, biflavones, and proanthocyanidins.**! Grape seed proanthocyanidins are unique in that they contain a high level of galloyl derivatives, about one per each monomeric unit.!*°! Estimates of masses of these components range from monomers through Dp17-28 depending on the analytical method employed.'****! Relative to quantification, one report indicated that monomers (catechin and epicatechin) constituted about 15% of the mass, galloylated monomers, dimers, trimers, and tetramers approximately 80%, and pentamers, hexamers, heptamers, and their galloylates around 5% 87) Pycnogenol has been characterized as having proanthocyanidins that range from monomers of catechin and taxifolin to oligomers or higher.®*! Witch hazel (extract of bark of H. virginiana) contains about 5% proanthocyanidins, characterized by polymers of Dpl7—29 as measured by thiolysis and Dp11-20 when gel permeation chromatography was employed.'**! A unique characteristic of these compounds was complete 3-O-galloylation of chain extension units. Extracts of loquat (E. japonitca) leaves contained procyanidin B2 as one active ingredient,7" whereas Crataegus sinaica isolates were characterized as having a series of dimers through pentamers, some of which had A-type interflavan linkages.?° Preparations from Ecdysanthera utilis contained two procyanidin trimers that each had an A-type linkage between monomeric unit T (top) and M (interior)

Proanthocyanidin content of selected foods (mg/100 g food)

Food/spice Apples, red delicious, with peel Blueberries

Chocolate, baking Chocolate, milk Cinnamon, ground Cranberries

Grape seed (dry) Grapes, red

Pecans

Dimers*

Dp3-10*

Dp > 10°

Total

Type”

14

64

38

116

By EC

7

40

129

176

B, PC

207

680

Soll

1438

B, PC

26

105

33

164

B, PC

256

5319

2509

8084

Ao BPC

26

152

234

412

A, B, PC

417

1354

1100

2871

B, PC

2

19

59

80

B, PC

223

476

42

2A

aee

B, PC, PD

Plums, black 16 100 115 231 A, B, PC i 6 nnn a Ee eee “Dimers—Dp2; Dp3-10—trimers through decamers summed; Dp > 10 indicates values for polymers larger than decamers which eluted as a single chromatographic peak. Linkage type (A, B) and proanthocyanidin subclasses (PC—procyanidin, PD—prodelphinidin, PP—propelargonidin) identified.

(Adapted from Ref.77!,)

Proanthocyanidins

563

as well as several common

A2, B2),P4

procyanidin dimers (A1,

Dietary Intake Foods Based on proanthocyanidin content for over 60 U.S. foods and daily food intake data [USDA Continuing Survey of Food Intakes by Individuals (CSFII) for 1994-1996], consumption by individuals in the United States has been calculated.!’) The mean intake for all ages (>2yr old) was estimated at 54mg/day/person for all proanthocyanidins. of Dp > 2. Proanthocyanidin consumption among adults ranged from 46mg/day (20-39 yr, female) to 66mg/day (>60yr, male). As outlined above, these data do not include proanthocyanidins that might be included in the consumption of red wines or other commonly consumed foods that have substantial polymer content but

were

not

analyzed.

Nonetheless,

these

results

provide the scientific community with the first estimates of proanthocyanidin consumption. Supplements Because of the dearth of analytical data for the proanthocyanidin content of supplements, botanicals, and herbals, data on levels of intakes from these diet-

ary sources are not available. A recent survey of over 23,000

women

in the

United

States

indicated

that

nearly one-sixth took at least one herbal supplement (including G. biloba) in the year 2000.7! Based on this study, those women characterized as more likely to consume herbal supplements were non-Hispanic white, were more educated, had higher income, had residence in South and West, and had functional limitations and

chronic conditions. Plans and approaches have been discussed for future inclusion of nutrients and healthrelated components provided by all supplements into USS. National Nutrition Surveys.?*!

pycnogenol has been shown to bind selected purified intracellular enzymes,”®! the precise role of these polymers in the alteration of the metabolic equilibrium in the GI tract of human beings is unknown. Toxicological studies on long-term (90 days) oral administration of GSPE to rats established a no-observed-adverseof 1.4g/kg body weight effect level (NOAEL) (BW)/day for males and 1.5g/kg BW/day for females.” Similarly, the LDso of a single oral dose of grape seed extract IH636 was greater than 5g/kg BW for both male and female rats.*! Feeding [H636

at the rate of 100mg/kg/day to male B6C3F1 mice for a year or 500 mg/kg/day to female mice for 6mo had no detectable adverse effects on the pathologies of vital organs or on serum chemistries. In terms of dermal

irritation,

IH636

was

rated

irritating and the no-observed-effect for systemic toxicity was set at 2g/kg female albino rats.?*! Observations in human subjects consuming FastOne, a

as

moderately

level (NOEL) for male and both rats and herbal supple-

ment containing extracts of kola nut, grape, green tea, and G. biloba, suggested an increased risk of colorectal cancers as substantiated by the induced activity of cytochrome P450 1A2.%! In a review of potential drug—dietary supplement interactions, about one-haif of patients taking prescription medication and at least one dietary supplement had potential for an “interaction of significance’, Of these patients, only 6% had the potential of a severe interaction. Spontaneous bleeding in the anterior chamber of the eye (hyphema) has been reported, which was resolved when consumption of G. biloba was stopped.''°'! Review of several clinical trials that investigated pycnogenol indicated that tolerance of the supplement was very good, with only rare side effects, most referring to gastric discomfort.!”! Similar observations were reported for Enzogenol, a combination of an extract of Pinus radiata bark and vitamin C07]

COMPENDIAL/REGULATORY

STATUS

Not applicable.

ADVERSE

BIOLOGICAL EFFECTS REFERENCES

Traditionally, condensed tannins (proanthocyanidins) have been considered antinutrients in animal nutrition due to their astringency (reduced feed intake) and ability to bind several macronutrients, thus reducing their digestion and absorption.©*! However, specific digestive advantages, e.g., reduced incidence of bloating in ruminants, have been realized for some of the unique chemical traits of proanthocyanidins (protein binding), which are now being pursued.?*">! Although

1.

2.

3.

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Pygeum africanum Extract Francois G. Brackman Alan Edgar Fournier Pharma, Garches, France

INTRODUCTION Pygeum africanum (also called Prunus africana) is a tree belonging to the Rosaceae family. It grows in tropi-

(opposite quarters) that does not compromise the tree’s survival. Reforestation and forest enrichment could contribute to conservation and sustainability.

cal and humid equatorial mountain zones, at altitudes

between 1000 and 2400 m. The tree is commonly found in countries such as Cameroon, Kenya, Madagascar, Congolese Democratic Republic, Equatorial Guinea, Uganda, Tanzania, Angola, South Africa, Ethiopia, Burundi, Rwanda, Malawi, and Nigeria.

The origin of the use of P. africanum bark is documented back to at least the early 19th century. The ground bark was used in a water, tea, or milk mixture as a drug, the use being triggered by its flavoring effects (hydrocyanic acid). The bark was used by the Zulus, who had observed beneficial effects on urinary symptoms. Other tribes from Africa and Madagascar used it for the relief of symptoms such as gastric pain, urinary disorders, and also for aphrodisiac properties. Such uses are, however, poorly documented,

and are

based on extracts whose contents and properties might differ both between modes of extraction and from the pharmaceutical standardized extract. The standardized P. africanum extract is used to alleviate lower urinary tract symptoms (LUTS) including those accompanying benign prostatic hyperplasia (BPH). Recent pharmacological and clinical studies have demonstrated that P. africanum extract quantitatively and qualitatively improves bladder as well as prostaterelated parameters and symptoms causing micturitional disorders. P. africana is featured in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) II list of endangered species, imposing strict regulations on its harvest and trade. The exploitation of the bark is done within the frame of durable development. Only adult trees, with a diameter of at least 30cm, undergo partial bark harvest

Dr. Francois G. Brackman, M.D., is Vice President, Department of Global Development and Medical Affairs at Fournier Pharma,

p Garches, France. Alan Edgar, Ph.D., is Head of Pharmacology Department of Drug Discovery at Fournier Pharma, France.

Studies, Garches,

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022124

Copyright © 2005 by Marcel Dekker. All rights reserved.

PHARMACEUTICAL

DESCRIPTION

Since the origin of the P. africanum bark and the extraction and standardization processes play a role in the final quality and content of the extract, results obtained with a given preparation might not necessarily apply to others. Most investigations performed and published were done using a standardized extract, which allows easier comparison of results. This extract is obtained by a process of maceration and lixiviation of the P. africanum bark in organic solvent. The solvent is eliminated, and the extract is

purified. The extract has a soft-to-hard consistency, dark brown color, and very strong aromatic odor. It is freely soluble in chloroform and is insoluble in water. The P. africanum bark extract features numerous constituents, including saturated and unsaturated fatty acids (C12—C22), phytosterols (B-sitosterol, B-sitosteryl glucoside, and f-sitosterone), pentacyclic triterpenoids (ursolic acid, 2a-hydroxyursolic acid, and oleanolic acid), alcohols (n-tetracosanol and _ n-docosanol), carbohydrates (triacontane and nonacosane). The pharmaceutical properties are documented in the European Pharmacopoeia (Monograph no. 07/2002: 1986). The American Pharmacopoeia mentions P. africanum bark, extract, and capsules in its Projects Pharmacopeum Forum 29 (4) July-August 2003.

CLINICAL STUDIES Benign Prostatic Hyperplasia Benign prostatic hyperplasia is a very common finding in aging men. Its prevalence above the age of 50 varies from 50% to 75% in most cases. Transurethral and open surgical adenomectomy are the most widely used treatments for BPH patients with severe symptoms. However, because of the clinically significant incidence of complications

such

as

blood. loss, urinary

tract 569

Pygeum africanum Extract

570

infections, urethral stenosis, incontinence, impotence, and the need for reintervention after prostatic surgery,!!] the management of BPH and LUTS has been rapidly changing. In addition, a large proportion of patients with LUTS do not have prostate enlargement and do not need surgical intervention. Medical approaches used in the treatment of BPH include «-adrenoceptor-blockers, 5-o-reductase inhibitors, and plant extracts. a-Blockers partially alleviate symptoms of BPH [2] by reducing the o-adrenergic tone of the smooth muscle. However, they may have significant cardiovascular side effects that may limit their therapeutic application. 5-a-Reductase inhibitors result from an elegant series of medicinal chemistry studies, and through a reduction in prostate size, are supposed to act on symptoms by a direct effect on the mechanical component of obstruction. However there is certain delay in the onset of the improvement (6-12 mo) and an important incidence of side effects, in particular, on sexual function.! The limitations of 5-c-reductase inhibitors and a-blockers in treating bladder outlet obstruction (BOO) might be related to the importance of the functional and structural remodeling of the bladder that also develops as the disease progresses. Benign prostatic hyperplasia is largely considered to be pathologically a disease of the prostate, while being symptomatically a disease of the bladder. While the analogy of the enlarged prostate impinging upon bladder function still holds true in general, numerous individuals see no improvement in urinary function in spite of objective reduction in prostate size. Still others see clear improvement in LUTS independent of any discernable change in prostate size.) Modifications in the prostate do not necessarily evolve in parallel with bladder remodeling. The efficacy of the various treatment options depends, in part, on the judicious use of the appropriate treatment corresponding to the stage of advancement of the disease. Treatment directed at improving bladder function may be more efficacious in older individuals, while that designed to solely affect the prostate may be more beneficial in relatively younger patients. Benign prostatic hyperplasia results from progressive enlargement of the transition zone of the prostate and involves both glandular and stromal prostate tissue. Stromal elements contain smooth muscle, and contraction is mediated by a-1l-adrenergic receptors. Hyperplasia of the transition zone is responsible for the organ enlargement. Prostate enlargement also mechanically and physically affects bladder dysfunction by a progressive denervation via damage to intramural nerves and synapses as seen in animal models and in obstructive dysfunction in men.” The involvement of specific bladder components indicates that effective treatment should not

solely target the prostate, but must also be directed at the bladder. Still, the origin of the modification of bladder function in BPH remains the prostate, as

witnessed by the paucity of symptoms in castrated individuals.®! More than 2000 patients were enrolled in clinical trials with P. africanum extract.”! These studies were conducted either in a double-blind placebo-controlled manner"?! or as open-labeled studies.!13] There are 18 published randomized controlled trials comparing preparations of P. africanum with placebo or medical therapy for >30 days in men with symptomatic BPH. Thirteen trials included comparison of P. africanum extract with placebo.!'*'°! Most often, the treatment regimen was 100 mg/day (50mg twice a day) for 1-2 mo. The persistence of the effect and the long-term safety profile over more than 5 yr have been investigated in an observational study.4 The effects observed in those studies were convergent with:

e e

e e e

e

e

More than 67% of patients reported “‘excellent,”’ “very good,’’ or “good’’ results. Mean maximum urinary flow rate was improved in all studies in which it was measured. Nocturia was improved in 50-100% of the patients in whom it was measured. Daytime frequency was improved in 50-95% of patients in whom it was measured. Hesitancy, urgency, weak stream, and dysuria were improved in the majority of studies. Quality of life scores were improved. Standardized assessment scores such as International Prostate Symptoms Score (IPSS) confirmed these results. Both clinical and urodynamic improvements were maintained during long-term treatment in a high proportion of patients. Both clinical and urodynamic improvements were maintained after 12 mo of treatment.

The most frequently used dosage of P. africanum extract is 50 mg b.i.d. One prospective clinical trial!!?!7] demonstrated the equivalence of effects of the extract 100 mg/day given either as 50mg twice daily, or as a single daily dose of 100mg in men with moderate-tosevere urinary symptoms associated with BPH. Both daily dosage modalities of P. africanum extract (100mg/day and 50mg twice daily) decreased the total IPSS according to an equivalent pattern and to a similar extent (41% and 37.5%, respectively). Mean maximum flow rate increased after 2mo of treatment by 16% in the 50mg bid. group and by 18.5% in the 100mg q.d. group. Similar effects were observed on all components of the condition and on quality of life scores.

Pygeum africanum Extract

571

MECHANISM OF ACTION Effects on In Vitro and In Vivo Growth Factor-Mediated Prostate Growth

Normal prostate growth is controlled by an orchestration of growth factor-mediated autocrine and paracrine communication acting on epithelial and stromal prostatic tissue. Mutually distinct biochemical and suborganelle perturbations within the prostate can pre-

dispose toward BPH or prostatic carcinoma.!'*! The

BPH affects predominantly stromal and glandular cell growth, where basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are major regulators along with insulinlike growth factor (IGF). Keratinocyte growth factor (KGF) is expressed in epithelial tissue and has a paracrine effect on stromal cells, whereas transforming growth factor-B1 (TGF-f1) has been shown to inhibit prostatic fibroblast proliferation. Finally, testosterone via dihydrotestosterone (DHT) has a causal role in controlling prostate growth and affects the expression of the aforementioned growth factors and their receptors indirectly. In advanced BPH, the role of androgens may be no more than permissive, while in prostatic carcinoma, androgen receptor signaling is not strictly a function of DHT levels.

between the in vitro ICsy value with a Cy,ax value for the same metabolite following oral administration. Therefore, in an attempt to confront this problem,

subsequent in vivo studies!**! focusing on the rat

ventral prostate, which

is approximately

equivalent

to the transition zone of the human prostate, showed

that P. africanum extract affected adenyl cyclasemediated cross talk in cell signaling pathways also at _ the level of protein kinase C. Finally, ventral prostate hyperplasia induced by DHT treatment in vivo in rats was reversed by oral administration of the extract, while the latter had no effect on dorsal prostate enlargement.) The molecular mechanism(s) underlying the in vitro and the in vivo effects of P. africanum extract are similar. The extract exhibited an effect on in vitro fibroblast proliferation of both rodent as well as human cells, which

correlated

with

a growth

factor profile

signature corresponding to stromal cell proliferation, and this was paralleled by a preferential in vivo effect of P. africanum extract on ventral (stromal) prostate hyperplasia in rodents.

Effects on Bladder Function

Furthermore, in men 50 yr old or more, the role of testo-

sterone relative to estrogen appears to diminish."”! Following an initial observation of an effect of P. africanum extract on bFGF- and EGF-mediated proliferation of 3T3 fibroblasts in vitro,?"! a detailed

series of experiments were performed comparing the potential of the extract to affect prostatic stromal cell proliferation mediated by various prostate-derived growth factors in rats.?!! At concentrations devoid of cytotoxicity, P. africanum extract was shown in vitro to affect stromal fibroblast cell proliferation induced by EGF, bFGF, and IGF-I as well as with protein kinase C activators,

but not by KGF.

Furthermore,

the inhibition of cell proliferation was observed with ICs) values in the range of 10 ug/ml. The similarity of the individual ICs) values of P. africanum extract on the various growth factors suggests that a locus common to the three growth factors was being affected.?7] Furthermore,

the in vitro studies demon-

strated that stromal fibroblast proliferation was inhibited via a site that converged near the level of protein kinase C. The results were not limited to rodent prostatic cells, as P. africanum extract was also shown to inhibit proliferation of fibroblasts from human hyperplastic

prostate and bladder as well.!?*! The precise molecular component(s) of P. africanum extract responsible for the in vitro effects is not known. Therefore, these results cannot infer in vivo activity per se, as

it is not

possible

to establish

a correlation

In man

as well as in select animal

models,

bladder

obstruction induced by enlargement of the prostate or partial outlet obstruction of the urethra leads to a progressive increase in urethral resistance. The latter is initially compensated by an increase in bladder wall thickness,

increased

pressure

generation,

and altera-

tions in flow parameters that limit the immediate deterioration in bladder function. This phase of compensated bladder function is slowly, though inexorably, followed by a decompensated bladder with a marked loss of contractility. The initial phase displays impaired detrusor smooth muscle function characterized by neuronal degeneration leading to reduced postsynaptic innervation, mitochondrial dysfunction, and loss of intracellular Ca7* homeostasis that collectively compromise myosin contractility. The preceding section described in vitro data corroborated by in vivo results that demonstrated an effect of P. africanum extract on molecular aspects of prostate growth mediated by prostate-derived growth factors. Growth factor-mediated fibroblast proliferation is common to both the hyperplastic prostate as well as the hypertrophic bladder secondary to BOO?®? similarly involving bFGF, EGF, and TGF-f. This common link was the rationale for investigating the effect of P. africanum extract in various models of impaired bladder function.'*! Indeed, initial results showed that the extract inhibited bFGF-stimulated fibroblast proliferation originating from

Pygeum africanum Extract

572

obstructed bladder in vitro?*! and modestly reduced bladder weight in vivo in a preliminary study that was further confirmed in larger subsequent studies. Pretreatment of rabbits with P. africanum extract prior to partial outlet obstruction dose- and timedependently reduced contractile dysfunction determined by measuring the contractile responses to field stimulation,

bethanechol

and

KCl.

At the dose

of

100 mg/kg, it improved the response to field stimulation by 50% and to KCl by 70%, and completely when to bethanechol the response normalized subsequently determined 3 weeks following partial outlet obstruction. Of the three parameters measured,

partial outlet obstruction affects to a greater extent field stimulation,

reflecting marked

deterioration

of

synaptic function. Therefore, the initial experiment was complemented by a series of investigations that first determined the time course of the effects of P. africanum extract and correlated this to specific perturbations in synaptic and postsynaptic membranes coupled to alterations in key mitochondrial enzymes and calcium homeostasis. The results of the time course study indicated that P. africanum extract was able to normalize the response to field stimulation after 2weeks of treatment. These results were superior to those obtained after 3weeks and may indicate a reduced efficacy of P. africanum extract after prolonged obstruction or an unremedial deterioration of synaptic function over time in this model. In an attempt to mimic the clinical situation, wherein bladder function is already compromised

Contractile dysfunctions of the obstructed bladder are directly related to ischemia (reduced blood flow) and detrusor hypoxia.? ! Thus, short-term ischemia is

a relevant model that recapitulates pathological aspects of contractile dysfunction inherent in partial outlet obstruction originating surgically or via prostatic In this model, unilateral ischemia enlargement. and irrevocable ischemic insult to one direct provokes while partially compromising the bladder, side of the t with what was observed agreemen In mic side. nonische on, P. africanum obstructi outlet partial following nonischemic side of the protected ment pretreat extract ent of contractile dysdevelopm the from the bladder further correlated was e effect protectiv This function. to an enhanced expression of Hsp70 and c-myc. The clinical pharmacological relevance of these animal

data

was

established

by Valentini

et al.,22)

who demonstrated in a blinded clinical study that P. africanum extract improved detrusor contractile function after 2 mo of treatment. ; The contractile dysfunctions induced by partial outlet obstruction in animal models, and by BPHinduced obstructive dysfunction in men, are secondary to

denervation,

mitochondrial

dysfunction,

and

calcium-storage dysfunction, which in turn is mediated partially by ischemia-generated free radicals and calcium-activated hydrolytic enzymes. One hypothesis is that P. africanum extract acts in part by protecting neuronal and subcellular membranes from ischemiainduced damage, and by this means protects the contractile function of the bladder.!7**)

that

Testosterone, in addition to its well-known action on

the efficacy of P. africanum extract was maintained when administered only after the application of partial outlet obstruction. Specifically, the effects of P. africanum extract in restoring the contractile

stimulating prostate growth, also affects the bladder, and in the rat, administration of DHT significantly affects urodynamic parameters including frequency and volume. In DHT-stimulated rats, P. africanum

response to field stimulation, carbachol and KCI were

extract,

qualitatively identical when administered before or after partial outlet obstruction.?*77! Contractile dysfunction ultimately results in reduced force generation and alterations in myosin isoforms. In parallel with the improved contractile dysfunction, P. africanum extract was able to partially normalize the expression of myosin isoforms in line with improved contractility. These studies were based on the observation that alternative post-transcriptional splicing of myosin mRNA generates two isoforms of myosin, SM-A and SM-B, with lower and greater actin-activated adenosine triphosphate (ATP) hydrolysis, respectively, and hence force generation. Following obstruction, detrusor smooth muscle SM-A myosin isoform expression increases threefold corresponding to reduced force generation, while treatment

effect on ventral prostate growth, also significantly improves bladder frequency and volume.&4) . Collectively, these results clearly demonstrate that P. africanum extract directly affects bladder function. Among the limitations of the animal models employed is the lack of a concerted pathophysiology strictly representative of human BPH. The anatomic | separation of prostate and bladder function in these models is, nonetheless, an advantage when attempting to demonstrate independent effects of P. africanum extract on the two organs. Furthermore, animal studies have the obvious advantage of being devoid of a placebo effect that.complicates the design and interpretation of clinical trials in this indication. Treatment of LUTS is complicated by the multifactorial and multiorganelle origin, the slow evolution of the disease process, as well as the high placebo response in this patient population, which collectively

when

treatment

is initiated, it was

with P. africanum

then shown

extract normalizes

the SM-B/

SM-A ratio in parallel with the improvement in field stimulation.b°

in addition

to the aforementioned

selective

Pygeum africanum Extract

limit the perceived efficacy of monotherapy in short-term clinical trials. P. africanum extract has demonstrated a reproducible efficacy in a variety of pharmacological studies addressing key aspects of lower urinary tract pathophysiology, thus altering the often encountered perception of plant extracts as poorly defined mixtures acting in an ambiguous manner to that of a reproducible molecular effector. The precise molecular component(s) responsible for the effects of P. africanum extract have not been identified. A limitation of the animal data described is the use of short-term treatment periods and the rapid instauration of the pathology to investigate what in man is a chronic disease. The treatment duration of most clinical trials has also been limited, and the patient population not always ideally chosen to demonstrate beneficial effects on the bladder. The optimal patient population for showing the efficacy of an a-blocker, a 5-a-reductase inhibitor and P. africanum extract could vary, reflecting different stages of the disease process and related symptoms. The sum of the in vitro and in vivo pharmacological studies delimits a pharmacological mechanism of action of P. africanum extract affecting independently: e

Prostate hyperplasia via a downstream target common to bFGF, EGF, as well as androgen-mediated

e

cell proliferation at or near the level of protein kinase C. The signature of growth factor-mediated inhibition by P. africanum extract on in vitro and in vivo prostatic cell proliferation suggests that in vitro results are predictive and correlated to in vivo activity. Bladder function with improvement in contractile dysfunction mediated via myosin isoform expression, lessened synaptic denervation, and improved mitochondrial function.

ADVERSE EFFECTS Routine preclinical safety trials performed in various animal species by oral and parenteral routes, with single and repeated administrations, studied P. africanum at doses greater than 50 times the therapeutic doses. In such studies, no target organ could be identified as to potential toxic effects. P. africanum extract is devoid of any mutagenic potential. Most published open-label and placebo-controlled studies mentioned. a good tolerance of the extract. Of particular note is the absence of any hormonerelated adverse effects, confirming that the extract does not exert any hormonal effect. No interactions with concomitant medications such as antihypertensive

573

agents, lipid lowering agents, or antiarrhythmics were reported. No significant changes were observed in biochemical safety parameters in those published studies

where this is documented.!°!713] In a recently published review, 13 of 18 randomized

controlled trial (RCT) studies provided information on specific adverse events. Side effects due to P. africanum were generally mild in nature and similar in frequency to placebo. The most frequently reported were gastrointestinal and occurred among seven men in five trials."! In the most recently completed study,'!7! the type and overall frequency of adverse effects had similar distributions between dosage modalities during the comparative phase and were comparable for both phases of the trial. Light-to-moderate

gastrointestinal effects, such as

nausea, constipation, or dyspepsia, which are known as treatment specific, were reported most frequently (5.4% and 9.8% of patients who participated in the comparative and the extension phase, respectively). The majority of the serious adverse effects regarded the urogenital system (1.3% and 3.5% of patients who participated in the comparative and the extension phase, respectively) and appeared more related to the natural evolution of the disease than to the medication itself. It has been observed that very few severe emergent adverse effects (SEAE) appeared during the study, and the risk of presenting an SEAE during the longterm follow up was very low and constant in time. The rate of the SEAE-free patients at l yr was 90% with the 95% CI [85%, 95%]. Similar observations were made for the treatment-related emergent adverse events (TREAE), with the rate of the TREAE-free patients at lyr equal to 92% [95% CI (88%, 96%)]. Few side effects were responsible for patients’ withdrawal from the study (15 patients during the comparative phase and eight patients during the long-term phase). No significant changes were noted in blood or urine analyses in either group or during the two phases of the study. There was no statistically or clinically significant variation of the prostate-specific antigen (PSA) level at 12mo compared to the baseline value. There is no report of any unwanted effect on sexual function with P. africanum. The cardiovascular effects were studied after a trial of 12 mo of treatment with P. a fricanum 7} The treatment was not associated with any unwanted cardio vascular effects in this study.

PRODUCTS AND DOSAGE P. africanum extracts are available worldwide under various formulations. One of them, P. africanum extract V1326, is the most frequent presentation

Pygeum africanum Extract

574

(50 countries) and is marketed under the Tadenan™ trade name, as a prescription drug. Other preparations are available in various countries, containing P. africanum extract either as a single component (such as Pronitol™, Bidrolar™, Foudaril™, Kunzle™, Neo Urgenin™, Normobrost™, Normoprost™, Prolitrol™,

Provol™,

etc.) or in combination

10.

Mazeman, E.; Mermon, R.; Reichelt, H.; Schonmetzler, F.; Suhler, A. Efficacy of Pygeum africanum extract for the treatment of micturi-

with other com-

tional disorders due to BPH: evaluation of objective and subjective parameters. A multicentre, placebo-controlled double blind clinical trial.

ponents such as vitamins or minerals (such as Pro ™ to Flow™, Potenzia™, Super Prostate Formula”, name a few).

De

Mebust,

W.K.;

Holtgrewe,

H.L.; Cockett, A.T.;

Peters, P.C. Transurethral prostatectomy: immediate and post operative complications. A cooperative study of 13 participating institutions evaluating 3885 patients. J. Urol. 1989, /41, 243-247. Lowe, F. Alpha-l-adrenoceptor blockade in the treatment of benign prostatic hyperplasia. Prostate Cancer Prostatic Dis. 1999, 2 (3), 110-119.

12:

Chatelain, C.; Autet, W.; Brackman, F. Therapeu-

133

tic equivalence of efficacy and safety of once and twice daily dosage forms of Pygeum africanum extract in patients with symptomatic benign prostatic hyperplasia. A prospective, randomised, double-blind study. Urology 1999, 54 (3), 473-478. Breza, J.; Dziurny, O.; Borowka, A.; Hanus, T.; Petrik,

Kato,

K.; Wein,

A.J.

Effect

of bladder

Levin,

R.W.;

Brading,

A.F.;

Mills,

14.

15:

6g

obstruction. In Prostatic Disease; Lepor, H., Ed.;

W.B. Saunders Co.: Philadelphia, 2000; 169-196.

as

Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol. Urodyn. 2000, 79 (5), 609-629. Levine, A.C.; Kirschenbaum, A.; Gabrilove, J.L.

Chadha-Boreham,

H.

Wilt, T.; Ishani, A.; Mac Donald, R.; Rutks, L;

UK, 2004; Issue 1. Ishani, A.; Mac Donald, R.; Nelson, D.; Rutks, L.;

meta-analysis. Am. J. Med. 2000, 109, 654-664. _ Moya-Prats, P.P.; Salva Verd, A ORR

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- genistein), 5-a-reductase type 2 (genistein, daidzein, and biochanin A), and

17-B-hydroxysteroid dehydrogenase (coumestrol, biochanin A, and genistein). Daidzein sulfates inhibit the

sulfatase and sulfotransferase enzymes that: 1) regulate sulfation of endogenous and environmental estrogens; 2) help determine the availability of hormones to tumors; and 3) have been hypothesized to minimize bioactivation of xenobiotic procarcinogens. All four isoflavones—biochanin A being the most potent— expedite the elimination of inactive hormone conjugates through stimulation of UDP-glucuronsyltransferase. Genistein stimulates the production of sex hormone binding globulin (SHBG) in human hepatocarcinoma cells. Equol producers exhibit enhanced urinary ratios of 2-hydroxyestrone to 16a-hydroxyestrone, a metabolic measure that is negatively correlated with breast cancer risk.

notrophic outcomes in the ovariectomized rat model,

but it did not stimulate breast cell proliferation. In a rat model of endometrial cancer, genistein upregulated estrogen-responsive genes but did not promote tumor growth. Consistent with ER-f selectivity, isoflavone preparations improve bone density and protect against cardiovascular disease in most ovariectomized rat and prepubertal rhesus monkey models, although bone results may vary with concentration, timing, and length of exposure. Isoflavone concentrations in humans routinely exceed endogenous estradiol concentrations. Yet RC is not overtly estrogenizing, suggesting that alternate molecular targets must mediate the clinical actions of the isoflavones.

Cancer-Related

Effects

Antioxidant

Crude RC extracts display antioxidant activity in several experimental systems. Known antioxidant compounds in RC include genistein, daidzein (equol), biochanin

A, genistin, daidzin, formononetin,

clova-

mide, and texasin. In addition to exerting their influence through direct chemical interactions, isoflavones

lower oxidative stress via indirect mechanisms such as induction of antioxidant scavenging enzymes. In certain biological environments, genistein may act as

Red Clover (Trifolium pratense)

592

a pro-oxidant with potential genotoxic consequences.

mutagenic

and/or

Antiproliferative At pharmacological doses, isoflavones inhibit the proliferation of both hormone-dependent and hormone-

independent cancer cells. This phenomenon has been differentially attributed, in part or in whole, to a wide

range of ER-independent effects:!! the inhibition of enzymes such as tyrosine kinase (genistein, biochanin A) and DNA topoisomerases I and II (genistein, biochanin A, equol, and orobol); regulation of growth factors and their receptors (genistein); effects on cell cycle regulatory proteins (genistein and biochanin A); regulation of stress response genes (genistein); apoptosis (genistein); and inhibition of nitric oxide synthesis (biochanin A). Biochanin A is largely responsible for the inhibition by RC of benzo(a)pyrene metabolism in hamster embryo cell culture. The isoflavones generally exert a chemoprotective effect if administered to rodents before puberty, although genistein will stimulate or suppress tumor growth depending on the cancer model employed. Other

Tumor progression and metastasis may be checked by the following actions of genistein: inhibition of angiogenesis, cell adhesion effects; tyrosine phosphorylation of membrane proteins that mediate cellular invasion; and collagenases/metalloproteases.

and in vivo findings."! In vitro, genistein and daidzein competitively inhibit thyroid peroxidase (TPO) to form iodinated isoflavones and irreversibly inhibit the enzyme in the absence of iodide, albeit at IC59s above typical circulating levels of free isoflavones. Rat studies have confirmed the relevance of these in vitro findings. Dietary exposure of Sprague-Dawley rats to genistein that achieved serum concentrations comparable to

those seen in humans caused intrathyroidal accumulation of genistein and inactivation of TPO. Para; doxically, the rodents did not present as hypothyroid. Thyroxine, tri-iodothyronine (T3), and thyroid stimulating hormone levels remained unchanged; and thyroid sections revealed no pathologies. In the ovariectomized ewe model, however, exposure to RC silage resulted in elevated free and total T3 levels, increased

thyroid follicle size, reactivity to ER-a.

and

thyroid

gland

immunoj

Glucose and Lipid Metabolism Isoflavone supplements and RC extracts have been postulated to benefit obesity and diabetes mellitus, and in vitro and in vivo experiments provide some support for this contention. Genistein increases glucose-induced insulin release from either pancreatic B-cells or insulinoma cell lines. Also contributing to the antidiabetic effect, genistein and, to a lesser extent,

daidzein inhibit insulin-stimulated glucose uptake from the intestine and other cells, and isoflavones

protect

against glucose-induced oxidation of low-density lipoproteins (LDL). Anabolic effects of RC isoflavonoids have been observed in mice, rats, and cattle.

Inflammation and Immune

Function

Several lines of evidence suggest that the isoflavonoids in RC modulate inflammatory!’ and immune responses."'! In the ovariectomized mouse model, genistein

in the diet or by injection induces thymic atrophy and suppresses cell-mediated immunity. Injected genistein also decreases humoral immunity. In the hairless mouse model, genistein, equol, isoequol, and dihydroequol protect against UV-induced inflammation and immunosuppression. An aqueous extract of RC strongly inhibited (82% at 0.25mg/ml) platelet activating factor-induced exocytosis in human neutrophils. The isoflavones modulate anti-inflammatory responses in animal models of chronic ileitis, inflammation-induced

corneal injury.

neovasculation,

and

ischemic

reperfusion

Thyroid Function

Speculation that the isoflavones, and RC by extension, trigger thyroid disease has been based on both in vitro

At least three rabbit studies have demonstrated beneficial effects of isoflavones or RC"! on lipid metabolism and/or progression of atherosclerosis, although reduced LDL peroxidation rather than changes in serum lipids was hypothesized to account . for improvements in one study. Genistein exerts lipolytic/antilipogenic effects in ovariectomized mice.

Other Biological Activities of RC Compounds Genistein weakly antagonizes the Al, A2a, and A3 adenosine receptors at low micromolar concentrations. Genistein and daidzein inhibit GABA, receptormediated chloride currents. The two isoflavones, and certain structural analogues, also block calcium chan-

nels in human platelets, inhibiting thrombin-induced [Ca**], elevation. The isoflavone actions at ligandgated ion channels occur independent of tyrosine kinase inhibition at low- to midrange micromolar concentrations. The RC isoflavones serve as effective antimicrobials (genistein, daidzein, biochanin A, and formononetin) and fungicides (genistein, biochanin

Red Clover (Trifolium pratense)

593

A, and formononetin). See Table 2 for a noncomprehensive list of selected RC compounds with interesting biological activity.

USAGE Human

Clinical Studies

Caveats

Inferences about RC based on soy isoflavone studies may not be valid. Red clover and soy not only differ in their balance of individual isoflavones, but soy foods and SPIs also contain a unique protein fraction that is not present in RC semipurified isoflavone extracts. To date, no reports have been published regarding the clinical activity of RC protein. Research of dietary soy isoflavone effects on hormone status is complicated by the relative amounts of fiber and nonstarch carbohydrates in the diet, as these influence the gut flora populations responsible for metabolism of isoflavones

in the colon. Additionally, it is currently assumed that the isoflavones are the only “active’’ constituents present in RC extracts. This may or may not eventually be shown to be true, as 20 or more minor compounds may be present in the semipurified extracts used in clinical trials. Studies reviewed here are limited to clinical trials of RC isoflavone supplements and/or pure isoflavones, and the abovementioned caveats should be borne in mind when interpreting the results presented. Refer to Table 3 for details regarding specific RC isoflavone products.

Menopause

Studies administering semipurified RC preparations to women to relieve menopausal hot flashes have generally been of short duration (80 mg/day are considered to be higher than isoflavone exposure received by eating a diet containing soyfoods and isoflavone-containing legumes.

SAFETY, TOXICITY, AND ADVERSE

EFFECTS

Adulteration Issues Heavy metal contamination and pesticide residues

differs slightly from

specifications)

for 4weeks/dose

and also found no effect on plasma lipids.?7! A 3-mo study in peri- and postmenopausal women taking 2 tablets Promensil (24.5 mg biochanin A, 8 mg formononetin, 4mg genistein, 5 mg daidzein/tablet) or Rimostil (2mg biochanin A, 25mg formononetin, trace genistein + daidzein/tablet) had no effect on plasma lipids, but did decrease triglycerides in women with high baseline levels.'! Another study administered 28.5, 57, or 85.5 mg of RC isoflavones/day as Rimostil for 6mo. High-density lipoprotein for all treatment groups increased at least 15%. Apolipoprotein B declined in all groups by at least 9%. One-year treatment with 43.5mg RC isoflavones daily decreased triglycerides and plasminogen activator inhibitor type I (PAI-1) in perimenopausal but not in postmenopausal women.*! Differences between RC extract

Although RC does not have a particular tendency to | preferentially absorb heavy metals under normal conditions, when it is grown on contaminated soil, it . can accumulate high levels of Cd, Cu, Pb, As, and

Zn to varying amounts, depending on soil pH and metal solubility. It is recommended that source material and/or any resultant extracts be assayed for presence and level of Pb, As, Cd, and Hg, and the country of origin be required to provide quality control documentation. Imported products containing the ingredients realgar (arsenic) and cinnabar (mercury) should be avoided.!”?°"! The first supplement to the U.S. Pharmacopoeia/ National Formulary 2003 recommends a limit of not more than 10 ppm of heavy metals be present in RC products. The following pesticides have been designated by the United Nations as hazardous and are banned by some

Red Clover (Trifolium pratense)

599

countries, including the United States: aldrin/dieldrin, chlordane, dichloro diphenyl trichloroethane (DDT),

Inhibition of Cytochrome P450 (CYP450) Enzymes

heptachlor, lindane, malathion, and parathion. These

chemicals were used on RC fields in the United States during the 1950s and 1960s. Residues persist in the soil for extended periods of time and are still present in everyday foods at low but detectable levels. It is unknown whether proprietary extraction processes may concentrate these residues in the botanical extracts used to make dietary supplements. Some of these pesticides remain in use in other countries, and

are a potential contaminant of imported plant material and extracts. Limits (mg/kg) of 34 specific organophosphorus, organochlorine, and pyrethroid pesticides for RC are given in Table 3 under method section (561) of the U.S. Pharmacopoeia/ National Formulary

2004.84

In vitro experiments with human microsomes have shown that RC extracts exhibit selective inhibition of CYP2C9, marginal inhibition of CYP1A2 CYP3A4, and nominal inhibition of CYP2A6

and and

CYP2D6. Genistein and daidzein, as well as genistin and daidzin, inhibit CYP1A1 as measured by reduction of enzyme activity in a mouse hepatoma cell culture system. In other experiments, genistein and equol did not cause significant induction of xenobiotic-metabolizing enzymes in mouse (ethoxyresorufin O-deethylase, p-nitrophenol oxidase, glutathione S-transferase, CYP1A2, CYP2E1, or CYP3A1) or human hepatic cells (CYPIAI, glutathione S-transferase 4a, or xenobiotic response elements). There are no reports of clinically significant RC : drug interactions.

Botanical misidentification

Thyroid Function T. pratense shares the common name “sweet clover’ with the plants M. alba Medikus and M. officinalis (L.) Pall. This shared common name is unfortunate but physical misidentification is avoidable; the flowers of RC are pinkish-purple, while those of M. alba and M. officinalis are white and yellow, and can be easily distinguished from one another. See the “Botanical description’’ section for more botanical characteristics of M. officinalis.

Red clover products are used by menopausal women, a group that is prone to hypothyroidism and autoimmune thyroiditis and could be particularly susceptible to the antithyroid actions of the isoflavones. Individuals in this patient population on chronic RC regimens should be monitored for thyroid function. In terms of potential benefit, the San Francisco Bay Area Thyroid Cancer Study recently concluded that isoflavone intake is associated with reduced thyroid cancer risk in both pre- and postmenopausal women.?!

Presence of Coumarins

There are over 3400 naturally occurring coumarins present throughout at least 160 plant families. Many do not have anticoagulant effects in vivo; many more have unknown effects. RC has been reported to contain some coumarins, but it has never been evaluated for long-term anticoagulant effects, or herb—drug interactions with blood-thinning drugs such as warfarin. Usual clinical doses of RC extracts are such that exposure to any particular coumarin present would likely be below the threshold where any (hypothetical) clinical anticoagulant effects should manifest. Dicoumarol, a 4-hydroxycoumarin derivative that is known to inhibit blood coagulation, was isolated in 1941 from M. alba Medikus and/or M. officinalis

(L.) Pall.87! It is a fungal metabolite formed by Penicillium species growing in diseased M. alba and M. officinalis. Although Melilotus and Trifolium species are closely related, there are no reports of dicoumarol occurring in Trifolium species. Coumestrol, daphnoretin, fraxidin, xanthotoxol,

medicagol,

are present in trace amounts

((PLA>) can be developed as potential anti-inflammatory agents for the treatment of rheumatic arthritis, asthma, and psoriasis. Ganoderic acid T was found to inhibit secreted PLA, from pig pancreas, human synovial fluid, and bee venom, but no such effect was observed with ganoderic acids

MITOGENIC ACTIVITY

IMMUNOMODULATING

EFFECTS

the CsA-induced

effects

which might be due to the inhibition of the protein kinase C signal pathway and acceleration of the CsA signal pathway.

AA, O, R,S,T-OH, and T-OH-H,.!"4

The immunomodulating shown in Fig. 5.

of G.

lucidum

are

Extracts from G. lucidum (e.g., polysaccharide fractions, methanolic extracts, and LZ-8) have mitogenic effects on mouse splenocytes and human peripheral blood mononuclear cells (PBMCs) in the presence of various immunostimulating or immunosuppressive agents (e.g., PHA and 12-O-tetradecanoylphorbol 13acetate).4*-7] Treatment of the PBMCs with cyclosporin A (CsA) led to blockage of the cell proliferation. The methanolic fraction from G. lucidum recovered inhibition

of the cell proliferation,

Reishi or Ling Zhi (Ganoderma lucidum)

—

611

Ganoderma constituents

NK cell

3

Serre TNF

IL-1 iL-2 IL-5 IL-6 IL-12 IFN-gamma cet

Histamine

Igs

Immunosurveillance Antiviral Antibacterial

Anti-histamine

_ Anti-inflammatory Anti-aging

Fig. 5 The immune-modulating effects of G. lucidum. The major immunomodulating effects of active substances derived from G. lucidum include mitogenecity and activation of immune effector cells such as T lymphocytes, macrophages, and NK cells leading to the production of cytokines including ILs, TNF-a, and IFNs. Other effects, such as inhibition of mast cells, activation of B lymphocytes, and the complement system have also been reported.!*!

EFFECTS ON IMMUNE EFFECTOR CELLS

T Cells

Splenocytes

Extracts from G. lucidum are potent activators of T cells, inducing the production of a number of cytokines, in particular IL-2. In human PBMC (primarily

In vitro and in vivo studies in mice indicated that G. luci-

T cells) in vitro, the crude G. lucidum water extract

dum water extract stimulates the production of IL-2 by

induced

splenocytes in the presence of hydrocortisone.°71°!

the

expression

of

cytokines

including

IL-10 and TNF-o«, IL-1B, IL-6, and IL-2! Crude

Reishi or Ling Zhi (Ganoderma lucidum)

612

polysaccharide fractions isolated from fresh fruiting bodies potentiated the release of IFN-y from human T cells.°” A polysaccharide fraction (GL-B) promoted the production of IL-2 in a dose-dependent manner and markedly enhanced the cytotoxicity of cytotoxic T lymphocytes, which was increased by 100% at a concentration of 200ug/ml. GL-B also restored the mixed lymphocyte response to alloantigen, automatic proliferation, and IL-2 production of splenocytes in aged mice declined as compared with that in young adult mice in vitro. LZ-8 is also a potent T-cell activator mediating its effects via cytokine regulation of integrin expression. Stimulation of human peripheral blood lymphocytes with LZ-8 resulted in the production of IL-2 and a corresponding upregulation of IL-2 receptor expression.'4! In addition to T-cell proliferation, microscopic examination of LZ-8-stimulated peripheral blood lymphocytes revealed that LZ-8 induced cellular aggregate formation. This formation correlated with a dramatic rise in ICAM-1 expression and an increased production of IFN-y, TNF-a, and IL-1B, molecules associated

with regulation of ICAM-1 expression. Both the aggregate formation and the proliferative effects of LZ-8 were blocked by the addition of a monoclonal antibody to either CD18

or CD11la, the counter—receptor

complex components for ICAM-1. Furthermore, addition of neutralizing antibodies to both IL-2 receptor and TNF-a blocked aggregate formation, cellular proliferation, and ICAM-1 expression.

Natural Killer (NK) Cells A water-extracted polysaccharide fraction from G. lucidum enhanced the cytotoxicity of splenic NK cells in tumor-bearing mice.?:1637]

Macrophages

Macrophages are responsible for killing pathogens in the body. Activation of macrophages by substances from

G. lucidum

results in the release of cytokines,

NO, and other mediators.8’**! All of these responses are associated with the antitumor, antimicrobial, and anti-inflammatory effects of G. lucidum. Polysaccharides from G. lucidum, in particular B-p-glucans, are potent stimulators of murine and human macrophages in vitro and in vivo.2745! CR3 receptors on macrophages are bound by f-p-glucans and internalized, priming a series of molecular events. Crude water-extracted polysaccharides isolated from fresh fruiting bodies of G. lucidum potentiated the production of cytokines including IL-1, IL-6, IFN-y, and TNF-« by human macrophages, which

were

antiproliferative,

differentiated

and

apoptosis

inductive to the HL-60 and the U937 leukemic cells.°7

IFN-y and TNF-« released from macrophages act synergistically to inhibit the growth of leukemic cells as shown by the antibody-neutralization studies. GLB7,

a G.

lucidum

polysaccharide,

decreased

the

production of oxygen-free radicals and antagonized the respiratory burst induced by PMA in murine peritoneal macrophages. These observations suggest that GLB7-decreased production of oxygen-free radicals in murine peritoneal macrophages plays an important role in the anti-aging effect of G. lucidum polysaccharides.!>! Ganoderan (GAN), a f-p-glucan isolated from G. lucidum, enhanced the production of NO in the

RAW 264.7 macrophages.'*! The ability of GANs to produce NO was based on differences in the chemical composition of GANs obtained from the mycelium on various carbon sources and mycelial fractionation. The highest NO production was observed in the polysaccharide, which was extracted from the mycelial wall. Partial removal of the protein in the extracellular GAN by TCA treatment did appreciably reduce its capacity to secrete NO. The cell proliferation of GAN-treated RAW 264.7 cell lines was inhibited compared to its control. Of the culture supernatant of macrophage activated by this glycan, the percentage of cytotoxicity against mouse leukemia L1210 cells was slightly dependent on the amount of NO in the culture supernatants of the activated macrophages. These results indicate that the B-glucan-related polysaccharides of the higher fungus activate macrophages and release NO, which is an important chemical messenger for the induction of many biological responses. A protein—polysaccharide fraction (GLB) from the growing tips of G. lucidum is a strong stimulator to the macrophages." When analyzed using a flow cytometer, GLB (100 g/ml) increased the phagocytic activity of the BALB/c mouse peritoneal macrophages as well as chicken macrophage BM2CL cells against FITC-labeled Candida albicans by 55.2% and 21.2%, respectively. It also enhanced the spreading and expression of MHC class IT molecules of BM2CL cells as well as the mouse peritoneal macrophages.

Mast Cells

Some substances from G. lucidum can act on mast cells. A water extract of the fruit body had inhibitory activity on histamine release from rat peritoneal mast cells, induced by compound 48/80 or antigen (egg white albumin)-antibody reaction and on passive cutaneous anaphylaxis reaction in guinea pigs and rats. Two ganoderic acids (C and D) isolated from the fruit body by methanol inhibited the histamine release from

Reishi or Ling Zhi (Ganoderma lucidum)

rat mast cells, induced by compound 48/80 and concanavalin A. A chloroform extract from G. lucidum broth also significantly inhibited histamine release from rat peritoneal mast cells induced by A-23187

and compound 48/80. The mechanism for the inhibitory activity on histamine release from mast cells was further studied. Palmitic acid, stearic acid, oleic acid,

and linoleic acid were isolated from the active fractions. Of these, oleic acids induced membrane stabilization in model membrane systems. Cyclo-octasulfur extracted from the culture medium of G. lucidum may decrease calcium uptake from the extracellular medium by a disulfide exchange reaction in the cell membrane leading to inhibition of histamine release

from mast cells.2:10.1!.14.161

COMPLEMENT

SYSTEM

An alkali extract isolated from cultured mycelium of G. lucidum activated classical and alternative pathways of a complement system. Activated complement C3 was observed by crossed immunoelectrophoresis in mice. This fraction also activated the reticuloendothelial system of mice in the carbon clearance test and increased hemolytic plaque forming cells of the spleen. The alkali extract consisted of 10% carbohydrate and 49% proteins. A clinical study in elderly patients with insomnia and palpitations recently showed that taking G. Iucidum essence for 4—6 weeks increased their serum C3 levels.'°70!

HISTAMINE RELEASE INHIBITION The fruiting bodies have been traditionally used as anti-inflammatory agents for the treatment of asthma or allergy. In the course of a screening test for the inhibition of histamine release from rat mast cells, it was found for the first time that ganoderic acids

C and D inhibited histamine release from rat mast cells (that were induced by compound 48/80 and concanavalin A). Other than the triterpenoid compounds, cyclo-octasulfur from this fungus also effectively inhibited histamine release from rat peritoneal mast cells and interacted with membrane proteins to inhibit Ca uptake causing a blockade of histamine release.!7:1399]

HEPATOPROTECTIVE ACTIVITY G. lucidum has been widely used for the treatment of chronic hepatopathy of various etiologies. Data from

613

in vitro and animal studies indicate that G. lucidum extracts (mainly polysaccharides or triterpenoids) exhibit protective activities against liver injury induced by toxic chemicals (e.g., CCl4) and Bacillus Calmette-Guerin (BCG) plus __ lipopolysaccharide (LPS). Reishi also showed antihepatitis B-virus (HBV) activity in a duckling study. Recently, a rando-

mized placebo-controlled clinical study!'*'7 showed that treatment with G. lucidum polysaccharides for 12 weeks reduced hepatitis B e antigen (HBeAg) and HBV DNA in 25% (13/52) patients with HBV infection. The mechanisms of the hepatoprotective effects of G. lucidum have been largely undefined. However, accumulating evidence suggests several possible mechanisms. These include antioxidant and radicalscavenging activity, modulation of hepatic Phase I and II enzymes, inhibition of B-glucuronidase, antifibrotic and antiviral activity, modulation of NO production, maintenance of hepatocellular calcium homeostasis, and immunomodulating effects (Fig. 6). The mushroom could represent a promising approach for the management of various chronic hepatopathies. Further studies are needed to explore the kinetics and mechanisms of action of its constituents with hepatoprotective activities. The polysaccharide fractions and _ triterpenes isolated from G. lucidum have shown protective effects on the liver in animal and human studies. Ninety patients with chronic hepatitis B, hepatitis B viral (HBV) DNA positivity, and aminotransferase elevation were included in this multicenter prospective randomized Phase I/II study. Subjects were randomized to be given Ganopoly (n = 60) or a placebo (n = 30) for 12 weeks, then followed up for 13 weeks. Effect of therapy on levels of HBV DNA and aminotransferase activities in serum and HBeAg status were investigated. There were 78 assessable patients who entered the trial for efficacy and safety; 13 of 52 (25%) receiving Ganopoly responded by reducing HBeAg and HBV DNA compared to 10 of 26 (4%) patients in the control group (P < 0.05). Among those with serum aspartate aminotransferase (AST) values 100U/L (n = 23), 65% (15/23) responded. Within the 6-mo study period, 33% (17/52) of treated patients had normal aminotransferase (ALT) values, and 13% (7/52) had cleared hepatitis B surface antigen (HBsAg) from serum, whereas none of the controls had normal ALT values or had lost HBsAg. Eight of the 60 patients in the Ganopoly group and 4 of the 30 in the controls were unable to be followed up due to loss or withdrawal. Our study indicates that Ganopoly is well tolerated and appears to be active against HBV patients with chronic hepatitis Bie

Reishi or Ling Zhi (Ganoderma lucidum)

614

GSH reductase

0,

NADH

Collagen

NAD"

L-Arginine

SOD ¥ H.02

Catalasey

H,0

iNOS GSH

Rapier

GSa

NO

Beta-glucuronidase |

77

GSH peroxidase

Fig. 6 Possible mechanisms for the hepatoprotective effects of G. lucidum. These include antioxidative and radical-scavenging effects, downregulation of activating enzymes, and upregulation of detoxifying enzymes, antiviral activities, inhibition of Bglucuronidase, enhanced hepatic nucleic acid and protein synthesis, inhibition of hepatic collagen synthesis, immunomodulating effects, and modulation of nitric oxide (NO) production. GSH = glutathione; GSSG = oxidized glutathione; INOS = induinducible NO synthase; SOD = superoxide dismutase; CYP = cytochrome P450; UGT = uridine diphosphate glucuronosyltransferases. (From Ref."'”!,)

ANTIDIABETIC EFFECT Animal studies have demonstrated that the polysaccharide fractions of G. lucidum have potential hypoglycemic and hypolipidemic activities. A water extract of reishi reduced the increase in blood glucose and blood insulin levels-in rats (50 mg p.o.) following oral glucose test. Following adrenaline (i.v.) or oral glucose in rats, the mushroom inhibited increases in blood glucose without raising blood insulin levels. Glycans (ganoderans B and D) have shown significant hypoglycemic activity in mice. A clinical study aimed at evaluating the efficacy and safety of polysaccharide fractions extracted from G. lucidum (Ganopoly) by a patented technique!'®! in 71 patients with confirmed type II diabetes mellitus (DM) was carried out. Eligibility criteria included

type II DM of >3 mo duration during which patients did not receive insulin; age >18 yr; normal vital signs for age and disease state; normal electrocardiogram (ECG); and fasting plasma glucose (FPG) level of 8.9-16.7 mmol/L in sulfonylurea-naive patients or an FPG < 10mmol/L before washout in sulfonylureatreated patients. They were randomly grouped and given either Ganopoly or an oral placebo of 1800 mg three times daily for 12 weeks. The subjects underwent 4weeks of dose adjustment, followed by 8 weeks of dose maintenance. Fasting and stimulated glycosylated hemoglobin (HbA,), plasma glucose, insulin, and C-peptide were monitored at predetermined intervals. Adverse events and hypoglycemic episodes were recorded. Treatment with Ganopoly significantly decreased the mean HbA,, from 8.4% at baseline to 7.6% at 12 weeks. Significant changes in mean FPG

Reishi or Ling Zhi (Ganoderma lucidum)

and PPG levels at the last visit paralleled the changes in mean

HbA,,levels. At baseline, the mean FPG and

PPG values for patients treated with Ganopoly were 12.0 and 13.6mmol/L, respectively. At week 12, mean PPG values had decreased to 11.8 mmol/L. However, these parameters did not change or slightly increased for patients receiving placebos. The between-group difference in PPG levels at week 12 was significant (P < 0.05). Changes in fasting insulin, 2-hr postprandial insulin, fasting C-peptide, and 2-hr postprandial C-peptide were consistent with the between-group differences in these end points being significant at the last visit. Overall, Ganopoly

administered administrated G. lucidum extract tablets (2 tablets bid. or 220mg/day), systemic blood pressure significantly improved in 82.5%, with capillary and arterial blood pressure showing significant improvements in as little as 14days. No changes of any significance were found in the placebo group. According to Soo!”! in treating hypertension, G. lucidum was shown to be highly effective in a very large number of treated cases. In the more successful cases, blood pressure was back to normal within 2mo, and in some

cases, within 2 weeks.

was well tolerated. This

study demonstrated that Ganopoly is efficacious and safe in lowering blood glucose concentrations.!"*! A 2-mo open label comparative clinical study of a reishi powder extract (lg t.id.) for eight diabetic patients (four with NIDD and four with IDDM) found hypoglycemic effects comparable to those found in controls who were administered insulin (1001U/ml for 60 days) or oral hypoglycemic agents (250 mg/day for 60 days).2:1011-18!

CARDIOVASCULAR FUNCTIONS

615

AND CIRCULATORY

Cholesterol and Lipid Metabolism

The powdered mycelium of reishi, at 5% of the diet of spontaneously hypertensive rats for 4.weeks, caused plasma total cholesterol to decrease significantly (by 18.6%) compared to controls. Total liver triglyceride and total liver cholesterol levels were also significantly lower in the reishi-fed group (by approximately 46%

and 56%, respectively).P!>7!

Hypertension

A water extract of the mycelium administered to rats and rabbits (3-30mg/kg iv.) produced significant hypotensive effects; an activity the researchers suggested is secondary to the primary effect that suppresses sympathetic outflow of the central nervous system, 7! The powdered mycelium of reishi, at 5% of the diet of spontaneously hypertensive rats for 4 weeks, caused systolic blood pressure to be significantly lower (approximately 10 mmHg) without causing a significant difference in the heart rate"! Jin et al." conducted a double-blind, placebo-controlled clinical study of G. lucidum in 54 patients with primary stage-II hypertension who had not responded to previous drug treatment (captopril 25mg tid. or nomodipine 20mg t.id.). In the group which was

ANTIBACTERIAL AND ANTIVIRAL VALUE Antibacterial Effect of Ganoderma lucidum on Gram-Positive and Gram-Negative Bacteria Recently, more studies demonstrated that G. lucidum contained antibacterial constituents that are able gram-positive and/or gram-negative ‘to inhibit bacteria.2>:10!1:17-5556) The aqueous extract from the carpophores of G. lucidum inhibited 15 types of grampositive and gram-negative bacteria. Further studies indicate that the antimicrobial combinations of G. lucidum extract with four antibiotics (ampicillin, cefazolin, oxytetracycline, and chloramphenicol) resulted in additive effects in most instances: synergism in two instances when combined with cefazolin against Bacillus

subtilis

and

Klebsiella

oxytoca,?))

and

antagonism in two instances.

Helicobacter pylori

Helicobacter pylori is associated with human gastroduodenal diseases such as gastritis, peptic ulcer, and gastric carcinoma. The extracts of many mushrooms inhibited the growth of this bacterium."7°8! The extract of G. lucidum and some other species of higher Basidiomycetes arrested the growth of this pathogen. When their extracts were fractionated, the ether fractions of G. lucidum and Agaricus bisporus (J. Lge) Imbach were the most effective. Among seven components separated from the ether fraction of G. lucidum extract by silica gel column chromatography, P3 was the most potent with a minimum inhibitory concentration of 200 ng/ml. It appears that some constituents such as ganomycin, triterpenoids, and aqueous extracts from Ganoderma species have a broad spectrum of in vitro antibacterial activity against gram-positive and gramnegative bacteria and H. pylori. Thus, it is possible that the antibacterial activity of Ganoderma species may be beneficial for those patients with chronic infection (¢.g., chronic bronchitis) and those with H. pylori-positive

Reishi or Ling Zhi (Ganoderma lucidum)

616

peptic ulcer diseases, though required to confirm this.

clinical

studies

are

Antihuman Immunodeficiency Virus (HIV) Activity HIV was isolated as an etiological agent of acquired immunodeficiency disease syndrome in 1983.67] Acquired immunodeficiency syndrome caused by HIV infection has recently become an important social and medical problem. Anti-HIV therapy by nucleoside analogues, such as 3’-azido-thymidine, is the major effective approach for the treatment of acquired immunodeficiency syndrome.) These agents are potent inhibitors of HIV reverse transcriptase (RT) and protease.'*!] However, the emergence of drug-resistant variants of HIV and toxicities severely limits the long-term effectiveness of these drugs. Recent studies have indicated that many natural products are active as anti-HIV agents. These compounds belong to a wide

a human T lymphoblastoid cell line.©3] The ICs and ECs, values were 125 and 11 pg/ml, respectively, resulting in a therapeutic index of 11.4. This aqueous low-molecular-weight extract was further fractionated to eight subfractions by methanol: GLA (methanolic extract), GLB (hexane soluble), GLC (acetic ether soluble), GLD (water soluble), GLE (neutral), GLF (acidic), GLG (alkaline), and GLH (amphoteric). All subfractions except GLD, GLF, and GLH exhibited anti-HIV activity with ICs and ECso values of

range of different structural classes, e.g., coumarins, flavonoids, tannins, alkaloids, lignans, terpenes,

22-44 ug/ml and 14-44 1g/ml, respectively. GLC and GLG inhibited HIV RT. Showing consistency, incubation of GLC at 50ug/ml or GLG (100 pg/ml) with Jurkat T cells gave a 75% and 66% inhibition of HIV growth, respectively. However, the high-molecularweight fraction did not inhibit any HIV-induced cytopathic effect. Both low-molecular-weight and high-molecular-weight fractions from G. lucidum had negligible toxicities to CEM cells. The results indicate that the aqueous low-molecular-weight fraction from the fruiting bodies of G. lucidum, and the neutral and alkaline subfractions from the methanolic extract might contain small molecular weight polysaccharides.

naphtho- and anthraquinones, and polysaccharides.!~) In vitro studies indicate that various triterpenoids from G. lucidum had potent inhibitory activity against

Epstein-Barr Virus

HIV. Lucidenic acid O and lucidenic lactone, isolated

from the fruiting body of G. lucidum, not only inhibited the activities of calf DNA polymerase-a and rat DNA polymerase-f, but also those of HIV-1 RT."7 Ganoderiol F and ganodermanontriol isolated from the fruiting bodies of G. lucidum are active against

HIV-1

growth with an ICjo9 of 7.8 pg/ml.01!17]

Ganoderic acid B and ganoderiol B showed potent inhibitory effect on HIV protease with an ICso value of 0.17mM. Other triterpenoids including ganoderic acid Cl, 3f-5a-dihydroxy-6B-methoxyergosta-7,22diene, ganoderic acid-«, ganoderic acid H, and ganoderiol A had moderate activity against HIV-1 protease

Virus-induced carcinogenesis is considered a complicated process with multiple steps involving a number of cellular signaling pathways. A few polyoxygenated lanostanoid triterpenes isolated from G. applanatus inhibited the 12-O-tetradecanoylphorbol-13-acetate induced Epstein-Barr virus early antigen in Raji cells. Similar effects have been observed with Zingiberaceae rhizomes, a commonly used traditional medicine in Malaysia. These results indicate that herbal medicines, such as Ganoderma species, may behave as antitumor promoters.!!7]

with ICs9 values of 0.17-0.23mM.201!1175°l Tn addition, ganoderic acid-B, lucidumol B, ganodermanondiol,

ganodermanontriol,

and

ganolucidic

acid

showed significant anti-HIV-1 protease activity with ICsq values of 20, 59, 90, 70, and 70uM, respec-

tively.7! Ganoderic acid A, B, and Cl had minor inhibitory activity against HIV protease with ICs values of 140-430 uM. It appears that there is a structure— activity relationship for triterpenoid showing antiHIV

protease

activity. The

C3, C24, or C25

atoms

are vital for the anti-HIV activity.?7! The aqueous low-molecular-weight fraction extracted from G. lucidum also exhibited anti-HIV activity using the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) —carbonyl]-2H-tetrazolium hydroxide] antiviral assay, which can quantitatively measure

cytopathic effects of HIV-1

on CEM

Other Viruses

A

cells,

The antiviral effects of two water-soluble substances (GLhw and GLIw) and eight methanol-soluble substances (GLMe-1-8) isolated from the carpophores of G. lucidum, were investigated on influenza A virus strains and vesicular stomatitis virus Indiana and New Jersey in vitro. These activities were evaluated by the cytopathic effect inhibition assay and plaque reduction assay using Vero and HEp-2 cells. Five substances, GLhw, GLMe-1, -2, -4, and -7 significantly inhibited the cytopathic effects of vesicular stomatitis virus. GLMe-4 did not exhibit cytotoxicity up to 1000 g/ml, while it displayed potent antiviral activity on the vesicular stomatitis virus New Jersey strain with a therapeutic index of more than 5.43.[6+6]

Reishi or Ling Zhi (Ganoderma lucidum)

617

Mechanism Consideration The mechanisms for the antibacterial and antiviral activity of Ganoderma species are largely undefined. Gao et al.''” suggest that multiple mechanisms may be involved. For example, the Ganoderma species constituents (e.g., polysaccharides and triterpenoids)

cerebrosides hardly influenced the activities of DNA polymerase-B, prokaryotic DNA polymerases, terminal deoxynucleotidyl transferase, HIV RT, RNA polymerase, deoxyribonuclease

I, and ATPase.

Linoleic acid

from G. lucidum inhibited the activities of mammalian

DNA polymerases.&*°¢

may inhibit viral replication of HSV, HBV, HIV, and

Immunomodulating effects of G. lucidum are considered to play a role in antimicrobial activity.!!>'7]

other types of viruses by interfering with their adsorp-

Activation

tion, virus—hepatocyte

macrophages, and natural killer cells) by both pathogen infection and G. lucidum administration caused

fusion

and

endocytosis,

viral

integration, assembly, and release (Fig. 7). Data from in vitro studies indicate that Ganoderma polysaccharides have direct anti-HBV activity through inhibition of HBV DNA polymerase. The extract from G. lucidum inhibited the HBV DNA polymerase activity in PLC/PRF/5 cells by 80%, 70%, and 60%, respectively, with a 28-41% decrease in HBV DNA contents. Some constituents isolated from G. lucidum showed inhibitory effect on eukaryotic DNA polymerase. For example, two cerebrosides from the fruiting bodies selectively inhibited the activities of replicative DNA polymerases (especially the « and 6 type) with a ICs9 of 12-57M. However, these

Transcription of early mRNA DNA replication

an

enhanced

of immune

production

effector

cells

of cytokines,

(e.g., T cells,

radicals,

and

NO facilitating the killing of viruses and bacteria. For example, activation of Kupffer cells by G. lucidum polysaccharides and triterpenoids within the liver facilitate the killing of HBV. In addition, a study of the mouse indicates that a proteoglycan with a carbohydrate protein ratio of 11.5: 1 isolated from G. lucidum stimulated the proliferation of mouse spleen lymphocytes, resulting in a three-to-four fold increase in the percentage of B cells. These B cells were enlarged, expressed CD71 and CD25 on the cell surface, and showed an increase in the production of

~.'7 Concentrations of fatty acids in prostatic tissue of patients with benign or malignant prostatic disease are different, with a significant reduction in arachidonic acid and docosapentaenoic acid in malignant prostatic tissue phospholipids. It has been suggested that the decrease in arachidonic acid concentration may be due to its increased metabolism via the cyclo-oxygenase and/or lipoxygenase pathways to produce eicosanoids such as prostaglandins and leukotrienes.°°! In one study, malignant prostatic tissue converted radiolabeled arachidonic acid to prostaglandin PGE2 at an almost 10-fold higher rate than BPH tissue. PGE2 production from [*H]arachidonic acid by malignant prostatic tissue was investigated in the presence of oleic acid, eicosapentaenoic acid, docosahexaenoic acid, dihomo-y-linolenic acid, eicosatetra-

ynoic acid, and ketoprofen. Oleic acid was found to

be the most effective inhibitor.?”! Saw palmetto extract, when administered orally in rodents, has shown antiedematous activity in a diversity of animal models, including centrifugationinduced

tail edema

in mice, histamine-and

dextran-

induced increase in microvascular permeability and edema in rats, IgE-dependent passive cutaneous anaphylaxis in rats, and UV erythema in guinea pigs. Since the antiedematous activity was also observed in an adrenalectomized

rat model,

glucocorticoids

cannot

be the source of activity.!*! Extracts of saw palmetto have shown potent noncompetitive inhibition of human prostatic «, adrenoceptors in vitro."*! Studies performed on isolated organs, including the rat deferential duct, and guinea

castration to remove the source of endogenous testosterone and were then given a subcutaneous (s.c.) injection of the hormone. Saw palmetto extract given orally for 12 days (300 mg/mouse) antagonized the stimulant effect of exogenous testosterone, reducing the weights of the ventral prostate and seminal vesicles by 46% and the weight of the preputial glands by 24% in comparison to the control mice treated only with testosterone. Rats given saw palmetto extract (200 mg/animal) orally for 6days showed similar results. The body weight and the weights of the levator ani muscle, thymus, adrenal glands, and spleen were unaffected by the extract. In prepubertal mice and rats treated with gonadotropin, orally administered saw palmetto extract (200mg/animal for 3days) antagonized the effect of androgens on the weight of the ventral prostate, seminal vesicles, and preputial glands. No effect was observed on the weight of the testicles, thymus, or adrenal glands.'©! The antiandrogenic activity in castrated rats treated orally for 10 days with 150 and 300 mg of a saw palmetto extract has been shown to depend on both the temperature and pressure conditions used for the CO, extraction of the fruits and the dose of the extract (Table 2).!"7! The absence of estrogenic properties has been demonstrated by studying the effect of saw palmetto extract on the growth of the prepubertal female mouse uterus and on the changes in the estrus cycle of adult female mice. The absence of progestational activity has been investigated in ovariectomized female mice that were sensitized with estrone and treated daily with 100-400 mg saw palmetto extract orally or 100 ug of progesterone subcutaneously. On day 10 of treatment, histamine was administered in one of the uterine horns. In contrast to progesterone, the extract of saw palmetto did not cause an increase in the weight of the uterus.'© Antispasmodic activity has been reported in vitro for saw palmetto extracts. A 90% ethanol extract reduced norepinephrine-induced contractions of rat deferential duct, and potassium chloride-induced contractions of guinea pig ileum and bladder smooth muscle tissue. Both lipid and saponifiable fractions of saw palmetto reduced norepinephrine-induced contractions

of rat aorta, in vitro as well as potassium

pig ileum and bladder, have shown the extracts to have

chloride-induced contractions of rat uterus. Vanadate-induced contractions of the rat uterus have also been reduced by a lipophilic (90% ethanol) saw

both « adrenoceptor antagonistic and calcium blocking properties.! Despite these in vitro reports, the typical side effects of a-blocker drug therapy do not seem to be frequently reported in clinical trials of saw palmetto. Nl The ‘peripheral antiandrogenic activity of the saw palmetto extracts has been studied in vivo in mice and prepubertal rats. The animals underwent

Saw palmetto has both individual compounds and specific extracts with pharmacological activity that could be related to mechanisms that would provide symptomatic relief for LUTS associated with BPH. The lack of systemic hormonal and «-blocker activity should be important for the safe use of saw palmetto and points to the need for more detailed research on

palmetto extract.!"41

Saw Palmetto (Serenoa repens)

642

Table 2

rats Effect of orally administered (10 days) hypercritical CO. S. repens fruit extract in castrated prepuberal

Body weightight (g)

>Dose (mg/day)

Treatment Normal control (olive oil)

—

Castrated control (olive oil)

—

TP (olive oil)

—

TP + Extract A

300

TP + Extract B

Initial

58.1 56.2 62.3 65.1 63.1 64.1 66.2

150

300 TP + Extract C

300

+ + + + + + +

Prostate weight (mg)

Final

91.1 87.2 95.2 92.3 94.2 93.6 94.1

2.6 18 3.1 2.7 2.7 1.7 2.2

+ + + + + + +

20.6 + 2.4 3.0 + 1.24 17.4 + 18 11.7413 11.9 + 1.6°, 6.5 + 1.2° 11.1 + 1.1°

3.2 2.2 2.5 2.5 2.5 2.4 2.5

TP = testosterone proptonate (15 mg/day, s.c.). Extract A: 35°C/250 bar; B: 45°C/220 bar; C: 50°C/280 bar. Values are mean

+ S.E.; n =

15.

*P < (0.01 vs. normal control.

pee 0.05: ©P < 0.01 vs. TP; Duncan’s test. (Adapted from Ref.!'7!,)

activity in prostate cells, tissues, and animals, as well as in humans, to determine whether multiple mechanisms of action are working in synergy or whether the main mechanism of action has yet to be discovered.

PHARMACOKINETICS Unlike pharmacodynamic studies that measure physiological changes, pharmacokinetic studies in humans with saw palmetto are difficult to conduct because the most active compounds that would logically be measured are fatty acids and sterols that are common in the normal Western diet. The pharmacokinetics has been investigated in an open, randomized,

cross-

over study of 12 healthy males who ingested one 320mg capsule or two 160mg capsules per day. The extract was absorbed rapidly, with a peak time (tax) of 1.50-1.58hr

and

peak

plasma

levels

(Cyax)

of

2.54-2.67 pg/ml. The area under curve value (AUC) ranged from 7.99 to 8.42p1g hr/ml. Since the plasma concentration—time profiles of both preparations were very similar, the preparations were considered bioequivalent, but the validity of the methodology has been questioned .!°"41 In another study, the bioavailability and pharmacokinetic profile of a rectal formulation containing 640mg of S. repens extract were determined in 12 healthy male volunteers. The rectal formulation was similar to the oral one but showed a slower absorption, with a tmax of 2.96 hr.!*! In a study of the administration of a radioactive lipophilic sterol extract of saw palmetto in rats, tissue concentrations of lauric acid, oleic acid, and f-sitosterol were highest in abdominal fat tissue, the prostate,

and the skin with lower concentrations in the liver and urinary bladder")

REGULATORY STATUS Saw palmetto products are regulated in the United States as dietary supplements since the passage of the Dietary Supplement Health and Education Act (DSHEA) in 1994. Under the regulations of DSHEA, saw palmetto is sold without FDA premarket approval since dietary supplements are sold as a category of food. Although clinical testing is not required for inclusion in the United States Pharmacopeia or National

Formulary,

identity, chemical

content,

and

quality standards for saw palmetto fruits, powders, extracts, and capsules have recently been reintroduced into the dietary supplement section of the USP27-

NF22.”'! Saw palmetto products sold as dietary supplements are not allowed to make a drug claim (such as for the treatment of urological symptoms associated with BPH) but are allowed to make structure function claims (such as “to support prostate health’’ or “‘sup-. port healthy prostate function’’). Saw palmetto is _ included in the Homeopathic Pharmacopoeia of the United States and as such may be sold as a homeopathic over-the-counter (OTC) drug at strength of 1x, which is similar to a full strength alcoholic (65%) extract.77]

The

Federal

Trade

Commission,

the US

govern-

ment agency that regulates advertising claims for all products, has issued a guidance document on truthful and nonmisleading advertising claims for dietary supplements that includes opinions from experts in botanical medicine and the claim being made.?! Saw palmetto is*sold in Canada as a natural health product that must be manufactured to pharmaceutical standards and meet other monograph standards, including the amount of the product to be taken and the form of the product that can be used, and is sold

with labels that can state the more

accurate

claim

Saw Palmetto (Serenoa repens)

643

“helps relieve the urologic symptoms associated with benign prostatic hyperplasia’’.4! In France, it is a prescription drug reimbursable by the national health insurance. In Italy, a lipophilic hexane extract is sold as a prescription drug. The dried fruit, simple galenical preparations, and lipophilic extracts are approved as nonprescription drugs in Germany. In Belgium, saw palmetto is a prescription adjuvant for BPH, while in Sweden, it is sold as a natural remedy for self-medication after premarketing authorization. In Switzerland, it is sold without a prescription, but sales are limited to pharmacies, and in the United Kingdom, saw palmetto is a herbal medicine on the General Sales List that requires full product licensing.""*!

America Editorial Committee, Eds.; Oxford University Press: New York and Oxford, 2000; Vol. 22. Flora of North America Online:

http://flora.huh.harvard.edu:8080/flora/browse.

do? flora_id=1&taxon_id=222000361 &key.no=1

(accessed May 20, 2004). Godfrey, R.K. Trees, Shrubs, and Woody Vines of Northern Florida and Adjacent Georgia and Alabama; University of Georgia Press: Athens,

1988. Bennett, B.C.; Hicklin, J.R. Uses of saw palmetto (Serenoa repens, Arecaceae) in Florida. Econ. Bot. 1998, 52 (4), 381-393. Wilt,

CONCLUSIONS Saw palmetto fruits and extracts, based on the known chemistry, pharmacology, clinical trial data, and the low number of adverse event reports, appear to be very safe for men to consume in the amounts used in clinical trials and available in dietary supplement products on the market. Mild nausea or mild GI upset appear to be the most frequent side effects from ingesting amounts currently consumed as dietary supplements or as drugs. Saw palmetto products that have not been tested for systemic estrogen and androgen effects pose a theoretical risk to the proper development of the

Bombardelli,

R.

E.; Morazzoni,

P. Serenoa repens

Ganzera, M.; Croom, E.M.; Khan, I.A. Determi-

nation of fatty acid content of Pumpkin seed, Pygeum, and Saw Palmetto. J. Med. Food 1999, 2 (1), 21-27. Peng, T.-S.; Popin, W.F.; Huffman, M. Systema-

tic investigation of quality management of saw palmetto products. In Quality Management of Nutraceuticals; American Chemical Society Symposium Series, No. 803; American Chemical Society: Washington, DC, 2002. Supplement Business Report. Nutr. Bus. J. 2002, 3-6.

10.

Schulz,

V.;

Hansel,

R.;

Tyler,

V.E.

Rational

Phytotherapy, 3rd Ed.; Springer: Berlin, 1998. AUA Practice Guidelines Committee. AUA guideline on management of benign prostatic hyperplasia (2003). Chapter 1: Diagnosis and treatment recommendations. Urology 2003, 170, 530-547.

ACKNOWLEDGMENTS

12)

Supplement Business Report.

REFERENCES Magnoliophyta: Alismatidae, Arecidae, Commelinidae (in Part), and Zingiberidae. Flora of North America North of Mexico; Flora of North

MacDonald,

(Bartram) J.K. Small. Fitoterapia 1997, 69 (2), 99-113.

metto is popular as self-treatment in the United States,

1.

A.;

(1993 printing)].

in the WHO monograph for pregnant or lactating women and children under 12 yr of age. Specific lipophilic saw palmetto hexane and CO, extracts produced to high quality manufacturing and chemical content standards have been shown in clinical trials to be safe and effective for lower urinary tract symptoms associated with BPH. Although saw pal-

Thanks to Renato Iguera and Indena S.p.A., Milan, Italy, for the photographs of saw palmetto. Thanks to Tom Aarts for permission to include the saw palmetto sales figures in 2001 from the Nutrition Business

Ishani,

American Dispensatory; 18th Ed.; 3rd revision; 1898; Vol. 2, 1750-1752 [Reprinted by Eclectic Medical Publications: Sandy, Oregon, 1983

fetus and infants; their use is therefore contraindicated

a large majority of the products on the market have not been evaluated in clinical trials for efficacy.

7T.;

Serenoa repens for benign prostatic hyperplasia (Cochrane Review). In The Cochrane Library, Issue 2; John Wiley & Sons, Ltd.: Chichester, UK, 2000. Felter, H.W.; Lloyd, J.U. Serenoa. In King’s

13f

Debruyne, F.; Koch, G.; Boyle, P.; Calais da Silva, F.; Gillenwater, J.G.; Hamdy, F.C.; Perrin, P.; Teillac, P.; Vela-Navarrete, R.; Raynaud, J.P.;

PERMAL Study Group. Comparison of a phytotherapeutic agent (Permixon) with an alphablocker (tamsulosin) in the treatment of benign prostatic hyperplasia: a 1-year randomized international study. Eur. Uro. 2002, 4/ (5), 497-507. Committee on the Framework for Evaluating the Safety of Dietary Supplements, Food and Nutrition

Board,

Board

on

Life

Sciences.

Dietary

Saw Palmetto (Serenoa repens)

644

Tissue effects of saw palmetto and finasteride: use of biopsy cores for in situ quantification of prostatic androgens. Urology 2001, 57 (5), 999-1005.

supplement ingredient prototype monographs developed as examples for the report, saw palmetto. Dietary Supplements: A Framework for Evaluating Safety; National Academies Press:

19:

14.

World Health Organization. Fructus Serenoae Repentis; WHO Monographs on Selected Medic-

bie

18.

Breu, W.; Hagenlocher, M.; Redl, K.; Tittel, G.; Stadler, F.; Wagner, H. Anti-inflammatory activ-

ity of sabal fruit extracts prepared with supercritical carbon dioxide. In vitro antagonists of cyclooxygenase and 5-lipoxygenase metabolism. Arzneimittelforschung 1992, 42 (4), 547-551. Cristoni, A.; Morazzoni, P.; Bombardelli, E. Chemical and pharmacological study on hypercritical CO, extracts of Serenoa repens fruits. Fitoterapia 1997, 68 (4), 355-358. Marks.) 12528 Hess) Dilys. Dorey) Macairan, M.; Cruz Santos, P.B.; Tyler,

Luz V.E.

H.-U.;

Zanker,

Chaudry,

AN

A.A.;

Pharmacopeia

Wahle,

K.W.;

27—-National

McClinton,

S.;

Formulary 22, USP

27th revision. United States Pharmacopeial Con-

2003; 311-319. 16.

Schweikert,

Moffat, L.E. Arachidonic acid metabolism in ° benign and malignant prostatic tissue in vitro: effects of fatty acids and cyclooxygenase inhibitors. Int. J. Cancer 1994, 57 (2), 176-180. Saw palmetto monographs. In United States

Brinckmann, J.; Wollschlaeger, B. Saw palmetto. In The ABC Clinical Guide to Herbs; Blumenthal, M., Hall, T., Goldberg, A., Kunz, T., Dinda, K., Eds.; American Botanical Council: Austin, TX,

H.-J.;

20.

inal Plant. 2001. www.who.int/medicines /library/ trm/medicinalplants/vol2/285to299.pdf (accessed May 31, 2004). 1S:

Niederpriim,

K.S. Testosterone 5 «-reductase inhibition by free fatty acids from Sabal serrulata fruits. Phytomedicine 1994, /, 127-133.

Washington, DC, 2004. http://www.iom.edu/ subpage.asp?id=19554 (accessed May 21, 2004).

22,

23.

vention, Inc.: Rockville, MD, 2004; 2034-2037. Homeopathic Pharmacopoeia of the United

States Official Compendium; Pharmacopoeia Convention of the American Institute of Homeopathy: Boston, MA, 1997. Dietary Supplements: An Advertising Guide for Industry; United States Federal Trade Com-

mission. 24.

http://www.ftc.gov/bcep/conline/pubs/

buspubs/dietsupp.htm (accessed May 31, 2004). Saw Palmetto Monograph; Health Canada, Natural Health Products Directorate. http://www. he-sc.gc.ca/hpfb-dgpsa/nhpd-dpsn/mono_saw_ palmetto_e.html (accessed May 31, 2004).

Selenium Raymond F. Burk Brooke K. Norsworthy Clinical Nutrition Research Unit, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A.

INTRODUCTION Selenium is an essential micronutrient that is incorporated into the primary structure of proteins in the form of selenocysteine. Selenoproteins facilitate redox reactions that underlie a number of biochemical processes. Among them are protection against oxidative damage, metabolism of thyroid hormones, support of DNA synthesis, and regulation of transcription factors. Selenium deficiency occurs in China and some other countries but not in the United States. The recommended dietary allowance (RDA) for selenium is 55,1g/day for adults.

BACKGROUND Selenium was discovered by Berzelius in 1817 as a by-product of sulfuric acid production. Its importance in biology was established in the 1930s, when it was identified as the toxic principle in plants that poisoned grazing animals in certain parts of the Great Plains of the United States. In response to this problem, the U.S. Department of Agriculture (USDA) mapped the selenium content of forage from all regions of the United States and produced a “selenium map’’ to help farmers avoid grazing their animals in areas that

might produce toxicity." In addition, the metabolism of the nutrient was studied over the next two decades with the aim of determining its mechanism of toxicity. Studies carried out during that period established that its metabolism was intertwined with that of sulfur.”! The essentiality of selenium in animals was recog-

development of dietary liver necrosis.?! Vitamin E could also prevent the condition, and this fact has linked these two nutrients since that time. A number of naturally occurring animal diseases are caused by combined selenium and vitamin E deficiency. They include mulberry heart disease, nutritional muscular dystrophy, and nutritional liver necrosis of swine, exudative diathesis of chickens, and white muscle disease of sheep. Reference to the selenium map produced by the USDA shows that these conditions occur in areas where plant selenium concentrations are low.!!] Geological studies led to the recognition that selenium is distributed irregularly in soil. Soils derived from volcanic rock are generally poor in selenium, as are those subject to leaching from high rainfall. Alkaline soils yield their selenium, and acid soils withhold it from plants. Thus, alkaline soils derived from

sedimentary rock that are in arid regions tend to transmit high levels of selenium to plants. Plants grown on acidic soil or volcanic soil tend to have low selenium content. One of the areas of low selenium

in the United

States is the Pacific Northwest,

and grazing animals require selenium supplementation there. The South Island of New Zealand and much of Scandinavia also have low soil selenium levels. Human selenium deficiency was firmly demonstrated in 1979, when Chinese scientists provided evidence that a cardiomyopathy occurred only in seleniumdeficient children living in regions of China where the nutrient content in plants was very low. Blood and hair selenium content of inhabitants of regions where Keshan disease was found were the lowest levels repor-

nized in 1957, when provision of the element to rats

ted in human beings.“! Thus, selenium is an essential

fed a yeast-based

nutrient for human beings. In recent years, supplementation of selenium in pharmacological doses has been touted for a variety of health purposes. These include chemoprevention of cancer, delay of aging, and prevention of heart disease. These potential actions do not appear to relate to its nutritional effects. This brief review will focus on nutritional (physiological) functions of selenium. Other uses of the element will be addressed only briefly.

diet was

shown

to prevent

the

Raymond F. Burk, M.D., is Professor at the Division of Gastroen-

terology, Department of Medicine and Pathology, Clinical Nutrition Research Unit, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A.

Brooke K. Norsworthy, M.Sc., is Research Assistant at the Division of Gastroenterology, Department of Medicine, Clinical Nutrition Research Unit, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A.

Encyclopedia of Dietary Supplements DOI: 10.1081 /E-EDS-120022063 Copyright © 2005 by Marcel Dekker. All rights reserved.

645

Selenium

646

BIOCHEMISTRY AND FUNCTION

NH>

| CH-Se-CH)-CH»-C-COOH H selenomethionine

NH) H-Se-CH)-C-COOH H selenocysteine

Fig. 1 The amino acid forms of selenium that account for most of the element in the body.

CHEMICAL FORMS Selenium occupies a spot in the periodic table of elements just below sulfur and shares many chemical properties with that element. Its chemistry is covalent in biological systems. Most selenium in biological material is present in amino acids (Fig. 1). Plants are not known to require selenium and incorporate the element nonspecifically into selenomethionine. A major source of selenium in the diet is selenomethionine. Once ingested by an animal, it enters the methionine pool and is not distin-

guished from methionine. Thus, much of the selenium in animal tissues is selenomethionine that is nonspecifically incorporated in proteins at methionine positions. When selenomethionine is catabolized, its selenium becomes available to the selenium metabolic pool. Thus, selenomethionine apparently has no biological function related to selenium other than serving as a dietary source of the element. Selenocysteine is present in stoichiometric amounts in selenoproteins and is necessary for their function. This is synthesized from the selenium present in the metabolic pool. It is often considered to be a “supercysteine’’ because its hydrogen is dissociated at physiological pH, making it very reactive. Presence of selenocysteine at active sites of enzymes such as glutathione peroxidases increases their activities several orders of magnitude above that when cysteine is present. The urinary metabolites of selenium that have been identified are trimethylselenonium ion and a methylated selenosugar.”! They are synthesized in the liver and kidney to regulate whole-body selenium. Inorganic forms of selenium such as selenite and selenate are biologically available and are often used to supplement selenium intakes. They are well tolerated and inexpensive.

Selenoprotein Synthesis Animal selenoproteins contain stoichiometric amounts of selenocysteine in their primary structures. The selenocysteine that is used for this process is synthesized in the cell by modification of a serine residue that has already been ligated to tRNA‘, The resulting selenocysteinyl-tRNA®"* recognizes a UGA in the open reading frame of the selenoprotein mRNA with the aid of at least two trans-acting proteins. The process of selenoprotein synthesis is complex and costly to the organism. It requires at least 5 gene products in addition to the ones normally used for protein synthesis. Reviews describing selenoprotein synthesis are

available.!*71

Selenoproteins and Their Functions Twenty-five genes that code for selenoproteins have been identified in the human genome."! Because some genes produce more than one protein product, the number of selenoproteins in humans is probably between 25 and 50. While most of these selenoproteins have not been characterized, a few of them have been

subjected to considerable study (Table 1).

Table 1 Human selenoproteins* Group/name Selenoproteins involved in thiol redox reactions Glutathione peroxidases Cellular glutathione peroxidase Gastrointestinal glutathione peroxidase Extracellular glutathione peroxidase Phospholipid hydroperoxide glutathione peroxidase Odorant metabolizing glutathione peroxidase Thioredoxin reductases Cytosolic thioredoxin reductase Mitochondrial thioredoxin reductase Testicular thioredoxin reductase

Methionine sulfoxide reductase B

Abbreviation

GPX1 GPX2 GPX3 GPX4 GPX6

TrxR1 TrxR2 TrxR3 MsrB

Selenoproteins involved in thyroid hormone metabolism

Type I iodothyronine deiodinase Type II iodothyronine deiodinase Type III iodothyronine deiodinase

D1 D2 D3

Selenoprotein involved in selenium supply to the brain and testes Selenoprotein P

Se-P

“For other selenoproteins, see Ref.!*!,

Selenium

647

Selenoproteins involved in thiol redox reactions There are two major systems that regulate thiol redox status in cells: The glutathione and the thioredoxin systems. Both of them depend on NADPH for their reducing equivalents and contain selenoproteins as components. The glutathione peroxidases utilize reduced glutathione (GSH) to catabolize hydroperoxides of various kinds. The active sites of these enzymes typically contain

selenocysteine,

although some

of them func-

tion with cysteine in the active site. Of the 6 glutathione peroxidase genes in the human genome, 5 code for selenoproteins. At least one of these genes produces 3 different protein products by the use of alternative translation start sites. Two glutathione peroxidase genes have been knocked out in mice without obvious effects on health and reproduction.”-'°! Only when these knockout mice are stressed do they show increased injury compared with wild-type animals.) A knockout of another glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, causes embryonic lethality.!7! One isoform of this latter enzyme has structural functions in spermatozoa.''?! The considerations abovementioned indicate that these enzymes have a variety of biological functions and each will have to be evaluated separately. A full discussion of this enzyme family is beyond the scope of this review and can be found elsewhere.'4) Thioredoxin reductases maintain thioredoxin and some other substances, including ascorbate, in a reduced state. These enzymes contain selenium in mammals,

but

lower

life

forms

sometimes

have

cysteine homologs. Thioredoxin is responsible for providing reducing equivalents to ribonucleotide reductase and to a number of other enzymes and transcription factors. Therefore, this family is important for gene expression, signaling, and oxidant

defenses.!'>!] Knockout of thioredoxin in mice causes

embryonic lethality.'7!

Selenoproteins involved in thyroid hormone metabolism

Thyroxine, or T4, is the hormone produced by the thyroid gland. It must be converted to triiodothyronine, or T3, to exert biological activity. T3 is inacti-

vated by conversion to T2. All these reactions are carried out by 3 selenoproteins known as deiodinases.!'8] In animals, selenium deficiency leads to decreased activity of the deiodinases, but this is compensated

for by a rise in T4 levels. When T4 production is compromised by iodine deficiency, the addition of selenium deficiency is not well tolerated.!'?! There have been

reports of cretinism occurring in infants living in areas where combined selenium and iodine deficiency is found.?°! Selenium transport Recently, selenoprotein P has been shown to have selenium transport properties. This extracellular selenoprotein contains 10 selenocysteine residues in its full-length form. It is produced in most tissues, but selenoprotein P in plasma appears to originate largely in liver. Mice with selenoprotein P knocked out have been produced.?'7] Males have abnormal spermatozoa and have sharply reduced fertility. Both males and females develop neurological impairment when fed a diet containing normal amounts of selenium. They can be rescued by provision of a high-selenium diet.?7! These studies appear to indicate that the brain and testes rely on selenoprotein P for their supply of selenium.

Other selenoproteins Most of the other selenoproteins appear to be present in low abundance. They will have to be studied individually to determine their biological functions.

Antioxidant Properties of Selenium Several lines of investigation indicate that selenium has antioxidant properties. Already mentioned is the relationship with vitamin E, which is a free radical scavenger. When rats that are vitamin E deficient are also made selenium deficient, they undergo massive lipid peroxidation and liver necrosis.?*! This indicates that selenium-dependent functions, presumably reactions catalyzed by selenoproteins, partially compensate for the lack of vitamin E, but that loss of both nutrients

leads to severe oxidative damage in the liver. A number of oxidant defense enzymes that do not contain selenium become induced in selenium deficiency. These include glutathione transferases, glutamate-cysteine ligase, NAD(P)H quinone reductase, and heme oxygenase- 1/4] Loss of thioredoxin reductase activity appears to be the cause of induction of heme oxygenase-1,'°! and this selenoprotein might be responsible for many of the oxidant defense properties of selenium. Cancer Chemoprevention A great deal of attention has been directed to the use of selenium to prevent cancer. The first data suggesting a correlation between low selenium intake and increased

Selenium

648

cancer incidence were presented in the 1970s. Since then, other observational studies have supported this correlation. However, they were unable to isolate selenium as the only factor responsible for the lower cancer incidence. Intervention studies have attempted to evaluate the effect of selenium administration on cancer incidence. One such study was carried out in China.?° An overall reduction in cancer mortality of 13% was achieved in a rural population by giving combined supplements of selenium, vitamin E, and carotene. Unfortunately, no

subjects were given selenium alone; so the protection cannot be ascribed to it. Another study using high-selentum yeast that provided 200 yg of selenium per day was carried out in subjects who had a nonmelanoma skin cancer.?7! The primary endpoint was development of another skin cancer. Secondary endpoints of other cancers were introduced after the study had been initiated. The initial analysis (10 yr of supplementation) of the primary endpoint showed no effect of the highselenium yeast on skin cancer development, but it showed a decrease of 37% in overall cancer development. The diagnosis of prostate cancer was decreased 63% in the high-selenium yeast group. Concern was raised recently by a report on the entire blinded period (12yr) of high-selenium yeast supplementation.?*! It showed that the supplemented group had a higher incidence of squamous cell carcinoma of the skin than did the placebo group. The initial report of this study stimulated a great deal of interest in selenium as a chemoprevention agent. A very large trial of selenium and vitamin E administration against the development of prostate cancer has been initiated with NIH support. Because of the uncertainty that exists about the effect of pharmacologic amounts of selenium on cancer development, selenium supplementation cannot be recommended at present to prevent cancer. Studies that are in progress and planned should provide guidance in this matter.

Thus, absorption of selenium is very high and not subject to regulation by selenium status. The selenium pool in the liver appears to be the site of homeostatic regulation. Absorbed selenium is removed from the portal blood by the liver, and selenomethionine is catabolized there by transsulfuration,

releasing its selenium to the selenium metabolic pool. Liver selenium is used to synthesize liver selenoproteins and selenoprotein P for export. Selenium in excess of that needed for these processes appears to be converted into excretory metabolites (trimethylselenonium ion and a methylated selenosugar) that appear in the urine. When

toxic amounts

of selenium

are present,

dimethyl selenide appears in the breath. Thus, excretion is responsible for regulating the selenium content of the body.

The only transport form of selenium that has been identified is selenoprotein P.”'?7] However, other form(s) must exist, because knockout of selenoprotein P does not affect selenium levels of many tissues. Selenium storage is accomplished through two mechanisms. One is unregulated and consists of selenomethionine that is present in the methionine pool. As selenomethionine is catabolized, its selenium is fed into the selenium metabolic pool. The other storage mechanism relates to the most abundant glutathione peroxidase enzyme. This protein contains a greater fraction of whole-body selenium than any other selenoprotein. When selenium is in short supply, the mRNA of this glutathione peroxidase is degraded more rapidly, reducing synthesis of this selenoenzyme. This allows selenium to be directed to other selenoproteins that are presumably more important for survival. In summary, selenium homeostasis is maintained by excretion of the element when it is present in amounts greater than what can be utilized for selenoprotein synthesis. When insufficient selenium is present for synthesis of all selenoproteins, hepatic cytosolic glu- | tathione peroxidase is downregulated so that selenium can be directed to other selenoproteins. |

DIETARY INTAKE AND DEFICIENCY METABOLISM Sources and Regional Variation of Selenium Selenium has chemical properties that account for its function in selenoproteins. Because these can also lead to catalysis of unwanted reactions, homeostatic control of selenium in the organism is necessary. The major dietary forms of selenium are selenomethionine derived from plants and selenocysteine from animal selenoproteins. Both amino acids appear to be virtually completely absorbed by the mechanisms used for absorption of their sulfur analogs. The inorganic forms that are often used for supplementation, selenite

and

selenate,

are

also well

absorbed.

The amount of selenium in plants depends on the availability in the soil on which the plants are grown. This fact leads to a single food plant such as wheat having a selenium content that can vary by a factor of 10 or more, depending on where it is grown. This variation renders food tables for selenium in plants of little value. Animals, on the other hand, require selenium and

have homeostatic mechanisms to maintain predictable concentrations in their tissues. Thus, foods of animal

Selenium

649

origin are more reliable sources of selenium. In areas where the soil is poor in selenium, animal foods contain more selenium than plant foods. The lowest and the highest intakes of selenium in the world have been reported in China. They vary from less than 10 g/day to over 1 mg/day.”°! The cause of this wide variation is the reliance of the population on plant foods and the extreme variation in available soil selenium in different regions. Intakes in other countries generally vary from around 30pug/day in New Zealand and parts of Scandinavia to around 100 g/day in North America. Intakes in Europe are in the range of 30-60 pg/day.

Keshan

Disease

The only human disease that has been clearly linked to selenium deficiency is Keshan disease. It is a childhood cardiomyopathy that occurs in low-selenium regions of China, where the intake of the element is approximately 10pg/day. A double-blind placebo-controlled study that was carried out in the 1970s showed that selenium supplementation could prevent the development of Keshan disease.) Because not all selenium-deficient children developed Keshan disease, a second stress was considered. Subsequent studies in mice have suggested that the second stress might be a viral infection.2°! The incidence of Keshan disease has declined in the last few decades and it is now rare. This is likely due to the

improvement

of

increased meat intake between regions.

the

and

Chinese

exchange

economy,

with

of foodstuffs

in the methionine

pool.

In the United

States,

the

amount of selenium in this form ranges from 1 to 12g per 100ml of plasma. This leads to the total plasma selenium concentration in the United States varying from about 9 to 20g per 100ml. The mean serum selenium level of 18,597 persons reported by the Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) was 12.5ug per 100 ml, with 5th and 95th percentiles of 10 and 15 pg

per 100ml, respectively.7!

Based on these results, plasma or serum selenium concentrations of 8g per 100 ml or higher in healthy people should indicate optimization of the plasma selenoproteins. Concentrations greater than this merely indicate that the subjects are consuming selenomethionine. People with diseases may have alterations in their selenoprotein concentrations caused by their conditions;”?! so this value may not apply to them. For reference purposes, plasma concentrations of selenium in low-selenium regions of China are generally 2g per 100 ml or less. In New Zealand, they are 5-8 ug per 100ml, and in Europe, 5-10 pg per 100 ml. Concentrations greater than 50g per 100ml of plasma occur in high-selenium regions of China.

Dietary Reference Intakes Estimates of the human selenium requirement have been based on two studies. One was performed in

China in the early 1980s.°4) Men with a dietary intake

Assessment of Selenium Status The endpoint usually used to set recommended intake of selenium is optimization of selenoprotein concentrations. This is based on the assumption that full expression of selenoproteins will promote optimal physiological function and health. Inadequate dietary supply of selenium limits selenoprotein synthesis and results in depressed selenoprotein concentrations. When selenoproteins are optimized, provision of additional selenium will not cause their concentrations to increase. Instead, the additional selenium is excreted. Thus, optimization of the plasma _ selenoproteins (as representatives of all selenoproteins) has been used to determine the selenium requirement. There are two selenoproteins in plasma, selenoprotein P and extracellular glutathione peroxidase. When optimized, these proteins contain 6.4 and 1.7yg of

selenium per 100ml of plasma, respectively.!’"! Thus, the total selenium in the plasma selenoproteins approximately 8 1g per 100 ml of plasma.

A third pool of selenium is present in plasma proteins. It is selenomethionine distributed nonspecifically

is

of 11 ug of selenium per day were supplemented with selenium as selenomethionine for several months. The group that received a supplement of 30ug of selenium per day optimized its plasma glutathione peroxidase activity. Thus, a total intake (diet plus supplement) of 41 g/day optimized plasma selenoproteins. The other study was carried out in New Zealand in a group with a dietary selenium intake of 28 pg/day. Its results were more difficult to interpret because of the

high

basal

selenium

intake.

indicated a similar requirement that found in China.

However,

results

for optimization

to

In 2000, based on these two studies, the Institute of Medicine set the RDA for selenium at 55 g/day for adults of both sexes.?°! Table 2 shows the corresponding values for other subjects as well. These recommendations are for healthy people and are meant to satisfy the biochemical selenium requirements of the body. Further research will be necessary to determine whether there are special populations that need a greater selenium intake. Also, this RDA does not take into consid-

eration the possible pharmacologic use of selenium.

Selenium

650

Table 2 Recommended dietary allowances (RDAs) for selenium Group

Amount (g/day)

Children 1-3 yr old 4-8 yr old 9-13 yr old

20 30 40

Adolescents (14-18 yr old)

3D

Adults (=19 yr old)

55

Pregnant women

60

Lactating women

70

high-selenium yeast contains selenomethionine, it shares this property. Scientific experiments generally use inorganic selenium or selenomethionine, depending on the design of the experiment. Research is being performed on other forms of selenium such as Se-methylselenocysteine to evaluate their chemoprevention activity. Such forms are not generally available at present and cannot be recommended except for research purposes. f Toxicity

(From Ref.)

SUPPLEMENTATION Several forms of selenium are available for use as supplements. The two inorganic forms, selenite and selenate, are often used in animal experimental diets because they cannot be converted to selenomethionine,

and therefore tissue selenium concentrations reflect only selenoproteins. These forms have similar bioavailability (50-90%), but selenite is subject to reaction with intestinal contents. Also, selenite is an oxidant and can be

quite damaging when given in large quantities. Selenite is added to salt in some selenium-deficient regions of China and to fertilizer in Finland. Both these methods of supplementation have been shown to be effective in improving the selenium status of human populations. Selenomethionine makes up a large fraction of the normal dietary selenium. It is virtually completely bioavailable. It is less likely to cause acute toxicity than are the inorganic forms, although its toxicity is approximately the same as that of those forms under steady state conditions. Administration of selenium as selenomethionine can complicate interpretation of tissue selenium levels, and it is more expensive than the inorganic forms. High-selenium yeast preparations are available for selenium supplementation. These are proprietary products in which yeast is grown in high-selenium medium. Much of the selenium in the yeast is in the form of selenomethionine, but many minor forms are also present in variable amounts. Because of the relatively uncharacterized nature of the selenium in these yeast products, their usefulness in scientific experiments is minimal. Producers and marketers of these yeast preparations make various claims, but there is no good evidence for their special efficacy. These are often expensive. All these forms of selenium are effective as supplements in delivering selentum to human beings and animals. The inorganic forms have the advantage of being inexpensive. Selenomethionine allows the person to incorporate surplus selenium into protein. Because

Selenium can be toxic. Manifestations range from severe acute multiorgan failure after ingestion of milligram-togram quantities of selenious acid (selenite) to loss of hair and nails caused by chronic ingestion of more than a milligram of selenium per day for long periods. Studies carried out in a high-selenium region of China indicated that brittle nails and hair loss did not occur at intakes below 1 mg/day. Based on this result, the Institute of Medicine set a safe upper limit of 400ug/day for chronic selenium intake for

adults.°°! In practical terms, this is about 300 1g above the dietary intake of selenium in the United States. Thus, it allows room for supplements to be taken by those who believe they might be efficacious.

CONCLUSIONS Selenium is an essential element that has a variety of biochemical functions. It is lacking in the food supply of many

countries,

but there is no such evidence

in

North America. There is a need for research into the effects of marginal selenium intakes such as those in New Zealand, Scandinavia, and Europe. Also, studies

are needed to determine whether genetic and/or dis- ease conditions raise the selenium requirement. Finally, _ claims have been made that ingestion of pharmacologic amounts of selenium can prevent cancer. Studies are underway to assess that possibility.

ACKNOWLEDGMENTS The authors are grateful to Kristina E. Hill, Ph.D., for helpful discussions. The work of the authors is supported by NIH grant DK58765.

REFERENCES 1. Allaway, W.H. An overview of distribution patterns of trace elements in soils and plants. Ann. N. Y. Acad. Sci. 1972, 199, 17-25.

Selenium

Zz

651

Painter, E.P. The chemistry and toxicity of selenium compounds, with special reference to the selenium problem. Chem. Rev. 1941, 28, 179-213. Schwarz, K.; Foltz, C.M. Selenium as an integral

part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 1957, 79, 3292— 3293: Keshan Disease Research Group. Observations on effect of sodium selenite in prevention of Keshan disease. Chin. Med. J. 1979, 92, 471-476. Kobayashi, Takayama,

16.

Arnér, E.S.J.; Holmgren, A. Physiological func-

is

tions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 2000, 267, 6102-6109. Matsui, M.; Oshima, M.; Oshima, H.; Takaku,

K.; Maruyama, T.; Yodoi, J.; Taketo, M.M, Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. Dev. Biol. 1996, 178, 179-185.

18.

Y.; Ogra, Y.; Ishiwata, K.; H.; Aim, N.; Suzuki, K.T. Seleno-

sugars are key and urinary metabolites for selenium excretion within the required to low-toxic range. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15,932-15,936. Driscoll, D.M.; Copeland,

P.R. Mechanism

regulation of selenoprotein Rev. Nutr. 2003, 23, 17-40.

synthesis.

molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr. Rev. 2002, 23, 38-89.

19:

hormone

21t

Zo.

Vanderpas, J.B.; Contempre, B.; Duale, N.L.; Goossens, W.; Bebe, N.; Thorpe, R.; Ntambue, K.; Dumont, J.;: Thilly; .CHS%.-Diplock, A:T,

Iodine and selenium deficiency associated with cretinism in northern Zaire. Am. J. Clin. Nutr. 1990, 52, 1087-1093. Schomburg, L.; Schweizer, U.; Holtmann, B.; Flohe, L.; Sendtner, M.; Kohrle, J. Gene disrup-

tion discloses role of selenoprotein P in selenium delivery to target tissues. Biochem. J. 2003, 370, 397-402. Hill, K.E.; Zhou, J.; McMahan, W.J.; Motley, A.K.; Atkins, J.F.; Gesteland,

Deletion of selenoprotein P alters distribution of selenium in the mouse. J. Biol. Chem. 2003, 278, 13,640-13,646.

Chu,

F.F.;

Doroshow,

J.H.;

Esworthy,

R.S.

Cheng, W.-H.; Ho, Y.S.; Valentine, B.A.; Ross, PVAs Conhs,0G.E,, Jt; Leg ox.G. Cellular

Imai, H.; Hirao, Mizukura, Y.;

Res. Commun. 2003, 305, 278-286. Ursini, F.; Heim, S.; Kiess, M.; Maiorino, M.; Roveri, A.; Wissing, J.; Flohé, L. Dual function

of

the

23,

selenoprotein

PHGPx

during

sperm

maturation. Science 1999, 285, 1393-1396.

Cell

14.

Arthur, J.R. The glutathione peroxidases. Mol. Life Sci. 2000, 57, 1825-1835.

IS;

Mustacich, D.; Powis, G. Thioredoxin reductase. Biochem. J. 2000, 346, 1-8.

R.F.; Burk, R.F.

Hafeman, D.G.; Hoekstra, W.G. Lipid peroxida-

tion in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. J. Nutr. 1977, 107, 666-672.

24.

Burk, R.F. Biological activity of selenium. Annu. Rev. Nutr. 1983, 3, 53-70.

a:

Mostert, V.; Hill, K.E.; Burk, R.F. Loss of activity of the selenoenzyme thioredoxin reductase causes induction of hepatic heme oxygenase-1. FEBS Lett. 2003, 541, 85-88. Taylor, P.R.; Li; B.; Dawsey, S.M-; Li, J.Y;; Yang, C.S.; Guo, W.; Blot, W.J. Prevention of

F.; Sakamoto, T.; Sekine, K.; Saito, M.; Kitamoto, T.;

Hayasaka, M.; Hanaoka, K.; Nakagawa, Y. Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene. Biochem. Biophys.

1.

thyroid

16,651.

glutathione peroxidase is the mediator of body selenium to protect against paraquat lethality in transgenic mice. J. Nutr. 1998, 128, 1070-1076.

12:

on

ity to hyperoxia. J. Biol. Chem. 1997, 272, 16,644—

Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI. J. Biol. Chem. 1993, 268, 2571-2576.

eh

deficiency

240S—243S.

20.

Lescure, A.; Fagegaltier, D.; Carbon, P.; Krol, A. Protein factors mediating selenoprotein synthesis.

10.

selenium

metabolism in rats. Am. J. Clin. Nutr. 1993, 57,

Annu.

deficient in cellular glutathione peroxidase develop normally and show no increased sensitiv-

Beckett, G.J.; Nicol, F.; Rae, P.W.; Beech, S.; Guo, Y.; Arthur, J.R. Effects of combined iodine

and

and

Curr. Protein Pept. Sci. 2002, 3, 143-151. Kryukov, G.V.; Castellano, S.; Novoselov, S.V.; Lobanov, A.V.; Zehtab, O.; Guigo, R; Gladyshev, V.N. Characterization of mammalian selenoproteomes. Science 2003, 300, 1439-1443. Ho, Y.-S.; Magnenat, J.-L.; Bronson, R.T.; Cao, J.; Gargano, M.; Sugawara, M.; Funk, C.D. Mice

Bianco, A.C.; Salvatore, D.; Gereben, B.; Berry, M.J.; Larsen, P.R. Biochemistry, cellular and

26.

esophageal

out

cancer:

the

nutrition

intervention

trials in Linxian, China Linxian Nutrition Intervention Trials Study Group. Cancer Res. 1994, 54, 2029s—203 1s. Clark, L.C.; Combs, G.F., Jr.; Turnbull, B.W.; Slate, E.H.; Chalker, D.K.; Chow, J.; Davis, L.S.; Glover, R.A.; Graham, G.F.; Gross, E.G.; Krongrad, "A.; Lesher, J.L., Jr; “Park, H.K.; Sanders, B.B., Jr.; Smith, C.L.; Taylor, J.R.

Effects of selenium supplementation

for cancer

Selenium

652

prevention

in patients with carcinoma

of the

skin. A randomized controlled trial. Nutritional prevention of cancer study group. J. Am. Med.

28.

Assoc. 1996, 276, 1957-1963. Duffield-Lillico, A.J.; Slate, E.H.; Reid, M.E.; Turnbull, B.W.; Wilkins, P.A.; Combs, G.F., Jr.; Park, H.K.; Gross, E.G.; Graham, G.F.; Stratton, M.S.; Marshall, J.R.; Clark, L.C. Selenium

supplementation

29:

30.

ilk

secondary

prevention

in

specific

and

non-specific

forms.

Biofactors 2001, 74, 107-114. Niskar, A.S.; Paschal, D.C.; Kieszak, S.M.; Flegal, K.M.; Bowman, B.; Gunter, E.W.; Pirkle, J.L.; Rubin, C.; Sampson, E.J.; McGeehin, M.

Serum selenium levels in the US population: third national health and nutrition examination

Biol. Trace Elem. Res. 2003,

33.

Burk, R.F.; Early, D.S.; Hill, K.E.; Palmer, LS.;

34.

Boeglin, M.E. Plasma selenium in patients with cirrhosis. Hepatology 1998, 27, 794-798. Yang, G.-Q.; Zhu, L.-Z.; Liu, S.-J; Gu, L.-Z.; Qian,

P.-C.;

selenium

Huang,

J.-H.;

requirements

Lu,

in China.

M.-D.

Human

In Selenium

in Biology and Medicine; Combs, G.F., Jr., Spallholz, J.E., Levander, O.A., Oldfield, J.E., Eds.; AVI: New York, 1987; 589-607.

of

nonmelanoma skin cancer in a randomized trial. J. Natl. Cancer Inst. 2003, 95, 1477-1481. Combs, G.F., Jr.; Spallholz, J.E.; Levander, O.A.; Oldfield, J.E. In Selenium in Biology and Medicine; AVI: New York, 1987. Beck, M.A.; Kolbeck, P.C.; Rohr, L.H.; Shi, Q.; Morris, V.C.; Levander, O.A. Benign human enterovirus becomes virulent in selenium-deficient mice. J. Med. Virol. 1994, 43, 166-170. Burk, R.F.; Hill, K.E.; Motley, A.K. Plasma

selenium

SDs

and

1984-1994.

survey,

91, 1-10.

35.

Institute of Medicine. In Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids; National Academy Press: Washington, DC, 2000.

FURTHER READINGS Burk, R.F.; Nutrition

Levander, O.A. Selenium. In Modern in Health and _ Disease, 9th Ed.;

Shils, M.E., Olson, J.A., Shike, M., Ross, A.C., Eds.; Lea and Febiger: Philadelphia, 1999; 265-276. Sunde, R.A. Selenium. In Present Knowledge in Nutrition, 8th Ed.; Bowman, B.A., Russell, R.M., Eds.; ISLI Press: Washington, DC, 2001; 352-365.

Shiitake (Lentinus edodes) Solomon P. Wasser University of Haifa, Mount Carmel, Haifa, Israel

INTRODUCTION Shiitake mushroom,

popular in nutritional and medicinal products throughout Asia, Europe, and North America. the common

Japanese name

for

Lentinus edodes (Fig. 1), derives from the mushroom associated with the shii tree (Castanopsis cuspidate Schottky) and take, the Japanese word for mushroom (Table 1). Because Japan is the world leader in production of this type of mushroom, the mushroom is now widely known by this name. These mushrooms are renowned in Far East countries (e.g., Japan, China, Korea) as a food and medicine for thousands of years. In the year 199 a.p., Kyusuyu, a native tribe of Japan, offered the Japanese Emperor Chuai a shiitake mushroom. Even older documents record its use in ancient China, where it was referred to as “ko-ko’’ or “hoang-mo.’"!! The cultivation of this mushroom has been practiced for a thousand years, with its cultivation originating in China during the Sung Dynasty (960-1127). Both history and legend credit Wu San Kwung as the originator of shiitake cultivation. Almost every mushroom-growing village in China

has a temple in his honor.”! In 1313, Chinese author Wang Cheng recorded shiitake-growing techniques in his Book of Agriculture. He described how to select a suitable site, choose appropriate tools, and cut down the trees on which one could cultivate the mushrooms. He outlined the basic methods as follows: Cut the bark with a hatchet and cover the logs with soil. After lyr, top the soil and water frequently. Beat the logs with

a wooden club to induce mushroom production. The mushrooms will appear after a rain.) Shiitake mushroom cultivation techniques were probably introduced to Japanese farmers by the

Chinese between 1500 and 1600 ap“! At present, shiitake is one of the five most cultivated

edible mushrooms in the world.) Its production (2 million tons) is second only to button mushroom Agaricus bisporus. Grown mainly in East Asia, shiitake is now arousing interest worldwide. Increasing markets have been spawned, partly by the exotic and wellappreciated taste of shiitake, and partly by advances in research that has demonstrated its significant medicinal properties. Shiitake mushroom is becoming

Solomon P. Wasser is at the University of Haifa, Mount Carmel, Haifa, Israel.

Encyclopedia of Dietary Supplements DOK: 10.1081 /E-EDS-120024880 Copyright © 2005 by Marcel Dekker. All rights reserved.

HABITAT AND DISTRIBUTION Gregarious on fallen wood of a wide variety of deciduous trees, especially shii, oak, chestnut, beech, maple, sweet gum, poplar (aspen, cottonwood), alder, hornbeam, ironwood, chinquapin, mulberry (Castanopsis cuspidate, Quercus, Castanea, Fagus, Acer, Liquidamber, Populus, Diospyros, Alnus, Carpinus, Morus) in a warm, moist climate. Most of these are raised for artificial cultivation of shiitake mushroom. L. edodes occurs naturally throughout Southeast Asia. It has been reported from China, Japan, Korea, Vietnam, Thailand, Burma, North Borneo, the Philippines, Taiwan, and Papua New Guinea.!7"’]

EDIBILITY AND NUTRITIONAL VALUE Shiitake

are traditionally

well-known

edible mush-

rooms of high nutritious value. Raw or dried forms, used in Chinese curative powers of shiitake mushroom,

are legendary. It was stated in Ri Youg Ben Cao, Vol. 3 (1620), written by Wu-Rui of the Ming Dynasty, “shiitake accelerates vital energy, wards off hunger, cures colds, and defeats body fluid energy.’’ In later years, it was found that the mushroom contained various important nutrients. Moreover, recent scientific investigations have isolated many compounds and have found evidence of their health promotion activities,{1-7-101 Shiitake mushrooms have excellent nutritional value. Their raw fruit bodies include 88-92%

water,

protein, lipids, carbohydrates as well as vitamins and minerals. It should be noted that amounts of nutrients and biologically active compounds differ in various strains and are affected by substrate, fruiting conditions, and methods of cultivation. On a dry weight basis, they have a relatively high nutritional value when compared to commonly consumed vegetables. Dried shiitake mushrooms are rich in carbohydrates and protein. They contain 58-60% carbohydrates, 20-23% protein (digestibility of 80-87%), 9-10% fiber, 3-4% lipids, and 4-5% ash. The mushroom

isa

provitamin

D»

good

source

of vitamins,

especially

653

Shiitake (Lentinus edodes)

654

LC

eat

Fig. 1 Shiitake mushroom [L. edodes (Berk.) Singer]. a—fruit body, b—spores, c—basidia, d—cheilocystidia, e—elements of pileal cuticle.

(ergosterol), 325mg%, which under ultraviolet (UV) light and heat yields calciferol (vitamin Dz). It also contains B vitamins, including B, (thiamine), B> (ribo-

flavin),

By.

(niacin),

and

pantothenic

acid.!1,3.9.10]

Minerals found include Fe, Mn, K, Ca, Mg, Cd, Cu,

P, and Zn. Analysis of dried cultured shiitake mycelium gives the following mineral concentrations (in mg/g of dry weight): K, 15.1; Ca, 22; Mg, 44-78; Matai. [email protected]; Bepe2.36;iNi, 152.5;[email protected]; 281; Zn, 282; Ge, 3; Br, 11.4; and Sr, 164.

Water-soluble polysaccharides amount to 1-5% of the dry weight of the shiitake mushroom. In addition to glycogen-like polysaccharides, (1-4)-,(1-6)-a-b-glucans and antitumor polysaccharides, lentinan, (1-3)-, (1-6)-B-bonded heteroglucans, heterogalactans, heteromannans, xyloglucans, etc., have been identified. The mushrooms’ indigestible polysaccharides, which serve as dietary fiber, include heteroglycan, polyuronide, B-glucan as well as chitin. Among the free sugars present are trehalose, glycerol, mannitol, arabitol,

mannose, and arabinose.!',’-'°l In shiitake mushrooms, dietary fiber consists of water-soluble materials such as B-glucan and protein and water-insoluble substances extractable only with

salts, acids, and alkalies such as polyuronide

(acidic

polysaccharide), hemicellulose, B-glucan with heterosaccharide chains, lignin, and chitin present as cell wall constituents. The fatty acids account for 3.38% of the total lipids.?-'©! Their composition is as follows: linoleic acid _ (18:2), 72.8%; palmitic acid (16:0), 14.7%; oleic acid (18:1), 3.0%; tetradecenoic acid (14:1), 1.6%; stearic | acid (18:0), 0.9%; and myristic acid (14:0), 0.1%. The aroma

components

include alcohols, ketones,

sulfides, alkanes, fatty acids, etc. The major volatile flavor contributors are matsutakeol (octen-1-ol-3) and ethyl-n-amyl ketone. The characteristic aroma of shiitake

mushrooms

was

identified

as

1,2,3,5,6-

pentathiepane. According to Mizuno,”! the components responsible for the delicious flavor are monosodium glutamate,

5’-nucleotides,

free

amino

acids,

lower

molecular weight peptides, organic acids, and sugars. Their relative ratios are responsible for the variation in flavor naturally seen in this mushroom. Organic acids contributing to the flavor of shiitake mushroom include malic acid, fumaric acid, «-keto-glutaric acid, oxalic acid, lactic acid, acetic acid, formic acid, and

glycolic acid.

Shiitake (Lentinus edodes)

Table 1

655

Mycological data for shiitake

Basidiomycotina Homobasidiomycetes Agaricomycetiae Agaricales Family: Pleurotaceae Genus: Lentinus

Lentinus edodes (Berk.) Singer, Mycologia, 1941 33, 451 (Fig. 1) Basyonym Agaricus edodes Berk., J. Linn. Soc. Bot. 1877, 16, 50. Synonyms Collybia shiitake Schroet., Garenfl. 1886, 35, 105. Armillaria edodes (Berk.) Sacc., Syll. Fung. 1887, 5, 79. Agaricus russaticeps (Berk.) apud Cooke, Grevillea 1889, 16, 106. Lepiota shiitake (Schroet.) Tanaka, Japan Bot. Mag. 1889, 3, 159.

Lentinus tonkinensis Pat., J. Bot. Paris 1890, 4, 14. Mastaleucomyces edodes (Berk.) O. Kuntze, Rev. Gen. Pl. 1891, 2, 861. Pleurotus russaticeps (Berk.), Sacc., Syll. Fung. 1891, 9, 48. Cortinellus shiitake (Schroet.) P. Henn., Not. Knigl. Bot. Gard. Mus. Berl. 1899, 2, 385.

Tricholoma shiitake (Schroet.) Singer, Ann. Mycol. 1936, 34, 332. Cortinellus edodes (Berk.) S. Ito et Imai, Journ. Fax. Agr. Hokkaido Imp. Univ. 1938, 43, 55.

Lentinula edodes (Berk.) Pegler, Kavaka 1975, 3, 20. English names

Black forest mushroom, black oak mushroom, golden oak mushroom, snake butter, pasania mushroom, oakwood mushroom, Japanese forest mushroom

Japanese name

Shiitake

Chinese names

Shiang-gu, Shing ku, Hua Gu, Xiangu, Hoang-mo

A detailed description of the shiitake mushroom can be found in Refs.

RELATED

SPECIES

[1.4.7]

stages. The first stage involves the preparation of the fruiting culture, stock culture, mother spawn, and plant-

Artificial Cultivation

The shiitake mushroom is commonly considered ‘as a species of the genus Lentinus Fr. As noted in the below synonymy, L. edodes has at various times been assigned to 10 genera (Agaricus, Collybia, Armillaria, Lepiota, Lentinus, Mastaleucomyces, Pleurotus, Corti-

nellus, Tricholoma, and Lentinula). While specialists in medicinal mushrooms and cultivation are familiar with shiitake mushrooms as L. edodes, some alternain are discussed classifications tive taxonomic Refs."!-9! Although the mushroom had been grown

ing spawn, and the second stage entails the preparation of the growth substrates for cultivation. Currently, the methods most widely adopted for commercial production are wood log and synthetic sawdust bag b816 A discussion of the cultivation methods used is beyond the scope of this review. Interested readers may refer to the references cited above; growth parameters for cold- and warm-weather strains are given in Ref.)

PRECLINICAL STUDIES

in Asian countries for centuries, the interest there, as

Therapeutic Applications

well as in the Western countries, has increased rapidly since World War II, especially in the last 15-20 yr. Its cultivation is now a worldwide multimillion dollar industry. The process for producing shiitake mushroom fruiting bodies (Fig. 2) is the same as for other cultivated edible mushrooms and can be divided into two major

This section mainly discusses preclinical in vitro and in vivo (animal) studies. Shiitake is one of the best-known and best-characterized mushrooms used in medicine. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide .

Shiitake (Lentinus edodes)

656

Fig. 2

lentinan,

shiitake

Shiitake mushroom [L. edodes (Berk.) Singer]: cultivated fruiting bodies.

mushroom

mycelium,

and culture

media extracts (LEM, LAP and KS-2),!7?-!®11

Anticarcinogenic and Antitumor Effects Using methods

of fractionation

and purification of

a right-handed triple helix.'””'7'®! It is water soluble, heat stable, and alkali labile. That is, B-p-glucan binds to lymphocyte surfaces or serum-specific proteins, which activate macrophage, T-helper cells, natural killer (NK) cells, and other effector cells. All these increase the production of antibodies as well as interleukins (IL-1, IL-2) and interferon (IFN-y) released

polysaccharides, Chihara et al.?°°7! isolated a water-

upon activation of effector cells.!'?:*4] Thus, the carci-

soluble antitumor polysaccharide from fruiting bodies of shiitake, which was named “lentinan’’ after the genus Lentinus to which the shiitake mushroom belongs. Chihara was one of the first to report on the antitumor properties of the mushroom, stating that lentinan “was found to almost completely regress the solid type tumors of Sarcoma 180 and several kinds of tumors including methylchloranthrene-induced

nostatic effect of lentinan results from the activation of the host’s immune system. In animal testing of carcinostatic activity, intraperitoneal (i.p.) administration is used, but oral administration (p.o.) is occasionally effective. The purified polysaccharide has been shown in animal studies to produce strong tumor regression and even the disappearance of sarcoma tumors in 5 weeks,

fibrosarcoma in synergic host-tumor system A.’’?!77]

ascite hepatoma

The antitumor effect of lentinan was originally confirmed by using Sarcoma 180 transplanted in CD1/ICD mice.?°! Later, it showed prominent antitumor activity not only against allogenic tumors such as Sarcoma 180, but also against various synergic and autochthonous tumors, and it prevented chemical and viral oncogenesis.?*! The molecular formula of B-b-glucan lentinan is (C6H;9Os),, and the mean mole-

as well as a number of other experimentally induced cancers in allogenic, syngeneic, and autologous hosts. It also exhibits preventive activity against chemical carcinogenesis. Injections of lentinan into mice produced either an 80% reduction in tumor size or complete regression in most of the animals tested. Moreover, an intact immune system and a functioning thymus gland were found to be requisite for its anticancer effect.!!!:'7] When immunosuppressive agents such as §-benzylthioguanosine or X-radiation were given with lentinan, the antitumor effect decreased. The polysaccharide has also been found to restore the enzyme

cular weight is about one million (—5 x 10°Da); [a]pb + 20-22° (NaOH). Its structure was confirmed as fB-(1-3)-p-glucopyranan with a branched chain of B-(1-6)-monoglycosyl (branching degree: 2.5°), showing

134,U8195]

and

Ehrlich

carcinoma

Shiitake (Lentinus edodes)

657

activity of X-prolyl-dipeptidyl-aminopeptidase, which can be depressed in cancer patients and in mice with implanted tumors,7° Laboratory tests seem to indicate a role for the adrenal-pituitary axis and central peripheral nervous system including serotonin, SHT, histamine, and cate-

cholamies in lentinan’s antitumor activity.{1-10!7241 The oral administration of the polysaccharide to AKR mice exerted strong antitumor activity resulting in raised levels of lymphocytokines, such as IFN-y, tumor necrosis factor (TNF-«) and IL-1 «. Tissue cultures of murine macrophages CRL-2019, B-lymphocytes HB-284, and T-lymphocytes DRL-8179, which were

treated

with

lentinan,

showed

high

levels

of

activation using flow cytometry. Lentinan-activated immunocytes, particularly the T-helper cells, might render the physiological constitutions of the host highly cancer- and infection resistant. Adoptive immunotherapy of the immunodeficient mice such as the nude (athymic) mice, B-cell deficient mice, and severe combined immunodeficient (SCID) mice via the transfer of the lentinan-activated immunocytes resulted in the inhibition of tumor growth. Lentinan appeared to represent a unique class of host defense potentiators (HDP), protecting the hosts from the side effects of conventional therapeutic measures and improving various kinds of immunological parameters with no toxic side effects in animal models.!!9-7427781

LEM and LAP Extracts from Shiitake Mushroom

Mycelium and Culture Media L. edodes mycelium (LEM) is prepared from an extract of the powdered mycelia of the shiitake mushroom. Its yield is about 6-7g/kg of medium. The precipitate obtained from a water solution of the mycelium by adding four volumes of ethanol was named LAP. The yield of LAP is ~0.3 g/g of LEM. L. edodes mycelium and LAP are glycoproteins containing glucose, galactose, xylose, arabinose, mannose, and fructose.!! The former also contains various

nucleic acid derivatives, vitamin B compounds especially B, (thiamine), B> (riboflavin), and ergosterol.!”"®) In

1990,

an

immunoactive

substance,

EP3,

was

obtained by fractionation of LEM. EP3 is a lignin complex composed of about 80% lignin, 10% carbohydrates, and 10% protein. After removal of the last two components, biological activity was not affected, but when lignin is removed, activity was reduced. Therefore, the active substance is believed to be a water-soluble

lignin containing numerous carboxyl groups.”"°! Both LEM and LAP have demonstrated strong antitumor activities orally and by injection to animals and humans. They were shown to act by activating the

host’s

immune

system

and

are

treatment of hepatitis B.?:1414

also

useful

for the

KS-2-a-Mannan Peptide Polysaccharide KS-2 (MW 6-9.5 x 10* [aJp + 62°: C = 0.5, water) was obtained by extraction of cultured mycelia of shiitake mushroom (strain KSLE 007) with hot water, followed by precipitation with ethanol.?:!9! The product is an o-mannan peptide containing the amino

acids serine, threonine, alanin,

and proline (as well as residual amounts of the other amino acids). The polysaccharide was shown"! to be effective

on

Sarcoma

180 and

Ehrlich’s

carcinoma,

either i.p. or p.o., and to act via interferon-inducing activity. The acute LCs) or KS-2 was found to be extremely low in mice, more than 12,500mg/kg when administered orally. The mechanism of action of KS-2 is not yet clear, but the results showed no direct KS-2 cytocidal effect against the tumor cells in vitro. Its antitumor activity was observed to be higher at the lower inoculum size of tumor cells, regardless of the routes of KS-2 admin-

istration (60% survival rate at 5 x 10° tumor cells/ mouse,

10% survival at 1 x

10° tumor cells /mouse).

The results also showed that the antitumor activity of KS-2 in mice was always accompanied by the induction of interferon in the sera. Furthermore, preliminary findings indicated that macrophages obtained from KS-2 treated mice exhibited tumoricidal activity,1°-16°l and it was reported that macrophage activation became tumoricidal when incubated in vitro with interferon. Considering these findings, the antitumor activity of KS-2 may be explained by macrophage activation with or without interferon induced by KS-2.

Immune-Modulating Effects As was stated earlier, lentinan and other polysaccharides from shiitake mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. Lentinan, for example, appears to act as an HDP, which is able to restore or augment the responsiveness of host cells to lymphocytokines, hormones, and other biologically active substances by stimulating maturation, differentiation, or proliferation of cells involved

in host defense mechanisms."'?4! Host defense potentiators are functionally different from biological response modifiers. Thus, lentinan is able to increase host resistance against various kinds of cancer and infectious diseases, including acquired immuno deficiency syndrome (AIDS).!”78 The initial interactions of lentinan in the human body or animals are not presently known. However,

Shiitake (Lentinus edodes)

658

T cell-, B cell-, and macrophage-dependent immune reactivities. Its antitumor effect is abolished by neonatal thymectomy and decreased by the administration of antilymphocyte serum, supporting the concept that the polysaccharide requires immunocompetent T-cell compartments. The effect of lentinan was also inhibited by antimacrophage agents, e.g., carrageenan. Unlike other

the disappearance of malignancy, while in another group their condition deteriorated or diminished.®?! The polysaccharide can activate the normal and alternative pathways of the complement system and can split C3 into C3a and C3b enhancing macrophage activation.?4) Many biological reactions are accelerated and induced by lentinan, including the very important phenomenon of infiltration of eosinophils, neutrophils, and granulocytes around target tissues. Fig. 3 shows early responses initiated by it and possible pathways: for inflammatory reactions. Lentinan’s immune-activating ability may be linked

well-known immunostimulants, lentinan is in a unique

with its modulation

class of distal tubular (DT)-cell-oriented assistant, in which macrophages play some part.!7119*4]

known to play a role in tumor growth. Aoki! showed that the antitumor activity of lentinan is strongly reduced by administration of thyroxin or hydrocortisone. It can also restore tumor-specific antigen-directed delayed-type hypersensitivity reaction (DTHR). Lentinan is not formally included among the nonspecific immunostimulants (RES stimulants), but it augments the induction of antigen-specific cytotoxic T-lymphocytes, macrophages, and other nonspecific immune responses. Possible immune system regulating actions of lentinan were summarized by Chihara et al.°*! and are seen in Fig. 4.

there is a transitory but notable increase in several serum protein components in the a- and B-globulin region, namely, complement C3, hemopexin, and ceruloplasmin.!71019*4] Lentinan

stimulates

various

kinds

of NK

cell-,

For example, lentinan can activate NK cells in vitro

in the same concentrations that are achieved in the blood plasma of patients treated clinically with lentinan.°!624] Natural killer-cell activity is involved in tumor suppression, and while these cells do not stimulate T-killer cell activity or do so only under certain conditions, they are strong T-helper cell stimulants both in vitro and in vivo.":”'!%194] Using the blood of healthy donors and cancer patients, some authors have shown that the polysaccharide is able to stimulate peripheral blood lymphocytes in vitro to increase IL-2mediated lymphokine-activated killer cell (LAK-cell) and NK cell activity at levels achievable in vivo by administration of clinical doses of lentinan. It has been shown to inhibit suppressor T cell activity in vivo and to increase the ratio of activated T cells and cytotoxic T-cells in the spleen when administered to gastric cancer patients undergoing chemotherapy.!710~4! Many interesting biological activities of lentinan have been reported including: a) an increase in the activation of nonspecific inflammatory responses such as acute phase protein (APP) production, b) vascular dilation and hemorrhage in vivo, c) activation and generation of helper and cytotoxic T-cells, d) augmentation of immune mediators like IL-1 and IL-3, colony stimulating factor(s), and migration inhibitory factor, and e) increasing the capacity of peripheral blood mononuclear (PBM) cells of patients with gastric cancer and producing IL-la, IL-1, and a TNF-a.!7-1019.24.271 In an in vivo study of rats with peritonitis, combined lentinan-gentamicin treatment had a significantly better survival rate than the controls. Lentinan activated the peritoneal macrophages’ secretory activity of active oxygen and produced cytokines, thus enhancing the ability of polymorphonuclear leukocytes (PMNs) to produce active oxygen, which has a bactericidal effect.°" It also increases peritoneal macrophage cytotoxicity against metastic tumor cells in mice, but not against a highly metastic tumor type.27! Some patients treated with lentinan for carcinomatous pleuritis or carcinomatous peritonitis have improved with

Cardiovascular

of hormonal

factors, which

are

Effects

The major cause of death in Western countries is coronary artery disease, a primary risk factor for which hypercholesterolemia is a factor contributing to hardening of the arteries. In humans, 50% or more of the total cholesterol is derived from de novo synthesis.183>761 It is known that shiitake mushroom is able to lower blood serum cholesterol (BSC) via a factor known as eritadenine (also called “‘lentinacin’’ or “lentysine’’). Apparently, eritadenine reduces BSC in mice, not by the inhibition of cholesterol biosynthesis, but by the acceleration of the excretion of ingested cholesterol and its metabolic decomposition. It has been shown to lower blood levels of cholesterol and lipids in animals. When added to the diet of rats, eritadenine (0.005%) caused a 25% decrease in total cholesterol in as little as one week. The cholesterol-lowering activity of this substance is more pronounced in rats fed a highfat diet than in those on a low-fat diet. Although feeding studies with humans

have

indicated

a similar effect,

further research is needed. Hobbs!!!° and Yang et al, have shown that shiitake mushrooms lowered BSC levels. Various studies have confirmed!'7!°!9 that the mushroom can lower blood pressure and free cholesterol in plasma, as well as accelerate the accumulation of lipids in the liver by removing them from circulation.

Shiitake (Lentinus (Lenti, dd edodes)

659

Macrophage

APPIF, / CSF, IL-1, GAF, SAA-inducer

IL-3, MIF, IPA

Vascular

O

Synovial

fy

fibroblast

T-cell

Hepatocyte

NN

“/- \e

Prostaglandin

collagenase

endothelial

cell

Acute-phase proteins

CTL NK Activated

macrophage

Fig. 3. Early responses initiated by lentinan and a possible pathway for inflammatory reactions. APPIF, acute phase proteininducing factor; VDHIF, vascular dilatation and hemorrhage-inducing factor; CSF, colony stimulating factor; MIF, migration inhibitory factor; GAF, glucocorticoid antagonizing factor; SAA, serum amyloid A; IPA, plasminogen activator inducer; VPF, vascular permeability factor; CTL, cytotoxic T-lymphocytes; NK, natural killer cells. [16]

Hepatoprotective Effects

important to protect AIDS patients from these various

The injection of LEM slowed the growth of cancerous liver tumor in rats."°!837] A polysaccharide fraction from shiitake mushrooms demonstrated liver protection in animals as well as the ability to improve liver function and enhance the production of antibodies to

hepatitis B.!”7>!

.

Lentinan improved serum glutamic pyruvic transaminase (SGPT) and completely restored GPT levels in the livers of mice with toxic hepatitis. Crude extracts of shiitake mushroom cultures have demonstrated liver-protecting actions.!!0!'8°I

Antiviral, Antibacterial, and Antiparasitic Effects

Lentinan various

and kinds

its derivatives of bacterial,

are

viral

effective

against

(including

AIDS),

and parasitic infections.!”!%'*8] An important area of this polysaccharide research deals with its ability to mobilize the body’s humoral immunity to ward off bacterial infections resistant to antibiotics.!”) Many cancer and AIDS patients die of opportunistic infections due to immunodysfunction.!”771 It is extremely

infections. According to Ref.°*!, in vitro studies show that lentinan, when used in combination with azido-

thymidine (AZT), suppressed the surface expression of human immunodeficiency virus (HIV) on T cells more so than did AZT alone. Lentinan and the sulfated form exhibited potent in vitro anti-HIV activity resulting in inhibition of viral replication and cell fusion. AIDS therapy must include a strategy to enhance the immune system. Among the various therapeutic approaches used, prevention of the development of AIDS symptoms in carriers should be stressed. Based on these in vitro studies, it is possible that such prevention may be realized by the use of HDPs such as lentinan or its related substances. For example, LEM is also useful in the treatment of AIDS.

It has been shown to inhibit HIV infections of cultured human T cells, and it potentiates the effects of AZT against viral replication in vitro. The mechanism of its action is not known for certain, but the extract was found to activate macrophages and stimulate the production of IL-1.!7197°° I Water-soluble lignins from EP3 and EPS4 from shiitake mushroom mycelium have shown antiviral and immunomodulating effects.“°! A water-soluble extract

Shiitake (Lentinus edodes)

660

General Potentiation of Host Resistance

- Colony Stimulating

C3a Anaphylatoxin

Factor

Serum Protein Ane bene Grete:

non-specific

TARGET TUMOR

Precursor.

Cytotoxic-T

Le Suppressor T-lymphocyte

DESTRUCTION

thats

Q v a

:

B-Lymphocytes

Natural Killer Cells Fig. 4 Possible mode of action of lentinan as HDP. (From Ref.3!_)

of mycelium known as JLS and JLS-18 has the ability to block the release of herpes simplex virus type | in animals JLS-18-consisting of 65-75% lignin, 15-30% polysaccharides, and 10-20% protein has inhibited the herpes virus both in vitro and in vivo.44

It should be noted that a protein fraction of shiitake mushroom fruiting bodies, labeled fruiting body protein (FBP), prevented the infection of plants with tobacco mosaic virus (TMV). The binding of the virus to the plant cells was inhibited by FBP.!7?-!°

In addition, lentinan has shown: a) antiviral activity

in mice against vesicular stomatitis virus (VSV) encephalitis virus, Abelson virus, and adenovirus type 12; b) stimulated nonspecific resistance against respiratory viral infections in mice; c) conferred complete protection against an LD75 challenge dose of virulent mouse influenza A/SW15; d) enhanced bronchoalveolar macrophage activity; e) increased resistance against the parasites Schistosoma japonicum and S. mansoni; f) exhibited activity against Mycobacterium tuberculosis bacilli resistant to antituberculosis drugs, Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Candida

albicans,

and

Saccharomyces

cerevisiae;

and h) increased host resistance to infections with the potentially lethal Listeria monocytogenes. Antibacterial polyacetylene compounds, centinamycin A and B, have also been identified in shiitake

mushroom.

Eritadenine,

cholesterol

a compound

that

affects

metabolism, also processes antiviral properties.!”!°>>!

HUMAN CLINICAL STUDIES AND MEDICINAL USES In the last 15-20 yr, shiitake mushroom has been subject to various clinical studies in humans and is thought to be beneficial for a wide variety of disorders including

different

types

of cancer,

heart

disease,

hyperlipidemia (including high blood cholesterol), hypertension, infectious disease, and hepatitis. The mushroom is used medicinally for diseases involving depressed immune function (including AIDS), cancer, environmental allergies, fungal (especially Candida) infection, frequent flu and colds, bronchial inflamma-

tion, and regulating urinary incontinence. It was shown that the success of immune adjuvant in therapy depends on the type of cancer (location) being

treated,

the

individual’s

general

health,

Shiitake (Lentinus edodes)

immunological and hormonal status as well as the individual’s constitution. Lentinan was demonstrated to have antitumor activity as well as to increase the survival time of patients with inoperable gastric cancer!!! and women with recurrent breast cancer following surgical therapy (for details on protocols, see Refs.!”'°!*!), According to Refs.!*-*9], when the polysaccharide is administered once or twice a week with chemotherapy to a patient with progressive cancer but with no serious liver, kidney, or bone marrow dysfunction, it produced a statistically significant improvement in immune and anticancer activity when compared to chemotherapy alone. Two hundred seventy-five patients with advanced or recurrent gastric cancer were given one of two kinds of chemotherapy (mitomycin C with 5fluorouracil or tegafur) either alone or with lentinan injections. Statistically, the best results were obtained when lentinan was administered prior to chemotherapy

and in patients with a basis of prolongation of life, regression of tumors or lesions, and the improvement of immune responses. According to Ref.'*!, lentinan was injected into malignant peritoneal and/or pleural effusions of a group of 16 patients with advanced cancer. Eighty percent of the lesions showed probable clinical responses, with an improvement in performance demonstrated in seven subjects. The survival time for those who responded immunologically to the treatment was 129 days and 45 days for those who did not respond. Shiitake mushrooms have _ cancer-preventative properties and can be a beneficial dietary supplement. Compounds that block the formation of carcinogenic N-nitroso compounds from nitrates (which occur in vegetables and meats) are produced in dried and heated mushrooms.!”*:'°l The uncooked form contains no detectable amounts of the nitrite-scavenging compound thiazolidine-4-carboxylic acid, while the dried variety has 134mg/100g (dry weight basis) of* this compound, and the boiled form holds 850 mg/100 g. In vitro studies have indicated that LEM from shiitake mushroom may be more effective than AZT in the treatment of AIDS (see discussions in the section on “Preclinical studies’’), because it inhibits the cytopathic effect of giant cell formation in a cell-free system with MT-4 cells, or a cell-to-cell infection system with

MOLT-4 cells, both of which induce multinucleated giant cells very efficiently. L. edodes mycelium may

work by blocking the initial stages of HIV infection,!

Azidothymidine inhibits cell-free infection of HIV, but

it is ineffective in preventing the formation of multinucleated giant cells. It is also expensive and is known to cause severe bone marrow toxicity and a host of other side effects. Furthermore, it may become less effective during long-term use or may not offer any long-term survival advantages even with early use. Mycelium

661

extract, however, is nontoxic and less expensive. Its encapsulated form is recommended as a daily dietary supplement primarily for prevention of disease and maintenance of health. It must be stressed that more clinical trials will be necessary to assess the long-term

benefit of the extract for HIV and AIDS. Another use is to boost the immune response in AIDS patients.!”!'®] When it was used to treat HIV-positive patients with AIDS symptoms, the T-cell count rose from a baseline of 1250/mm? after 30days up to 2550/mm° after 60 days. An improvement in clinical symptoms was also noted. Lentinan has shown favorable results in treating chronic persistent hepatitis and viral hepatitis B.!°! Forty patients with chronic viral hepatitis B and seropositive for Hbe antigenemia were given 6g of LEM daily (orally) for 4mo. The study focused on the number of patients seroconverting from Hbe antigen positive to antiHbe positive, which was 25% after LEM therapy, and was higher in patients with chronic active hepatitis (36.8%). In addition, 17 patients (43%) became seronegative for Hbe antigen. Liver function tests improved even for patients who remained seropositive, and they had raised plasma albumin, and adjusted protein metabolism. Dried shiitake mushroom (9g/day) decreased 7-10% serum cholesterol in patients suffering with hypercholesterolemia. For many patients 60 years of age or older with hyperlipidemia, consuming fresh shiitake mushroom (90 g/day in 7 days) led to a decrease in total cholesterol blood level by 9-12% and triglyceride level by 6-7%.!!°7°! Lentin, a novel protein isolated from shiitake mushroom, exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells.?77*)

Antifungal Activity From the fruiting bodies of the shiitake mushroom, a

novel protein designated lentin with potent antifungal activity was isolated in 2003.28! It was unadsorbed on

DEAE-cellulose,

and

adsorbed

on

Affi-gel blue

gel and Mono S. The N-terminal sequence of the protein manifested similarity to endoglucanase. Lentin, which

had a molecular

mass

of 27.5kDa,

inhibited

mycelia growth in a variety of fungal species including Physalosporia piricola, Botrytis cinerea, sphaerella arachidicola"*)

and Myco-

Toxicity and Side Effects Shiitake mushroom is edible, but some individuals may experience minor side effects or allergic reactions.

Shiitake (Lentinus edodes)

662

Literature describes!”:'°!84°! cases of shiitake-induced toxicodermia and shiitake dermatitis. Allergic reactions to the spores of shiitake mushrooms have been reported in workers picking mushrooms indoors, who are prone to an immune reaction to spores called “mushroom worker’s lung.’’ Symptoms include fever, headache,

congestion,

coughing,

sneezing,

nausea,

and general malaise.*°! A water extract of the fruiting body was found” to decrease the effectiveness of blood platelets in initiating coagulation. So people who bleed easily or who take blood thinners should be closely monitored when under long-term treatment with shiitake mushroom or its water-soluble fractions. L. edodes mycelium has shown no evidence of being acutely toxic, even in massive doses of over 50mg/day for 1 week, though mild side effects such as diarrhea and skin rash may occur.* As a rule, symptoms disappear after a short period, when the body has adapted to the extract. Lentinan has no known serious side effects. However, in clinical trials of patients with advanced cancer, minor side reactions occurred such as

a slight increase in glutamate-oxaloacetate transminase (GOT) and GPT liver enzymes and a feeling of mild pressure on the chest. But these changes disappeared after lentinan administration was stopped.&4!

COMMERCIAL PREPARATIONS SHIITAKE MUSHROOMS

OF

Dosage and Preparation

therapeutic doses are so great that its consumption could cause digestive upset. That is why LEM, which is concentrated

and

easily

absorbed,

for

is preferred

medicinal use.!”"*:1°1 Fresh and dried shiitake mushrooms are used in medicinal mushroom dishes (“Yakuzen’’). Certain medicinal effects have been recently studied"© and found to reduce the ill effects of certain gourmet diets. These dishes can be prepared in many ways, limited only by one’s ingenuity: boiled, grilled, skewered, or, on aluminum foil with different types of seasoning. To obtain a concentrate, whole fruit bodies or powdered mushrooms are boiled in water. The extract is then concentrated, and is used as a drink. It can also be consumed as a tea: canned “shiitake tea,’’ which

contains a concentrated extract, or many other shiitake “healthy tea’’ products sold as mushroom containing

tea bags.

;

Shiitake mushroom concentrate can be freeze-dried or spray-dried to form a granular powder. There are many products containing powdered shiitake mushroom

extract, such as a mixture

of this powder with

vitamin C crystals or with medicinal plants such as ginseng. In Eastern countries, the mushroom is mainly used as a concentrate when extracted with boiling water. Residues from these processes still contain substantial amounts of useful polysaccharide substances, including those effective as antitumor compounds such as B-glucans, nucleic acids, dietary fiber, etc. : An alcohol extraction product is obtained by preserving fresh or dried shiitake mushroom in alcohol, which has been mixed with sugar or molasses. Some products, including “healthy shiitake wine,’’ are sold

Shiitake mushroom is prescribed in various forms. It may be injected as a solution (1 mg/Vvial) or ingested as a sugar-coated tablet, capsule, concentrate,

as a nightcap or as a tonic drink.®*"!

powdered extract, syrup, tea, wine, and/or as a medic-

Drug Interactions

inal dish. Lentinan’s anticancer effect is highly dosedependent. The standard dose of the dried fruiting body in tea or in mushroom dishes is given as 6-16 g, equivalent to about 90g of fresh fruiting body. As a tablet, the dosage is usually in the form of 2g tablets

24x /day. Commercial

preparations can be found in many countries in health food stores and supermarkets. The tablets are usually made from a dried waterextract of the mycelia or fruiting bodies because drying concentrates the lentinan and other active principals. Standardized extracts are also available, and they are preferred because the amount of lentinan present is certified and clearly stated on the bottle. Although the fresh form can be a valuable dietary supplement, the quantities one would require for

A watery extract of the whole fruiting body of L. edodes is reported to lessen the effectiveness of the blood platelets during the process of coagulation. People who bleed easily or who take blood thinners should use caution when chronically using L. edodes extracts in therapeutic amounts or in its water-soluble

fractions (LEM).!1°47]

hepatitis, the best oral dose of LEM “The author does not consider these massive doses.

.

For cancer patients, smaller doses of intravenous and intramuscular lentinan are more. effective than larger ones (e.g., | mg per injection is considered safe, whereas 10mg may produce marked depression in the host immune response). Aoki! notes that what is considered an excessive dosage intravenously may be a favorable dosage when using oral administration. For treating the initial stages of AIDS or chronic is between 2 and

6g/day in 2-3 divided doses. If the disease is stable, the dosage may be decreased to 0.5-1 g/day.!”!°

Shiitake (Lentinus edodes)

663

REFERENCES

by Cancer

research

UK.

The

University

of

Strathclyde in Glasgow, 2002; 256.

1.

Hobbs, Ch. Medicinal Mushrooms:

An Explora-

19;

Yap, A.T.; Ng, M.L. Immunopotentiating properties of lentinan (1-3)-B-p-glucan extracted from culinary-medicinal shiitake mushroom Lentinus edodes (Berk.) Singer (Agaricomycetideae). Int. J. Med. Mushr. 2003, 5, 352-372.

20.

Chihara, G.; Maeda, Y.Y.; Hamuro, J.; Sasaki, T.; Fukuoka, F. Inhibition of mouse Sarcoma

tion of Tradition, Healing, and Culture, 2nd Ed.; Botanica Press, Inc.: Santa Cruz, CA, USA, 1995.

Miles, P.G.; Chang, S.T. Mushroom Biology: Concise Basics and Current Development; World Scientific: Singapore, 1997; 193. Przbylowicz, P.; Donoghue, J. Shiitake Grower's Handbook: The Art and Science of Mushroom Cultivation; Hunt Publ. Co.: Dubugue, Kendall,

1990; 199. Singer, R.; Harris, B. Mushrooms Botany,

Cultivation,

and

180 by polysaccharides from Lentinus edodes (Berk.). Sing. Nature 1969, 222, 687-688.

an. and Truffles:

Utilization,

2nd

Fukuoka, F. Antitumor polysaccharide derived chemically from natural glucan (pachyman). Nature (London) 1970, 225, 943-944.

Ed.;

Koeltz Sci. Books: Koenigstein, 1987. Chang, S.T. World production of cultivated edible and medicinal mushrooms in 1997 with emphasis on Lentinus edodes (Berk.) Sing. in

YH

Royse, D. Specialty mushrooms and their cultivation. Horticult. Rev. 1997, 19, 59-97.

Stamets,

P. Growing

Mushrooms,

3rd

Gourmet

Ed.;

Ten

and

Speed

pS),

Medicinal Press:

CA,

24.

USA, 2000. Mizuno, T. Shiitake, Lentinus edodes: functional

properties for medicinal and food purposes. Food Rev. Int. 1995, 7/7, 7-21. 10.

Hobbs, Ch. Medicinal value of Lentinus edodes

(Berk.) Sing. A literature review. Mushr. 2000, 2, 287-302.

Int. J. Med.

11.

Pegler, D. The classification of the genus Lentinus

bes

Fr. (Basidiomycota). Kavaka. 1975, 3, 11-20. Earle, F.S. The genera of the North American gill-fungi.

13.

Bull.

N.

Y.

Bot.

Gard.

1909,

25.

5,

373-451. Murrill, W.A. Additions to Florida fungi. 1. Bull.

26.

Singer, R. The Agaricales in Modern Taxonomy, 4th Ed.; Koeltz Sci. Books: Koenigstein,

b>.

16.

Li.

1986.

Pegler, D. The genus Lentinula (Tricholomataceae tribe Collybiaeae). Sydowia 1983, 36, 227-239. Mizuno, T. A development of antitumor polysaccharides from mushroom fungi. Food Food Ingred. J. Jpn. 1996, 167, 69-85.

18.

2s

28.

Wasser, S.P. Medicinal mushrooms as a source of

antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 2002, 60, 258-274. Smith, J.; Rowan, N.; Sullivan, R. Medicinal mushrooms. ‘Their therapeutic properties and current medical usage with special emphasis on

cancer treatment; Special Report Commissioned

Chihara, G.; Hamuro, J.; Maeda, Y.Y.; Shiio, T.; Suga, T.; Takasuka, N.; Sasaki, T. Antitumor and

metastasis-inhibitory activities of lentinan as an immuno-modulator: an overview. Cancer Detect. Rev. Supp. 1987, 7, 423-443. Yap, A.T.; Ng, M.L. An improved method for the isolation of lentinan from the edible and medicinal shiitake mushroom, Lentinus edodes (Berk.) Sing. (Agaricomycetideae). Int. J. Med. Mushr. 2001, 3, 9-20. Moriyama, M. et al. Anti-tumor effect of polysaccharide lentinan on transplanted ascites hepatoma-134 in C3H/He mice. In Manipulation of Host Defence Mechanisms, International Congress Series 576; Excerpta Media: Amsterdam, 1981. Mori, K.; Toyomasu, T.; Nanba, H.; Kuroda, H.

Antitumor activity of fruit bodies of edible mushrooms orally administrated to mice. Mushr. J. Trop. 1987, 7, 121-126.

Torrey Bot. Club 1939, 66, 29-37.

14.

Chihara, G.; Hamuro, J.; Maeda, Y.Y.; Arai, Y.; Fukuoka, F. Fractionation and purification of the

polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom). Cancer Res. 1970, 30, 2776-2781.

China. Int. J. Med. Mushr. 1999, 7, 387-409.

Wasser, S.P.; Weis, A.L. Medicinal Mushrooms. Lentinus edodes (Berk.) Singer; Nevo, E., Ed.; Peledfus Publ. House: Haifa, Israel, 1997; 95.

Chihara, G.; Hamuro, J.; Maeda, Y.Y.; Arai, Y.;

DeVere White, R.W.; Hackman, R.M.; Soares, S.E.; Beckett, L.A.; Sun, B. Effect of a mushroom

mycelium extract on the treatment of prostate cancer. Urology 2002, 60, 640-644. Ngai, P.H.K.; Ng, T.B. Lentin, a novel and potent antifungal protein from shiitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells. Life Sci. 2003, 73,

3363-3374. Pak.

Fujii, T.; Maeda, H.; Suzuki, K.; Ishida, N. Isola-

tion and characterization

of a new

antitumor

polysaccharide, KS-2, extracted from culture mycelia of Lentinus edodes. J. Antibiot. 1978,

31, 1079-1090.

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A.G.; Corrochategui,

Suzuki, C. Killing activity of experimental tumor cells given to macrophage by new antitumor immunopotentiator,

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1978, 33, 78-85. 34:

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Kurashige, S.; Akusawa, Y.; Endo, F. Effects of Lentinus edodes, Grifola frondosa, and Pleurotus ostreatus administration on cancer outbreak, and activities of macrophages and lymphocytes in mice treated with a carcinogen, N-butyl-Nbutanolnitrosoamine. Immunopharm. Immuno-

3oe

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Ladanyi, A.; Timar, J.; Lapis, K. Effect of lentinan on macrophage cytotoxicity against metastatic tumour cells. J. Cancer Immunol. Immunother. 1993, 36, 123-126. Yoshino, S.; Tabata, T.; Hazama, S.; lizuka, N.; Yamamoto, K.; Hirayama, M.; Tangoku, A.; Oka, M. Immunoregulatory effects of the anitumour polysaccharide lentinan on Thl/Th2 balance in patients with digestive cancers. Anti-

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Yang, Q.Y.; Jong, S.C. Medicinal mushrooms in China. Mushr. Sci. XII. 1989, 631-642.

St. John’s Wort (Hypericum perforatum) Jerry M. Cott Food and Drug Administration, Rockville, Maryland, U.S.A.

INTRODUCTION

for atopic dermatitis.7! In 2002, SJW was the seventh

St. John’s wort (SJW) is one of the best known yet least understood of the modern herbals. Though clearly a favorite medicinal among herbalists and European physicians, its reputation has been sullied by reports of drug interactions and lack of efficacy in two USS. trials. Safe use of this herb requires some familiarity with its effects on intestinal P450 enzymes and p-glycoprotein. Understanding the efficacy data requires a critical analysis of depression trials that have been performed with this antidepressant and an appreciation of why clinical studies fail. SJW is a well-studied botanical extract that appears to have a clinically significant therapeutic activity in mild-to-moderate major depressive disorder. While side effects are rare and mild, interactions with other medications may occur, particularly with those that are substrates of

best-selling herbal supplement (at just under US$15 million) in mainstream retail stores in the United States, though sales have declined somewhat in recent years (down 18% from 2001). Causal factors may include reports of poor quality control, herb—drug interactions, and concern that SJW may be ineffective due to the publication of the first “‘negative’’ clinical trial data in the Journal Association.“>!

of the American

Medical

CHEMISTRY AND PREPARATION OF PRODUCTS SJW has long been known to contain red pigments that have been postulated to be the primary active

both intestinal cytochrome P450 3A4 (CYP3A4) and the p-glycoprotein transporter.

BACKGROUND Hypericum perforatum L. Hypericaceae (St. John’s wort; SJW) has been used for millennia for its many medicinal properties, including wound healing, treatment

for kidney

and

lung ailments,

insomnia,

and

depression (Fig. 1). The SJW has become increasingly popular in Germany where it is approved for use in the treatment of mild-to-moderate depression, and has remained a first-line treatment for many years. It is named for its flowering time at the end of June, around the birth day of John the Baptist. Originally brought from Europe to North America, the plant can be found growing wild along roadsides and in fields and pastures. Current uses are primarily in treating central nervous system (CNS) indications such as depression, anxiety, and insomnia. Oil-based preparations are used for stomach upsets and are also applied topically to treat bruises, muscle aches, and

first-degree burns." A cream-based formulation was recently shown to be more effective than the vehicle

St, Iohris Wert

Hypericum perforat wm.

Jerry M. Cott, Ph.D., is a Pharmacologist at the Center for Drug Evaluation and Research, Food and Drug Administration, Division of Anesthetic, Critical Care & Addiction Drug Products, Rockville,

Maryland, U.S.A. Encyclopedia of Dietary Supplements DOI: 10,1081 /E-EDS-120022129

Copyright © 2005 by Marcel Dekker. All rights reserved.

anno otimins

aSSS Sea a

Fig. 1 St. John’s wort. (Compliments of Peggy (View this art in color at www.dekker.com)

Duke.)

665

St. John’s Wort (Hypericum perforatum)

666

constituent(s) in this plant genus, though there is insufficient evidence for this assumption. They are the naphthodianthrones hypericin and pseudohypericin (Fig. 2), and other derivatives that make up approximately 0.1-0.15% by weight. It also contains flavonoids and flavonols (2-4%) such as_hyperoside, quercitrin,

isoquercitrin,

quercetin,

biapigenin,

rutin,

and the biflavonoid, amentoflavone; and the phloroglucinols, primarily hyperforin (24%) (Fig. 3). Other constituents include volatile oils, tannins (6.5—15%), and caffeic acid derivatives. While originally believed to be a necessary component for antidepressant activity, hypericin is considered a marker compound for purposes of botanical identification. The early SJW studies from Germany examining the antidepressant activity were based on extracts standardized to hypericin only. More recent research suggested that hyperforin might also be important, and that this constituent may degrade (oxidize) under normal

manufacturing

conditions.

Therefore,

some

companies began to stabilize their formulations to prevent oxidation and standardize the hyperforin content at 3-5%. The necessity for this was called into question when the relatively hyperforin-free (20. Information related to

duration of illness was not provided. Meta-analysis showed that SJW was significantly more effective than placebo but not remarkably different in efficacy from active antidepressants. A subanalysis of six placebocontrolled trials and four active comparator trials that met stricter methodological criteria also showed SJW to be more effective than placebo and similar to standard antidepressants. There was no evidence of publication bias. Kim, Shreltzer, and Goebert”!

performed a meta-

analysis utilizing only well-controlled, double-blind studies with strictly defined depression criteria. There were six randomized double-blind trials, which included a total of 651 outpatients. As with the other meta-analyses, there was little mention of such factors as mean duration of depression, family history of affective disorders, previous episodes of depression, and exclusion of bipolar illness. The trials appear to have been performed in psychiatric and general practice sites. Five of the studies were carried out in Germany, and Hamilton scores ranged from 15 to 24. SJW was found to be 1.5 times more likely to result in an antidepressant response than placebo and was equivalent to tricyclic antidepressants (TCAs). Most clinical trials (including the two recent American studies) were performed with a standardized

St.

John’s Wort (Hypericum perforatum)

SJW extract such as LI160. This is one of the several officially recognized formulations in Germany for the treatment of depression. SJW remains a first-line treatment for mild-to-moderate depression in Europe.?! So, why did the two clinical trials conducted in the United States [Shelton et al.) (funded by Pfizer); and the Hypericum Depression Trial Study Group,"! (funded by the National Institutes of Health, NIH)] fail to show efficacy? Was there something different about these trial designs from other trials conducted with SJW? While both studies were purported to be the first rigorous and definitive studies of the herb, a closer examination reveals a shared weakness. The primary difference in these experiments is their departure from standard entry criteria for patient inclusion into a depression trial. While most previous studies of SJW targeted patients with ‘mild-to-moderate’ depression [approximately 15-20 on the 17-item Hamilton Depression Scale (HAM-D)], both of these trials required a score of at least 20. In the final demographics, the mean severity score for both studies was greater than 22; this is more toward the ‘“‘moderateto-severe’’ range of severity. Although it is commonly believed that more severely depressed patients will be less likely to respond to placebo—and that their inclusion in a clinical trial would increase the power to detect a true drug effect—published data do not

support this.°'! Antidepressant trials performed by the pharmaceutical industry routinely use a severity score of approximately 17 on the 17-item Hamilton scale as a criterion for entry to trials. For example, Walsh et al.8"! recently reported a mean Hamilton entry score of 16.7 for published depression trials. Given that 50% of industry antidepressant trials fail

669

sions regarding efficacy can be drawn from it. In order to determine efficacy, there must be a statistically significant difference between two groups on the primary outcome measure. Failure to show a difference could simply mean that the trial lacked the sensitivity to detect a difference (as with the Pfizer study). This is certainly the case for the NIH study where sertraline failed to differentiate from placebo on either of the primary efficacy endpoints. Thus, the two American trials are not useful for determination of efficacy. Safety information might reasonably be derived from them however, and is considered in a later section.

Anxiety Disorders SJW has also shown efficacy in placebo-controlled trials in somatoform disorders (medical conditions that appear to be psychological in origin).°?! The authors found a pronounced and relatively rapid onset of activity and a notable lack of adverse effects when compared to other treatments of somatoform disorders. They also noted that efficacy was not dependent on the presence of depressive symptoms, since patients without significant depression improved as much as those who did. The National Institute of Mental Health (NIMH) is currently supporting a placebocontrolled trial of SJW in social phobia in Madison. This same group has previously reported success in treating obsessive-compulsive disorder (OCD) in an open-label trial with SJW.©*! There is also a case report of three patients with generalized anxiety disorder (GAD) who responded to treatment with SJw.2*

to show a significant therapeutic effect,°”! it is clearly in the corporate interest to design studies with adequate statistical power. To do otherwise would not make economic sense. In the 40-plus years of experience in designing and implementing antidepressant trials, it seems doubtful that the pharmaceutical industry would have overlooked such an easy fix (increasing severity scores required for entry). Thus, the use of a more severely depressed population is unlikely to improve the quality of a study, and may even contribute to its failure. Of perhaps greater significance for these studies than the severity of the patients’ symptoms was the duration of their illness. The average length of the current depressive episode in both these trials was quite long, often greater than 2 yr. It seems likely that this patient population would be relatively unresponsive to any brief treatment within a clinical trial setting. While both trials claimed to exclude “treatment refractory’’ patients, it is doubtful that this was the case. The failure of sertraline to improve mood scores in the NIH study is consistent with this notion. The Pfizer study contained no active comparison, and thus no conclu-

Adverse Effects

Tolerability of SJW has been found to be very good with few adverse drug reactions (ADRs) reported (Table 3). The extensive use in Germany has not resulted in any serious drug interactions or overdose toxicity. While SJW is often said to be contraindicated in severe depression, this may have more to do with the need for medical supervision and the inappropriateness of self-treatment rather than a true lack of efficacy. In their meta-analysis of 22 trials, Whiskey, Werneke, and Taylor'*! reported that adverse effects occurred more frequently with standard antidepressants than with SJW. The meta-analysis by Kim, Streltzer, and Goeber?*! showed that there was

a higher dropout rate due to side effects in the tricyclic groups and that these drugs were nearly twice as likely to cause side effects—including those that were more severe—than did SJW. Linde and Mulrow™*! report that extensive use of SJW in Germany has thus far not resulted in published cases of serious drug interactions or toxicity after overdose.

St. John’s Wort (Hypericum perforatum)

670

Table 3

Incidence of adverse events Reference

Adverse event rate

Type of study

26% SJW; 45% standard drug

Meta-analysis

Linde and Mulrow

4% STW; 4.8% PBO 20% SJW; 36% standard drug

Meta-analysis

Linde et al. (1996)

3% SJIW

Review

Kasper (2001)

1-3% SJW; photosensitivity ~1 in 300,000

Review

Schulz?”

26% SJIW; 47% TCAs

Meta-analysis ; Meta-analysis

28 Whiskey, Werneke, and Taylor 7

Common ADRs: gastrointestinal symptoms, dizziness/confusion, tiredness/sedation

Review

Ernst et al. (1998)

Insomnia, vivid dreams, restlessness, anxiety,

Review

Upton et al. (1997), Schulz,&71 Anon!)

Case reports

Nierenberg (1999)

18% SJW; 16% PBO 28% SJW, 47% standard drug

agitation, irritability, dry mouth, headache

Mania in bipolar disorder

The two American trials found SJW to be well tolerated. In the NIH study, the number of withdrawals due to side effects was 2 for SJW, 3 for placebo, and

5 for sertraline. Patients reaching the maximum dose during the trial were 54% for SJW and placebo and 36% for sertraline (p < 0.005). The significant side effects of sertraline were diarrhea (38%), nausea (37%), anorgasmia (32%), forgetfulness (12%), frequent urination (21%), and sweating (29%). Those of SJW were anorgasmia (25%), frequent urination (27%), and swelling (19%). While the adverse events reported for sertraline are very consistent with literature reports, those for SJW are not. Specifically, anorgasmia has not been reported to occur in any of the German studies. Since this side effect is one of the most common complaints associated with SSRIs, it is tempting to speculate that it might have somehow “‘contaminated’’ the side effect profile of SJ)W. No explanation was provided by the authors. Finally, both the patients and the physicians correctly guessed the sertraline treatment in the majority of patients, while placebo and SJW were correctly guessed by no more than chance. In the Pfizer study, headache was the only adverse event that occurred with greater frequency in the SJW group (41%) than with placebo (25%). Patients discontinuing due to adverse events comprised only 1% in both the placebo and SJW groups. There were

no reports of anorgasmia. Photosensitivity The potentially serious adverse effect, photosensitivity, occurs very rarely. Photosensitivity from SJW preparations appears to be due to the naphthodianthrones, hypericin and pseudohypericin. These compounds are photoactive quinones that produce singlet oxygen and free radicals when exposed to light. For most people receiving high doses of SJW, the extent of photosensitivity is a slight reduction in the minimum

[25]

29 Kim, Streltzer, and Goeber tP 9]

tanning dose. This was demonstrated in a randomized, placebo-controlled clinical trial in which fair-skinned subjects who burned easily were given metered doses of SJW extract (LI160), and exposed to UVA and UVB irradiation.2° Volunteers received placebo or 900, 1800, or 3600 mg SJW extract, containing 0, 2.8,

5.6, and 11.3mg of total hypericin prior to testing. Sensitivity to selective UVA light was increased slightly (~20%) after the highest dose of SJW. Another group received multiple dosing at twice the recommended dose [600 mg three times a day (t.i.d.)]. In the SJW group, there was a slight increase in SSI (both UVA and B) sensitivity (~9%), and a larger increase to UVA light (~21%). The authors concluded that photosensitization was without clinical relevance at the recommended dosages. While the German Commission E notes that photosensitization is possible, especially in fair-skinned

individuals,

it concludes

that

animal

and human research indicates that photosensitization is not likely to occur at recommended dosages."! Reproduction The potential cognitive effects of prenatal exposure of SJW were tested in mice. SJW or a placebo was given in food bars (estimated to contain ~182 mg/kg/day) for 2weeks before mating and throughout gestation. The SJW did not affect body weight, body length, or head circumference measurements; physical milestones

(teeth eruptions, eye opening, external genitalia); reproductive capability, perinatal outcomes, or growth and development of the second-generation offspring.°7! In an identically designed study,®*! one offspring per gender from each litter (SJW: 13; placebo: 12) was tested on various learning and memory tests. In the majority of the tests, there were no differences between groups. Occasional minor discrepancies were found in favor of placebo-treated mice, but the authors questioned whether this was biologically significant.

St. John’s Wort (Hypericum perforatum)

671

The safety of SJW to nursing mothers and their infants was examined in a Canadian study”! Thirtythree breastfeeding women receiving SJW were followed between May 1999 and April 2001 and compared with matched controls. There were no statistically significant differences found in maternal or infant demographics or maternal adverse events. None was observed in the frequency of maternal report of decreased milk production, or in infant weight over the first year of life.

A mother with postnatal depression was admitted to a healthcare center in Germany. Her pharmacist had recommended taking an SJW preparation three times a day (LI160). Four breast milk samples during an 18-hr period were analyzed for hypericin and hyperforin. Only hyperforin was excreted into breast milk at sufficient levels to enable detection (0.6—-18 ng/ml). Neither was found in the infant’s plasma. No side effects were seen in the mother or infant.”! Mutagenicity The mutagenic potential of S)W was determined in an Ames test and the unscheduled DNA synthesis (UDS) assay. High concentrations of the extracts showed an increase in the number

of revertants, both with and

without metabolic activation. The authors ascribed the mutagenic effects to the flavonols found in SJW. Of the substances present in SJW, quercetin has generated the most controversy with respect to mutagenic potential. The genotoxicity of a standardized aqueous ethanolic extract (Psychotonin M) was examined in different in vivo and in vitro test systems with mammalian cells. The in vitro investigations were performed with the hypoxanthine guanidine phosphoribosyl transferase (HGPRT) test, UDS test, and the cell transformation test using Syrian hamster embryo (SHE) cells. In these studies, both the in vitro tests as well as the in vivo tests—fur spot test of the mouse and the chromosome aberration test with the Chinese hamster

bone

marrow

cells—were

negative.

Thus,

there was mixed evidence regarding the mutagenic potential of SJW (see Ref.4") for review).

Drug Interactions

Studies have shown that SJW can reduce plasma levels of several drugs.”*! Current knowledge regarding the metabolism of these drugs indicates that the liver cytochrome P450 drug metabolizing enzyme systems cannot,

by itself, account

for these effects. There

is

fairly substantial evidence that SJW induces both intestinal CYP3A4 and the P-glycoprotein (Pgp) after chronic administratrans-membrane pump tion.!4243] While drugs that are substrates of only one of these systems may show a measurable decrease,

these are not generally clinically relevant.

However,

medications with a narrow therapeutic index that are substrates of both CYP3A4 and Pgp (e.g., cyclosporine and indinavir) are of concern due to the possibility that SJW may result in substantial decreases in plasma concentrations.) While anecdotal reports have suggested that other medications or enzyme systems may be affected, these studies must be carefully evaluated in light of the specific methodology and the total body of evidence. For example, Obach™! reported that crude SJW methanolic extracts showed inhibition of all CYP enzymes when tested at very high concentrations [50% enzyme inhibition (ICs59) ranged from 10 to 1000 pg/ml]. Hyperforin inhibited 2D6, 2D9, and 3A4 with ICs of 1.6, 4.4, and 2.3 uM, respectively. The significance of these data is uncertain since the concentrations were higher than those attained clinically, e.g., hyperforin maximum plasma level (Cmax) was reported to be approximately 280nM (150ng/ml)."*! In addition, the activities of isolated chemical constituents may not be relevant to whole or crude plant extracts. However, within physiologically relevant concentrations, the SJW constituent, hyperforin, induces CYP3A4 in hepatocyte cells via the pregnane X nuclear receptor (K; = 27nM)'**! and the steroid X receptor.“°

Artificially high concentration of test substances in in vitro assays can result in false positives. Direct (in vivo) evidence of interaction with CY P450 is more useful for predicting clinical interactions. Clinical trials designed specifically to test for CYP450 enzymes show induction of CYP3A4 but not of other major

enzymes, CYP1IA2 and 2D6.47>!! SJW has recently been reported to induce Pgp as well as CYP3A4. The administration of the extract to rats or humans for 14days resulted in a 3.8-fold and 1.4-fold increase, respectively, of intestinal Pgp expression.47] This would explain the report that SJW reduces the plasma levels of digoxin,"?°"! which is not a substrate of P450 enzymes, but rather of Pgp. Of possible importance to this discussion, the lowhyperforin, low-hypericin formulation, Zel17, lacks interaction potential with digoxin (Table 4).©7] Hypericin has been reported to induce Pgp, though not as markedly as the whole extract.P3] Reduced levels of phenprocoumon (an anticoagulant closely related to warfarin) in 10 subjects were suggested to be caused by an interaction between SJW and CYP2C9 (the primary liver enzyme associated with warfarin metabolism) although there was no direct evidence for this. Another possible explanation for the interaction is reduced intestinal absorption due to induction of Pgp. In support of this possibility, rats treated orally with SJW did not show changes in liver enzyme activity but did exhibit reduced plasma

St. John’s Wort (Hypericum perforatum)

672

Table 4

Drugs reported to interact with SJW based on solid (pharmacokinetic) data Effect

SJW treatment

Drug (subjects)

57%

300 mg t.i.d. 14 days

Indinavir (n = 8)

~50%

300 mg t.i.d. 21 days Lil60

Cyclosporine (n = 2)

| AUC

| AUC

Reference

Piscitelli et al. (2000)

Ruschitzka et al. (2000)

900 mg/day 10days Lil60 300 t.id. 14 days 250 mg b.i.d. 14days (Ze 117)

25% | AUC 18% | AUC No effect

Johne et al.6!! Diirr et al.) Brattstrém>7)

300 mg t.i.d. 11 days Lil60

17% | AUC

Maurer et al. (1999)

3-Ketodesogestrel (n = 3/17)

600/900 mg/day 2 cycles Lil160

Pfrunder et al.*!

Norethindrone/ethinyl estradiol 771)

300 mg t.i.d. 2 cycles (Rexall-Sundown)

42-44% | AUC 14% | clearance/

3-Ketodesogestrel/ethinylestradiol (m= 116)

250 mg b.i.d. 14 days (Zel17)

No effect

Carbamazepine (n = 5)

300 mg t.i.d. 14 days

No effect

Burstein et al.64

22% | AUC

Johne et al. (2002)

Digoxin (n = 13) (n = 8) Digoxin Digoxin (n ~ 8) Phenprocoumon (n =

Amitriptyline (n =

12)

10)

900 mg/day 14days Li160

levels of orally administered warfarin.°*! These data also suggest (in rats at least) that the warfarin induction by SJW takes place in the intestine, rather than in the liver. Intermenstrual bleeding has been reported in women who had been taking long-term oral contraceptives and recently started taking SJW. Induction of 3A4 by SJW could be responsible, since steroids are known substrates of CYP3A4. A recent study?! on the pharmacokinetics of ethinylestradiol and 3-ketodesogestrel found no evidence of ovulation during low-dose oral contraceptive and SJW combination therapy. There were no significant changes in follicle maturation, serum estradiol, or progesterone concentrations when compared with oral contraceptive treatment

alone. There was, however,

an increase in

intracyclic bleeding episodes and a decrease in serum 3-ketodesogestrel concentrations, suggesting that SJW might increase the risk of unintended pregnancies. The hyperforin-poor formulation, Ze117, did not have this effect on 3-ketodesogestrel or ethinylestradiol, however (Table 4). Another study examined the effects of SJW on the anticonvulsant, carbamazepine.*! There were no significant differences before and after the administration of SJW in carbamazepine concentrations at peak, trough, or AUC. This suggests that SJW is either not a particularly powerful CYP3A4 inducer or that it cannot induce carbamazepine metabolism beyond the extent to which it induces itself. The concern about interactions of SJW with other antidepressants probably stems from reports about its inhibition of MAO and SHT uptake. The theory suggests that combination of an antidepressant and an MAOI could cause in a hypertensive crisis, and combination with an uptake inhibitor could result in

48% |

t

Hall et al. (2003)

ty, Brattstrém>7)

SHT syndrome. There have been no reports suggesting that side effects resembling MAOIs have occurred with SJW. This is consistent with current evidence suggesting that MAO inhibition may be an in vitro artifact.?! There are a few case reports of “SHT syndrome’’ in the United States that implicate SJW. However, there have been none in Europe where SJW has been used extensively for many years.°”! One case report concerned four elderly patients described as having “mild SHT syndrome’’ but were consistent with exaggerated side effects of sertraline, namely nausea, vomiting, and rest-

lessness.©*! All of them were stable on sertraline and experienced these effects within 34days of adding SJW. There are many conflicting literature references to drug metabolism, and sertraline is certainly an example. While most references do not list sertraline as a substrate of CYP3A4, there is a case report of a 12-yr-old boy on sertraline who experienced a 5HT syndrome when erythromycin, a known CYP3A4 inhibitor, was added.*! There is evidence that acute doses of SJW have a mild inhibiting action on CYP3A4. Since all these patients were stable on sertraline at the time they initiated SJW, this response can be explained by an increase in sertraline plasma levels—a pharmacokinetic effect, rather than a pharmacodynamic effect. A fifth elderly patient in the Lantz, Buchalter, and Giambanco’*! report was stable on nefazodone when she added SJW. A similar exaggerated serotonergic response resulted that is consistent with increased blood levels of nefazodone due to acute inhibition of CYP3A4.5"! The opposite effect could be predicted if the SJW had been started first, followed by the antidepressant. This is in fact the result of a clinical trial of amitriptyline and SJW (Table 4). In this study, 12 depressed patients received 900 mg SJW extract along with 75mg twice daily of amitriptyline

St. John’s Wort (Hypericum perforatum)

673

for 14days. Reductions in AUC of 21.7% were seen for amitriptyline and 40.6% for nortriptyline. Levels of amitriptyline and its metabolite continuously decreased over the 14day period, consistent with enzyme induction. Amitriptyline is another drug for which considerable contradiction exists in the literature regarding

Schempp, St. John’s

2C19, 2C9, and 2D6, while Feucht and

cyclosporine absorption. REFERENCES 1.

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plentiful, but often contradictory, in vitro data.

Of interest in the context of herb-drug and food—drug interaction is a recent report showing that red wine causes metabolic effects comparable to SJW on the pharmacokinetics of cyclosporine. In a randomized, two-way crossover study of 12 healthy individuals, red wine caused a 50% increase in the oral clearance of cyclosporine. Systemic exposure as measured by the area under the concentration-vs.-time curve (AUC) and peak concentration (Cmax) was significantly decreased by red wine. However, half-life was not affected, suggesting that the wine decreased

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Assoc. 2001, 285 (15), 1978-1986. Hypericum Depression Trial Study Group. Effect of Hypericum perforatum (St. John’s wort) in major depressive disorder. J. Am. Med. Assoc. 2002, 287, 1807-1814. Draves, A.H.; Walker, S.E. Analysis of the hypericin and pseudohypericin content of commercially available St. John’s wort preparations. Can. J. Clin. Pharmacol. 2003, /0 (3), 114-118.

acutely, and moderate induction of intestinal

CYP3A4 and CYP2E1l activity after repeated dosing. Chronic administration of SJW also induces the drug transporter protein, Pgp. Drugs that are substrates of both systems (e.g., indinavir and cyclosporine) are of particular concern as they would be predicted to be doubly affected by SJW. While the currently popular constituent, hyperforin, appears to be responsible for the enzyme induction, it may not be necessary for the therapeutic effect. This is evidenced by the low hyperforin, low hypericin formulation, Ze117, showing efficacy in major depression.'*?! Of particular interest in this context is that clinical pharmacokinetic studies have shown this formulation to lack interaction potential with either the CYP3A4 system or the Pgp transporter.) More in vivo data are needed to make sense of the

cream—a

Blumenthal, M. Herbs continue slide in’ mainstream market: sales down 14 percent. HerbalGram 2003, 55, 71. Shelton, R.C.; Keller, M.B.; Gelenberg, A.J. et al. Effectiveness of St. John’s wort in major depression: a randomized controlled trial. J. Am. Med.

Weissman! also describe it as a substrate for CYP3A4 and glucurony]l transferase. Recent studies have included the previously unstudied enzyme, CYP2E1. It was found that chronic treatment with SJW in both mice"! and humans!*”! leads to induction of this enzyme. The most commonly associated substrate for CYP2E1 is ethanol, but anesthetic gases also use this route of elimination (http: //medicine.iupui.edu/flockhart/). Thus, SJW is capable of weak inhibition of CYP3A4

wort

controlled, double blind half-side comparison. Phytomedicine 2003, /0 (Suppl. 4), 31-37.

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ADDITIONAL READINGS Bennett, D.A., Jr.; Phun, L.; Polk, J.F.; Voglino, S.A.; Zlotnik, V.; Raffa, R.B. Neuropharmacology of

St. John’s wort (Hypericum). Ann. Pharmacother. 1998, 32 (11), 1201-1208. Bladt, S.; Wagner, H. Inhibition of MAO

by fractions

and constituents of hypericum extract. J. Geriatr. Psychiatry Neurol. 1994, 7 (Suppl 1), S57-S59. Chatterjee, S.S.; Biber, A.; Weibezahn, C. Stimulation

of glutamate, aspartate and gamma-aminobutyric acid release from synaptosomes by hyperforin. Pharmacopsychiatry 2001, 34 (Suppl 1), S11-S19. Chatterjee, S.S.; Nolder, M.; Koch, E.; Erdelmeier, C.

Antidepressant activity of Hypericum perforatum and hyperforin: the neglected possibility. Pharmacopsychiatry 1998, 37 (Suppl 1), 7-15. Eckert, G.P.; Miiller, W.E. Effects of hyperforin on the fluidity of brain membranes. Pharmacopsychiatry 2001, 34 (Suppl 1), S22—S25. Ernst, E.; Rand, J.I.; Barnes, J.; Stevinson, C. Adverse

effects profile of the herbal antidepressant St. John’s wort (Hypericum perforatum L.). Eur. J. Clin. Pharmacol. 1998, 54 (8), 589-594. Gobbi, M.; Valle, F.D.; Ciapparelli, C.; Diomede, L.;

Morazzoni, P.; Verotta, L. et al. Hypericum per foratum L. extracts does not inhibit 5-HT transporter in rat brain cortex. Naunyn Schmiedebergs Arch. Pharmacol. 1999, 360 (3), 262-269. Hall, S.D.; Wang, Z.; Huang, S.-M.; Hamman, M.A.; Vasavada, N.; Adigun, A.Q.; Hilligoss, J.K.; Miller, M.; Gorski, J.C. The interaction between St. John’s

wort and an oral contraceptive. Clin. Pharmacol. Ther. 2003, 74, 525-535. Huntwe:)] demonstrated the alkylating ability common to epoxides evinced by valtrate and didrovaltrate at the same rate as the epoxide epichlorohydrin, and the chlorinated tertiary amine, N,N-dimethyl-N-(2-chloroethyl) amine.

Valerian

693

An assessment of their effect on DNA synthesis in cultured hepatoma cells® indicated that the diene valtrate had a rapid and extensive inhibitory effect on incorporation of 3H-thymidine and 3H-leucine and that the monoenes, didrovaltrate, and deoxydidrovaltrate, while being less active, nonetheless exhibited considerable cytotoxicity. Structure—activity considerations indicated that the C5—C6 (cyclopentenyl) double bond was important for cytotoxic activity but that the epoxide group is not essential. A later comparative evaluation of valepotriate toxicity showed that the diene types were about 2-3 times more toxic than the monoenes. The decomposition products of diene valepotriates baldrinal and homobaldrinal were 10-30 times less toxic than their parent compounds, while isovaltral, like homobaldrinal a pro-

duct of isovaltrate, was more cytotoxic than its parent. Monoene valepotriates were found to be remarkably stable under storage, while dienes, as noted earlier, are very labile.©7] However, in vitro observations of

valepotriate cytotoxicity against mouse bone marrow cells®) could not be replicated in vivo on the same type of cell, whether orally or intraperitoneally.©7! These results indicate that human in vivo toxicity is likely considerably less than that observed in in vitro experiments, probably as a result of poor absorption and/or distribution and metabolism of these iridoids. It has been discovered that valepotriates and baldrinals, the

latter likely produced in the gastrointestinal (GI) tract, are rapidly absorbed in the body, the latter more so. Baldrinal glucuronide esters have been isolated, which did not give positive reactions in both the Ames test and the SOS chromotest.'" At most, the administration of valepotriates would seem to pose only a slight but potential genotoxic risk to the GI tract and liver.?9! However, until this is confirmed by long-term in vivo studies in humans, it would be prudent for children!!4!

as well as for adults not interested in the antianxiety effect to avoid consumption of valepotriates. Tinctures of Valeriana species stored at ambient room temperature for 2mo were found to have lost most of their valepotriate content, a recommended precautionary

treatment for reducing valepotriate toxicity.!°!! CLINICAL STUDIES

A much smaller number of clinical trials have been conducted with valepotriate-rich extracts for treatment of generalized anxiety disorder (GAD) and affective disorders.

Sleep Disorders More than a dozen double-blind placebo-controlled trials of valerian have been conducted, most also ran-

domized and/or of crossover design. The results of those studies have been summarized in great detail in

the second edition of Botanical Medicines; a summary of salient details of the most recent rigorously designed trials is presented in Table 1. Generally, 400-450 mg of an aqueous extract taken at bedtime produced a significant decrease in subjectively evaluated sleep-onset latency, as well as improved sleep quality of poor or irregular sleepers, without causing any hangover the following morning.!°'! The earlier of these two studies’) compared the effects of an aqueous extract of valerian to those of a commercial preparation (Hova™) of combined valerian root and hop flower extract, against placebo. Hova affected neither sleep latency nor sleep quality but generated a marked increase in reports of feeling more sleepy than usual upon awakening. The reason for the inferiority of the commercial preparation cannot be speculated upon without knowledge of the details of its preparation. A much later study with Hova compared its effects to those of the benzodiazepine,

bromazepam,

in 37

middle-aged women and 9 men with nonpsychiatric/ nonchronic sleep disorders;!°*! the randomized, doubleblind, placebo-controlled, parallel-group designed trial found that the health of patients improved only after 2 weeks and that the valerian—hop combination product was equally effective as the benzodiazepine, therefore presenting a sensible alternative to the latter. A combination concentrated standardized valerian— lemon balm extract was compared with triazolam, another benzodiazepine, and placebo in 20 healthy volunteers, poor sleepers aged 30-50.!*4! Both treatment groups showed a significant increase in sleep efficiency especially those in the subcategory of “bad’’ sleepers. In contrast to the triazolam group, however, no daytime sedation, impairment of concentration, or performance capabilities were observed in

Most of the clinical studies with valerian relate to sleep disorders, mainly in treatment of insomnia. Those trials have generally employed valepotriate-free aqueous extracts of root/rhizome, although hydroalcoholic extracts with substantial sesquiterpene content and very low content in valepotriates have also been tested. Combinations of extracts of valerian root with extracts

of hop

flowers,

lemon

balm

leaves,

and

passionflower aerial parts have also been evaluated.

the valerian/lemon balm group. Interestingly, a proprietary 70% alcohol extract of valerian root (Lichter AG, LI 156) produced significant improvement in sleep characteristics only after

2 weeks of treatment.!*! Kamm-Kohl,

Jansen, and Brockmann,” [66] in a ran-

domized placebo-controlled double-blind study with 80 hospitalized elderly patients with sleep disturbances and/or rapid fatigue, investigated the efficacy of an

Valerian

694

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aqueous extract of valerian root over 14days (45 mg, 3-9 times daily) . Statistically significant improvements were observed in both subjective well being and behavioral disturbances in the treated group, but not in the placebo group. Patients in the verum group reported improved ability to fall asleep and sleep through the night, as well as reduced feeling of fatigue. More recently, three trials were conducted with the commercial preparation, Sedonium®, in the form of

coated tablets each containing 300 mg of a dry extract of valerian root.!©”-°! Each study involved 16 patients in a randomized double-blind, placebo-controlled, crossover design. The first of these!®” examined the effects on electroencephalogram (EEG) in single (1.2 g) and multiple-dose (600 mg/night) for 2 weeks; the observed changes in EEG approximated those produced by psychosedative anxiolytic drugs. The second! used polysomnographic tests to measure slow-wave sleep (SWS) and SWS latency, among other parameters.

In objective measurements,

valerian

decreased

SWS latency, compared to placebo, while increasing the duration of SWS compared to baseline; subjectively, the time to sleep onset was reduced by valerian as compared to placebo. The third!) assessed the short-term (single dose of 2 x 300mg tablets) and long-term (14days with multiple dosage) treatment being administered 1 hr before bedtime. No effects on sleep structure and subjective sleep assessment were observed after a single dose of valerian. After the multidose treatment,

however, as in the second trial,

significant differences were noted in SWS and SWS latency between verum and placebo groups. Interestingly, polysomnographic comparisons with baseline values indicated significant increases in sleep efficiency for both placebo and multiple-dose treatment groups.

Lindahl and Lindwall!’" conducted a randomized double-blinded crossover comparison of two combination preparations. The first, a commercial product (Valeriana Natt) containing valerian extract equivalent to 400mg of root, standardized to and containing primarily sesquiterpenes, and with only traces of valepotriates, combined with hop and lemon balm extracts. The second contained valerian extract equivalent to only 4mg of root, combined with “‘a full dose of Flores

humuli and Lemon melissa.’’ Any observed differences would be attributed to the difference in valerian content. Eighty-nine percent of the 27 subjects reported improved sleep and 44% “‘perfect sleep’’ from Valeriana

Natt.

No

which nightmares, customary sedatives.

side effects

had

been

were

reported,

experienced

nor

with

SAFETY In all the clinical trials of valerian preparations conducted so far, only mild side effects such as stomach

upset, headaches and itching have occasionally been noted.!”"! No subchronic or chronic toxicity data are available. A recent case report, involving consumption of more than 20 times the recommended dose of powdered valerian root by an 18-yr-old female college student, in an apparent

suicide attempt,

revealed

only

mild symptoms, all of which resolved within 24 hr.!77! The symptoms noted were fatigue, abdominal cramping, chest tightness, lightheadedness, and foot and hand tremor. A study aimed at assessing possible delayed adverse effects of valerian overdose treated 10 males and 14 females after allegedly taking an overdose of a purported valerian-containing OTC product, Sleep-Qik, a valerian dry extract combined with hyoscine hydrobromide and cyproheptadine hydrochloride.!7*! No clinical evidence of acute hepatotoxicity or subclinical liver damage was detected, and delayed liver and other adverse effects were judged unlikely. Concern for valerian hepatotoxicity has been misdirected and misguidedly associated in its indictment,

along with skullcap (Scutellaria lateriflora), in a case of hepatotoxicity,!”4! almost certainly due to germander (Teucrium chamaedrys) substituted for skullcap. The case of a high-output cardiac failure, in a 58-yrold man who had been consuming a plethora of medications including protracted high dosage valerian, following withdrawal of valerian was suspected to be a

“‘benzodiazepine-like withdrawal syndrome.’’!’*! However, while a causal association could not be estab-

lished, similarities of mechanism of action between valerian and benzodiazepines strike a cautionary note. Studies of valerian’s influence on vigilance have revealed no effects that would impair coordination or

decrease alertness.!”°! However, while alcohol does not potentiate the effects of valerian,!’’! as do many synthetic tranquilizers, because of additive effect caution should be exercised with joint consumption during driving and operating heavy machinery.

Contraindications and Drug Interactions European clinical monographs list no contraindications to its use in pregnancy or during lactation. Also, studies with pregnant rats indicate no deleterious

effects of oral consumption of valepotriates.!”*! Nonetheless, erring on the side of caution, ingestion of appreciable quantities of valepotriates is generally disavowed, recommending either removal by extraction or sufficient length of storage to promote degradation of the iridoids (see earlier). While no drug interactions have been reported in humans, studies in rodents indicate a potential for reaction with barbiturates and benzodiazepines.” In mice, sleeping time induced by thiopental was extended and thiopental anesthesia prolonged, after treatment

Valerian

698

with both aqueous and alcoholic extracts of valerian, perhaps contraindicating valerian while undergoing treatment with barbiturates. On the other hand, the demonstrated affinity of valerian extracts and valepotriates for GABA and benzodiazepine receptor sites, as well as the diminution of diazepam withdrawal effects observed in rats treated intraperitoneally with valepotriates, suggests that valepotriate-rich preparations may be helpful in easing withdrawal from benzodiazepines.

14.

Schilcher, H. Phytotherapy in Paediatrics; Medpharm Scientific Publishers: Stuttgart, Germany, 1997; 59-62.

ba:

McKenna, D.J.; Jones, K.; Hughes, K. Botanical The Desk Reference for Major Medicines: Herbal Supplements; 2nd Ed.; The Haworth Press

16.

Inc.: New York, 2002; 1007-1037. The ABC Clinical Guide to Herbs; Blumenthal, M., Goldberg, A., Kunz, T., Dinda, K., Eds.; American Botanical Council: Austin, TX, 2003;

357-358. fie

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Gerhardt, U.; Linnenbrink, N.; Georghiadou, Ch.

Effects of two plant-based sleep remedies on vigilance. Schweiz. Rundsch. Med. Prax. 1996, 85, 473-481. Von Eickstedt, K.-W. Die Beeinflussung der Alkohol—Wirkung durch Valepotriate. Arzneim Forsch. 1996, 19, 995-997. Tufik, S.; Fujita, K.; de Lourdes, M.; Seabra, V.;

Lobo, L.L. Effects of prolonged administration of valepotriates in rats on the mothers and their offspring. J. Ethnopharmacol. 1994, 4/7, 39-44.

P.

Modern valerian therapy of nervous disorders in elderly patients. MedWelt 1984, 35, 1450-1454.

delayed

Cardiac complications and delirium associated with valerian root withdrawal. J. Am. Med. Assoc. 1998, 280 (18), 1566-1567 (Letter).

J.

Therapie von Insomnien: Wirksamkeit und Vertraglichkeit eines Baldrianpraparats. Psychopharmacotherapie 1996, 3, 109-115.

Hum. Toxicol. 1996, 37 (4), 364-365. Chan, T.Y.K. An assessment of the

remedies. Br. Med. J. 1989, 229, 1156-1157.

Dressing, H.; Reimann, D.; Low, H.; Schredl, M.; Reh, C.; Laux, P.; Miiller, W.E. Baldrian-—

Vorbach,

Lindahl, O.; Lindwall, L. Double-blind study of a

valerian preparation. Pharmacol. Biochem. Behav. 1989, 32, 1065-1066. Schmidt-Voigt, J. Treatment of nervous sleep disorders and unrest with a sedative of purely vegetable origin. Therapiewoche 1986, 36, 663-667. Willey, L.B.; Mady, S.P.; Cobaugh, D.J.; Wax, P.M. Valouad overdose: a case report. Vet.

Cobden, I.; Hayes, P.C. Hepatotoxicity of herbal

zur

Melisse-Kombinationen versus Benzodiazepin; Beischlafst6rungen gleichwertig? Therapiewoche 1992, 42, 726-736.

objective and subjective sleep in insomniacs. Eur. J. Clin. Pharmacol. 1997, 52 (Suppl.), A 170 (abstract 548). Donath, F.; Quispe, S.; Diefenbach, K.; Maurer,

effects associated with valerian overdose. J. Clin. Pharmacol. Ther. 1998, 36 (10), 569 (Letter).

of valerian reduced latency to fall asleep in man.

Untersuchung der Lebensqualitat von Patienten mit exogenen Schlafst6rungen (voriibergehenden Ein- und Durchschlafst6rungen) unter Therapie mit einem Hopfen-Baldrian-Praparat und einem Benzodiazepin-Praparat. Wien. Med. Wochenschr. 1998, 148, 291-298.

Quispe Bravo, S.; Diefenbach, K.; Donath, F.; Fietze, I.; Roots, I. The influence of valerian on

A.; Fietze, I.; Roots, I. Critical Svalnatianl of the

vies

62.

1996, 50, 541

effect of valerian extract on sleep structure and sleep quality. Pharmacopsychiatry 2000, 33, 47-53.

1/7,

65-71.

Eur. J. Clin. Pharmacol.

(abstract).

valepotriates on mouse early hematopoietic progenitor cells and human T-lymphocytes. Arch.

59.

Donath, F.; Roots, I. Effects of valerian extract

(Sedonium®) on EEG power spectrum in male healthy volunteers after single and multiple appli-

79.

Brinker, F. Herb Contraindications and Drug Interactions, 2nd Ed.; Eclectic Medical Publica-

tions: Sandy, OR, 1998; Vol. 134, 162-163.

.

Vitamin A A. Catharine Ross The Pennsylvania State University, University Park, Pennysylvania, U.S.A.

INTRODUCTION Vitamin A (all-trans-retinol, vitamin A alcohol) is the parent molecule of a family of compounds with shared biological activities. Collectively, these compounds are essential for visual functions and for the maintenance of healthy epithelial tissues (skin, immune system organs, gastrointestinal tract, reproductive organs, lungs, and others). The nutritional term vitamin A encompasses both preformed vitamin A and provitamin A. Preformed vitamin A (retinol and its esters) occurs naturally in foods of animal origin and is added in the preparation of some fortified foods and nutritional supplements. Provitamin A compounds (B-carotene,

o-carotene,

and

f-cyptoxanthin),

which

are

Retinol in the diet serves as a precursor for the regulated production of several essential metabolites. Among these are 11-cis-retinaldehyde, the form of vitamin A that is essential for vision, and all-transRA, the form that is most potent in cell and tissue differentiation assays. However, numerous other retinoids (cis-trans isomers, ring-oxidized derivatives,

and conjugated forms) have been isolated from plasma, tissues, bile, and excreta. It is likely that several dozen different natural retinoids exist, many with

some detectable biological activity. Some of these can best be categorized as precursors (f-carotene, retinol) that may give rise to active metabolites (11-cis-retinaldehyde,

RA),

and

some

products (ring-oxidized

are

mainly

catabolic

end

retinoids; conjugated water-

synthesized by plants as accessory photosynthetic pig-

soluble

ments, can be metabolized by humans and animals to

example, retinoyl-B-glucuronide, a water-soluble conjugated form present in bile, shows bioactivity in some assays, perhaps as a result of the release of RA. B-Carotene is a fat-soluble hydrocarbon, C4oHs¢, molecular mass 536g/mol, with two B-ionone rings (identical to those in retinol) connected by a methylsubstituted polyene chain (Fig. 1B). During absorption, the molecule is cleaved either centrally at the

retinol. The essential metabolites of vitamin A include 11-cis-retinaldehyde, the form required for vision, and all-

trans-retinoic acid (RA), which functions as a hormone and an essential regulator of gene transcription.

NAME AND GENERAL DESCRIPTION Retinol is a fat-soluble compound, C2)H390, molecular weight 286.44, which can be converted in vivo to all

A.

forms).

However,

Retinol (all-trans)

of the other essential forms of vitamin A. The structure of retinol includes a methyl-substituted cyclohexenyl (B-ionone) ring and a conjugated tetraene side chain ending in a terminal hydroxyl group (Fig. 1). The predominant retinyl ester in foods as well as in most human tissues is retinyl palmitate. The retinol in supplements is usually of synthetic origin and is often esterified with palmitic or acetic acid for greater product stability. Vitamin A> (3,4-didehydroretinol) is a minor variant found in some freshwater fishes. Its biological activity is qualitatively similar to that of

R R R R

there

are

exceptions;

for

= CH20OH, retinol = CHe0-fatty acyl group, retinyl ester = CHO, retinaldehyde =COOH, retinoic acid

retinol, but quantitatively about half as much.!!!

Fig. 1 (A) Retinol. R indicates the various substituents at carbon 15 giving rise to retinol, retinyl esters, retinaldehyde, and retinoic acid. Some of these molecules also exist A. Catharine Ross, Ph.D., is Professor at the Department of Nutritional Sciences, The Pennsylvania State University, University Park, Pennysylvania, U.S.A.

Encyclopedia of Dietary Supplements DOK: 10.1081 /E-EDS-120022048

Copyright © 2005 by Marcel Dekker. All rights reserved.

naturally

in several

isomeric

forms,

such as 9-cis,

11-cis,

and 13-cis isomers. (B) B-Carotene. The position of central (symmetrical) cleavage at the 15,15’ double bond is shown. 701

Vitamin A

702

15,15’ bond (see figure) or off-center (eccentrically) to yield up to two, but often only one, molecule(s) of retinol. The efficiency with which humans convert the B-carotene contained in supplements or foods to retinol varies considerably among subjects (see section on “Intestinal Vitamin A Absorption’’). The provitamin A carotenoids «-carotene and -cryptoxanthin, each having a single B-ionone ring, have about half the bioactivity of B-carotene. Several carotenoids that are relatively abundant

in foods (lutein, zeaxanthin,

lycopene, phytofluene) lack the B-ionone structure, which is crucial to the functions of vitamin A.

BIOCHEMISTRY

AND

FUNCTION

Activation

Neither retinol nor B-carotene is directly bioactive. Both compounds must be metabolized to generate the bioactivity of vitamin A.7! Key reactions in the conversion of retinyl esters and B-carotene to retinol, and in the oxidation of retinol to RA, are shown in Fig. 2. Biosynthesis and catabolism are both important as a means for controlling the cellular levels of retinoids, especially hormonally active forms such as RA. For most of the reactions shown, several enzymes have been described. It is uncertain at this time which of them are critical and which may serve ancillary or redundant functions. As shown in Fig. 2, the oxidation of retinaldehyde to RA is irreversible. The regulatory enzymes involved in these steps, and how they differ in different tissues, are being investigated intensively because they may determine the capacity of various tissues to use retinol in vivo.

Hormonal

Functions

The hormonal functions of vitamin A are mediated mostly, if not entirely, by RA, which acts both as a

Dietary forms: Absorption: ism: Metabolism:

Preformed

B-carotene and

vitamin A

provitamin A carotenoids intestinal

Intestinal

hydrolysis

RE. [chylomicron

in turn, affects the health of the skin, immune system,

reproductive organs, and other organ systems. Retinoic acid exists in at least three isomeric forms:' all-trans-RA, 9-cis-RA, and 13-cis-RA. All-trans-RA,

the most potent form in most cell differentiation assays, is a specific ligand for the nuclear retinoic acid receptor, RAR (described below). 9-cis-RA binds to both RAR and retinoid X receptor (RXR), although its main physiological target is considered to be the activation of the RXRs. 13-cis-RA is an enigmatic retinoid—while it is present in human plasma, possesses bioactivity in some assays, and is used clinically, this

isomer has not been shown to be a significant ligand for nuclear retinoid receptors. It is possible that it undergoes slow conversion to all-trans- or 9-cis-RA and therefore acts as a precursor.

Nuclear Receptors

The RARs and RXRs are members of the steroid/thyroid hormone receptor superfamily. There are three genes in each family, alpha, beta, and gamma, and their gene products are differentially expressed in various tissues. Nearly all cell types express at least one form of RAR and RXR. Each RAR and RXR protein has a DNA-binding domain (DBD) that interacts with specific DNA sequences (response elements) present in retinoid-responsive genes. Each receptor also has a ligand-binding domain (LBD) capable of binding all-trans-RA (the RARs) or 9-cis-RA (prinicipally the RXRs). Binding of a retinoid ligand induces a conformational change in the receptor protein that

cleavage

,

oxidation

esterification 90% of total body vitamin A is present as retinyl esters stored in the liver, most of it in perisinusoidal stellate cells."! B-Carotene is stored in liver and in fat at relatively low concentrations. When vitamin A is needed, retinol is released from

storage by hydrolysis. The released retinol is bound to newly synthesized RBP, as has been shown in cultured hepatocytes, and then secreted into plasma. If a person’s intake of vitamin A is inadequate, nearly all of the liver’s vitamin A stores can be mobilized. Once

Vitamin A is present in plasma as retinyl esters transported in chylomicrons during absorption, and as retinol bound

to RBP,

which

circulates

at a nearly

constant level throughout the day. After ingestion, chylomicron vitamin A peaks in lymph and plasma at about 2-6 hr; the magnitude of the peak is directly related to the quantity of vitamin A ingested. Chylomicrons are cleared from plasma with a half-life of

30 nmol/L*

Plasma pyridoxal Plasma total vitamin Bg

NV >40 nmol/L

Erythrocyte pyridoxal-5’-phosphate

NV

Urine

4-Pyridoxic acid

>3.0 pmol/day

Total vitamin Be

>0.5 umol/day

Indirect

Blood

S725 >1.80°

Erythrocyte alanine aminotransferase Erythrocyte aspartate aminotransferase

Urine 2g Tryptophan load test; xanthurenic acid

0.02

Pyridoxine-B-glucoside

NV

Other

NV

EEG pattern

NV, no value established; limited data available, each laboratory should establish its own reference with an appropriate healthy control population. *Reference values in this table are dependent on sex, age, and protein intake and represent lower limits. (22] >For each aminotransferase measure, the activity coefficient represents the ratio of the activity with added PLP to the activity without PLP added.

with the younger women, the older women had a lower

mean plasma PLP and plasma and urinary total vitamin Bg and slightly higher urinary 4-pyridoxic acid excretion with the 2.3-mg intake. Interestingly, there was no difference in urinary excretion of xanthurenic or kynurenic acid following a 2-g L-tryptophan load. Thus, while there may

be age-related

differences

in

vitamin Bs metabolism, there is no significant age effect on functional activity of vitamin Be when intake is adequate. The metabolism of vitamin Bes has been studied in elderly men and women older than 60 yr. While younger individuals were not examined in the same study, the researchers concluded that the elderly had an increased vitamin Bg requirement, indicative of increased

metabolism.

Kant,

Moser-Veillon,

and

Reynolds®*! observed no age-related impairment in the absorption or phosphorylation of vitamin Be. However,

there was

an increase

phosphatase activity with hydrolysis of PLP.

in plasma

alkaline

age that would

increase

The use of plasma PLP as a status indicator has been questioned?*! and the determination of plasma PL recommended. Others have also suggested that plasma PL may be an important indicator of status. When Barnard et al.°7! studied the vitamin Be status in pregnant women and nonpregnant controls, they

found that plasma PLP concentration was 50% lower in the pregnant women but that the concentration of the total of PLP and PL was only slightly lower. When concentrations of PLP and PL were expressed on a per-gram-albumin basis, there was no difference between groups. In contrast, in pregnant rats, both plasma PLP

and PL decreased, as did liver PLP, in

comparison with nonpregnant control rats.2*! These studies are in direct opposition to each other but do provide support for the need to determine several indices of vitamin Bg status.2?743¢ Urinary 4-pyridoxic acid excretion is considered a short-term indicator of vitamin Bg status. In deficiency studies in males!**! and females, the decrease in

Vitamin Bg

719

urinary 4-pyridoxic acid paralleled the decrease in plasma PLP concentration. As reflected in the studies in which dietary intake was assessed or known, 4-pyridoxic acid excretion accounts for about 40-60% of the intake (see Table 10 in Ref.””!). Because of the design of most studies and the limited number of studies done to determine whether there is a significant difference

in one-carbon metabolism. Alteration in one-carbon metabolism can then lead to changes in nucleic acid synthesis. Such changes may be one of the keys to the effect of vitamin Bs on immune function.>?%! Studies in animals have shown that vitamin Be deficiency adversely affects lymphocyte production”! and antibody response to antigens.'°! Additional studies in animals support an effect of vitamin Bs on

between

cell-mediated immunity." A review, though dated,

with females compared males

with males, it is not possible

and females.

However,

males

consis-

tently had higher plasma PLP and total vitamin Bg concentrations as well as higher excretion of 4-pyridoxic acid and total vitamin Bs. Urinary total vitamin (all forms, including phosphorylated and glycosylated) excretion

is not a sensitive indicator

of the vitamin,

except in situations where intake is very low.°”*! Erythrocyte transaminase activity (alanine and aspartate) has been used to assess vitamin Be status

in a variety of populations,??404!424°47) including oral-contraceptive users.?**44°! Transaminase activity is considered a long-term indicator of vitamin Beg status. Most often, it has been measured in the presence and absence of excess PLP."*! While this index is used to assess

status,

there

is no

unanimous

agreement,

and some consider it to be less reliable than other indicators.°?47] The long life of the erythrocyte and tight binding of PLP to hemoglobin may explain the lack of a consistent significant correlation between plasma PLP and transaminase activity or activity coefficient. An additional consideration that complicates the use of aminotransferases is the finding of genetic polymorphism of erythrocyte alanine aminotransferase.*! Urinary excretion of tryptophan metabolites following a tryptophan load, especially excretion of xanthurenic acid, has been one of the most widely used

tests for assessing vitamin Be status.49°°] The use of this test has, however, been questioned,?!” 2] especially

in disease states or in situations in which hormones may alter tryptophan metabolism independent of a

direct effect of vitamin Bs metabolism." Other tests for status include the methionine load,>*! oxalate excretion, electroencephalographic

tracings,°! and lymphocyte proliferation.°*! These

tests are used less often but under appropriate circumstances provide useful information. The review by Reynolds®* provides an excellent critique of methods currently in use for assessment of vitamin Be status.

FUNCTIONS Immune System Functions The involvement of PLP in a multiplicity of enzymatic reactions”! suggests that it serves many functions in the body. PLP acts as a coenzyme for serine transhydroxymethylase,? 8] one of the key enzymes involved

of vitamin Bs and immune competence is all that is currently available.!©!

Gluconeogenesis Gluconeogenesis is key to maintaining an adequate supply of glucose during caloric deficit. Pyridoxal-5’phosphate is involved in gluconeogenesis via its role as a coenzyme for transamination reactions”! and for glycogen phosphorylase.!*?! In animals, a deficiency of vitamin Bg, results in decreased activities of liver alanine

and aspartate

aminotransferase.!“4!

However,

in humans (females), a low intake of vitamin Be (0.2mg/day), as compared with an adequate intake (1.8mg/day), did not significantly influence fasting

plasma glucose concentrations.'©*! Interestingly, the low vitamin Bg intake was associated with impaired glucose tolerance in this study. Glycogen phosphorylase is also involved in maintaining adequate glucose supplies within liver and muscle and, in the case of liver, is a source of glucose for adequate blood glucose levels. In rats, a deficiency of vitamin Bg has been shown to result in decreased activities of both liver!®*! and muscle glycogen phosphorylase.!® 66.67] Muscle appears to serve as a reservoir for vitamin Bz hee but a deficiency of the vitamin

does not result in mobilization of these stores. However, Black, Guirard, and Snell!®) have shown that a

caloric deficit does lead to decreased muscle phosphorylase content. These results suggest that the reservoir of vitamin Bg (as PLP) is only utilized when there is a need for enhanced gluconeogenesis. In male mice, the half-life of muscle glycogen phosphorylase has been shown to be approximately 12 days.!°"! In contrast to rats with a low intake of vitamin B,, those given an

injection of ahigh dose of PN, PL, or PM (300 mg/kg) show a decrease in liver glycogen and an increase in serum glucose.!”°! This effect is mediated via increased secretion of adrenal catecholamines. The extent to which lower intake of Be vitamers has this effect or

whether this occurs in humans remains to be determined.

Erythrocyte Function

Vitamin

Bs has

an

additional

function and metabolism.

role in erythrocyte

The function of PLP

as a

Vitamin Be

720

coenzyme for transaminases in erythrocytes has been mentioned. In addition, both PL and PLP bind to

hemoglobin.'”!! The binding of PL to the « chain

of hemoglobin'”?! increases the O, binding affinity,” while binding to the B chain of hemoglobin S or A

lowers it.!”*! The effect of PLP and PL on O, binding

may be important in sickle cell anemia.!7¢! Pyridoxal-5'-phosphate serves as a cofactor for 6-aminolevulinic acid synthetase,!”7! the enzyme that catalyzes the condensation of glycine and succinyl-CoA to form 5-aminolevulinic acid. This latter compound is the initial precursor in heme synthesis.!”*! Therefore, vitamin Bg plays a central role in erythropoiesis. A deficiency of the vitamin in animals can lead to hypochromic microcytic anemia. Furthermore,

in humans,

there are several reports of patients with pyridoxineresponsive anemia.!’”*! However, not all patients with sideroblastic anemia (in which there is a defect in 5-aminolevulinic acid synthetase) respond to pyridoxine therapy.8! Niacin Formation

One of the more extensive functions of vitamin Be that has been researched is its involvement in the conversion of tryptophan to niacin." This research is in part related to the use of the tryptophan load in evaluating vitamin B, status. While PLP functions in at least four enzymatic reactions in the complex tryptophan—niacin pathway, there is only one PLP-requiring reaction in the direct conversion of tryptophan to niacin. This step is the transformation of 3-hydroxykynurenine to 3hydroxyanthranilic acid and is catalyzed by kynureninase. Leklem et al. have examined the effect of vitamin Be deficiency on the conversion of tryptophan to niacin.®" In this study, the urinary excretion of Nmethylnicotinamide and N’-methyl-2-pyridone-5carboxamide, two metabolites of niacin, was evaluated

in women. After 4 weeks of a low-vitamin Bg diet, the total excretion of these two metabolites following a 2-g L-tryptophan load was approximately half that when subjects received 0.8-1.8mg vitamin Be per day. This suggests that low vitamin Bg has a moderate negative effect on niacin formation from tryptophan.

Nervous System Functions In addition to the effect of vitamin B, on tryptophanto-niacin conversion, there is another tryptophan pathway that is vitamin Bg dependent. The conversion of 5-hydroxytryptophan to 5-hydroxytryptamine is catalyzed by the PLP-dependent enzyme 5-hydroxytryptophan decarboxylase.*”! Other neurotransmitters, such as taurine, dopamine, norepinephrine, histamine,

and

y-aminobutyric

acid,

are

also

synthesized

by

PLP-dependent enzymes.®7! The involvement of PLP in neurotransmitter formation and the observation that there are neurological abnormalities in human

infants'®384] and animals®*! deficient in vitamin Beg

provide support for a role of vitamin Bs in nervous system function. Recent reviews on the relationship between nervous system function and vitamin Beg are

available.*°*71

In infants fed a formula in which the vitamin Bg was lost during processing, convulsions and abnormal observed.) were (EEGs) electroencephalograms Treatment of the infants with 100mg of pyridoxine produced a rapid improvement in the EEGs. In these

studies reported by Coursin, the protein content of the diet appeared to be correlated with the vitamin Bg deficiency and the severity of symptoms. Other evidence for a role of vitamin Bs comes from studies of pyridoxine-dependent seizures, an autosomal recessive disorder. Vitamin Be dependency, though a rare cause of convulsions, has been reported by several investiga- . tors.8°:8°] The convulsions occur during the neonatal period, and administration of 30-100 mg of pyridoxine is usually sufficient to prevent them and correct an abnormal EEG.®?°l However, there are atypical patients who present a slightly different clinical picture and course but are responsive to pyridoxine?! Vitamin Be deficiency in adults has also been reported to result in abnormal EEGs,">?”! especially in individuals on a high-protein (100 g/day) intake. In one study,”*! subjects received a diet essentially devoid of vitamin Be (0.06mg). Grabow and Linkswiler fed to 11 men a high-protein diet (150g) and 0.16mg of vitamin Bg, for 21days.?3! No abnormalities in EEGs were observed; nor were there changes in motor nerve conduction times in five subjects who had this measurement. Kretsch, Sauberlich, and

Newbrun! observed abnormal EEG patterns in two of eight women after 12 days of a low (0.05 mg/day) vitamin Be diet. Feeding 0.5mg/day corrected the abnormal pattern. While there were differences in the length of the period of deficiency in these studies, which may explain the differences observed, it appears that long-term very low vitamin Bg intakes are necessary before abnormal EEGs are observed in humans. Another aspect of the relationship of vitamin Bg (as PLP) to the nervous system is the development of the brain under conditions of varying intakes of the vitamin. Kirksey and coworkers have conducted numerous well-designed studies in this area. These have utilized the rat model to examine the development of the brain, especially during the critical period when cells undergo rapid mitosis. Early experiments showed that dietary restriction of vitamin B, in the dams was associated with a decrease in alanine aminotransferase and glutamic acid decarboxylase activity and low brain weights of progeny.“

Vitamin Bg

721

Alterations in fatty acid levels, especially those involved in myelination,?*! decreases in cerebral sphingolipids and in the area of the neocortex and cerebellum, as well as reduced molecular and granular layers of the cerebellum have all been noted, One of the more intriguing and controversial aspects of vitamin Bg is its role in lipid metabolism.?”! Studies conducted more than 60 years ago suggested a link between fat metabolism and vitamin B,.°*! Subsequent research showed that liver lipid levels were significantly lower in vitamin Be-deficient vs. pair-fed rats.?°! The changes were due mainly to lower trigylceride levels, whereas cholesterol levels were not

clinical implications. The effect, if any, of vitamin Bg on cholesterol metabolism remains controversial. Studies by Lupien and coworkers have shown that the rate of incorporation of ['*C]-acetate into cholesterol was increased in vitamin Be¢-deficient rats as compared to controls.!!°*! However,

the amount

of cholesterol

in plasma and liver of rats and other species has been reported to be increased, not changed, or even decreased."'°*! Significant positive correlation between plasma PLP and high-density-lipoprotein (HDL) cholesterol and negative correlations with total cholesterol and low-density-lipoprotein (LDL) cholesterol have been reported in monkeys fed atherogenic Western

that

diets and a “prudent’’ Western diet.!'°”! However, the

rats fed a high-protein (70%), vitamin Be-deficient diet developed fatty livers and suggested that this was due to impaired lysosomal degradation of lipid.!!°°! The synthesis of fat in vitamin Be-deficient rats

diets fed to the monkeys contained distinctly different amounts of vitamin Bs. The use of supplemental vitamin Beg in reduction of blood cholesterol has not been definitively tested. Serfontein and Ubbink reported decreased serum cholesterol (0.8 mmol/L) in 34 subjects given a multivitamin containing 10 mg of pyridoxine.!'!°! The reduction was mainly as LDL cholesterol. In another study, pyridoxine (50mg/day) administration prevented the increase in serum cholesterol seen when disulfiram was administered!!!" Controlled trials of pyridoxine are needed to resolve the role of vitamin Beg in modifying serum cholesterol levels. The role of vitamin Bg in lipid metabolism remains unclear. Evidence to date suggests a role in modifying methionine metabolism and thus an indirect effect on phospholipid and fatty acid metabolism. This effect and one on carnitine synthesis!'!7! appear to be the primary effects of vitamin Bg on lipid/fatty acid metabolism.

different. In contrast, Abe and Kishino

showed

has been reported to be greater,!°!) normal,” or depressed.!'°"! The observed differences may be related to the meal pattern of the animals.!!%4! The effect of vitamin Bg deprivation on fatty acid metabolism has also received attention. A pyridoxine deficiency may impair the conversion of linoleic acid to arachidonic acid.'?'°°! Cunnane and coworkers!!! found that phospholipid levels of both linoleic and y-linolenic acid were increased in vitamin Be-deficient rats, but the level of arachidonic

acid was decreased

as compared with that of control levels in plasma, liver, and skin. They suggested that both linoleic desaturation and y-linoleic acid elongation may be impaired by

a

vitamin

Be, deficiency.

She,

Hayakawa,

and

Tsuge!!°*! have observed decreased activity of terminal A°-desaturase in the linoleic acid desaturation system in rats fed a vitamin Be-deficient diet and a positive correlation between phosphatidylcholine (PC) content and A°-desaturase activity in liver microsomes. Subsequent work by She et al. suggests that alteration of S-adeno-

sylmethionine (SAM) to S-adenosylhomocysteine is involved in these changes.!'°7! In one of the few studies of vitamin

B, and fatty acid metabolism

in humans,

desoxypyridoxine was utilized to induce a vitamin Be deficiency."*"! Xanthurenic acid excretion following a 10-g p,L-tryptophan load indicated a moderate vitamin B,-deficient state. Only minor changes in fatty acid levels in plasma and erthyrocytes were observed as a result of the deficiency produced. The pattern of fatty acids observed was interpreted by the authors to support the findings of Witten and Holman.!'! The work of She, Hayakawa, and Tsuge!!°! supports this. This provides a plausible mechanism, because the primary metabolic steps in fatty acid metabolism do not involve nitrogen-containing substrates, a feature common to most PLP-dependent enzymatic reactions. The change observed in arachidonic acid levels and the role it plays in cholesterol metabolism may have

Hormone Modulation/Gene Expression One of the more intriguing functions of PLP is as a modulator of steroid action.!!'*!'*! Reviews of this

interaction are available!!'>!'®] PLP can be used as an effective tool in extracting steroid receptors from the nuclei of tissues on which the steroid acts.''7) Under conditions of physiological concentration of PLP, reversible reactions occur with receptors for

estrogen,"!!®! androgen,!'!”! progesterone,!'?"! and glucocorticoids."*!] PLP reacts with a lysine residue on the steroid receptor. As a result of the formation of a Schiff base, there is inhibition of the binding of the

steroid-receptor complex to DNA.!'!?! Holley et al. found that when female rats were made vitamin Be deficient and injected with [H]-estradiol, a greater amount of the isotope accumulated in the uterine tissues of the deficient animal than in the tissues of controls.!!77] Bunce and Vessal studied the dual effect of zinc and vitamin Beg deficiency on estrogen uptake

by the uterus.''*?!

They

found

that there

was

an

Vitamin Be

722

increased uptake of the harmone in both the vitamin Be- and the zinc-deficient animals. A combined deficiency of the two nutrients resulted in even greater retention of estrogen. The number of estrogen receptors was not altered by the deficiency of vitamin Be. This study suggests that there might be increased sensitivity of the uterus (or other end-target tissues) to steroids when vitamin Bg, status is abnormal. Sturman and Kremzner found enhanced activity of ornithine decarboxylase in testosterone-treated vitamin Bg-deficient animals as compared to control animals.!'*4] DiSorbo and Litwack observed increased tyrosine aminotransferase activity in hepatoma cells raised on a pyridoxine-deficient medium and treated with triamcinolone acetonide as compared to pyridoxinesufficient cells treated with the same steroid.!'?°! Allgood and Cidlowski ''**! have used a variety of cell lines and a range of intracellular PLP concentrations to show that vitamin Bs modulates transcriptional activation by several (androgen, progesterone, and estrogen) steroid hormone receptors. This supports the role of vitamin Bg as a physiological modulator of steroid hormone action. Oka et al.!!?7| have found that in vitamin B,-deficient rats, the level of albumin mRNA was sevenfold that of control rats. They suggest that PLP modulates albumin gene expression by inactivation of tissuespecific transcription factors. Oka and coworkers have also observed a sevenfold increase in the level of mRNA for cystosolic aminotransferase in vitamin Be-deficient rats as compared to that of vitamin Besufficient rats.'?*] Subsequent work by Oka et al.!'??! shows an inverse relationship between intracellular PLP concentration and albumin in mRNA in rats given amino acid loads. Thus, PLP may be a modulator of gene expression in animals, especially under conditions of altered amino acid supply. Given the intimate relationship of vitamin Beg and amino acid metabolism, these investigations open up for study a new area of metabolic regulation via altered intracellular nutrient (PLP) concentration.

VITAMIN Bg REQUIREMENTS Considering the numerous functions in which vitamin Be is involved, assessment of the requirement for this

vitamin becomes important. Reviews of vitamin Be requirements are available.!'30-!97] Several relevant studies have been conducted. These have been carried out in both young and elderly adults and in males and females.?7!73-!5] While some of them are similar to previous ones in that they employed depletion/repletion design!'**-'**! and diets with high Be bioavailability, others have used diets more representative of the usual U.S. diet.?7

What is also different about some of these studies is that they have included additional measurements that may be indicative of intercellular function of PLP: Meydani et al.!'*°! examined the effect of different levels of vitamin Be (pyridoxine added to a low-Be food diet) on immune function. They observed that adequate immune function in elderly women was not achieved until 1.9mg/day of vitamin B, was fed. Men required 2.88 mg/day to return function to baseline levels. In addition, several indices of vitamin Be

status were measured. Based on when these values for these indices returned to predepletion levels, the requirement for vitamin Bg was estimated to be 1.96 and 1.90 mg/day for men and women, respectively. Kretsch et al.'°°! fed four graded doses of vitamin Bs to eight young women following a depletion diet (for 11-28 days). Based on this and other studies, less than 0.5mg/day is needed to observe clinical signs of vitamin Bg deficiency. Functional signs, such as abnormal EEGs, were only seen with an intake lower than 0.5mg/day. Various biochemical measures, including the functional tests of tryptophan metabolite excretion (xanthurenic acid) and erythrocyte aspartate transaminase (EAST) stimulation, were normalized at the 1.5 and 2.0mg/day level, respectively. The authors stated that if all currently used biochemical measures were to be normalized, then more than 0.020mg of vitamin Be per gram of protein is required. Hansen et al.?*! used a different approach in evaluating the effect of graded doses of vitamin Be on status. First, rather than feeding a diet deficient in vitamin

Be, a diet containing

a level that was

low but

within the realm of what individuals might normally consume was fed. Various levels of pyridoxine (as an oral solution) were then added to the basal diet (range 0.8-2.35 mg B,/day). Based on both direct and indirect measures (including tryptophan metabolite excretion), it was concluded that a B¢/protein ratio greater than 0.20 was required to normalize all vitamin Bg status indices. Ribaya-Mercado et al.''*3! evaluated the vitamin Be requirements of elderly men and women in a depletion/repletion study. The authors concluded that the vitamin Bg requirements of elderly men and women are about 1.96 and 1.90 mg/day, respectively. The vitamin Be (pyridoxine) fed to these subjects was in a highly bioavailable form. A metabolic study in young women evaluated the requirement for vitamin B,.!'*4! Again, a depletion/ repletion design was used and several indices of vitamin Be status were measured. These included urinary 4-PA excretion, plasma PLP, erythrocyte PLP, and erythrocyte alanine aminotransferase (EALT) and EAST activity coefficients. Using predepletion baseline levels (after 9days of feeding 1.60mg/day) of these indices as a basis for comparison in determining adequacy, the amount of vitamin B¢ required to normalize

Vitamin

723

Be

these indices was 1.94mg/day (Bg to protein ratio of 0.019). An important consideration relative to many of these metabolic studies that have been used in establishing the adult vitamin Bs recommended dietary allowance (RDA) is the composition of the diets used. Most were ones in which the amount

of vitamin Be

from food was low and of relatively high bioavailability. Vitamin Bg was added back to the diets in the form of pyridoxine hydrochloride and thus is considered

disorders,!'4! alcoholism,"**! heart disease,"* pregnancy,>!47?l breast cancer,''48! Hodgkin’s disease, !!*7! and sickle cell anemia.!”*! Hypophosphatasia is an example of a condition in which plasma PLP levels are markedly elevated in some individuals." Relatively few of these studies have exhaustively evaluated vitamin Bs metabolism.

Coronary Heart Disease

100% bioavailable. Therefore, the total vitamin Bg in

the diets is probably 95-100% bioavailable. Taken together, these four recent metabolic studies support a higher vitamin Bg requirement for women and men than is currently employed. A value of 1.9mg/day for women and 2.2mg/day for men is recommended. Since the vitamin Bg in these studies was highly available, the inclusion of a factor for bioavailability would further increase the RDA'37] The above discussion has focused on the vitamin Be requirement for adults aged 18-70. There has been little research to support a statement of recommendations for children (aged 1-10) or adolescents (aged 11-18).

The relationship between vitamin Bg and coronary heart disease can be viewed from both an etiological perspective and that of the effect of the disease state on vitamin Bg metabolism. With respect to an etiological role, altered sulfur amino acid metabolism has been suggested to result in vascular damage. A poor vitamin Be status can result in an increased circulating concen-

tration of homocysteine."°"! In the trans-sulfuration pathway, serine and homocysteine condense to produce cystathionine. This reaction is catalyzed by the PLP-dependent enzyme cystathionine B-synthase. In genetic disorders of this enzyme, homocysteine accu-

mulates in the plasma."'°*! An increased incidence of Food Sources

There are various forms of vitamin Bg in foods. In general, these forms are a derivative of pyridoxal, pyridoxine, and pyridoxamine. Pyridoxine and pyridoxamine (or their respective phosphorylated forms) are the predominant forms in plant foods such as lima beans, spinach, broccoli, avocados, white beans, lentils, nuts,

and brown rice. Although there are exceptions, pyridoxal, as the phosphorylated form, is the predominant form in foods. (Data on the amount of each of the

three forms are listed in Table 4 of Ref.?71) DISEASE AND TOXICITY Several books*°7] and reviews’! have examined the

relationship between specific diseases and vitamin Bg nutrition in detail. There are numerous diseases or pathological conditions in which vitamin Bg metabolism is altered. The primary indicator of an alteration in vitamin Bg metabolism has been an evaluation of tryptophan metabolism or the plasma PLP concentration. The first of these is an indirect measure of status and the second is a direct measure. Conditions in which tryptophan metabolism has been shown to be altered and in which vitamin Be (pyridoxine) administration was used include asthma,!!**! diabetes,!*”! certain cancers,7! pellagra,!!*°! and rheumatoid arthritis.!!4!! Diseases and pathological conditions in which plasma PLP levels have been shown to be depressed include asthma,"7!_ diabetes,"'**!_ renal

arteriosclerosis has been associated with this enzyme defect.!'°7! In addition, elevated levels of homocysteine in the plasma have been observed in people with ischemic heart disease.!!°*!°*! There has been an explosion in the number of papers suggesting that elevated plasma homocysteine is a risk factor for heart disease and stroke. While folic acid is most effective in redu-

cing the plasma concentration of homocysteine,!'°!°71 vitamin Bs has been shown to be most effective in reducing plasma homocysteine when a methionine load is given. A European study of 750 patients with vascular disease and 800 control subjects found that increased fasting homocysteine (more than

12.1 pmol/L) was associated with elevated risk of vascular disease.''>*! In this study, a plasma concentration of PLP below the 20th percentile (less than 23 pmol/L) for controls was associated with increased risk. This relationship between plasma PLP and atherosclerosis was independent of homocysteine levels. Other studies have also found an increase in coronary artery disease risk and low PLP levels in plasma.!!°°!>7! While some animal experiments have shown that rhesus monkeys made vitamin Beg deficient develop atherosclerotic lesions," other studies do not reveal any pathological lesions.“°'! In humans at risk for coronary heart disease, a negative correlation between dietary vitamin Bg and bound homocysteine has been observed."©! For some people with homocysteinuria, treatment with high doses of vitamin Be reduces the plasma concentration of homocysteine in certain patients but does not totally correct methionine metabolism,''®! especially when there is an increased

Vitamin Be

724

methionine intake. Thus, if vitamin B, therapy is to be successful in reducing vascular lesions, diet modifica-

tion with a lowered methionine intake may be necessary. The extent to which supplemental vitamin Be intake (beyond normal dietary intakes) may reduce the risk of coronary heart disease is not known. A second aspect of coronary heart disease is the relationship between the presence of the disease and vitamin B, status. Several recent studies have found that the plasma PLP concentrations in people with coronary heart disease are significantly lower (2141 nmol/L) than those in healthy controls (32-46 nmol / jy ees However, Vermaak et al. have found that the decrease in plasma PLP concentration is only seen in the acute phase of myocardial infarction."°°! Unfortunately, other measures of vitamin Bg, status have not been evaluated in this disease. In one study,"™! giving cardiac patients vitamin Be supplements (amounts not given) resulted in plasma PLP levels well above normal. The effect of long-term vitamin Bs therapy on recurrence of coronary artery disease has not been evaluated. Elevated plasma cholesterol concentration has been strongly associated with an increased risk of coronary heart disease. As previously reviewed, vitamin Bs may influence cholesterol metabolism. Serfontein and Ubbink!!'" have found that use of a multivitamin supplement containing about 10mg of pyridoxine for 22 weeks by hypercholesterolemic adult men resulted in a significant decrease in cholesterol levels, with most

of the reduction due to a decreased level of LDL cholesterol. Smoking is an additional risk factor for coronary heart disease. Interestingly, smokers have decreased plasma levels of PLP."''°!°° Evidence to date suggests a link between several risk factors for coronary heart disease and altered vitamin Be status and a potential beneficial effect of increased vitamin Bs intake on cholesterol levels. Furthermore, wellcontrolled studies are needed before the therapeutic effect of vitamin Bg can be evaluated for this disease.

HIV/AIDS Vitamin

Bg status''®”!®!

and,

to a limited

extent,

metabolism!'®™! have been examined in persons with human immunodeficiency virus (HIV). Because of the link between immune function and vitamin B,,!°7! one would expect that maintaining an adequate vitamin Be status is critical for HIV patients. Several studies have evaluated vitamin Be intake,!!°8!7°!7 and the progression of the disease as related to intake of nutrients, including vitamin B,.!'’"! These studies generally found low intakes of vitamin B,, and one study”! reported an inverse relationship between vitamin Bg intake and progression.

Biochemical

assessment

of vitamin

Bg status has

been done in several studies!!®”'°!7"! and has revealed it to be poor. In most of these studies, (1°71) a-EAST

activity and status.

In the

stimulation

was

studies,

samples

used were

as an

index

frozen,

of

which

may have compromised the data and subsequent evaluation. Although other researchers have measured and reported low levels of “serum vitamin Bg,’ they failed to specify what form was being measured,!171177] Therefore, given the complexities of nutritional wellbeing in HIV/AIDS patients and methodological problems in these studies, it is difficult to assess the role of vitamin Bg in this syndrome. In vitro studies suggest that PLP may play a role in

HIV/AIDS. Salhany and Schopfer!!”*! found that PLP binds to the CD4 receptors at a site that is competitive with a known antiviral agent (4,4’-diisothiocyanato2,2'-stilbenedisulfonate). Other investigators have found that PLP is a noncompetitive inhibitor of HIV1 reverse transcriptase.!'’*!7°! Based on these in vitro . studies, clinical trials with vitamin Bg appear warranted.

Premenstrual Syndrome Premenstrual syndrome (PMS) is another clinical situation for which vitamin Bg supplementation has been suggested.!'”° Estimates indicate that 40% of women are affected by this syndrome.” Using a wide variety of parameters, no difference in vitamin Be, status was observed in women with PMS compared

to those not reporting symptoms.*!!78! Nevertheless, beneficial effects of Bg administration on at least some aspects of PMS have been reported. Treatment of PMS with vitamin B¢ has been based in part on the studies of Adams et al..'7°l in which PN was

used to manage the depression observed in some women taking oral contraceptives. Of the several stu-

dies in which PN was used to treat PMS, there have been open-type studies that were double-blind and placebo-controlled. Open studies are prone to a placebo effect error, often as high as 40%. Of the well-controlled type, one study showed no effect of pyridoxine therapy,''®°! whereas three studies reported significant improvement of at least some of the symptoms associated with PMS. In one study, 21 of 25 patients improved."'8") Another study found that about 60% of 48 women showed improvement with pyridoxine (200 mg/day) and 20% showed improvement with placebo.!'®7] The fourth study"'®3! reported improvement in some symptoms in 55 women treated daily with 150mg of pyridoxine. Brush!!”*! has reported results of studies he has conducted using vitamin Bg alone and vitamin Bes plus magnesium. His data suggest that doses of 150-200 mg of vitamin Bg are necessary before a significant positive effect is observed. In addition, the

Vitamin B,

725

combination of vitamin Bs plus magnesium appears to be beneficial. The complexity of PMS and the subjective nature of symptom reporting continue to result in contradictions and controversy in the lay and scientific literature. Kleijnen et al.!'*4! have reviewed 12 controlled trials in which vitamin Bg was used to treat PMS. They concluded that there is only weak evidence of a positive effect of vitamin Bs. There may be a decrease in the availability of vitamin Bs during PMS, possibly due to cell transport competition, from fluctuating hormone concentrations. An increase in vitamin Bg concentration could overcome competition and may explain the relief of symptoms seen in some women following high-dose vitamin Bg supplementation.

Sickle Cell Anemia

Low levels (18 pmol/L) of plasma PLP have been reported in 16 persons with sickle cell anemia.!7®! Treatment of 5 of these patients with 100 mg of pyridoxine hydrochloride per day for 2mo resulted in a reduction of severity, frequency, and duration of painful crises in these persons. The mechanism by which vitamin Bg acts is not known, but it may be related to pyridoxal and PLP binding to hemoglobin.

Asthma

Depressed levels of plasma and erythrocyte PLP have also been reported in persons with asthma.!!47! Of significance was the fact that all persons were receiving bronchodilators. Treatment of seven asthmatics with 100 mg of pyridoxine hydrochloride per day resulted in a reduction in the duration, occurrence, and

severity of their asthmatic attacks. Subsequent work by one of these authors has not fully supported the earlier findings.!'**) Treatment of 15 asthmatics with vitamin Bg did not result in a significant difference in symptom scores, medication usage, or pulmonary function tests as compared to placebo treatment. Ubbink et alee have shown that theophylline lowers plasma and erythrocyte PLP. Pyridoxal kinase is inhibited by Table 4

theophylline and was responsible for the decreased PLP level in the plasma and presumably intracellularly.

Carpal Tunnel Syndrome At least five placebo-controlled trials from four different laboratories have shown that administration of PN relieved the symptoms of carpal tunnel syndrome (pain and/or numbness in hands).!'87-!88] In one study, no significant improvement was observed.!'®”! Since supplementation with vitamin Bg well in excess of the RDA was required for improvement (generally 50150 mg), it would seem that individuals with this disor-

der have a high metabolic demand or that the vitamin is active in some non-coenzyme role. Two recent studies examined the relationship between plasma PLP and carpal tunnel syndrome. One study"'””! found no relationship between symptoms of carpal tunnel syndrome and plasma PLP, but a study by Keniston

et al.!’?" found a significant inverse univariate relationship between plasma PLP concentration and the prevalence of pain, the frequency of tingling, and nocturnal

awakening.

Drug—Vitamin B, Interaction Treatment of persons with various drugs may also compromise vitamin Bg status and hence result in an increased need for the vitamin. Table 4 lists several drugs and their effect on vitamin Bs, status. Bhagavan

has reviewed these interactions in detail.!!°7) A common feature of these drug interactions is their adverse effect on central nervous system function. In addition, many of these drugs react with PLP via Schiff base formation. This reaction can result in decreased levels of PLP in tissues, such as the brain, leading to a functional defi-

ciency. In most cases, supplemental vitamin Bg reverses the adverse consequences of the drug. Oral contraceptives do not react directly with PLP but do induce enzyme synthesis. Some of these enzymes are PLP dependent,

and

as

a

result,

PLP

is metabolically

trapped in tissues. This may then lead to a depressed

plasma

PLP

concentration"?!

In

addition,

Drug—vitamin Bg interactions

Drug

Examples

Mechanism of interaction

Hydrazines

Iproniazid, isoniazid, hydralazine

React with pyridoxal and PLP to form a hydrazone

Antibiotic

Cycloserine

Reacts with PLP to form an oxime

L-DOPA

L-3,4-Dihydroxyphenylalanine

Reacts with PLP to form tetrahydroquinoline derivatives

Chelator

Penicillamine

Reacts with PLP to form thiazolidine Ethinyl estradiol, mestranol, increased enzyme levels in liver

Oral contraceptives -

and other tissues; retention of PLP Alcohol

the

Ethanol

Increased catabolism of PLP; low plasma levels

Vitamin Bg

726

Table 5

Toxicity symptoms reported to be associated with chronic use of high-dose pyridoxine

Symptoms

Ref. Coleman et al.!!90!

Motor and sensory neuropathy; vesicular dermatosis on regions of the skin exposed to sunshine

Schaumburg et al,[°°O

Peripheral neuropathy; loss of limb reflexes; impaired touch sensation in limbs; unsteady gait; impaired or absent tendon reflexes; sensation of tingling that

proceeds down neck and legs Brush!!7¢!

Dizziness; nausea; breast discomfort or tenderness

Bernstein and Lobitz!!>!

Photosensitivity on exposure to sun

synthetic estrogens specifically affect enzymes of the tryptophan-niacin pathway, resulting in abnormal

tryptophan metabolism.®"! There may bea need for extra vitamin Be above the current EDA in a small proportion of women using oral contraceptives and consuming low levels of the vitamin. Any drug that interacts with the reactive molecule PLP in a Schiff base reaction should be considered an instigator of resultant adverse effects on vitamin Bg status and a subsequent negative influence on central nervous system function.

Hazards of High Doses With the therapeutic use of pyridoxine for various disorders and self-medication has come the potential problem of toxicity. Shaumburg et al. have identified several individuals who developed a peripheral neuropathy associated with chronic high-dose use of pyridoxine."**! Subsequent to this, other reports of toxicity related to pyridoxine ingestion have been pub-

lished.""°°! The minimal dose at which toxicity develops remains to be determined. Other toxicity symptoms have been identified. These symptoms and those reported by Schaumburg et al. are listed in Table 5. They are relatively rare, and the use of pyridoxine doses of 2—250mg/day for extended periods of time

appears to be safe,!1°! In rats given high doses of pyridoxine hydrochloride for 6 weeks, there was a decrease in testis, epididymis, and prostate gland weight at the 500- and 1000-mg/kg dose.!'°] There was also a decrease in mature spermatid counts. This high intake would be equivalent to 1.5-2.0g of vitamin B, for a human.!'?®! Thus, the application of these data to human nutrition is not clear. Additional safety evaluation is found in the DRI guidelines."°7!

active form, PLP, in such a wide spectrum of enzymatic reactions is an indication of the importance of this vitamin. In addition to the involvement of PLP in amino acid metabolism and carbohydrate metabolism, its reactivity with proteins points to the diversity of action of this vitamin. Further research is needed on the factors controlling the metabolism of vitamin Be and determination of vitamin Bs needs of specific populations. With knowledge of the functional proper- — ties of vitamin Be and quantitation of its metabolism under various physiological and nutritional conditions, the health and well-being of individuals can be improved.

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Witteman,

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Graham,

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folate and vitamin B¢ concentrations: risk factors for stroke, peripheral vascular disease and coronary artery disease. Circulation 1998, 97,

159:

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By

Swift, M.E.; Shultz, T.D. Relationship of vitamins Beg and By to homocysteine levels: risk

for coronary heart 1986, 34, 1-14.

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Barnard, H.C.; Potgieter, Plasma _pyridoxal-5’-phos-

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165.

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178.

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1988; 363-379. O’Brien, P.M.S. The premenstrual syndrome: a review of the present status of therapy. Drugs 1982, 24, 140-151. Mira, M.; Stewart, P.M.; Abraham, S.F. Vitamin

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180. 181.

menstrual tension. Lancet 1972, 7, 1177-1178. Abraham, G.E.; Hargrove, J.T. Effect of

1987,

63,

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Ubbink,

J.B.;

De

Villiers,

Baum, M.K.; Mantero-Atienza, E.; Shor-Posner, G. et al. Association of vitamin Be status with,

1323

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184.

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Kendall,

K.E.;

Schurr,

P.P.

The

effects

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vitamin Bs supplementation on premenstrual syndromes. Obstet. Gynecol. 1987, 70, 145-149. Kleijen, J.; Riet, G.T.; Knipschild, P. Vitamin Be in the treatment of the premenstrual syndrome— review.

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1990,

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R.A.; Reynolds,

R.D.

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307-315.

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a

1595-1600. MPs:

5'-phosphate inhibits the polymerase activity of a recombinant RNase H-deficient mutant HIV-1 reverse transcriptase. AIDS Res. Hum. Retroviruses 1992, 8, 597-604. Brush, M.G. Vitamin Be treatment of premenstrual syndrome. In Clinical and Physiological Applications of Vitamin Bs; Leklem, J.E.,

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et al. Dietary micronutrient progression to acquired syndrome (AIDS) in human virus type 1 (HIV-1)-infected

wee

Moen, L.K.; Bathurst, I.C.; Barr, P.J. Pyridoxal

L.S.; Becker, P.J. Depressed plasma pyridoxal5’-phosphate levels in tobacco-smoking men.

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168.

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carpal tunnel syndrome. J. Occup. Environ. Med. 1997, 39, 949-959. Bhagavan, H.N. Interaction between vitamin Be and drugs. In Vitamin Bg: Its Role in Health and

Leklem, J.E. Vitamin Bs requirement and oral use—a

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1986,

116, 475-477. 194.

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Cohen, M.; Bendich, A. Safety of pyridoxine—a review of human and animal studies. Toxicol. Lett. 1986, 34, 129-139.

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Mori, K.; Kaido, M.; Fujishiro, K.; Inoue, N.;

Koide, O. Effects of megadoses of pyridoxine on spermatogenesis and male reproductive organs in rats. Arch. Toxicol. 1992, 66, 198-203.

Franzblau, A.; Rock, C.L.; Werner, R.A. et al.

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Schaumburg, H.; Kaplan, J.; Windebank, A.; Vick, N.; Rasmus, S.; Pleasure, D.; Brown,

Cohen, P.A.; Schneidman, K.; Ginsberg-Fellner,

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Disease; Reynolds, R.D., Leklem, J.E., Eds.; Alan R. Liss: New York, 1985; 401-415.

193:

K.; Dalton,

Neurol. Scand. 1987, 76, 8—11.

Smith, G.P.; Rudge, P.J.; Peters, J.J. Biochemical

The relationship of vitamin Bg status to median nerve function and carpal tunnel syndrome among active industrial workers. J. Occup.

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Vitamin

B, 2

Lindsay H. Allen Katharine M. Jones United States Department of Agriculture—Western Human Nutrition Research Center, University of California, Davis, California, U.S.A.

INTRODUCTION Because vitamin Bj» is found only in animal source

foods (ASF), strict vegetarianism has long been associated with a greater risk of deficiency of this vitamin. The elderly, many of whom lose their ability to absorb vitamin Bj, and the small proportion of the population with pernicious anemia (PA) due to lack of intrinsic factor (IF) are also established high-risk groups for this vitamin deficiency. It is generally assumed that clinical symptoms of Bj deficiency take many years to appear after intake or absorption becomes inadequate. However, in recent years, it has become apparent that this deficiency is much more prevalent than previously assumed, affecting a high proportion of people in developing countries and even many lacto-— ovo-vegetarians. Considering the number and size of population groups at risk of deficiency, it is important that we develop the most sensitive and specific methods of assessing vitamin Bj) status and understand the potential adverse functional consequences of this deficiency across the life span.

VITAMIN

B,.2 STRUCTURE

cobalt atom above the plane of the ring account for the various forms of active Cbl (Table 1). The two forms of vitamin Bj). with metabolic activity are 5/-deoxyadenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). Hydroxycobalamin (OHCbI) and cyanocobalamin (CNCbI) are also biologically active after conversion to AdoCbl or MeCbl. Cyanocobalamin

is rare in nature, but after isolation

is used in the laboratory and is also the form used in vitamin Bj. supplements. AdoCbl and MeCbl are generally considered to be light sensitive, but CNCbl is relatively stable.

Coenzyme Function In humans, vitamin B,. functions as a coenzyme

for

only two reactions in the body, catalyzed by methylmalonyl CoA mutase and methionine synthase. AdoCbl transfers a hydrogen atom in the methylmalonyl CoA mutase reaction, which is required for the conversion

AND FUNCTION

Structure

Vitamin Bp, or cobalamin (Cbl), is a water-soluble vitamin with a molecular weight of 1355. Symptoms of Cbl deficiency were first described in the early 18th century, but the vitamin was not isolated until 1948. The molecule contains a corrinoid ring with a cobalt molecule at its center, beneath which is linked

a nucleotide (Fig. 1). Analogs of Cbl have different structures of the nucleotide and do not retain the active properties of Cbl. However, the ligands linked to the

Lindsay H. Allen is at the USDA—Western Human Nutrition Research Center and Department of Nutrition, University of fy California, Davis, California, U.S.A. Katharine M. Jones is at the USDA—Western Human Nutrition

Research

Center

and

Department

of Nutrition,

University

of

California, Davis, California, U.S.A.

Encyclopedia of Dietary Supplements DOT: 10.1081 /E-EDS-120022051

Copyright © 2005 by Marcel Dekker. All rights reserved.

HO

Fig. 1 The chemical structure of vitamin B). (From http:// www.engr.psu.edu/wep/EngCompSp98 /Aclausi/HodgkinD7. html.) (View this art in color at www.dekker.com. ) 735

Vitamin By

736

Table

binder found in the salivary and esophageal glands,

1 Cobalamins with vitamin Bj). activity

R-group

Biological role of cobalamin

—5/-Deoxyadenosyl

Adenosylcobalamin—coenzyme for methylmalonyl CoA mutase

—CH;

Methylcobalamin—coenzyme for methionine synthase

—CN

Cyanocobalamin—biologically active upon conversion to AdoCbl or MeCbl

—OH

Hydroxycobalamin—biologically active upon conversion to AdoCbl or MeCbl

of propionyl CoA to succinyl CoA, an integral step in odd-chain fatty acid breakdown (Fig. 2). Propionyl CoA is first converted to methylmalonyl CoA via a carboxylase, after which AdoCbl-dependent methylmalonyl CoA mutase converts methylmalonyl CoA to succinyl CoA. Methylcobalamin accepts and donates a methyl group in the second vitamin B,2-dependent reaction, in which methionine synthase converts methyltetrahydrofolate (CH3-THF) and homocysteine (Hcy) to tetrahydrofolate (THF) and methionine (Fig. 3). Methionine is then metabolized to S-adenosylmethionine (SAM), a universal methyl donor. THF is further metabolized to methylenetetrahydrofolate (CH>THF), which is a cofactor for thymidylate synthetase, the enzyme that converts uracil ((UMP) to thymidine

(dTMP).

As

a cofactor

for methionine

synthase,

vitamin Bj plays an important role in the synthesis of purines, pyrimidines, and amino acids, and in the transfer of methyl groups.

binds the vitamin. In the stomach, Cbl remains bound to Hc, while IF, a second By, binding protein, is

secreted by the parietal cells. Haptocorrin has a higher affinity for Cbl than IF, and must be degraded by proteolytic enzymes from the small intestine before IF can bind the vitamin. In the ileum, CbI-IF specific receptors bind the complex, and absorption occurs by endocytosis into endothelial cells, in a calctum-dependent but energy-independent process that takes about 3-4 hr. Percentage absorption decreases with the size of the dose."!! The recommended dietary intake values for the United States/Canada assume that vitamin By

in food is 50% bioavailable. One percent of free vitamin Bj, is absorbed passively, which is important because sufficient amounts of the vitamin can be absorbed from large doses (i.e., SOOpug/day) to restore and maintain Bj status even in individuals lacking IF. The complexity of the digestive process means that _ abnormalities can occur at several points. An inability to effectively degrade proteins in food, such as that occurring in achlorhydria or atrophic gastritis, often prevents release of vitamin B,, from food. Transfer of vitamin Bj from food to Hc, which is dependent on pH and pepsin secretion, may also be compromised.

Finally, lack of IF, due to the autoimmune condition PA, will prevent the uptake of Cbl by the ileal endothelial cells. Transport Proteins Transcobalamin II (TC Il) After absorption into the endothelium, IF is degraded, and free vitamin

VITAMIN Biz METABOLISM

B)> is bound

to transcobalamin

II

(TC I), which then transports Bj). through the plasma. TC HU, which has a half-life of several minutes, is pro-

Digestion and Absorption The digestion of vitamin Bj) is unique in its complexity (Fig. 4). When Cbl is released from the proteins to which it is attached in food, haptocorrin (Hc), a B;>

duced locally by many cells and is thought to play a role in cellular export of vitamin Bj», as well as plasma transport. It is the only one of the three plasma Bj) binding proteins (transcobalamins I, II, and III) that is responsible for receptor-mediated uptake of Bj. into

ATP +HCO,

=

Hye

Cn

ADP +B

© — [proionyl-CoA carbox ylase [biotin]

nC

CO,

ee

|

,;

SCoA

So

CO,

[methyimalonyl ~CoA racemase

SCoA

-0,C-—CHy—CHs-C YS aeact

SCoA

1-6

[jaa

va

methylmalonyl mutase ee B 12 )>] (vitamin

Fig. 2 5'-Deoxyadenosylcobalamin is a coenzyme for methylmalonyl CoA mutase, which converts methylmalonyl CoA to succinyl CoA. (From: y http://h p://heibeck.freeshell.org/NESA A / Biochem_Fall_2001/Lipid_Metab.pdf.)

Vitamin Bo

737

Synthesis of

5,10-Methylenetetrahydrofolate reductase

nucleotides

Cystathionine

5,10-le tetrahydrofolate

Cystathionine B-synthase Vitamin Bg

5-Methyltetrahyd aplere

Homocysteine erat

Tetrahydrofolate

Folate receptors

Folic Acid

Methionine synthase Vitamin By Methionine synthase reductase

=

S-Adenosylhomocysteine

Methionine

te

oe

Methylated DNA, proteins, and lipids

S-Adenosylmethionine DNA, proteins, and lipids

Fig. 3

Methylcobalamin is a coenzyme for methionine synthase, which transfers methyl-tetrahydrofolate and homocysteine to

tetrahydrofolate and methionine. (From Botto, L.D.; Moore, C.A.; Khoury, M.J.; Erickson, J.D. Neural tube defects. N. Engl. J.

Med. 1999, 347, 1515.)

cells, and constitutes about 10-20% of the total plasma

Bj2. Transcobalamin II is cleared from the plasma by the kidney, liver, heart, lung, spleen, and intestine?!

cell precursors, hepatoma cells, salivary glands, and granulocytes, and is largely unsaturated. An absence of Hc protein does not alter vitamin B)2 metabolism

detrimentally, but an inability to produce TC II results in symptoms of vitamin Bj» deficiency, including mega-

Haptocorrin

loblastic anemia

Approximately 75% of plasma vitamin Bj is bound to He (transcobalamins I + III), which has a half-life of 9-10 days, and can be thought of as a circulating store of the vitamin. Haptocorrin is produced in red blood

and neurological

abnormalities."

Several genetic polymorphisms have been identified in proteins involved

in the metabolism

of vitamin

Bj,

which may lead to reduced plasma concentrations of vitamin Bj.

Excretion and Storage

Salivary He Protein-bound B-12

Vitamin Bj. is excreted through the urine, bile, and feces. Enterohepatic recirculation of vitamin Bj is efficient, with 65-75% reabsorbed“! and therefore

plays an important role in maintaining adequate Cbl status. Vitamin Bj> is stored in the liver, which in an

adult human may contain 1-2mg of a 2-5 mg total body pool.

VITAMIN B,.—NUTRIENT INTERACTIONS Vitamin B,2 and Folate Fig. 4. Vitamin

Bj) digestion

requires adequate

stomach

acidity, and secretion of haptocorrin and intrinsic factor before receptor-mediated absorption can occur in the ileum.

Both

vitamin

B;2

and

folate

are

involved

in the

methionine synthase pathway (Fig. 3). According to the ‘folate trap’? hypothesis, CH3-THF builds up in

Vitamin By

738

excess when vitamin By deficiency prevents methionine synthesis from proceeding. While it was originally thought that vitamin Bj» deficiency trapped CH3-THF intracellularly, it is possible that Cbl-deficient cells fail to retain intracellular CH3-THF. This theory is supported by data showing a rise in plasma folate concentration in vitamin Bj) deficient animals and humans.”! Inadequate availability of folate coenzyme (CH>-folate) for DNA synthesis, due to folate or By2 deficiency, can produce megaloblastic anemia. However, folate deficiency does not produce the neuropathy that accompanies strict vitamin Bj deficiency. DIETARY REQUIREMENTS

AND SOURCES

but

not plants and animals, and humans depend on ASF,

fortified foods, and supplements for dietary Cbl. Organ meats, beef, pork, poultry, fish, shellfish, eggs, and dairy products are rich sources of vitamin Bj). (Table 2). Cobalamins from other sources including algae and yeast are probably not biologically active!!! Although dietary requirements are minimal when compared to those of other micronutrients, the facts that vitamin Bj). is present in a limited number of food sources and that the digestive process is complex make

deficiency a risk for certain populations. This certainly includes strict vegetarians, who lack sources of vitamin Bio in their diet,'©! and the elderly, who may have an impaired ability to absorb the vitamin from food sources due to gastric atrophy. There are also multiple reports of lower plasma vitamin B)2 concentrations in lacto—ovo-vegetarians than in omnivores.!”*! Populations in developing countries are also at risk for vitamin Bj. deficiency, due to the high cost and low availability of ASF, and a lack of fortified foods

Table 2 Animal source foods (ASF) rich in vitamin B,> By (ug per 100g)

Beef liver, fried

83.0

Chicken liver, fried

21.0

Beef, cooked

1.3-4.0

Pork, cooked

0.7

Turkey, cooked

0.4

Chicken, cooked

0.3

Tuna, canned in oil

aD)

Egg, fried

1.4

Cheese

Milk, whole (From USDA National Nutrient Database. gov/finic/cgi-bin/nut-search.pl.)

Bj.

Dietary Reference Intakes Daily loss of Cbl is estimated to be 0.1% of the total body pool.! Assuming a total body pool of 2-5 mg, the daily losses for adults would be 2—5ug. Due to the low ratio of daily losses to the total body pool, vitamin Bj). deficiency may take several years to develop after the removal of ASF, vitamin Bj» fortified foods, and supplements from the diet. This time could be substantially shorter in people who reabsorb less

lower output in bile as a result of depleted liver stores or malabsorptive disorders. It has been estimated that

Bj is synthesized by micro-organisms,

ASF

(See the section on “Vitamin

of the vitamin by enterohepatic recirculation, due to

Food Sources

Vitamin

and supplements. Deficiency.’’)

0.4-1.7

0.5 http.//www.nal.usda.

with a daily turnover rate of 0.1% per day, it can take from 1.5 to 1l.6yr to see signs of By. deficiency depending on initial liver Bj. stores."! The adult recommended dietary allowance (RDA) © of 2.4u1g is based on the intake levels required to maintain hematological status and normal vitamin Bj

plasma concentrations.) The RDA in pregnancy increases to 2.6ug due to transfer of newly absorbed Bj> to the fetus, and to 2.8 pg during lactation to cover secretion of B;2 into breast milk. There is no tolerable upper level of intake as no negative consequences have been associated with excessive vitamin B,7 consumption.

The recommended intake of 0.4ug for infants is based on the average intake (AI) of infants fed princi-

pally with breast milk."! The intake estimates assume that breast milk concentration averages 0.42 pg/L milk, based on a review of B,2 concentrations in the

milk of well-nourished women. However, as vitamin Biz is tightly bound to haptocorrin in human milk, and not all methods

for vitamin Bj, analysis release

the vitamin in milk, reported values vary widely and are uncertain. The AI of infants aged 7-12 mo is extrapolated from the requirement of 0-6-mo-old infants. The remaining RDAs for children and adolescents are extrapolated down from adult values, as sufficient data on intake within these groups are lacking. Table 3 summarizes the current daily recommended intakes for all age groups.

VITAMIN Biz STATUS THROUGHOUT THE LIFE CYCLE The Pregnant Woman

Intestinal absorption of Cbl is increased during pregnancy,”! although an overall fall in maternal plasma vitamin By> is observed and accompanied by a fall in

Cbl binders. As many as 15-30% of women may have

Vitamin B,>

Table 3.

739

Dietary recommended intakes of vitamin By

Age group

DRI B,2 (g/day)

Data used to determine DRI

Infants, 0-6 mo

0.4

Al of breastfed infants

Infants, 7-12 mo

0.5

Al extrapolated from AI of infants, 0-6 mo

Children, 1-3 yr

0.9

RDA extrapolated from RDA for adults

Children, 4-8 yr

12

RDA extrapolated from RDA for adults

Children, 9-13 yr

1.8

RDA extrapolated from RDA for adults

Adolescents, 14-18 yr

2.4

RDA extrapolated from RDA for adults

Adults, 19-50 yr

2.4

RDA based on intake required to maintain hematological status

Adults, over 50 yr

2.4

and plasma Bj» concentration

RDA based on intake required to maintain hematological status and plasma B)2 concentration

Pregnant women

.

2.6

RDA

based on adult RDA plus the amount of B;2 deposited into

the fetus daily Lactating women

2.8

RDA

based on adult RDA

plus the amount of Bj)» secreted into

breast milk daily DRI

= dietary recommended intake; AI = adequate intake; RDA

= recommended dietary allowance.

throughout pregnancy.” At birth, the total body

low plasma vitamin B,> during the third trimester of pregnancy, but concentrations rise sharply postpar-

content of vitamin Bj2 is approximately 50 ug, about half of which is stored in the liver. Plasma vitamin

tum; therefore, hemodilution, which increases plasma

volume by approximately 50%, may account for some

Bj2 concentration is usually twice that of the mother,

of this transient decrease. However, the fact that users of oral contraceptives also have lower plasma B)2 con-

but may be more than fourfold higher. When mothers suffer from vitamin Bj) deficiency, which may be highly prevalent in developing countries, their infants are also likely to have low vitamin stores, a phenomenon that can be improved by maternal supplementation with the vitamin. After the neonatal period, the plasma vitamin Bj? concentration of the infant begins to decline. Based on a requirement of 0.1 tg Cbl per day for tissue synthesis, the neonatal body stores can last approximately 8 mo even if vitamin Bj is completely absent from the diet. However, this assumes that the infant is born with

centrations"! suggests that hemodilution alone may not be entirely responsible. Low plasma Bj) during pregnancy is less likely to reflect a true deficiency in women with diets containing adequate ASF, and most pregnant women with low plasma Bj2 concentrations do not exhibit other signs of deficiency.""! Insufficient research has been conducted on the relationship between maternal vitamin Bj2 status and

pregnancy outcome. Fetal malformations were not associated with maternal plasma Cbl concentration in the first trimester of pregnancy in a French popula-

adequate stores, which may not be true of infants of malnourished and vitamin B,> deficient mothers.

tion.!'7] However, cord blood vitamin Bj. (but not maternal plasma vitamin Bj) was correlated with

birthweight

in a study of 188 pregnant

women.!!3]

Breast Milk as a Source of Vitamin Bi.

Moreover, homocysteinemia is a risk factor for num-

erous adverse pregnancy outcomes including birth defects!) and pre-eclampsia;""*! it is thus reasonable to assume, but not yet proven, that maternal vitamin By deficiency could have adverse effects on pregnancy outcome. In rural Nepal, where 65% of a group

Human milk has an impressive unbound Cbl binding capacity, approximately 1000 times greater than that of plasma, due to its high concentration of Hc. The large amount of Hc in milk may suppress Cbl-dependent intestinal microorganisms, such as

of pregnant women had low plasma B)2 concentrations, homocysteinemia and low plasma Bj2 were asso-

ciated with a doubling of pre-eclampsia and preterm delivery." The Neonate and Infant

Up to 60% of the Cbl absorbed in pregnancy is concentrated in the fetus, and the rate of transfer increases

E.

—

coli,

because

Hce-bound

Cbl

is unavailable

to

micro-organisms. Colostrum contains vitamin By) in excess, after which breast milk Cbl content decreases. Breast milk may contain 100-2000 pmol Bj2/L, but on average, a well-nourished woman’s milk contains 300-600 pmol By2/L throughout the lactational period. In women with a low Bj» intake, concentrations are lower and often correlated with maternal plasma Bj) values.

Vitamin

740

There are many case reports of neonates diagnosed with vitamin Bj» deficiency as a result of maternal veganism; both low infant stores at birth and low breast milk B;» would contribute to this serious situa-

tion. Breast milk Cbl < 362 pmol/L may be insufficient to meet infant requirements, as infant urinary methylmalonic acid (MMA) is inversely related to milk

VITAMIN Bi2 DEFICIENCY Methods for Evaluating Vitamin B;2 Status

The accepted cutoff point for a plasma vitamin Byi2 concentration that defines deficiency has been typically set as 148 pmol/L (200 pg/ml), and individuals with values below this point may show symptoms of deficiency. A plasma Cbl concentration between 148 and 220 pmol/L (200-300 pg/ml) is often used to designate marginal deficiency. In a study of infants, plasma MMA was markedly elevated (indicating vitamin Bj deficiency) when plasma vitamin B;2 was less than 220 pmol/L,"*! and in groups ranging from elderly adults!*"! to Guatemalan schoolers,’!! MMA increases when plasma Bj? falls below about 265 pmol/L

(350 pg/ml). Using a cutoff of 300pmol/L plasma Cbl to identify potential cases of B)2 deficiency, plasma Cbl had a diagnostic sensitivity of 0.40 and specifiof

0.98,

based

on

elevated

plasma

affluent settings, inadequate ingestion is less likely than poor absorption when consumption of ASF is relatively frequent. When ASF intake is limited, however, the risk of deficiency derives from low intake of the vitamin, although in the elderly the main cause of deficiency is usually poor absorption.

Inadequate absorption

B,2 at concentrations below this level, 18!

city

B, 2

MMA

Inadequate Cbl absorption is widely accepted to be the , principal cause of vitamin B,2 deficiency in affluent countries and may occur for several reasons. First, in PA, the lack of IF leads to an inability to absorb Cbl through the IF—Cbl receptor process. However, only about 24% of the elderly, for example, have PA.P*! Second, conditions that alter intestinal function, such

as achlorhydria and lack of enzymes such as pepsin, can lead to inefficient absorption of Bj. from food. Third, overgrowth of intestinal bacteria competing for the vitamin may lead to deficiency, a cause that is more important in conditions such as tropical sprue and after some types of intestinal resection. One study found that patients with bacterial overgrowth due to atrophic gastritis absorbed significantly less proteinbound Bj than control subjects, but that antibiotic therapy rapidly normalized absorption.?4) In the elderly, infection with Helicobacter pylori, in particular, may contribute to Bj deficiency by causing atrophic gastritis, lack of gastric acid, and, in its final stages, a lack of IF. Subsequently, there is impaired

(>0.34 umol/L) to confirm diagnosis.°7] Thus, plasma

absorption of food-bound Bj.7°! Vitamin By defi-

Bz is a reasonable, but not perfect, indicator of risk of vitamin B;> deficiency, and low concentrations should generate concern.

ciency associated with malabsorption is common in elderly populations and prevalence is often high in this group. For example, in 548 surviving participants of the original Framingham Study, the prevalence of Cbl deficiency was estimated to be more than 12% in free-living elderly Americans (women and men aged 67-94 yr).

Diagnosis of Bj. status using plasma Cbl can be supplemented with additional assays for the metabolites MMA

and Hcy, which become elevated in defi-

ciency. MMA increases in plasma and urine due to an inability to convert methylmalonyl CoA to succinyl CoA via methylmalonyl CoA mutase. Normal plasma MMA

concentrations are in the nanomole range, but

in Cbl deficiency they may be in the micromole range. Homocysteine may also be elevated in By deficiency due to the inability of methionine synthesis to proceed (Fig. 3). However, while elevated MMA is specific to vitamin Bj) deficiency, it is not analyzed routinely due to the need for specialized equipment and its high cost. Elevated Hcy may be a product of folate deficiency, or disease, and its use as a tool to specifically diagnose vitamin Bj) deficiency is limited.

Causes of Deficiency Poor absorption and inadequate ingestion are the chief causes

Inadequate ingestion

of vitamin

Bj)2 deficiency.

For individuals

in

Dietary vitamin Bj) deficiency has been described in affluent populations, traditionally in exceptional communities that practice religious dietary restrictions, or adhere to strict dietary guidelines, such as macrobiotic or vegan

diets.

Hindus,

for example,

often

restrict

intake of meat, or meat and eggs, and may suffer the consequences of deficiency. Some studies suggest that lacto-ovo- or lactovegetarians (who consume animal products, but not meat) are also at risk for developing deficiency. In a study of German vegetarians, 60% had evidence of elevated plasma Hcy and MMA*! Elevated MMA was found in 5% of the omnivores, 77% of the lacto— ovo-vegetarians, and 83% of the vegans. Mean plasma vitamin Bj, concentrations

of lacto-vegetarians were

741

Vitamin By 2

substantially lower than those of nonvegetarians in studies in India.?7! Case reports of dietary induced vitamin By deficiency have been made in teenagers, and incidence of severe infant deficiency associated with maternal dietary restriction has also been reported. In a study of macrobiotic children (mean age 6.4 yr) who had followed a strict macrobiotic diet in early childhood but had been omnivorous since that time, MMA and Hcy were elevated and cognitive

Hemoglobin develops at a normal pace but mitosis lags behind. As a result, RBC production is deranged and an abnormally large nucleus is extruded, leaving behind a large cytoplasm filled cell. A mean corpuscular volume (MCV) > 115 fl defines megaloblastic cells, which may be as large as 130-150 fl. Total erythrocyte

function was altered.?®! In developing countries, dietary induced vitamin B)2 deficiency may be more common, especially in low socioeconomic status groups, where ASF are inaccessible due to high cost. Widespread vitamin B)2 deficiency has

been

observed

in

several

countries

in

Latin

America and Southeast Asia, where a predominantly plant-based diet is consumed. The reported prevalence of low plasma B)2 values in various countries in Latin America was about 40% across the life span, and in

B)2 does not change as the blood cell matures because

the nucleus of the red cell is extruded and Bj is largely present in this organelle. Although megaloblastic anemia is recognized as a classic symptom of vitamin Bio deficiency, many individuals may not experience measurable hematological change. In addition, megaloblastic

anemia

is a nonspecific

outcome

of By

deficiency, as folate deficiency induces the same hematological changes through the same pathway. While the presence of megaloblastic anemia may alert clinicians to the need to assess folate and Bj status, it is important to recognize that the anemia occurs at

a much later and more severe stage of Bj2 depletion.

both sexes. More than one-half of pregnant Nepali women had elevated Hcy and MMA." In a group of vegetarian and nonvegetarian adults living in Pune,

Indicators of status, such as MMA

India, Bj deficiency was detected in 47% based on low

Neuropathies

and plasma Cbl,

are more sensitive.

plasma Cbl, and in 73% based on elevated MMA.?7]

Consequences of Deficiency Clinical symptoms of deficiency in adults are often nonspecific, and include fatigue, apathy, listlessness, diarrhea, and anorexia. Some patients experience oral discomfort, such as soreness of the tongue or ulcera-

tion. Although initial clinical symptoms may be vague, nevertheless,

dramatic

hematological,

neurological,

and immunological changes may occur (Table 4).

Hematological changes The hematological consequences of vitamin: Bj. deficiency include megaloblastic anemia due to a reduced capacity to synthesize DNA rapidly, caused by alterations in the methionine synthase pathway. Table

In the elderly, vitamin Bj. deficiency produces subacute combined degeneration (SCD), a syndrome of irregular spongiform demyelination of spinal cord (SC) white matter, and astrogliosis. Whereas deficiency in the elderly primarily affects change in the spinal cord, in infants the central nervous system (CNS) is damaged. In fact, Cbl deficiency in this age group can produce severe brain damage that may not be completely reversible upon Cbl therapy. Common domains of neural symptoms in vitamin Bj. deficient elderly are: 1) sensory (paresthesias, diminished proprioception, diminished vibratory sensation); 2) motor (weakness); 3) reflex disorder-related; 4) autonomic (incontinence, impotence); 5) gait-related (ataxia); 6) mental (intellectual/behavioral impairment); and 7) visual (impaired visual acuity).77! Deficiency is often resolved, and symptoms reversed, with Cbl therapy. Either megadoses of vitamin By?

4 Commonly used indicators of vitamin Bj, deficiency Cut-off

Marker

Specificity

Plasma vitamin Bj2

Deficient: 12 pmol/L

Nonspecific, also elevated in folate deficiency and some disease states

MCV

>95fL

Nonspecific, also elevated in folate deficiency

Neutrophil hypersegmentation

>5 five-lobed neutrophils

Nonspecific, also elevated in folate deficiency

dUST test

>10% suppression

Nonspecific, also elevated in folate deficiency

MMA

= Methylmalonic acid; Hey = homocysteine.

Specific to Bz status

Vitamin B,2

742

can be injected intramuscularly or large doses (500-1000 ug/day) of crystalline Byz can be taken orally. Because 1% of the vitamin can be passively absorbed without the need for IF, oral treatment is effective for many patients with PA or deficiency caused by gastric atrophy, and consumption of foods fortified with Bj. predicts higher plasma By in the

elderly.°" Demyelination of nerves associated with

SCD is likely responsible for the majority of symptoms experienced by the elderly. There

are

numerous

case

reports

of severe

By2

deficiency in infants of mothers with PA or mothers practicing a vegan/lacto-vegetarian diet.2) Symptoms include regression of mental development, abnormal pigmentation, hypotonia of muscles, enlarged liver

include the much higher global prevalence of this deficiency than is generally recognized and the fact that even those who avoid meat but eat other ASF are at higher risk of depletion. Deficiency also occurs more rapidly than was formerly believed, especially in people whose stores are relatively depleted or who malabsorb the vitamin. At the same time, our understanding of

the mechanisms that cause the adverse functional consequences of deficiency is at a relatively primitive stage, and previously unknown adverse consequences are being identified. Because vitamin Bj can be safely added as a fortificant to food, or taken orally in high doses, greater attention should be paid to ensuring that this nutrient deficiency is detected and treated in at-risk groups.

and spleen, sparse hair, tremors, irritability, anorexia,

failure to thrive, poor brain growth, refusal of solid foods, and diarrhea. Marked cerebral atrophy and ventricular enlargement may also be present.?7! Onset of deficiency in infants occurs within a few months of dietary absence and patients are often responsive to Cbl treatment. Biz deficiency in infancy may have long-term consequences. A follow-up study of six children with severe B,2 deficiency in infancy showed mixed outcomes.*! In the Netherlands, infants aged 4-18 mo who were born to macrobiotic mothers developed psychomotor skills later than omnivorous

ARTICLE OF FURTHER INTEREST Folate, p. 219

REFERENCES 1.

Institute of Medicine. Dietary Thiamin, Riboflavin, Niacin, Vitamin B12, Pantothenic Choline; National Academy DC, 2000.

2.

Schneider, R.J.; Burger, R.L.; Mehlman, C.S.; Allen, R.H. The role and fate of rabbit and

controls.&4) Immune function In

developing

countries,

both

immunomodulation

associated with malnutrition and repeated exposure to infection promote high rates of morbidity and mortality. Recently, an immunomodulating role specific to vitamin Bj has been reported. Markers that may be influenced by vitamin Bj status include: 1) complement component C3; 2) CD4 and CD8T cell counts, and CD4/CD8 ratio; 3) natural killer (NK) cell activity; and 4) TNF-a concentration. Low lymphocyte counts, elevated CD4 cells, decreased CD8

cells, ele-

vated CD4/CD8 ratio, and suppressed NK cell activity have been observed in Bj deficiency. Therapy restored several of these abnormal values.°! Vegans in the United States have signs of compromised immune function, including lower leukocyte counts and C3, even when micronutrient levels appear normal. The

human transcobalamin ITin the plasma transport of vitamin B12 in the rabbit. J. Clin. Invest. 1976,

57, 27-38. 3. Meyers, P.A.; Carmel, R. Hereditary transcobalamin II deficiency with subnormal serum cobalamin levels. Pediatrics 1984, 74, 866-871. 4. Grasbeck, R. Biochemistry and clinical chemistry of vitamin B12 transport and the related diseases. Clin. Biochem. 1984, 17, 99-107. 5.

6.

Parenter. Enteral Nutr. 2002, 26, 57-62. Alexander, D.; Ball, M.J.; Mann, J. Nutrient

intake and haematological status of vegetarians and age-sex matched omnivores. Eur. J. Clin. Nutr. 1994, 48, 538-546.

for such changes in immune function requires further exploration. 7.

Helman, A.D.; Darnton-Hill, I. Vitamin and iron

status in new vegetarians. Am. J. Clin. Nutr. 1987, 45, 785-789.

CONCLUSIONS 8. knowledge of vitamin B;7. The more important issues

Compher, C.W.; Kinosian, B.P.; Stoner, N.E.; Lentine, D.C.; Buzby, G.P. Choline and vitamin

B12 deficiencies are interrelated in folate-replete long-term total parenteral nutrition patients. J.

extent to which vitamin Bj) deficiency is responsible

This review has highlighted several new aspects of our

Reference Intakes: Vitamin B6, Folate, Acid, Biotin, and Press: Washington,

Herrmann,

W.; Schorr, H.; Obeid, R.; Geisel, J.

Vitamin B-12 status, particularly holotranscobalamin II and methylmalonic acid concentrations,

Vitamin Bu

743

and hyperhomocysteinemia in vegetarians. Am. J. Clin. Nutr. 2003, 78, 131-136.

20.

Hellegers, A.; Okuda, K.; Nesbitt, R.E., Jr.; Smith, D.W.; Chow, B.F. Vitamin B12 absorption

10.

i.

in pregnancy and in the newborn. Am. J. Clin. Nutr; 1957, 5, 327-331. Shojania, A.M. Oral contraceptives: effect of folate and vitamin B12 metabolism. Can. Med. Assoc. J. 1983, 126, 244-247.

12.

B(12)

in

the

non-anaemic

pregnant

Frery, N.; Huel, G.; Leroy, M.; Moreau, T.,; Savard, R.; Blot, P.; Lellouch, J. Vitamin B12

L5.

17.

aoe

Bondevik, G.T.; Schneede, J.; Refsum, H.; Lie, R.T.; Ulstein, M.; Kvale, G. Homocysteine and

Serum

vitamin

24.

J. Clin. Nutr.

iC.

Specker, B.L.; Miller, D.; Norman,

26.

2

R.;

Perez-Perez,

J.; Vollset, S.E.; Orning,

G.I;

Blaser,

L.; Guttormsen,

M.J.

A.B.;

Joglekar, A.; Sayyad, M.G.; Ulvik, A.; Ueland, P.M. Hyperhomocysteinemia and_ elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am. J. Clin. Nutr. 2001, 74, 233-241. 28.

Louwman, M.W.; van Dusseldorp, M.; van de Vijver, F.J.; Thomas, C.M.; Schneede, J.; Ueland, P.M.; Refsum, H.; van Staveren, W.A. Signs of

impaired cognitive function in adolescents with marginal cobalamin status. Am. J. Clin. Nutr. 2000, 72, 762-769.

E.J.; Greene,

PAY,

Schneede, J.; Dagnelie, P.C.; van Staveren, W.A.; Vollset, S.E.; Refsum, H.; Ueland, P.M. Methyl-

30.

infants on macrobiotic diets. Pediatr. Res. 1994,

Suter, P.M.; Golner, B.B.; Goldin, B.R.; Morrow, F.D.; Russell, R.M. Reversal of protein-bound

Helicobacter pylori infection and food-cobalamin malabsorption. Dig. Dis. Sci. 1994, 39, 309-314. Herrmann, W.; Geisel, J. Vegetarian lifestyle and monitoring of vitamin B-12 status. Clin. Chim. Acta 2002, 326, 47-59. Refsum, H.; Yajnik, C.S.; Gadkari, M.; Schneede,

H.; Hayes, K.C. Increased urinary methylmalonic acid excretion in breast-fed infants of vegetarian mothers and identification of an acceptable dietary source of vitamin B-12. Am. J. Clin. Nutr. 1988, 47, 89-92.

36, 194-201.

1996,

Carmel,

1985, 4/,

malonic acid and homocysteine in plasma as indicators of functional cobalamin deficiency in

Intern. Med.

Dine

330-335.

18.

in the elderly. Arch.

vitamin B12 malabsorption with antibiotics in atrophic gastritis. Gastroenterology 1991, /0/, 1039-1045.

B12 levels in parturients, in the

with folate levels. Am.

deficiency in general practice. Assessment of the diagnostic utility and cost-benefit analysis of methylmalonic acid determination in relation to current diagnostic strategies. Clin. Chem. 1999, 45, 189-198. Carmel, R. Prevalence of undiagnosed pernicious

156, 1097-1100.

methylmalonic acid levels in pregnant Nepali women. Should cobalamin supplementation be considered? Eur. J. Clin. Nutr. 2001, 55, 856-864. Giugliani, E.R.; Jorge, S.M.; Goncalves, A.L.

intervillous space of the placenta and in fullterm newborns and their interrelationships

Holleland, G.; Schneede, J.; Ueland, P.M.; Lund, P.K.; Refsum, H.; Sandberg, S. Cobalamin

anemia

Levine, R.J.; England, L.J.; Sibai, B.M. Elevated

plasma homocysteine in early pregnancy: a risk factor for the development of severe preeclampsia. Am. J. Obstet. Gynecol. 2002, 786, 1107.

16.

22.

Vollset, S.E.; Refsum, H.; Irgens, L.M.; Emblem, B.M.; Tverdal, A.; Gjessing, H.K.; Monsen, A.L.;

Ueland, P.M. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland homocysteine study. Am. J. Clin. Nutr. 2000, 77, 962-968.

Rogers, L.M.; Boy, E.; Miller, J.W.; Green, R.J.;

Allen, L.H. High prevalence of cobalamin deficiency in Guatemalan school children: associations with low plasma holotranscobalamin II, and elevated serum methylmalonic acid and plasma homocysteine concentrations. Am. J. Clin. Nutr. 2003, 77 (2), 433-440.

Reprod.

among parturients and their newborns and its relationship with birthweight. Eur. J. Obstet. Gynecol. Reprod. Biol. 1992, 45, 155-163.

14.

21.

patient. Hum. Reprod. 2000, 15, 224-226. Stoll, C.; Dott, B.; Alembik, Y.; Koehl, C. Maternal trace elements, vitamin B12, vitamin A,

folic acid, and fetal malformations. Toxicol. 1999, 13, 53-57.

13.

deficiency in the Framingham elderly population. Am. J. Clin. Nutr. 1994, 60, 2-11.

Pardo, J.; Peled, Y.; Bar, J.; Hod, M.; Sela, B.A.; Rafael, Z.B.; Orvieto, R. Evaluation of low serum

vitamin

Lindenbaum, J.; Rosenberg, I.H.; Wilson, P.W.; Stabler, S.P.; Allen, R.H. Prevalence of cobalamin

Healton, E.B.; Savage, D.G.; Brust, J.C.; Garrett,

T.J.; Lindenbaum, J. Neurologic cobalamin deficiency. Medicine 1991, 70, 229-245.

aspects of (Baltimore)

Campbell, A.K.; Miller, J.W.; Green, R.; Haan,

M.N.; Allen, L.H. Serum gastrin and intake of crystalline vitamin B-12 are associated with plasma vitamin B-12 in an elderly Latino population. J. Nutr. in press.

Vitamin By

744

31:

B25

Allen, L.H. Impact of vitamin B-12 deficiency during lactation on maternal and infant health.

CD8+ T lymphocytes and natural killer (NK) cell activity in vitamin B12-deficient patients by

Adv. Exp. Med. Biol. 2002, 503, 57-67. Wighton, M.C.; Manson, J.I.; Speed,

methyl-B12 treatment. Clin. Exp. Immunol.

33.

Graham,

S.M.;

Arvela,

O.M.;

Wise,

116, 28-32.

L;

Robertson, E.; Chapman, E. Brain damage in infancy and dietary vitamin B12 deficiency. Med. J. Aust. 1979, 2, 1-3. G.A:

Long-term neurologic consequences of nutritional

1999,

36.

Haddad, Hubbard,

E.H.; Berk, L.S.; Kettering, J.D.; R.W.; Peters, W.R. Dietary intake

and biochemical, hematologic, and immune status of vegans compared with nonvegetarians. Am. J. Clin. Nutr. 1999, 70, 586S—593S.

vitamin B12 deficiency in infants. J. Pediatr. 1992,

121, 710-714.

34,

Dagnelie, P.C.; van Staveren, W.A. Macrobiotic

nutrition and child health: results of a population-based, mixed-longitudinal cohort study in The

Netherlands.

Am.

J. Clin. Nutr.

1994, 59,

1187S-1196S.

30:

Stabler, S.P. Vitamin B-12. In Present Knowledge in Nutrition, 8th Ed.; ISLI Press: Washington, DC,

2001; 230-240.

Tamura, J.; Kubota, K.; Murakami, H.; Sawamura, M.; Matsushima, T.; Tamura, T.; Saitoh, T.; Kurabayshi, H.; Naruse, T. Immuno-

modulation

FURTHER READINGS

by vitamin

B12:

augmentation

of

_

Weir, D.G.; Scott, JM. Cobalamin. In Modern Nutrition in Health and Disease, 9th Ed.; Shils, M.E., Olson, J.M., Shike, M., Ross, C.A., Eds.; Lippincott, Williams & Wilkins: Baltimore, 1999; 447-458.

Vitamin C Mark Levine Arie Katz Sebastian J. Padayatty Yaohui Wang Peter Eck Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

Oran Kwon Korea Food and Drug Administration, Seoul, Korea

Shenglin Chen Jee-Hyuk Lee Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

INTRODUCTION

the gene encoding gulonolactone oxidase, the terminal enzyme in the biosynthetic pathway of the vitamin.

Vitamin C (L-ascorbic acid, ascorbate) is a watersoluble micronutrient essential for human health. It is a six-carbon lactone with a molecular weight of 176 (Fig. 1)."! Humans and other primates cannot synthesize ascorbate because of multiple mutations in

Thus, humans have to obtain vitamin C from food.

Mark Levine, M.D., is at the Molecular and Clinical Nutrition Section, Digestive Diseases

Branch,

National Institute of Diabetes

and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Arie Katz, M.D., is at the Molecular

BIOCHEMISTRY AND FUNCTIONS Vitamin C is an electron donor, and this property accounts for its known and postulated functions. As an antioxidant, or reducing agent, the vitamin sequen-

tially donates two electrons from the C2—C3 bond.

and

Clinical

Nutrition

Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Sebastian J. Padayatty, M.D., Ph.D., is at the Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Yaohui Wang, M.D., is at the Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Peter Eck, Ph.D., is at the Molecular and: Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Oran Kwon, Ph.D., is Senior Scientific Officer at the Division of

Dietary Supplement Standards, Korea Food and Drug Administration, Seoul, Korea.

Shenglin Chen, Ph.D., is at the Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes

and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Jee-Hyuk Lee, Ph.D., is at the Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Encyclopedia of Dietary Supplements DOI: 10.1081/E-EDS-120022052 Copyright © 2005 by Marcel Dekker. All rights reserved.

The

first intermediate,

formed

double

by the loss of

one electron, is the unstable free radical semidehydroascorbic acid. This intermediate is relatively e

Kod

G

“

L-Ascorbie Acid aie il Dehydro-c-Ascorbie Acid Enzyme Cofactor

2H

2e §

.

Collagen synthesis Carnitine synthesis

Norepinephrine synthesis

Peptide hormone synthesis Tyrosine metabolism

«

.

F

Chemical Reductant 1lron absorption in

ele”

al gastrointestintract

Antioxidant (Reduction of Harmful Free Radicals)

‘ Oxidative DNA and/or protein damage } Low-density lipoprotein oxidation

t Lipid peroxidation

‘ Oxidants and nitrosamines in gastric juice + Extracellular oxidants from neutrophils

t Endothelium-dependent vasodilation

Fig. 1 Actions of vitamin C. (From Ref.""].) (View this art in color at www.dekker.com. ) 745

Vitamin C

746

unreactive and does not interact with other compounds to form potentially harmful free radicals, and can be

reversibly reduced to ascorbate. Semidehydroascorbic acid undergoes further oxidation to form the more stable product dehydroascorbic acid (DHA) (Fig. 1), which can be reduced back to ascorbate by glutathione or by three distinct enzymatic reduction reactions.?7] If not reduced, DHA undergoes ring rupture and is irreversibly hydrolyzed to 2,3-diketogulonic acid. The latter is metabolized to xylose, xylonate, lyxonate, and oxalate, which is a clinically significant end product of vitamin C metabolism. Enzymatic Functions Vitamin C is a cofactor for eight different enzymes in mammals (Fig. 1)."! Three enzymes participate in collagen hydroxylation and two in carnitine biosynthesis; one is necessary for norepinephrine biosynthesis, another is required for amidation of peptide hormones, and one participates in tyrosine metabolism. It is assumed that scurvy, the disease caused by vitamin C deficiency, is due to impaired functioning of these enzymes, although direct experimental proof is lacking.

metal-catalyzed oxidation and by affecting monocyte adhesion and platelet aggregation. In vitro, it inhibits metal-catalyzed oxidation of LDL at concentrations above 40-50 uM.©! However, metal-catalyzed oxidation may not be important in vivo because the high oxidant and metal concentrations and the relatively long periods of time needed to induce oxidation in vitro are unlikely to occur in humans, especially since the relevant cations (iron, copper) in vivo are tightly bound to proteins. In depletion-repletion studies, no significant relationship was found between. vitamin C dose and plasma concentrations of F>-isoprostanes, which are considered as biomarkers of endogenous lipid peroxidation." Another potential protective mechanism is indirect, as vitamin C can regenerate oxidized «-tocopherol (vitamin E) in LDL in vitro. Additional effects of extracellular vitamin C in atherosclerosis could be due to its action on adhesion of monocytes to endothelium or aggregation of platelets and leukocytes. Again, these effects have not been shown in vivo, and their clinical relevance is

unclear. Although laboratory data show a possible pro- — tective role for vitamin C in atherosclerotic heart disease, epidemiologic data are inconsistent. Diets rich in fruits and vegetables, and therefore rich in vitamin C, protect

against However,

atherosclerosis

and

it is not known

many whether

other

diseases.!7-*]

this protection

is

due to the high vitamin C content of such diets or to other reasons.

Reducing (Nonenzymatic) Functions Vitamin C as an antioxidant in vitro

Vitamin C may have nonenzymatic functions due to its reduction—oxidation (redox) potential (Fig. 1). In vitro evidence suggests that it may have a role as a reducing agent both intra- and extracellularly. In the cell, ascorbate might protect intracellular proteins from oxidation. The vitamin and other antioxidants may regulate transcription or translation and may affect post-translational modification. Extracellular vitamin C might protect against oxidants and oxidantmediated damage. In vitro studies suggest that it may be the primary antioxidant in plasma for quenching aqueous peroxyl radicals as well as lipid peroxidation products. In vitro, vitamin C is preferentially oxidized before other plasma antioxidants such as uric acid, tocopherols, and bilirubin. However, these oxidation—

reduction reactions may not specifically require the vitamin in vivo. Vitamin C may quench oxidants that leak from activated neutrophils or macrophages that, in turn, may damage supporting tissues such as collagen or surrounding fibroblasts. Many antioxidant effects demonstrated in vitro have uncertain importance in the intact organism."

Effects on blood flow and endothelial function

In some, but not all, patients with coronary artery disease, administration of large doses of oral vitamin C (24g for acute administration and 500mg/day for chronic treatment) resulted in improved endotheliumdependent vasodilation.”! Such a vasodilatory effect may be compatible with enhanced bioactivity of endothelium-derived nitric oxide (EDNO). Vitamin C did not alter blood flow in healthy subjects and did not reverse endothelial dysfunction in hypertensive patients. Some studies have shown that the vitamin may ameliorate endothelial vasomotor dysfunction when administered intra-arterially in patients with chronic heart failure, type 2 diabetes, and coronary spastic angina, or when given intravenously to smokers. However, intra-arterial concentrations in these studies were far higher than can be achieved under physiological conditions. More data are needed to determine whether endothelium-dependent vasodilation mediated by vitamin C has clinical relevance. Effect on nitrate tolerance

Effects on atherogenesis Vitamin C can theoretically reduce by protecting low-density lipoprotein

atherogenesis (LDL) from

Due to its redox properties, ascorbic acid is a candidate to prevent nitrate tolerance.!'” Tolerance to nitrates, used to treat heart disease, develops within the first

747

Vitamin C

day of continuous exposure and makes treatment less effective. Vitamin C given orally at doses of 3-6 g/day prevented the development of tolerance in some healthy subjects and in patients with ischemic heart disease or heart failure. Because these studies were short term and involved small numbers of patients, the clinical utility of vitamin C treatment in the prevention of nitrate tolerance is not yet clear.

additional vitamin C, with intracellular concentrations

increasing approximately 10-fold. This accumulation is mediated by a process termed ascorbate recycling, of unknown function."*! Since ascorbate recycling does not occur in pathogens, and since neutrophils are the primary host-defense cells in human blood, the process may represent a eukaryotic defense mechanism against pathogens.

Effects in stomach and duodenum Tissue Accumulation

Vitamin C can quench reactive oxygen metabolites in the stomach

and duodenum,

and prevent the forma-

tion of mutagenic N-nitroso compounds. As its concentration in gastric juice is approximately three times higher than that in plasma, vitamin C appears to be an attractive candidate for the prevention of gastric cancer. Whether this suggested antioxidant action has significance in vivo is uncertain. Although high vitamin C dietary intake correlates with reduced risk of gastric cancer,

it is unknown

whether

the vitamin

itself or

other components in plant-derived foods are responsible for the protective effect.

lron absorption

Vitamin C promotes iron absorption in the small intestine by maintaining the element in the reduced (Fe**) form. It can increase soluble nonorganic iron absorption 1.5-10-fold depending on iron status, the vitamin dose, and the type of test meal. Amounts necessary for enhancing iron absorption (20-60 mg)! are found in foods that are good sources of the vitamin. However,

the effect of vitamin C on hemoglobin concentration is modest at best, at least in small, short-term studies.

Vitamin C is accumulated in tissues by two distinct pathways. One pathway is sodium-dependent transport. The other is termed ascorbate recycling,"'?! and dehydroascorbic acid (DHA, oxidized vitamin C) is transported independent of sodium and reduced intracellularly to ascorbate.

Sodium-dependent vitamin C transport Two sodium-dependent vitamin C transporters, SVCT1 and SVCT2, have been identified.!'+'>! Both of these car-

rier proteins couple the transport of 2 Na™: 1 vitamin C. SVCTI is a low affinity, high velocity transporter.!>! SVCT2 has a 10-fold higher affinity for vitamin C but exhibits a lower rate of uptake. SVCT1 is found in kidney, liver, small intestine, thymus, and prostate. In the

small intestine and kidney, SVCT1 is primarily localized in the epithelium, consistent with a role in intestinal absorption and renal reabsorption of vitamin C. SVCT2 has a more general distribution, with mRNA found in most tissues, including the brain, retina, placenta, spleen,

small intestine, and gonads.!'*! Neither of these transporters transports DHA"! Dehydroascorbic acid is transported by the facilitative glucose transporters

GLUTI, GLUT3," and GLUT4, which do not trans-

PHYSIOLOGY Tissue Distribution

Vitamin C is widely distributed in the human body, and many organs contain millimolar concentrations of the vitamin. The highest concentrations are found in adrenal and pituitary glands, at 30-50 mg/100g of tissue. Liver, spleen, pancreas, kidney, brain, and lens

contain 5-30 mg/100 g.7] The choroid plexus actively secretes the vitamin into the cerebrospinal fluid. Ascorbate in the cerebrospinal fluid is then concentrated by many parts of the brain. It is unknown why many of these tissues concentrate vitamin C. High ascorbate concentrations (10-50-fold higher than that in plasma) are also found in white blood cells such as neutrophils, lymphocytes, and monocytes. When activated by exposure to bacterial or fungal pathogens, human neutrophils rapidly accumulate

port vitamin C. The gene for human SVCT1 (hSVCT1) has been mapped to chromosome 5q23, and that for human SVCT2 (hSVCT2) to chromosome 20p12.3.

Sodium-independent transport of DHA (ascorbate recycling) Ascorbate recycling is a process in which extracellular ascorbate is oxidized to DHA, which, in turn, is transported into cells through glucose transporters and reduced back to ascorbate (Fig. 2)."31 Ascorbate recycling enables rapid accumulation of vitamin C in activated neutrophils and is induced by Gram-positive and Gram-negative bacteria, and Candida albicans. Neutrophils from patients with chronic granulomatous disease do not make oxidants due to defective superoxide generation, and these neutrophils cannot recycle vitamin C. The clinical importance of ascorbate recycling is not known. Possibilities include protection of

Vitamin C

748

Fig. 2. Mechanisms of vitamin C accumulation in human _neutrophils. Vitamin C accumulation in neutrophils occurs by ascorbic acid transport and recycling. Ascorbic acid (AA) is transported by sodium-dependent vitamin C transporters (SVCTs) that mainconcentrations millimolar tain inside resting neutrophils. Recycling occurs when bacteria, yeast, or pharmacologic agents activate neutrophils. Activated neutrophils secrete reactive oxygen species that oxidize extracellular AA to acid (DHA). dehydroascorbic

. rs, e

“ny S. % x? i 4

oe Diffusible

“:, Oxidants

_

wat

M,g

ee

_ » -PATHWAYS

"Reduced

3

Oxidants

:

4-

y: 7

£

—

j

%

13 f ?

i

.

Se “

DHA is rapidly transported into neutrophils by glucose transporters (GLUT1, GLUT3) and is then immediately reduced to AA by the glutathione-dependent protein glutaredoxin (GRX). Glutathione (GSH) utilized during DHA reduction is regenerated from glutathione disulfide (GSSG) by glutathione reductase (GRD) and NADPH. NADPH is a product of glucose metabolism through the pentose phosphate pathway. (Reproduced with permission from Padayatty, S.J.; Levine, M. Can. Med. Assoc.

Reduced Oxidants

neutrophil and surrounding tissues from oxidative damage, enhanced phagocytosis or bacterial killing, and activation of programmed cell death. Recycling does not occur in bacteria or in other pathogens, suggesting that it may be a_ host-specific protective mechanism.

Plasma and Cell Concentrations

Steady-state plasma concentrations in relation to dose Steady-state plasma concentration data as a function of dose were obtained in 7 healthy men and 15 healthy women aged 19-26 yr, each of whom was hospitalized for approximately 5-6mo.'*'7 A depletion—repletion design was used. Inpatient subjects consumed a diet containing less than 5mg of vitamin C per day, and all other nutrients in adequate amounts. After depletion of the vitamin (plasma concentrations of 6.9 + 2uM for men and 8 + 1uM for women), steadystate plasma concentrations were obtained for daily

Diffusible Oxidants

J. 2001, 164 (3), 353-355.)

vitamin C doses of 30, 60, 100, 200, 400, 1000, and

2500mg. The vitamin was measured using HPLC with coulometric electrochemical detection. The relationship between plasma steady-state concentration and dose was sigmoidal. At a dose of 30mg, there was only a small increase in plasma vitamin C concentration compared to nadir. At the dose range of 30-100 mg, small changes in daily vitamin C intake resulted in large changes in steady-state plasma concentrations (Figs. 3 and 4). Although curves for men (Fig. 3)'7 and women (Fig. 4)! were sigmoidal, the steep portion of the curve for women was shifted to the left compared to men. Women had higher steadystate plasma vitamin C concentrations than men in the dose range of 30-100 mg daily. These differences disappeared at doses of 200mg per day and higher. At a dose of 200mg, the curves for both men and women were near plateau. At doses of 400mg daily and higher, plasma was saturated with a vitamin C concentration of approximately 70-80uM. At these large doses, ingestion of the vitamin resulted in decreased absorption (i.e., decreased bioavailability) and increased urinary excretion as described later.

Vitamin C

749

——

120 0 mg

200 mg

100

fasting AM

(uM) CPlasma vitamin

35

14

9

10S

Duration of each phase (Days) Maximum duration of each phase is indicated

OF55910515;20)25 30 Scale: Days

Fig. 3 Vitamin C plasma concentration as a function of dose in men. Seven men were hospitalized for 4-6 mo as described in the text. The duration in days for each subject for the depletion phase (0 mg) and for receipt of 7 different vitamin C doses is shown on the X axis. The maximum duration of each phase is indicated numerically. Fasting vitamin C concentrations for all phases of the study are shown on the Y axis. Vitamin C doses for each phase are indicated at the top of the figure. Each subject is indicated by a different symbol. There was variation between subjects in the time taken to reach nadir and in the number of days required to reach steady state for each dose. Reproduced with permission from M Levine, Y Wang, A Katz, P Eck, O Kwon, S Chen, J Lee, SJ Padayatty Ideal Vitamin C Intake. Biofactors 2001; 75, 71-74.

Steady-state circulating blood cell concentrations in relation to dose Vitamin

C was

measured

in circulating neutrophils,

monocytes, and lymphocytes at steady state, at the same daily doses as for plasma. Utilizing active transport, these cells accumulated vitamin C 10—50fold compared to plasma concentrations. Intracellular concentrations increased substantially between 30 and 100mg daily doses.©!" Cells saturated before plasma, at daily doses of 100-200 mg. This is probably because the maximal transport velocity (apparent Vmax) of the tissue vitamin C active transporter SVCT2 is approximately 70 uM.

Bioavailability

Bioavailability of oral vitamin C was determined in depletion-repletion studies by comparison of plasma concentrations of. the vitamin after oral and after intravenous administration. The studies were done with doses of 15, 30, 50, 100, 200, 500, and 1250 mg.

The experiments were performed at steady state, since bioavailability is best studied at equilibrium for plasma and tissue.'©! At steady state for any given dose of vitamin C, its concentrations in plasma and in tissues are in equilibrium. When oral or intravenous doses of vitamin C are given acutely at this stage, plasma concentrations rise and then return to baseline for that steady state. This rise and return to baseline forms an area under the curve (AUC), which can be used to determine bioavailability of the vitamin. To measure bioavailability, the same doses of vitamin C were given orally and intravenously at different times during steady state, usually a day apart. After

administration

of

the

vitamin,

its

plasma

concentrations were measured at intervals of minutes to hours. Bioavailability was calculated as the area under the curve (AUC) for the oral dose (AUC,,) divided by the area under the curve for the intravenous dose (AUC;,,). For a single dose of vitamin C, it was calculated as (approximately) 100% for 200mg, 73% for 500mg, and 49% for 1250 mg. The AUC method could not be used for vitamin C doses of 200mg and lower. This method is only

Vitamin C

750

120

1000}

2500

100

80

60

Fasting AM (uM) CPlasma Vitamin 20

147

175201200

222

241

270

Days at dose Fig. 4 Vitamin C plasma concentration as a function of dose in women. Fifteen women were hospitalized for 4-6mo as described in the text. vitamin C concentrations are shown as a function of days at dose. Doses are indicated at the top of the figure. Each symbol represents a different subject. There is a 1 day gap between all doese for bioavailability sampling. Doses through 200 mg daily were received by 15 subjects, through 1000 mg daily by 13 subjects, and through 2500 mg by 10 subjects. (Reproduced with permission from Ref!)

accurate if volume of distribution and rate of clearance are constant. Vitamin C distribution differs between

Renal Excretion

plasma, circulating blood cells, and other tissues. The

Urinary excretion of vitamin C was measured at steady state for doses of 30-1250mg daily. At oral doses below 60 mg/day, no vitamin C appeared. At an oral dose of 100 mg/day, corresponding to a plasma concentration of approximately 604M in both men and women, approximately 25mg of vitamin C in men and 50mg in women was excreted in the urine.!®'7! As oral doses increased, the vitamin appeared in the urine in increasing quantity. While a larger quantity

differences in distribution are less pronounced at higher doses, when plasma and circulating cells are saturated with the vitamin. Moreover, renal excretion is not linear, as it only starts above the renal threshold, as described later. Therefore, a mathematical model

was developed to account for these factors. Using this, bioavailability was found to be 80% for 100mg and 46% for 1250 mg. The values calculated by the two methods mentioned above are for vitamin C administered as a chemically pure substance in an aqueous solution. When given as a supplement, bioavailability depends on the preparation and may be reduced substantially by factors such as supplement binders and supplement dissolution time in the gastrointestinal tract. When administered as plant-derived food, the bioavailability of vitamin C is currently unknown and might be altered by other substances in the food.

of vitamin

C was

absorbed

as doses

increased,

the

percentage of the absorbed dose decreased. This percentage decrease in absorption is decreased bioavailability. For example, when 1250 mg of vitamin C was given orally, approximately 600 mg was absorbed and subsequently excreted in the urine. Upon intravenous administration, without the confounding effects of intestinal absorption, virtually the entire administered dose was excreted at 500 and 1250 mg. Since vitamin C is not protein bound, it is presumably filtered at

Vitamin C

751

the glomeruli and reabsorbed in the renal tubules. When the ability of the kidney to reabsorb vitamin C is overwhelmed (the transport mechanism for reabsorption is saturated), the vitamin appears in the urine, analogous to glucosuria in patients with uncontrolled diabetes. In patients with end-stage renal disease, excess vitamin C cannot be excreted. In such patients, vitamin C doses above 200mg can accumulate and produce hyperoxalemia. On the other hand, many patients with end-stage renal disease on dialysis lose vitamin C during dialysis and have chronically low plasma concentrations.

Potential Variable Factors

Healthy subjects were studied for 5—6 mo to determine plasma and cell concentrations, bioavailability, and renal excretion of ascorbate in relation to dose (pharmacokinetics). It is possible that the pharmacokinetics findings will be different in subjects with acute and chronic diseases and in the elderly. Prolonged hospitalization for these subjects is not possible for the purpose of obtaining pharmacokinetics data, and to do so will require the development of new methods.

VITAMIN C DEFICIENCY AND SCURVY The earliest recorded descriptions of scurvy are in Egyptian hieroglyphics ca. 3000 B.c. James Lind published his A Treatise of the Scurvy in 1753, providing

Subclinical vitamin C deficiency may be much more common than overt disease, and is difficult to recognize, since the early symptoms of deficiency are unremarkable and nonspecific.

INDICATIONS AND USAGE Food Sources

Vitamin C is widely distributed in fruits and vegetables.

Fruits

rich

in vitamin

C

include

strawberry,

papaya, orange, kiwifruit, cantaloupe, grapefruit, mango, and honeydew melon. Vegetables with a high content of the vitamin are broccoli, Brussels sprout, cabbage, potato, sweet potato, cauliflower, red and

green pepper, tomato, snow pea, and kale!!! Fruit Juices such as orange juice, tomato juice, grapefruit juice, and fortified juices are also sources of the vitamin. Vitamin C is labile, and its content in plant foods may vary to some extent depending on season, transportation, shelf time, storage, and cooking practices. Five servings of a variety of fruits and vegetables per day, as recommended by the USS. Department of Agriculture (USDA) and the USS. National Cancer Institute (NCI), provide 210-280 mg of vitamin C. Fruit and vegetable consumption restricted to a narrow selection could provide smaller amounts.

Functions in Relation to Concentration

evidence that fruits could prevent the disease."®! As noted by Lind and later confirmed by others, the early symptoms of scurvy are weakness, fatigue, listlessness, and lassitude. The physical signs that follow include petechial hemorrhage; perifollicular hyperkeratosis; erythema and purpura; bleeding into the skin, sub-

Other than to prevent scurvy, there is dence that a particular vitamin C plasma centration is more beneficial than others. whether the various biochemical roles either known or postulated, are related

cutaneous

tissues,

muscles,

no direct evior tissue conIt is uncertain of vitamin C, to concentra-

and joints; coiled hairs;

tions of the vitamin in vivo. There are, however, hints

breakdown of wounds; arthralgias and joint effusions; swollen and friable gums; hypochondriasis and depression; Sj6gren syndrome; fever; shortness of breath; and confusion. In severe scurvy, irreversible changes may

that the optimum plasma concentration is higher than the minimum required to prevent clinical scurvy (approximately 10 mg/day). For example, in his Trea-

occur, including dental loss, bone damage, and seque-

sign of impending scurvy is fatigue, or “lassitude.’U®! In vitamin C depletion-repletion studies fatigue was also noted, and in one study, it appeared at vitamin C plasma concentrations of approximately 20 uM. There are also indirect findings suggesting that a higher plasma vitamin C concentration may be beneficial: The Vax Of the vitamin C transporter hSVCT2 is 70 uM; LDL oxidation in vitro is inhibited by vitamin C at 40-50 uM; plasma concentration is tightly controlled at 70-80 1M, and circulating blood cells saturate at approximately these concentrations. The optimal plasma concentration for vitamin C is yet to be determined by clinical studies.

lae of internal hemorrhage and infection. Untreated, the disease is uniformly fatal. The signs related to wound dehiscence and friable gums may reflect impaired collagen synthesis. As noted above, however, there is no experimental evidence that directly links these signs and low vitamin C concentrations to diminished enzyme actions. Frank scurvy is now rare and is seen in the United States primarily among malnourished populations, including patients with cancer cachexia and malabsorption, poor and elderly people, alcoholics,

and some

individuals

with

chemical

individuals consuming

dependency,

idiosyncratic diets.

tise, James

Lind

mentions

that the most

prominent

Vitamin C

752 Possible Benefits of Vitamin

C Consumption

Diets with 200mg or more of vitamin C from fruits and vegetables are associated with lower risk of cancer (especially cancers of the oral cavity, esophagus, stomach, colon, and lung)! and stroke, and with reduced overall mortality. Higher fruit and vegetable consumption and plasma vitamin C concentrations are inversely related to risk of ischemic heart disease, diabetic complications, and blood pressure in hypertensive patients. The USDA and NCI recommendations of consuming five fruit and vegetable servings daily are based on this extensive evidence. These studies, however, are correlational. Whether vitamin C in fruits and vegetables confers these benefits is not known. It may be a surrogate marker for fruit and vegetable consumption and, perhaps, for other healthy lifestyle practices. In the United States, approximately 30% of adults consume less than 2.5 servings of fruits and vegetables per day, and the estimated vitamin C intake is even lower among some groups, including children. Vitamin C as a food supplement was tested for primary prevention of cancer, cardiovascular disease, stroke, and age-related eye diseases. In epidemiological studies and in some large-scale interventional studies,

vitamin C was consumed in combination with other food supplements such as vitamins and antioxidants, and was partially obtained from foods. Under these conditions, it did not provide the health benefits seen with consumption of fruits and vegetables: Vitamin C supplements did not prevent cancer, heart disease, stroke, or cataract. To date, no large-scale interven-

tional studies have been reported where vitamin C was administered as a sole supplement. Vitamin C as a food supplement has also been tested for its effects on disease outcome for hypertension, diabetes, infectious diseases, and age-related eye diseases. Some small, short-term studies suggest that consumption of vitamin C supplements might lower blood pressure in hypertensive and diabetic patients. No large-scale studies are available to confirm these findings. Some experiments show an improvement in lipid profile and insulin sensitivity in diabetic patients who consumed vitamin C supplements, but others fail to do so. Contrary to what has been suggested by some, daily vitamin C supplementation did not decrease common cold incidence in most studies. Similarly, data are insufficient to conclude that vitamin C supplements have an effect on reducing severity of illness due to common cold, except, perhaps, in some people who are vitamin C deficient. Although some small studies suggested that supplementation might prevent cataract, larger studies showed no effect of vitamin C supplementation on the development or progression of cataracts.

Prevention of Deficiency

Steady-state plasma concentrations achieved by a vitamin C dose of 60mg/day can prevent deficiency for 10-14 days, and those achieved by 100 mg/day can probably prevent deficiency for approximately | mo. The above only applies to healthy people who, under otherwise normal conditions, are depleted of vitamin C alone. There is currently little knowledge of vitamin C metabolism in disease states. Deficiency might occur more rapidly in various clinical circumstances where low concentrations were reported, such as in smokers, and in patients with diabetes,

myocardial

infarction,

pancreatitis,

end-stage

renal

disease, and in critical illness requiring intensive care

unit support.

Treatment of Scurvy Upon diagnosis of scurvy, based primarily on clinical findings and confirmed by plasma concentrations of vitamin C, treatment can be initiated with doses of 100 mg given three times a day. An initial intravenous dose of 100mg may be administered. If diagnosis and treatment are prompt, permanent damage can be prevented.

Dietary Reference Intakes (DRIs) Dietary reference intakes are a set of nutrient-based reference values that can be used for planning diets and that are meant to expand the concept of recommended daily allowances (RDAs) in the United States.°°] DRIs have several categories: estimated average requirement (EAR), RDA, adequate intake (AI), and upper limit (UL). The EAR is the median usual intake value that is estimated to meet the requirements of half the healthy individuals in a life stage and gender group. This value is used to calculate the RDA, which is the average daily dietary intake level that is sufficient to-meet the nutrient requirements of nearly all healthy individuals in a life stage and gender group. The RDA is calculated as the EAR plus two standard deviations of the EAR measurement. AI, or adequate intake, is derived when data to establish EARs are insufficient and, therefore, an RDA cannot be set. It is based on experimentally derived intake levels or approximations of observed mean nutrient intake by groups of apparently healthy people. UL, or upper limit, is the highest level of continuing daily nutrient intake that is likely to pose no risk of adverse health effects in almost all individuals in a life stage and gender group.

Vitamin C

753

DRI Values for Vitamin C

Dietary reference intake values were published for ascorbic acid by the Food and Nutrition Board of the U.S. National Academy of Sciences in a report released in April 2000.7°! EARs were calculated based on data on neutrophil saturation and urinary excretion that were obtained’in a depletion-repletion study in men as described earlier. The EAR for men 19 yr and older was established as 75 mg/day. The requirement for women in the same age group was extrapolated based on body weight differences, and the EAR was set at 60mg/day. The RDAs for vitamin C in the United States were calculated from these EAR values. In this report from 2000, the RDAs were increased

from 60 to 90mg/day for men and to 75mg/day for women (Table 1). Using Food and Nutrition Board criteria, data published after the DRI recommendations were released suggest that the RDA for healthy young women should be increased to 90 mg/day, and that the RDA for men may have been underestimated and should be increased to 105mg/day. UL recommendations are discussed below under adverse effects. Use in Pregnancy The RDA for vitamin C during pregnancy is 80mg/day for women aged 14-18 yr and 85mg/day Table 1 Recommended dietary allowances (RDAs)* for vitamin C consumption Group

for women 19yr and older (Table 1). This increase, compared to the recommendations in nonpregnancy, is based on the assumption that additional vitamin C is required to provide adequate transfer to the fetus. Plasma vitamin C concentrations decrease during pregnancy, perhaps secondary to hemodilution or active transfer to the fetus, but this decrease has not been

shown to have clinical significance. Vitamin C deficiency during pregnancy is associated with increased risk of infection, premature rupture of membranes, premature

delivery,

and

eclampsia.

However,

it is

unknown whether vitamin C deficiency contributes to these conditions or is simply a marker of poor nutritional status. Precise data are lacking regarding fetal requirements and quantity of maternal vitamin C transferred to the fetus. Therefore, an increase of 10mg/day for pregnancy was recommended based on data that intakes of 7mg/day of vitamin C prevent young infants from developing scurvy.

Use in Disease

Some studies, as discussed above, suggest that vitamin C administration may have health benefits in those with endothelial dysfunction, such as patients with ischemic heart disease, diabetes, or hypertension, and that it may reduce tolerance to nitrates. However, there are currently not enough data to support specific vitamin C intake recommendations in such patients other than the RDA and the general recommendation for fruit and vegetable intake.

RDA (mg/day)

Infants” 0-6 mo 7-12 mo

Boys 40 50

Girls 40 50

Children 1-3 yr 4-8 yr 9-13 yr 14-18 yr

Boys 15 25 45 75

Girls als DS 45 65

Adults 19 yr and older

Men 90

Women W®

Pregnancy 14-18 yr 19-50 yr

80 85

Lactation 14-18 yr 19-50 yr

ibis) 120

4UJ.§. Food and Nutrition Board of the Institute of Medicine, 2000.

bvValues for infants are given as adequate intake (AI), since RDAs are unavailable. Note that AI values may be higher than RDAs due to the different methods of estimation. The data used for infant Als are milk composition’and amount of milk consumed. RDAs for children are based on assumed differences in body weight from adults, for whom data are available.

Optimum Vitamin C Intake Recommendations for optimum intake should be based on its dietary availability, steady-state concentrations in plasma and in tissue in relation to dose, bioavailability, urinary excretion, adverse effects, bio-

chemical and molecular function in relation to concentration, beneficial effects in relation to dose (direct effects and epidemiological observations), and prevention of deficiency. Although recent studies provided valuable data on some of these aspects, additional clinical reports are needed to provide definitive recommendations for optimal intake in health and disease. Some recommendations can still be made now using available data. Five or more varied servings of fruits and vegetables daily will provide approximately 200 mg of vitamin C and might offer protection against cardiovascular diseases and stroke. It is recommended that healthy people strive to meet this ingestion amount using fruits and vegetables, not supplements.

Vitamin C

754

ADVERSE

REFERENCES

EFFECTS

The toxic effects of vitamin C are few and are dose related. Ingestion of 3-5 g at once can cause diarrhea and bloating. The vitamin enhances iron absorption from

the small

intestine

and

may,

2.

3.

are no clinical indications at this time for such doses,

B.S.;

Orselli,

Rumsey, S.C.; Levine, M. Absorption, transport,

Padayatty, S.J.; Katz, A.; Wang, Y. et al. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J. Am. Coll. Nutr. 2003, 22, 18-35.

5.)

Jialal «1: eFoller, CJ. Effect. of vitamin oi) vitamin C and beta-carotene on LDL oxidation .and atherosclerosis. Can. J. Cardiol. 1995, JJ,

97G-103G.

6.

Levine, M.; Wang, Y.; Padayatty, S.J.; Morrow, J. A new recommended dietary allowance of vitamin C for healthy young women. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9842-9846.

7.

Joshipura,

K.J.; Hu,

F.B.;

Manson,

J.E.

et al.

The effect of fruit and vegetable intake on risk for coronary heart disease. Ann. Intern. Med. 2001, 134, 1106-1114. 8.

Khaw, K.T.; Bingham, S.; Welch, A. et al. Rela-

tion between plasmaascorbic acid and mortality in men and women in EPIC-Norfolk prospective study: a prospective population study. European Prospective Investigation into Cancer and Nutrition. Lancet 2001, 357, 657-663. 9.

Gokce, N.; Keaney, J.F. Jr.; Frei, B. et al. Long-

term ascorbic acid administration reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation 1999, 99, 3234-3240.

mia, rebound scurvy, infertility, mutagenesis, and destruction of vitamin B;>. None of these effects are

11.

McVeigh,

G.E.; Hamilton,

P.; Wilson, M. et al.

Platelet nitric oxide and superoxide release during the development of nitrate tolerance: effect of supplemental ascorbate. Circulation 2002, 106, 208-213. Hallberg, L.; Brune, M.; Rossander-Hulthen,

Is there a physiological

caused by vitamin C.

iron absorption?-Ann.

Not applicable.

The

T.S.

4.

10.

STATUS

Rex,

Biochem. 1998, 9, 116-130.

although some patients ingest these amounts for possible benefit despite little or inconclusive evidence. Vitamin C, at doses of 250 mg and above, may cause false-negative results for stool occult blood with guaiac-based tests. Intake of the vitamin should be reduced to less than 250 mg for several days prior to such testing. Several harmful effects have erroneously been attributed to the vitamin, including hypoglyce-

COMPENDIAL/REGULATORY

S.M.;

and disposition of ascorbic acid in humans. Nutr.

of the vitamin in some individuals, although the clinical

importance of this is unknown. Large-scale studies in healthy individuals with no prior history of kidney stones did not show an increased risk of formation of renal calculi with increased vitamin C consumption from food and supplements. However, vitamin C at doses of 1 g daily and higher may precipitate this problem in some individuals with occult hyperoxaluria. In patients receiving dialysis treatment, hyperoxalemia has been induced by repeated intravenous administration of 1 g, and it may also be promoted by daily doses of 500mg. Although adequate vitamin C intake for patients with end-stage renal disease on dialysis is not known, based on current evidence, it probably should not exceed 200 mg daily." In its latest published recommendations, the Food and Nutrition Board set the tolerable upper limit (UL) for vitamin C at 2g daily, based on gastrointestinal adverse effects at higher doses.°"! Of note, there

Winkler,

redox couple between glutathione and ascorbic acid: a chemical and physiological perspec1994, Biol. Med. 17, Free Radic. tive. 333-349.

thalassemia major, or sideroblastic

anemia, or patients who require multiple, frequent red blood cell transfusions). In healthy individuals, vitamin C most probably does not induce iron overabsorption in doses as high as 2g. In patients with glucose6-phosphate dehydrogenase (G6PD) _ deficiency, hemolysis was induced by intravenous vitamin C administration as well as by oral administration of single doses of at least 6g of the vitamin. Doses of 3 g may cause transient hyperuricosuria, but this does not occur at doses of less than | g. Likewise, oxalate excretion may be increased by ingestion of 1 g or more daily

Levine, M.; Rumsey, S.C.; Daruwala, R.; Park, J.B.; Wang, Y. Criteria and recommendations for vitamin C intake. J. Am. Med. Assoc. 1999,

281, 1415-1423.

in large doses,

increase the risk of iron overload in patients who are prone to that condition (such as patients with hemochromatosis,

1.

12.

role of vitamin N. Y. Acad.

L.

C in

Sci. 1987,

498, 324-332. Hornig, D. Distribution of ascorbic acid, metabolites and analogues in man and animals. Ann. N. Y. Acad. Sci. 1975, 258, 103-118.

—

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NM:

755

Wang, Y.; Russo, T.A.; Kwon, O.; Chanock, S.;

Rumsey, S.C.; Levine, M. Ascorbate recycling in human neutrophils: induction by bacteria. Proc. Natl. Acad.

Sci. U.S.A.

ascorbic acid. J. Biol. Chem. 18,989.

» 17.

1997, 94, 13,816—

Tsukaguchi,

A

family

L-ascorbic

H.; Tokui, T.; Mackenzie,

of

mammalian

acid transporters.

B. et al.

Na‘-dependent Nature

18.

1999, 399,

16.

Rumsey, S.C.; Kwon, O.; Xu, G.W.; Burant, C.F.;

Simpson, I.; Levine, M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydro-

Stewart,

C.P.,

Guthrie,

D.,

Eds.;

Edinburgh University Press: Edinburgh, 1953. Byers, T.; Guerrero, N. Epidemiologic evidence for vitamin C and vitamin E in cancer prevention. Am. J. Clin. Nutr. 1995, 62, 1385S—1392S.

Daruwala, R.; Song, J.; Koh, W.S.; Rumsey, S.C.;

Levine, M. Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett. 1999, 460, 480-484.

Lind, J. Lind’s Treatise on Scurvy. Bicentenary Volume.

70-75. 15.

Levine, M.; Conry-Cantilena, C.; Wang, Y. et al.

Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3704-3709.

13,819. 14.

1997, 272, 18,982—

20.

Food

and

Nutrition

Board,

Panel

on

Dietary

Antioxidants and Related Compounds. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, National Academy Press: Washington, DC, 2000; 95-184.

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RR

I 4

—

ROO’

+ RH—R’*

However,

+ ROOH

if vitamin

E (e.g., a-T) is present,

trienols have antioxidant activity, and in some systems the

hydroxyl group on the chromanol ring reacts with the peroxyl radical to form a tocopheroxyl radical and a lipid hydroperoxide. Thus, vitamin E acts as a chain-breaking antioxidant, thereby preventing further

autoxidation of lipids.!”! ROO’

+ a-T—«a-T°

patients with coronary artery disease."'*) as well as in brains collected postmortem from patients with Alzheimer’s disease.!"®! It is vital to observe that all tocopherols and toco-

+ ROOH

The tocopheroxyl radical («-T°) has a number of possible fates. It can react with another radical to form nonreactive products. Alternatively, it can be further

oxidized to tocopheryl quinone, a two-electron oxidation product. Another possibility is “vitamin E recycling,’’ where the tocopheroxyl radical is restored to its unoxidized form by other antioxidants such as vitamin C, ubiquinol, or thiols, such as glutathione."! This “‘recycling’’ process depletes other antioxidants; hence, an adequate intake of other dietary antioxidants is important to maintain vitamin E concentrations. In addition, the tocopheryoxyl radical, because it is relatively long lived and if there are no other coantioxidants with which it could react, can hypothetically re-initiate lipid peroxidation.”! Upston, Terentis, and Stocker"! have called this “TMP or tocopherolmediated peroxidation’’ and claim it can occur in vivo based on the detection of both oxidized lipids and unoxidized vitamin E in atherosclerotic lesions. In addition to its antioxidant activity, y-tocopherol and other non-«-vitamin E forms can also trap reactive nitrogen oxides because they have an unsubstituted position on the chromanol ring.!" Cooney et al.!!"! reported that y-T is more effective in detoxification of NO, than o-T. Furthermore, Hoglen et al.!'7! demonstrated that 5-nitro-y-tocopherol (2,7,8-trimethyl2-(4,8,12-trimethyldecyl)-5-nitro-6-chromanol; NGT) is the major reactive product between peroxynitrite and y-tocopherol. NGT has been reported in the plasma of zymosan-treated rats,!'*! cigarette smokers, "4!

many of these have been reported to have higher antioxidant activity than a-tocopherol.!'”'*! Nonetheless, it must be emphasized that the relationship between biologic activity and antioxidant activity is not clear. a-Tocopherol has the highest biologic activity, suggesting it shows some specific molecular function.

Biologic Activity Biologic activity is a historic term indicating a disconnection between molecules having vitamin E antioxidant activity and a relative lack of in vivo biologic function. Observations in rodent experiments carried out in the 1930s formed the basis for determining the “biologic activity’’ of this vitamin.!'?! Although the various molecules with vitamin E activity had somewhat similar structures and antioxidant activities, they differed in their abilities to prevent or reverse specific vitamin E deficiency symptoms (e.g., fetal resorption, muscular dystrophy, and encephalomalacia).?” a-Tocopherol, with three methyl groups and a free hydroxyl group on the chromanol ring with the phytyl tail meeting the ring in the R-orientation (Fig. 1), has the highest biological activity. This specific structural requirement for biological, but not chemical, activity is now known to be dependent upon the hepatic

a-TTP.P" As will be discussed below, a-TTP maintains

plasma,

and

indirectly

tissue,

a-tocopherol

concentrations.??77

Molecular Functions

In addition to antioxidant activity, there are specific a-tocopherol-dependent functions that normalize cellular signaling and metabolism in a variety of cells.) y-Tocopherol plays a critical role through its

ability to inhibit the activity of protein kinase C,25) a

central player in many signal transduction pathways.

_

Vitamin E

Specifically, it modulates pathways of platelet aggregation,?°*7! endothelial cell nitric oxide production,?*77! monocyte/macrophage superoxide production,?"! and smooth muscle cell proliferation.'! Regulation of adhesion molecule expression and inflammatory cell cytokine production by «a-tocopherol have also been ?7] reported. These have been reports regulation of the expression of lipoprotein receptors by «a-tocopherol. Both the scavenger receptor BI(SR-BI),&*! and its homolog, CD36,"*75! are decreased by high cellular «-tocopherol and increased by low concentrations. y-Tocopherol, as well as its metabolite (y-CEHC; y-carboxyethyl hydroxychroman), possesses antiinflammatory properties, because stimulated macrophages and epithelial cells, treated with y-tocopherol, have decreased cyclo-oxygenase-2 activity and lower

levels of prostaglandin E, (PGE>) synthesis.°°! Moreover, in rats fed a high y-T diet (33 mg/kg chow) and subjected to carrageenan-induced inflammation, PGE, and leukotriene B, synthesis were decreased by 46%

and 70%, respectively.°” Additionally, y-CEHC has been shown to increase sodium excretion.*! The in vivo significance of many of these various effects and the role of vitamin E in signaling pathways remain controversial because most of the information in this area has been obtained from in vitro studies. Additionally, microarray technology has been used to show changes in gene expression in response to vitamin E,?*° but the physiologic relevance has not yet been clearly documented. More studies in humans are needed to relate «-tocopherol intakes and tissue concentrations to optimal tissue responses and gene regulation.

PHYSIOLOGY Absorption and Plasma Transport Intestinal absorption of vitamin E is dependent upon normal processes of fat absorption. Specifically, both biliary and pancreatic secretions are necessary for solubilization of this vitamin in mixed micelles containing bile acids, fatty acids, and monoglycerides (Fig. 2). a-Tocopheryl acetates (or other esters) from vitamin E supplements are hydrolyzed by pancreatic esterases to a-tocopherol prior to absorption. Low fat diets limit vitamin E absorption, especially from supplements.!*”! Following micellar uptake by enterocytes, it is incorporated into chylomicrons and secreted into the lymph. Once in the circulation, chylomicron triglycerides are hydrolyzed by lipoprotein lipase (LPL). During chylomicron catabolism in the circulation, vitamin E is

nonspecifically transferred both to tissues and to other circulating lipoproteins.*7!

TES

INTESTINE Chylomicrons a-T

Remnants

Fig. 2 Intestinal vitamin E absorption and plasma lipoprotein transport. Vitamin E absorption requires both biliary and pancreatic secretions for solubilization of vitamin E in mixed micelles. Following micellar uptake by enterocytes, vitamin E (shown as a- and y-tocopherols, «-T and y-T) is incorporated into chylomicrons and is secreted into the lymph. During chylomicron catabolism in the circulation, it is nonspecifically transferred both to tissues and to other circulating lipoproteins (not shown). It is not until the vitamin E-containing chylomicrons reach the liver that discrimination between the various dietary vitamin E forms occurs. The hepatic «-TTP preferentially facilitates secretion of atocopherol from the liver into the plasma in very low density lipoproteins (VLDLs). In the circulation, VLDLs are catabolized to LDLs. During this lipolytic process, all of the circulating lipoproteins (e.g., LDL and HDL) become enriched with «-tocopherol.

It is not until the vitamin E-containing chylomicrons reach the liver that discrimination between the various dietary vitamin E forms occurs. The hepatic x-TTP preferentially facilitates secretion of «-tocopherol, specifically 2R-«-tocopherols, but not other tocopherols or tocotrienols, from the liver into the plasma in very low-density lipoproteins (VLDLs).434 In the circulation, VLDLs are catabolized to lowdensity lipoproteins (LDLs). During this lipolytic process, all of the circulating lipoproteins become enriched with a-tocopherol. There is no evidence that vitamin E is transported in the plasma by a specific carrier protein. Instead, the vitamin is nonspecifically transported in all of the lipoprotein fractions.*! An advantage of this transport is that oxidation-susceptible lipids are protected by the simultaneous transport of a lipid-soluble antioxidant. Similarly, delivery of vitamin E to tissues is dependent upon lipid and lipoprotein metabolism. Thus, as peroxidizable lipids are taken up by tissue, the tissues simultaneously acquire a lipid-soluble antioxidant. Plasma «a-tocopherol concentrations in humans range from 11 to 37pmol/L, while y-tocopherol

Vitamin E

760

concentrations are roughly 2-5 mol/L and tocotrienol concentrations are less than 1 pmol/L, even in subjects supplemented with tocotrienols.4°! When plasma lipids are taken into account, the lower limits of normal level are 1.6y:mol «-tocopherol/mmol lipid (sum of cholesterol and triglycerides), or 2.5 mol a-tocopherol/mmol cholesterol.47! The apparent half-life of RRR-a-tocopherol in plasma of normal subjects is approximately 48 hr, {#81 while that of SRR-a-tocopherol

that of y-tocopherol

is only 15 hr,**! and

is also similar to the SRR-a-

tocopherol, about 15 hr.?! This relatively fast turnover

of 2S-a-tocopherol is also accompanied by increased

metabolism.°! The comparatively fast disappearance of the 2S-«-tocopherols indicates that by 48 hr, nearly 90% of the 2S-forms have been removed from the plasma, while 50% of the 2R-forms remain. It is then

no wonder that the plasma disappearance curves of RRR- and all rac-a-tocopherols are parallel; they both

vitamin E from VLDL to HDL and from lipoproteins into cells.°! PLTP knockout mice compared with wild types have higher vitamin E in apolipoprotein B-containing lipoproteins (VLDL or LDL). The involvement of the plasma cholesteryl ester transfer was process in this transfer protein (CETP) ruled out.!®7! The regulation of tissue vitamin E is not well understood,

but it is seen

that

a-tocopherol

is the pre-

dominant form in tissues as a result of its plasma concentrations.7! The ATP-binding cassette transporter (ABCAI) has been shown to participate in the efflux of a-tocopherol from cells to HDL! Apparently, excess vitamin E could be removed from cells via ABCAI facilitating its transfer to apolipoprotein AI, and transport via HDL

to the liver where SR-BI

could mediate vitamin E transfer into a liver pool destined for excretion in bile. 7

trace the 2R-forms’ disappearance.©!~**) Metabolism and Excretion

Vitamin E is excreted as intact tocopherols or toco-

Tissue Delivery

trienols, oxidized forms, and a metabolic product.!7] Vitamin E is delivered to tissues by three methods,

none of which is specific for vitamin E. But rather its trafficking depends on mechanisms of lipid and lipoprotein metabolism. These include transfer from triglyceride-rich lipoproteins during lipolysis, delivery as a result of receptor-mediated lipoprotein uptake, and exchange between lipoproteins or tissues. With respect to lipolysis, LPL facilitates the delivery of a-tocopherol from triglyceride-rich lipoproteins to cells, as shown in vitro.“ The importance of this pathway was demonstrated in vivo when LPL was overexpressed in muscle, resulting in increased vitamin E

delivery to muscle.)>! Both low-and high-density lipoproteins (LDL and HDL, respectively) have been shown to deliver vitamin E to tissues. The LDL receptor-mediated uptake of LDL delivers the lipoprotein particle via an endocytic pathway, and vitamin E is released during lipoprotein degradation.©*! In contrast, HDL binds to the SR-BI allowing selective delivery of the HDL lipids, including vitamin E, to the cells.°” In SR-BI

knockout

mice,

plasma «-tocopherol concentrations are elevated. Some tissues (e.g., brain*! and lung?!) contain decreased a-tocopherol contents, while hepatic tocopherol concentrations are unchanged. But biliary tocopherol excretion is decreased.*! Apparently, SR-Bl-mediated hepatic uptake of HDL-associated «a-tocopherol is coupled to biliary excretion of vitamin E.>7! Although vitamin E spontaneously exchanges between lipoproteins, the phospholipid transfer protein (PLTP) facilitates the exchange of phospholipids between lipoproteins, as well as the transfer of

a- and y-Tocopherols as well as «- and y-tocotrienols are metabolized to a- and y-CEHCs [2,5,7,8-tetramethyl- and 2,7,8-trimethyl-2-(2’carboxyethyl)-6-hydroxychromans], respectively, by humans.?! CEHCs were first described in rats fed high amounts of 6tocopherols.*! About 1% of a dose of a-tocopherol or tocotrienol and 5% of a dose of y-tocopherol or tocotrienol are excreted in the urine as CEHCs.®! Based on studies in hepatocytes,'©°°”! it is likely that the liver synthesizes CEHCs. Studies in renal dialysis patients'*"! suggest that in addition to urinary excretion,! bile may be a major route for CEHC excretion. Similarly, CEHCs

have been found in both rat urine

and bile.!7°! The importance of vitamin E metabolism in the regulation of vitamin E status is unknown. The various forms of vitamin E appear to be metabolized similar to xenobiotics in that they are initially oxidized by P450s,

conjugated,

and

excreted

in urine

or

bile.

CEHCs have a shortened phytyl tail, resulting from ®-oxidation, a cytochrome P450 (CYP)-mediated process, followed by B-oxidation.!’!~7?! Hepatic CYP 4F2 is involved in w-oxidation of «- and y-tocopherols,!”*) and so could CYP 3A[%7!-”-741 Tt should be noted that a compound can stimulate CYPs other than those involved in its own metabolic pathway; thus, interactions with a variety of pathways are possible. CEHCs can be sulfated or glucuronidated.!’>-”7! Both free and conjugated forms have been detected in plasma,!”" urine,! and bile!78! All of the systems involved in vitamin E metabolism could be under PXR regulation.!”*!

Vitamin E

761

Dietary vitamin E forms, such as y-tocopherol!*°] or y-tocotrienol,®" are more actively metabolized to

CEHCs than «-tocopherol.°°>75! In fact, nearly all of the absorbed y-tocopherol has been estimated to

be metabolized

to y-CEHC!’*!

High a-tocopherol

intakes, e.g., supplements, lead to both increased o-CEHC"! and y-CEHC excretion.'©*! Thus, vitamin E metabolism may be a key factor in hepatic disposal of excess vitamin E.

HUMAN VITAMIN E DEFICIENCY Vitamin E deficiency was first described in children with fat malabsorption syndromes, principally abetalipoproteinemia, cystic fibrosis, and cholestatic liver disease. ®?) Subsequently, humans with severe deficit with no known defect in lipid or lipoprotein metabolism were described to have a defect in the «-TTP

84] gene,

Erythrocyte fragility, hemolysis, and anemia were described as vitamin E deficiency symptoms in various

animals fed diets devoid of this antioxidant.®*! How-

ever, in humans, the major symptom is a peripheral neuropathy characterized by the degeneration of large caliber axons in the sensory neurons.®¢

INDICATIONS

AND USAGE

E can

be readily obtained

o-

Treatment of Vitamin E Deficiency Overt vitamin E deficiency occurs only rarely in humans and almost never as a result of inadequate vitamin E intakes. It does occur as a result of genetic abnormalities in «-TTP!”! and various fat malabsorption syndromes.” Vitamin E supplementation halts the progression of the neurologic abnormalities caused by inadequate nerve tissue «-tocopherol, and in some cases, has reversed them.!°7!

Patients with these disorders require daily pharmacologic vitamin E doses for life to overcome and prevent the deficiency symptoms. Generally, subjects with Ataxia with Vitamin E Deficiency (AVED) are advised to consume 1000mg RRR-«-tocopherol per day in divided doses, those with abetalipoproteinemia 100mg/kg body weight, and for cystic fibrosis 400 mg/day. However, patients with fat malabsorption due to impaired biliary secretion generally do not absorb orally administered vitamin E. They are treated with special forms of vitamin E, such as a-tocopheryl polyethylene glycol succinate, which spontaneously form micelles, obviating the need for bile acids.?*) Chronic Disease Prevention

Food Sources Vitamin

o-TEs are used to estimate dietary «-tocopherol, tocopherol intakes may be overestimated.

from

food,

but

relatively few foods have high «-tocopherol concentrations.°”7] Generally, the richest sources are vegetable oils. Wheat germ oil, safflower oil, and sunflower oil

contain predominantly «-tocopherol, while soy and corn oils have mainly y-tocopherol. All of these oils are polyunsaturated. Good sources of monounsaturated oils, such as olive or canola oils, also have «-toco-

pherol to a large extent. Whole grains and nuts, especially almonds, are also good a-tocopherol sources. Fruits and vegetables, although rich in water-soluble antioxidants, are not good sources of vitamin E.

In individuals at risk for vitamin E deficiency, it is clear that supplements should be recommended to prevent it. What about vitamin E supplement in normal individuals? Dietary changes such as decreasing fat intakes,“ substituting fat-free foods for fat-contain-

ing ones, and increasing reliance on meals away from the home have resulted in decreased consumption of «-tocopherol-containing foods. Therefore, intakes of the vitamin E recommended dietary allowance (RDA)—15 mg «—tocopherol—may be difficult. The most recent estimates of «a-tocopherol intakes by Americans suggest that less than 10% consume adequate amounts

of the vitamin, and that women

have

lower intakes than men.?*! Increased consumption of

E in

nuts and seeds, as well as olive and canola oils, may

the American diet.*! In the past, it was assumed for the purpose of calculating dietary vitamin E intakes in «a-tocopherol equivalents («-TEs) that y-tocopherol can substitute for a-tocopherol with an efficiency of 10%." However, functionally, y-tocopherol is not equivalent to the latter. Caution should be exercised in applying a-TEs to estimates of a-tocopherol intakes when corn or soybean oils (hydrogenated vegetable oils) represent the major oils present in foods. These oils have high y-tocopherol contents, and if food tables reporting

be useful in increasing a-tocopherol intakes. The potential role of vitamin E in preventing or ameliorating chronic diseases has prompted many investigators to ask if supplements might be beneficial. When “excess’’ amounts of many vitamins are con-

Indeed, desserts are a major source

of vitamin

sumed, they are excreted and provide no added bene-

fits. Antioxidant nutrients may, however, be different. Heart

disease

and

stroke,

cancer,

chronic

inflam-

mation, impaired immune function, Alzheimer’s disease—a case can be made for the role of oxygen-free radicals in the etiology of all of these disorders, and

Vitamin E

762

even in aging itself. Do antioxidant nutrients counteract the effects of free radicals and thereby ameliorate these disorders? And if so, do large quantities of antioxidant supplements have beneficial effects beyond “required’’ amounts? The 2000 DRI Report on Vitamin C, Vitamin

E, Selenium,

and Carotenoids

stated

that there was insufficient proof to warrant advocating

supplementation with antioxidants." But it also stated that the hypothesis that antioxidant supplements might have beneficial effects was promising. Despite the lack of positive findings from various intervention studies,°°?" and some more positive findings from others,?*!°!! the consequences of a long-term increased antioxidant intake in healthy people are not known. Moreover, a study examining the relationship between the genetic background of diabetic women and the benefits of antioxidant supplementation found a marked beneficial effect on the minimum luminal diameter in haptoglobin 1 allele homozygotes, but not in those with the haptoglobin 2 allele."’°”! Thus, it would appear that subjects with high oxidative stress and the appropriate genetic background may benefit from antioxidant supplements, but not in those without these factors.

Dietary Reference Intakes In 2000, the Food and Nutrition Board of the Institute of Medicine, National Academy of Sciences published the Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and the Carotenoids."! Recommendations for vitamin E intakes are shown in Table 1. The requirements for vitamin E intakes are based primarily on its long-term (5-7 yr) depletion and repletion studies in humans carried out by Horwitt et al.!!°7) Serum a-tocopherol concentrations and the corre-

sponding hydrogen peroxide-induced erythrocyte hemolysis were determined at various intervals. Serum concentrations necessary to prevent in vitro erythrocyte hemolysis in response known levels of vitamin E intake in subjects who had undergone experimentally induced vitamin E deficiency were used to determine estimated average requirements (EARs) for vitamin E. The RDAs are levels that represent the daily «-tocopherol intakes required to ensure adequate nutrition in 95-97.5% of the population and are an overestimation | of the level needed for most people in any given group. —

Vitamin

E Units

The Food and Nutrition Board defined vitamin E for human requirements to include only a-tocopherol and specifically those forms with 2R-a-tocopherol stereochemistry.!"! According to the U.S. Pharmacopoeia (USP), 1 international unit (IU) of vitamin E equals | mg all rac-a-tocophery] acetate, 0.67 mg RRR-~ a-tocopherol, or 0.74 mg RRR-a-tocophery] acetate. These conversions were estimated on the relative “biologic activities’? of the various forms when tested in the rat assay for vitamin E deficiency, the fetal resorption assay. These USP IUs are currently used in labeling vitamin E supplements and food fortificants. It should be noted that the 2000 RDA does not use vitamin E USP units; rather the recommendation is set at 15mg 2R-a-tocopherols. To convert IU to milligram of 2R-a-tocopherols, the IU RRR-a-tocopherol (or its esters) is multiplied by 0.65, while the IU all rac-a-tocopherol (or.its esters) is multiplied by 0.45.

ADVERSE

EFFECTS

Upper Tolerable Limits Table 1 Estimated average requirements (EARs), recommended dietary allowances (RDAs), and average intakes (Als) (mg/day) for a-tocopherol in adults and children Lifestage

EAR

RDA

0-6 mo

4

7-12mo

1-3 yr

5

4-8 yr

6

7

9-13 yr

9

11

6

14-18 yr

2

15

Adult (male or female)

12

1)

Pregnancy

12

15

Lactation

16

19

(Adapted from Ref.)

Al

High vitamin E intakes are associated with an increased tendency to bleed. It is not known if this is a result of decreased platelet aggregation caused by an inhibition of protein kinase C by «-tocopherol,”® some other platelet-related mechanism,!!™! or decreased clotting due to a vitamin E interaction with vitamin K.!'° Tt has also been suggested that extraordinarily high vitamin E intakes may interfere with activation of vitamin K.!'°7 Individuals who are deficient in vitamin K or who are on anticoagulant therapy are at increased risk of uncontrolled bleeding. Thus, patients on anticoagulant therapy should be monitored when taking vitamin E supplements to insure adequate vitamin K intakes,!!°%! The 2000 Food and Nutrition Board of the Institute

of Medicine,

National

Academy

of Sciences,

Vitamin E

763

recommended 1000 mg as an upper limit (UL) of all forms of a-tocopherol in supplements taken by adults 19yr and older, including pregnant and _ lactating women. The vitamin E UL was set for only supplements because it is impossible to consume enough a-tocopherol-containing foods to achieve a daily 1000mg intake for prolonged periods of time. The UL was defined for all forms of a-tocopherol, not just the 2R-forms, because all eight of the stereoisometric

forms in all rac-a-tocopherol are absorbed and delivered to the liver and therefore potentially have adverse effects. The ULs for supplements containing either RRR- or all rac-a-tocopherol supplements are 1500 IU RRR-«-tocopherol

or its esters, or 1100 IU

of all rac-a-tocopherol or its esters. The UL for RRR-«-tocopherol is apparently higher because each capsule of RRR-a-tocopherol contains fewer milligram of a-tocopherol than does one containing all rac-atocopherol. ULs were set for children and adolescents by adjusting the adult limit on the basis of relative body weight. No UL was set for infants due to lack of adequate data. The 2000 Food and Nutrition Board did recommend that food be the only source of vitamin E for infants. However, a UL of 21 mg/day was suggested for premature infants with birth weight of 1.5 kg, based on the adult UL.

Adverse Interactions of Drugs and Vitamin E Drugs intended to promote weight loss by impairing fat absorption, such as Orlistat or olestra, can also impair vitamin E and other fat-soluble vitamin absorption. Therefore, multivitamin supplementation is recommended. Supplements should be taken with meals at times other than when these drugs are taken to allow adequate absorption of the fat-soluble vitamins. Findings from two clinical trials have suggested adverse

vitamin

E effects.

One

study

was

a 3-yr,

double-blind trial of antioxidants (vitamins E and C, B-carotene, and selenium) in 160 subjects on simvastatin—niacin or placebo therapy.°?!!°l In subjects taking antioxidants, there was less benefit of the drugs in rais-

antioxidant

(WAVE) Trial, a randomized, double-blind trial of 423

postmenopausal women with at least one coronary stenosis at baseline coronary angiography. In postmenopausal women on hormone replacement therapy, all-cause mortality was higher in women assigned to

compared

with placebo group

sons for these adverse effects, especially mortality, are unclear because a meta-analysis of more than 80,000 subjects taking part in vitamin E intervention trials did not find increased mortality in those’ taking vitamin E."''?] However, reports of adverse effects of vitamin E supplements in humans are so rare that the Food and Nutrition Board set the upper tolerance

level for vitamin E using data from studies in rats."”!

CONCLUSIONS One of the real difficulties in setting requirements or making recommendations for optimal vitamin E intakes is that the function of the antioxidant remains undefined. Certainly, its in vitro antioxidant function

has been agreed upon for decades, but questions remain as to whether this is the only function of vitamin E, or if indeed antioxidant activity is its in vivo function.!!!3!!41 Tn addition, if the vitamin functions

solely as an antioxidant, then biomarkers of oxidative stress will never be useful for setting requirements because oxidative damage certainly can be modulated by antioxidants in addition to vitamin E. Thus, one of the major thrusts is to establish the function of vitamin E. One important area that is currently under investigation is its role in inflammation"'*! and immune function.!!!® But, here again, the role of oxi-

dative stress confounds the findings because leukocytes release reactive oxygen species and this is attenuated by

vitamin E.°" Clearly, defining vitamin E function(s) is the goal of future studies.

REFERENCES 1.

Food and Nutrition Board; Institute of Medicine. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids; National Academy Press: Washington, 2000.

2.

Evans, H.M.; Bishop, K.S. On the existence of a

hitherto unrecognized dietary factor essential for

ing HDL cholesterol than was expected,|"””! while there

was an increase in clinical end points [arteriographic evidence of coronary stenosis, or the occurrence of a first cardiovascular event (death, myocardial infarction, stroke, or revascularization)}."'°! The other study was the Women’s Angiographic Vitamin and Estrogen

vitamins

(HR, 2.8; 95% CI, 1.1-7.2; P = 0.047)" The rea-

reproduction. Science 1922, 56, 650-651.

3.

Yamamoto, Takashima,

Y.; Maita, N.; Fujisawa, A.; J.; Ishii, Y.; Dunlap, W.C. A new

vitamin E (alpha-tocomonoenol) from eggs of the Pacific salmon Oncorhynchus keta. J. Nat. Prod. 1999, 62, 1685-1687. 4.

Yamamoto, Y.; Fujisawa, A.; Hara, A.; Dunlap,

W.C. An unusual vitamin E constituent (alphatocomonoenol) provides enhanced antioxidant protection in marine organisms adapted to

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764

Williamson, K.S.; Gabbita, S.P.; Mou, S.; West, M.; Pye, Q.N.; Markesbery, W.R.; Cooney, U.; P.; Reimann-Philipp, Grammas, R.V.;

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Floyd, R.A.; Hensley, K. The nitration product 5-nitro-gamma-tocopherol is increased in the Alzheimer brain. Nitric Oxide 2002, 6, 221-227.

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G.W.;

Traber,

M.G.

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E: anti-

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oxidant activity, biokinetics and bioavailability. Annu. Rev. Nutr. 1990, 70, 357-382. Ingold, K.U.; Webb, A.C.; Witter, D.; Burton, G.W.; Metcalfe, T.A.; Muller, D.P. Vitamin E

remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency. Arch. Biochem. Biophys. 1987, 259, 224-225. Packer,

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E is nature’s master

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Upston,

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Szewezyk,

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C.E.; Traber, M.G. 5-Nitro-y-tocopherol increases in human plasma exposed to cigarette smoke

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Terasawa, Y.;' Ladha, 4: "Leonard, S.W: Morrow, J.D.; Newland, D.; Sanan, D.; Packer, L.; Traber, M.G.; Farese R.V., Jr. Increased

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Traber, M.G.; Packer, L. Vitamin E: beyond antioxidant function. Am. J. Clin. Nutr. 1995, Zingg, J.M.; Azzi, A. Non-antioxidant activities of vitamin E. Curr. Med. Chem. 2004, /1/,

11t3=1133. Jialal, I.; Devaraj, S. Antioxidants and athero-

sclerosis: don’t throw out the baby with the bath water. Circulation 2003, 107, 926-928.

116.

Meydani, S.N.; Meydani, M.; Blumberg, J.B.; Leka, L.S.; Siber, G.; Loszewski, R.; Thompson, C.; Pedrosa, M.C.; Diamond, R.D.; Stollar,

B.D. Vitamin E supplementation and in vivo immune response in healthy elderly subjects. A randomized controlled trial. J. Am. Med. Assoc. 1997, 277, 1380-1386.

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Vitamin K J.W. Suttie College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.

INTRODUCTION

BACKGROUND, CHEMISTRY, AND DIETARY SOURCES

Vitamin K_ activity is exhibited by phylloquinone, found in green plants, and by a series of menaquinones, which are synthesized by a limited number of anaerobic bacteria. The metabolic role of this vitamin is as a substrate for an enzyme, the vitamin K-dependent carboxylase, which mediates a posttranslational modification of a small number of proteins by converting specific glutamyl residues to y-carboxyglutamyl (Gla) residues. These proteins include a number that regulate hemostasis: prothrombin, factor VII, factor IX, factor X, and proteins C, S, and Z. The bone protein, osteocalcin, and matrix Gla protein, several found in bone and other tissues, also require

vitamin K for their synthesis as do a small number of less well-characterized proteins. The human requirement

for vitamin

K is low, and the adequate

intake for adult men and women is currently set at 120 and 90pg/day, based on median intakes of the U.S. population. The classical symptom of a vitamin K_

deficiency,

a

hemorrhagic

event,

is essentially

impossible to produce in adults without some underlying factor influencing absorption of the vitamin. However, newborn infants are routinely supplemented with vitamin K to prevent a condition called hemorrhagic disease of the newborn. A small amount of the protein osteocalcin circulates in plasma, and this

protein is not maximally y-carboxylated at normal levels of intake. There is currently a great deal of interest in a possible role of vitamin K in promoting skeletal health. Supplementation of the diet with 45mg of menaquinone-4 is a widely used treatment for osteoporosis in Japan and other parts of Asia. The efficacy of this treatment in North America or Europe has not yet been established but remains a question of significant research interest.

J.W. Suttie, Ph.D., is at the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.

Encyclopedia of Dietary Supplements DOI: 10.1081/E-EDS-120022055

Copyright © 2005 by Marcel Dekker. All rights reserved.

In the early 1930’s, Henrik Dam observed that chicks

consuming very low lipid diets developed subdural or muscular hemorrhages and that blood taken from these animals clotted slowly. This hemorrhagic disease could not be cured by supplementation with any other known dietary factor, and Dam"! proposed the existence of a new fat-soluble factor, vitamin K. Sub-

sequent studies by Dam and others"! established that the antihemorrhagic factor was present both in the lipid extracts of green plants and in preparations of fish meal that had been subjected to bacterial action. The vitamin could be isolated from alfalfa as a yellow oil, and it was characterized as 2-methyl-3-phytyl-1,4naphthoquinone™! and synthesized by Doisy’s group at the St. Louis University. The Doisy group also isolated a crystalline form of the vitamin from putrefied fish meal and demonstrated that this compound contained an unsaturated polyprenyl side chain at the 3-position of the naphthoquinone ring. The term vitamin K is now used as a generic descriptor of 2-methyl-1,4-naphthoquinone (menadione) and all derivatives of this compound that exhibit an antihemorrhagic activity in animals fed a vitamin K-deficient diet (Fig. 1). The major dietary source of vitamin K, the form found in green plants, is commonly called vitamin K,, but is preferably called phylloquinone. The compound, 2-methyl-3-farnesylgeranylgeranyl-1,4-naphthoquinone, first isolated from putrefied fish meal, is one of a series of vitamin K com-

pounds with unsaturated side chains called multiprenylmenaquinones, which are produced by a limited number of anaerobic bacteria and are present in large quantities in the lower bowel. This particular menaquinone has 7 isoprenoid units in the side chain and was once called vitamin K>. That term is currently used to describe any of the vitamers with an unsaturated side chain, and this compound is more correctly identified as menaquinone-7 (MK-7). The predominant menaquinones found in the gut are MK-7 through MK-9, but smaller amounts of others are also present. Menadione is used as a source of vitamin K activity in poultry and swine rations, and a specific 771

Vitamin K

772

Table 1 Phylloquinone concentration of common foods* COMINOD ea (COC Food item

eee pg/100 g

Vegetables

Menaquinone-4

Fig. 1 Structures of vitamin K active compounds. Phylloquinone (vitamin K,) synthesized in plants is the main dietary form of vitamin K. Menaquinone-9 is a prominent member of a series of menaquinones (vitamin K ) produced by intestinal bacteria and menaquinone-4, while a minor bacterial product can be synthesized by animal tissues from phylloquinone.

compound, menaquinone-4 (2-methyl-3-geranylgerany]l1,4-naphthoquinone), is formed in animal tissues by its alkylation.“! This is the biologically active form of the vitamin present in animal tissues when menadione is used as the dietary form of vitamin K. Standardized procedures to assay the vitamin K content of foods, and sufficient values"! to provide rea-

sonable estimates of its daily intake are now available (Table 1). In general, foods with higher phylloquinone content are green leafy vegetables. Those providing substantial amounts of the vitamin to the majority of the population are spinach (380 upg/100 g), broccoli (180 pg/100 g), and iceberg lettuce (35 ug/100g). Fats and oils are also a major contributor to the vitamin K content of the diet. Soybean oil (190 pg/100 g) and canola oil (130pg/100g) are quite high, while corn oil (3 ug/100 g) is a very poor source. The source of fat or oil will influence the vitamin K content of margarine and prepared foods with a high fat content. The process of hydrogenation to convert plant oils to solid margarines or shortening converts some of the phylloquinone to 2',3’-dihydrophylloquinone with a completely saturated side chain. The biological activity of this form of the vitamin is not accurately known, but it has

been reported that the intake of this form of the vitamin by the American population may be 20-25% that of phylloquinone.!©

Collards Spinach Salad greens Broccoli Brussels sprouts Cabbage Bib lettuce Asparagus Okra Iceberg lettuce Green beans Green peas Cucumbers Cauliflower Carrots Tomatoes Potatoes

440 380 315 180 177 145 122 60 40 35 BS 24 20 2010 6 1

Protein sources

Dry soybeans Dry lentils Liver Eggs Fresh meats Fresh fish Whole milk

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CEST

Zinc Carolyn S. Chung Janet C. King Children’s Hospital Oakland Research Institute, Oakland, California, U.S.A.

INTRODUCTION

Catalytic, Structural, and Regulatory Functions

Zinc is the most abundant intracellular trace element. It is present in every living cell in the body and has many diverse biological functions. Because zinc is a component of many enzymes involved in the synthesis and degradation of carbohydrates, lipids, proteins, nucleic acids, and gene expression, as well as in the

Zinc functions as a catalytic, structural, and regulatory component of nearly 300 enzymes, in which it maintains structural integrity and plays a role in regulation of gene expression.“ In humans, the element is critical for the proper function of about 60 enzymes. Enzymes that require zinc for their catalytic function are found in all six enzyme classes (ligases, isomerases, lysases, hydrolases, transferases, and oxidoreductases).©! An enzyme is considered to be a zinc metalloenzyme if removal of zinc reduces enzyme activity without affecting the protein structure per se, and if reconstitution with zinc restores activity.! The metal may serve a catalytic function for these enzymes by serving as an electron acceptor. It is a catalyst for RNA polymerase, alkaline phosphatase, and carbonic anhydrase. In its structural role, zinc helps proteins fold into their three-dimensional configurations; this folding is required for these proteins to have biological activity. “Zinc fingers,’’ or loops formed by binding zinc to

metabolism of other nutrients," it has a multitude of

physiological and biochemical functions, notably in embryogenesis, immunity, and growth. The element is abundant in the food supply; however, its absorption from foods depends on the presence or absence of substances in foods that can bind zinc and make it unavailable for absorption. In 1961, the human requirement for zinc was shown by reports of hypogonadism and dwarfism of rural Iranian boys who consumed food that contained zinc, but which was not readily absorbed.”! The significance of the clinical and public health implications of zinc health is currently being addressed and deserves attention. In cases of inadequacy, primarily in developing countries, treatment with supplements (zinc sulfate, zinc gluconate, etc.) is effective.

BIOCHEMISTRY AND PHYSIOLOGIC FUNCTIONS

Zinc has a 2+ charge (Zn**). It has an atomic number of 30 and an atomic weight of 65.37 (isotopic mean). In the pure form, it is a bluish white metal. Zinc is a strong Lewis acid, meaning it is an electron acceptor. It has a particular affinity for thiol and hydroxy groups and for amine electron donors.4! The metal readily forms complexes with amino acids, peptides, proteins, and nucleotides.

It does

not exhibit redox

chemistry.

Carolyn S. Chung, Ph.D., is a Postdoctoral Fellow at Children’s Hospital, Oakland Research Institute, Oakland, California, U.S.A. Janet C. King, Ph.D., is a Senior Scientist at Children’s Hospital Oakland Research Institute, Oakland and Professor at UC,

Berkeley, Davis, California, U.S.A.

Encyclopedia of Dietary Supplements DOK: 10.1081 /E-EDS-120022064

Copyright © 2005 by Marcel Dekker. All rights reserved.

the amino

acids, cysteine,

and

histidine,

enable

the

folding to take place.!”! Examples of proteins requiring zinc for normal structure include DNA transcription factors (e.g., metal transcription factor 1), nuclear receptors (e.g., retinoic acid receptors), and enzymes (e.g., Copper—zinc superoxide dismutase). Zinc finger proteins are widely distributed in the cell and also bind to RNA molecules and other proteins. This interaction with other molecules allows for transcriptional and translational control and signal transduction. This ability to regulate gene expression may explain the tight homeostatic control of zinc metabolism. Zinc fingers and their basic functions may also explain the nonspecific symptoms that characterize zinc deficiency.! _ Zinc and sulfur join to form thiolate clusters in proteins such as metallothionein. These clusters allow metallothionein to exchange electrons and react with oxidants (nitric oxide, glutathione/glutathione reductase) in the body. When oxidized, thiolate clusters release zinc. The activity of nitric oxide synthase”! may explain the increased oxidative stress in cells

associated with zinc deficiency."”! As a regulatory agent, zinc controls the expression of various genes (e.g., the metallothionein gene), 791

Zinc

792

programmed cell death or apoptosis,"'*! and synaptic signaling.''*! Zinc binds to zinc finger domains on a protein called metal response element transcription factor (MTF1). The zinc-MTF1 complex then binds to metal response elements (MREs) on the promoter

area of the gene!'*! and thereby regulates gene expression."'*! Individual human responses to dietary zinc intake are highly variable and may be explained by. slight alterations in the regulation of the MTF1 gene."'*! Genes associated with apoptosis and immune function are also responsive to zinc regulation.!"7! Zinc regulates immune function by _ receptormediated signal transduction. Zinc bound to a T-cell receptor joins with the protein tyrosine kinase enzyme''®! to activate the T cell. The transfer of information between and within cells that activate or inhibit communication with other cells, or that initiate signal transduction, allows an ion channel or a

second messenger system to further activate immune cell

function,

differentiation,

and

proliferation.!'7!

The metal also functions as a signal molecule when released in the synaptic vesicles of “‘zinc-containing’’ nerve cells found almost exclusively in the brain!?”!. Zinc-containing neurons bind zinc and glutamate suggesting that zinc may regulate the function of glutamatergic synapses.?"! Protein and Nucleic Acid Metabolism

Zinc plays a fundamental role in protein and nucleic acid metabolism, and cell division and differentiation.

Deficiency has a greater effect on cell division, a function of nucleoprotein synthesis, than on cell growth, which is a function of protein synthesis. The adverse effects of inadequacy on gene expression and DNA or RNA metabolism impair cell division and cause growth failure.”"! The effect of severe zinc shortage on protein synthesis may increase amino acid oxidation. The metal is required for the normal synthesis of collagen (essential for wound healing), metallothionein (a protein involved in zinc homeostasis), and immune proteins. Lipid and Carbohydrate Metabolism Zinc-dependent enzymes are involved in the metabolism of carbohydrate and lipid, and release of energy. They also are involved in the synthesis of longchain fatty acids from their dietary precursors and the formation of various prostaglandins. Zinc also appears to stabilize membranes by binding directly to protein and lipid components. Impaired glucose tolerance is associated with zinc deficiency, possibly because the metal is required for insulin to control glucose uptake by adipocytes.

METABOLISM Zinc Distribution in Humans Zinc is present in all tissues, organs, fluids, and secre-

tions of the body. The total body content is 1.5g in females and 2.5g in males. The zinc concentration of various tissues of the body and proportion of total body Zn vary. Its concentration is highest in bone, muscle, prostate, and kidney tissue and is lowest in brain, skin, heart, and plasma.7} Bone and skeletal

muscle comprise

86%

of total body zinc. Approxi-

mately, 95% is found within cells.??!

Zinc Absorption Advances in knowledge regarding the molecular understanding of zinc absorption by the gastrointestinal tract contribute to and support previous studies of zinc metabolism,

absorption kinetics, and homeo-

static regulation. The discovery of zinc transporters suggests that zinc metabolism in human cells is strongly influenced by transporter genes regulated by diet and hormones/cytokines, by their tissue specificity of expression and interactions, as well as by mutations and polymorphisms in these genes and their phenotypic consequences. Two families of zinc transporter proteins exist. The ZnT (ZnT1—ZnT9) and ZIP family (at least 15 mammalian ZIP transporters) each have numerous members.”*7°! ZnTs facilitate zinc efflux across the plasma membrane or into intracellular vesicles. In contrast,

ZIPs

enable

zinc influx into cells or from

vesicles. Localization and expression of ZnT and ZIP genes may be a function of physiologic conditions and body zinc status.?°7"! Specific transporters, more than bind multiple divalent ions, may explain some metal-metal interactions (e.g., zinc and_ iron).?®) Human ZnT and ZIP genes respond to zinc: by up- or downregulation,"'7! and may contribute to the tight homeostatic control of zinc. Zinc is absorbed along the entire intestinal tract.?! The human-jejunum has the highest rate of absorption.°°! However, other studies with humans and animals suggest the duodenum is most important to the overall amount of zinc absorbed because the duodenal lumen has the highest initial zinc concentration after a meal.?"! Zinc resecreted into the digestive tract from sloughed-off intestinal and pancreatic cells or endogenous secretions contributes to the high concentration of zinc in the duodenal lumen. The estimated average requirement (EAR) and recommended dietary allowance (RDA) for zinc are now based on endogenous zinc excretion, to which intestinal secretion is the major contributor. Apparent absorption

Zine

averages 33% and is a factor used to compute the RDA.""! Factors that influence endogenous intestinal zinc secretion, such as pancreatic insufficiency and inflammatory bowel disease, have an impact on intestinal zinc absorption/retention. The relationship between endogenous zinc loss and absorbed zinc is the basis around which the new RDA was constructed.

Homeostatic Regulation Total body zinc is primarily controlled by intestinal absorption. When dietary zinc intake decreases, absorption increases.°?*] At the same time, endogenous losses decrease in response to low dietary intake.?7! During times of severe dietary zinc restriction, losses can be reduced to less than 1 mg/day.©°! Changes in physiologic state such as lactation increase absorp-

tion.??3* In contrast, states of stress and infectious disease may decrease zinc absorption." Zinc Turnover and Transport Plasma zinc comprises only 0.1% of total body zinc and is maintained within a narrow range (10-15 umol/L). Reduction of zinc status in humans results in decreased plasma zinc levels, but results are not consistent.27! Severe zinc depletion markedly reduces these levels.°*! Acute disease can decrease plasma zinc,?*! but acute

starvation increases the levels to above normal.®! After food is eaten, plasma zinc declines by about 15% over the next 2—3 hr, and may reflect hormonally regulated tissue zinc redistribution." An increase in plasma zinc occurs when consumption is above the RDA 42:43] Zinc in plasma is bound to the proteins, albumin,! 6]

and o-macroglobulin. Approximately, 70% of it in plasma is bound to albumin and exchanges easily (Kd = 7.5M) with it. %)-Macroglobulin, a protease inhibitor and carrier of growth factors, binds the metal tightly and represents most of the remaining protein-

bound zinc in plasma.'°“4! The flux of zinc through the plasma in the body is ~130 times/day.'*°! Zinc in blood is found mainly in leukocytes (6mg of Zn/10° cells) compared to erythrocytes (1 mg of Zn/10° cells).!4°! Leukocytes actively make proteins, and cDNA array analyses have shown that leukocyte genes are very sensitive to zinc."] Genes expressed in circulating leukocytes may respond to plasma zinc levels. Kinetic data, with both radioactive and stable iso-

topes, model how zinc pools turn over. Two metabolic pools have been identified in humans: a rapid pool that turns over in ~12.5days and a slow pool that turns over in 300 days.47-48! Zinc rapidly turns over in liver, pancreas, kidney, and spleen, while slow turnover is

793

found in muscle and erythrocytes, followed by bone and the nervous system. Using a comparable metabolic model in rats identified the thymus, skin, spleen, intestine, and, especially, the bone marrow as important organs for zinc metabolism.'*?! An exchangeable zinc pool (EZP) whose size is influenced by zinc: intake has been defined in human subjects.©°! It is decreased after severe dietary zinc restriction. This may reflect the pool of zinc available to tissues and may therefore provide a measure of zinc status. The EZP encompasses all of the zinc in the plasma compartment, and some in the hepatic compartment. Zinc Reserves

Zinc has no specific storage site. Conservation mechanisms are such that tissue zinc levels are maintained during depletion. Supplemental zinc, above normal requirements, may provide some reserve in animals during a state of deficiency.©'! Occurrence of a comparable reserve in humans has not been investigated. Retention of body zinc is accomplished through powerful retention mechanisms and recycling. Red blood cells contain between 20 and 40 pg Zn/g hemoglobin,°°] and the average circulating hemoglobin content is 750g in adults. This represents a red blood cell zinc pool of 15-30 mg. The average life span of a red blood cell is 120 days. Therefore, turnover for this zinc pool is between 0.12 and 0.25mg/day. This suggests that a meaningful amount of zinc needs to be supplied for erythropoiesis. A decline in erythropoiesis is known to occur with severe zinc deficiency in experimental animals.) Excretion

The major route of zinc excretion is secretion into the gastrointestinal tract. This is the combined contribution from the pancreatic secretions (enterohepatic circulation), sloughing of mucosal cells into the intestinal lumen, and transepithelial flux of intestinal zinc from the serosa to the mucosa.!*! The metal lost via pancreatic secretions comprises an ill-defined mixture, but certainly includes zinc metalloenzymes. It is not surprising, therefore, that pancreatic zinc secretion is stimulated by meals. Quantitatively, this may constitute the major route of fecal elimination. Gastrointestinal secretion is a function directly related to dietary

zinc intake. Estimates are as low as 20% of the population

Plasma or serum zinc

Proportion of population below nonfasting concentration of 65 pg/dl (70 pg/dl fasting)

>20% of the population

(Adapted from Ref.""!,)

Zine

Isolated outbreaks of acute toxicity have occurred as a result of consumption of food or beverages contaminated with zinc from galvanized containers or from accidental exposure to zinc oxide fumes. The overt toxicity symptoms include nausea, vomiting, epigastric pain, lethargy, and fatigue. Chronic overdosage of zinc (i.e., 100-300 mg zinc per day) is of greater concern than acute toxicity because it is more common and is less easily detected. The major consequence of chronic excess zinc is its negative effect on copper absorption and _ status. Intakes as low as 60 mg zinc per day have been shown to reduce erythrocyte copper—zine superoxide dismutase (ESOD) activity, a sensitive marker of copper status.P°! Doses between 50 and 100mg zinc per day have lowered high-density lipoprotein (HDL) cholesterol in some,”!?7! but not all, studies.°7! ULs of zinc intake, based on studies using ESOD activity, have been established by the U.S. Food and Nutrition Board of the Institute of Medicine?! to prevent the symptoms of zinc overdosage.

ADVERSE INTERACTIONS Zine deficiency may also be related to a competitive interaction with iron. The findings are varied, however,

vo

25mg of zinc may are used.

when

these drugs

CONCLUSIONS Groups at risk of zinc deficiency include those with high zinc needs such as preterm infants, growing children and adolescents, and pregnant and lactating women. Dietary zinc intakes in the United States are adequate for most groups, with the exception of vegans,

who

may

not consume

fortified cereals, and

possibly the elderly. Supplemental zinc has been used to treat a variety of diseases; however, not all disease outcomes are improved. Individuals experiencing trauma and tissue breakdown lose zinc from tissues, and zinc supplements are used to treat wound healing. Supplements may also be indicated for those taking drugs that chelate zinc. The WHO advocates providing 20mg/day of zinc supplementation for 10-14days (10mg/day for infants under 6months old) for management of acute diarrhea.!*°!

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2)”

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Index

Accutane® (vitamin A), 710

Ascorbate, ascorbic acid. See Vitamin C.

Acetylcholine, 105, 107 Acetyl-CoA carboxylase, biotin and, 32, 33 Acetyl-L-carnitine. See Carnitine.

Astragalus, 25-30

Acitretin® (vitamin A), 710

Acyl carrier protein (ACP), and pantothenic acid, 517, 518, D9, 320, 22, S20 AdoMet. See SAMe. Adrenarche and adrenopause, 169 a-Adrenoceptor blockers, and Pygeum africanum extract, Sil oye Afrodex™ (yohimbe), 784 Age-related macular degeneration. See AMD. Aglycone, and quercetin, 577, 578 Alanine, and glutamine, 291 Allium sativum. See Garlic. Alzheimer-type dementia, 107 AMD, and lutein, 409, 417 Anabolic steroids, 10, 11

Androgens, 7, 169 Androstenediol, 7, 10 Androstenedione, 7-13, 172 adverse effects, 11 biochemistry and physiology, 7-8 contamination, 11 detection, 12 and feminization, 7, 10, 11 hormonal effects, 8-11 in men, 8-10 in women, 10-11 metabolism, 10 mislabeling, 11-12

muscle size and strength, effects on, 10 pharmacokinetics, 8-11 in men, 8-10 in women,

10-11

prohormones, 7, 10 precursor, 7 purity of, 11 regulatory status, 7, 12 site of production, 7 toxicity, 11

usage, 8. See also Doping. Anemia iron and, 360, 361 Aneurin. See Thiamin. Angelica sinensis. See Dang gui. Anticonvulsants, biotin and, 35, 36, 37 Antiemetic, ginger, 241-248 Antispasmodic effect, valerian and, 687 Anxiolytic effect kava and, 376, 377 valerian and, 687,.691, 697 Arachidonic acid (AA), 494, 505, 506, 507, 509, 511, 512, 513 Arginine paradox, 19

biochemistry and function, 25-27 cardiovascular effects, 27 dosages, 28 driving, effect on, 28 hepatoprotective effects, 27 immunomodulatory effects, 25 indications, 28 interactions, 28 lactation, use in, 28 mutagenicity, 28 overdose, 28 pharmacodynamics, 25 pharmacokinetics, 25 precautions, 28 pregnancy, 28 regulatory status, 29

safety profile, 28 side effects, 28 toxicology, 28 Astragalus membranaceus. See Astragalus. Astragalus mongholicus. See Astragalus. Avidin, 31, 35 Awa. See Kava.

Azidothymidine (AZT), and shiitake, 659, 661

Bantu siderosis, 360 Basic fibroblast growth factor (bFGF), and Pygeum africanum extract, 571, 573 B-complex vitamins biotin, 31-40 folate, 219-228 niacin (vitamin B3), 483-491 pantothenic acid (vitamin Bs), 517-525 thiamin (vitamin B,), 677-686 vitamin Be, 715-734 vitamin Bj, 735-744 Benign prostatic hyperplasia. See BPH. Berberine. See Goldenseal. Berzelius, discoverer of selenium, 645 Betaine, and choline, 105, 106, 107, 109 Bidrolar® (Pygeum africanum extract), 574 Bifidobacteria. See Lactobacilli. Biotin, 31-40 absorption, intestinal, 31, 33, 34, 36

adequate intake (AI), 37 anticonvulsants and, 35, 36, 37 bioavailability, 33, 35 biochemistry, 31-33

blood, transport in, 34 carboxylases and, 31-33, 34, 35, 36, 37

as cofactor, 31 deficiency

801

Index

802

Biotin (contd.) diagnosis, 35-37 marginal deficiency, 36 occurrence, 35-36 treatment of, 37 symptoms of, 36 digestion of protein bound, 33 estimated average requirement (EAR), 37 excretion, urinary, 35, 36 gene expression and, 37 hepatic uptake, 34 history of, 31 intake, 37 pharmacology, 35 physiology of, 33-35 renal handling, 34-35 structure, 31 synthesis

Goldberg/Sternbach method, 31 microbial, 31 toxicity, 37 transport, 33-35 in blood, 34 in CNS, 35 in human milk, 35 in placenta, 35 transporters, 33, 34, 35 usage, 37 Biotinidase, 33, 34, 36 Bisphosphonates, 69

Black cohosh, 41-53 adverse effects, 50

biochemistry and functions, 48-49 cell proliferation, 49

CNS effects, 49 ER competitive binding, 48 estrus induction, 49

hormonal secretion, 49 neurotransmitter binding, 49 osteopenia inhibition, 49 plasma hormone levels, 48 receptor expression, 48 uterine weight, 49 chemistry, 41-42 aromatic acids, 41

triterpenes, 41 clinical studies, 42, 48 commercial products, 42 compendial/regulatory status, 50 description, botanical, 41

dosage, 42, 50 lactation, use in, 49 menopause, 42

pregnancy, use in, 49 regulatory status, 50

Remifemin®, 42, 48, 50

triterpene glycosides in, 41 Bone mineral density (BMD). See also Osteoporosis. DHEA and, 171 isoflavones and, 367

phosphorus and, 540 red clover and, 596 Borate, 55, 57. See also Boron. Boric acid, 55, 56, 57. See also Boron. Boromycin, HIV and, 56. See also Boron. Boron, 55-63 animal and human tissues, 57

bioavailability and excretion, 58

biochemical forms, 55 boroester, 55 cartilage and bone structure, 60 dietary sources, 59 environmental forms, 55 immune function, 60 indications and usage, 59 insulin and glucose metabolism, 60 mineral metabolism, 59 nonfood personal care products, 59 overdosage, 61 plant-based foods, 57 procaryotes, 56 sources and intakes, 58 dietary recommendations, 58 dietary supplements, 58 speciation, 55 steroid metabolism, 60 BPH and Pygeum africanum extract, 569, 570, 571 and saw palmetto, 638-639

Calbindin, 67 Calcitriol therapy, 70 Calcium, 65-71

absorption of, 65, 67-68, 69, 70 magnesium in, 447 adverse reactions to, 70 contraindications in use of, 70

in cytosol, 18, 66, 124 excretion, 68 in extracellular fluid (ECF), 65, 66, 67 indications and usage, 68-70 ancillary therapy, 70 osteoporosis therapy, 69 internal economy of, 66-68 magnesium in, 451-452 magnesium, in Ca channel blocking, 445-456 metabolic functions, 65-66 bone remodeling/resorption, 65, 66, 67 intraluminal effects, 66 “off-loop’’ effects, 66 overdosage, 70

pharmacology/physiology, 65-68 and phosphorus, 538, 539, 540, 541, 542 precautions, 70 preparations of, 69 protein tertiary structure and, 65 recommended intake levels, 68-69 regulatory status, 70 supplementation, 68, 70 thyroxin absorption, interference with, 70 Calcium paradox disease, 66 Camellia sinensis. See Green tea polyphenols. Cantoni, G., discoverer of SAMe, 1, 2 Carbidopa, and 5-HTP, 350, 351, 353 and peripheral L-aromatic amino acid decarboxylase (AADC) inhibitors, 350, 353 Carboxylases, biotin and, 31-33, 34, 35, 36, 37 Careseng® (Asian ginseng), 261 Carnitine, 73-80 adverse effects, 78 biochemistry and functions, 73-75 acyl-CoA/CoA ratio, modulation of, 74

ammonia intoxication prevention, 75

Index

803

fatty acids, transfer of, 73-74

glucocorticoid action, mimicking, 75 membrane phospholipid remodeling, 75 mitochondrial decay, reversal of, 75 thyroid hormone action, antagonism toward, 75 CoA, interrelations with, 522

compendial/regulatory status, 78 deficiencies, 76 dietary reference intake (DRI), 75 indications and usage, 76-78 cardiovascular disease, 76, 77 chronic fatigue, 77 depression and cognitive function, 77, 78 diabetes, 78 end-stage renal disease, 76 exercise performance, 77 human immunodeficiency virus (HIV) infection, 78 liver dysfunction with hyperammonemia, 78 replacement therapy, 76-78 supplementation, 76-78 weight reduction, 77 physiology, 75, 76 bioavailability, 75 biosynthesis, 75 dietary intake, 75 distribution, 75

homeostasis and renal handling, 76 tissue accumulation, 76 recommended dietary allowance, 75 Carnitine acetyltransferase (CAT), 74 Carnitine—acylcarnitine translocase (CACT), 73, 74 Carnitine palmitoyltransferase I (CPT I), 73 B-Carotene, 81-87. See also Carotenoids. absorption and metabolism, 81, 82-83 as antioxidant, 81, 84 bioavailability, 81-83 biochemistry and functions, 81 solar radiation, protection against, 81 vitamin A activity, 81, 83 contraindications, 84-85 indications and usage, 83-84 cancer prevention, 83 cardiovascular disease, 84 erythropoietic protoporphyria, 84, 85 and lung cancer risk, 81, 83, 84, 85 and lutein, 412, 416, 417 as provitamin A, 701, 702, 705, 706, 708 recommended intakes, 83 regulatory status, 85 safe upper level, 83 sources, 82, 83, 85

toxicity /adverse effects, 85 Carotenodermia, 85, 418 Carotenoids B-carotene, 81-85 lutein, 409-420 lycopene, 421-434 and vitamin A, 702, 705, 706, 707, 708 Cartilage boron and, 60 chondroitin and, 113, 114 glucosamine and, 279, 280, 283, 284 Cascara sagrada, 89-93 appearance, 89 chemistry, 89 glycosides, 89 commercial preparations, 89

laxative, use as, 90

pharmacology, 90 toxic activities, 90 use in humans, 91-92 adverse reactions, 91 contraindications, 91 dosage forms, 91

dose, 91 precautions, 91

regulatory status, 91 Cataract, and lutein, 409, 417 Chacon, and maca, 435 Chasteberry, 95-103 adverse effects, 100

chemistry, 95 agnuside, 95 casticin, 95 diterpenes, 95 flavonoids, 95

iridoids, 95 clinical studies, 96-99 acne and, 99

corpus luteum insufficiency and, 96, 98 hyperprolactinemia and, 98 infertility and, 98-99 mastalgia and, 97-98

menstrual cycle irregularity and, 98-99 PMS and, 96-97 dosage, 100 mechanism of action, 99 dopaminergic, 99 estrogenic, 99, 100 prolactin, 98, 99

products, 100 therapeutic indications, 96 Chitin, glucosamine precursor, 281, 282, 284 Cholesterol synthesis, and coenzyme Qjo, 121, 123, 127, 129 Choline, 105-111

adequate intake (AI), 105, 109 biochemistry and nutrients, 105-106 synthesis, 105, 106, 107, 108, 109

and brain, 107-108 development,

108

function, 107 deficiency of, and organ function, 107 and cancer, 107 cardiovascular disease, 107 renal dysfunction, 107 dietary recommendations,

109

homocysteine, 105, 107, 110 physiology, 106-107 metabolism,

106-107

requirement, 108-109 adverse effects, 109 food sources, 109 gender and, 108

in milk, 108-109 in plasma, 109 pregnancy and, 108 Chondroitin, 113-119 biochemistry, 113-114 and bovine spongiform encephalopathy (mad cow disease) risk, 117 clinical studies, 115-117 mechanisms of action, 115 and NSAIDs, 116, 117 pharmacology, 114

Index

804

Chondroitin (contd.) bioavailability, 114 pharmacokinetics, 114 physiology, 113-114 preparations, 115 safety, 117 side effects, 117 structure, 113-114 Chondroitin sulfate, 113, 114, 115, 116, 117

Chrysanthemum parthenium. See Feverfew. Cimicifuga racemosa. See Black cohosh. Climacteric symptoms. See also Menopause. black cohosh and, 41, 42

isoflavones and, 367 Clonidine, 213 Cobalamin. See Vitamin By>. Coenzyme A (CoA), and pantothenic acid. See Pantothenic acid. Coenzyme Qjo, 121-131 analysis, 121 biochemistry, 121-123 biosynthesis, 121, 126, 127

enzymatic reduction, 123 mevalonate pathway, 121, 126 deficiency, 127, 128-129 aging, 128 cardiomyopathy, 127, 129 exercise, 129 genetic disorders, 127, 128 neurological disorders, 129 statin therapy, 129 dosage, 130

extraction, 121 functions, 123-125 antiatherogenic effects, 124 antioxidant activity, 123, 124 apoptosis, role in, 124 electron transport chain, role in, 123 enzymatic functions, 123 lipid peroxidation, role in, 124 mitochondrial pore opening, role in, 123-124 nonenzymatic functions, 123-125 protonmotive Q cycle, role in, 123 thermogenesis, role in, 124 uncoupling protein function, 124 vascular effects, 125 physiology, 125-128 bioavailability, 126-128 catabolism, 127 circulation, 126 endoplasmic reticulum (ER)—Golgi system and, 126 intracellular distribution, 125-126 regulation of tissue content, 127

tissue distribution, 125, 128. See also bioavailability. transport, 126 uptake, 126, 127 semiquinone radical, 124 side effects, 130 Commensal flora, 381, 382, 383, 384, 385

Condensed tannins. See Proanthocyanidins. Contractile dysfunction, 572, 573

Copper, 133-141 absorption, 134 actions, 133-134 in enzymes, 133, 136

adequate intake (AI), 135 biochemistry, 133-134

deficiency, 133, 135. See also indications and usage. depletion experiments, 134, 135 estimated average requirement (EAR), 134, 135 glutathione metabolism and, 133

hypercholesterolemia and, 133 indications and usage, 134-137 anemia, 133, 135, 137 burn victims, use in, 136 cardiovascular abnormalities, 133, 135, 136 osteoporosis, 136 premature infants, use in, 136 iron and, 133 physiology, 133-134 phytates and, 134 recommended dietary allowance (RDA), 134, 135 sources, 134 supplements, 134 tolerable upper intake level (UL), 136, 137 toxicity, 136-137 vitamin C and, 137 © in Western diet, 134 zinc and, 136, 137 Copper gluconate, 134, 136 CoQ, CoQH>. See Coenzyme Qio. Cortisol, and DHEA, 168 Cranberry, 143-149 adverse effects, 146 chemistry, 143 flavonoids, 143, 145 phenolic acids, 143 proanthocyanidins, 144, 145 clinical studies, 146 efficacy, 146 contraindications, 147 drug interactions, 147 preclinical studies, 144 antiadhesion, 145 antioxidant, 145

atherosclerosis, 145 bioavailability, 144 cancer, 146 dental plaque, 145 Helicobacter pylori, 145 urinary acidification, 144, 147 urinary tract infection, 145, 146 regulatory status, 147 safety studies, 146 Crataegus. See Hawthorn. Creatine, 151-158 adverse effects, 157 biochemistry and function, 151-153 biosynthesis, 151-153 chemical synthesis, 152 creatine kinase reaction, 152 structure, 151, 152

deficiency and supplementation, 151 deficiency syndromes, 153-156 AGAT deficiency, 155 GAMT deficiency, 153-155 significance of, 155, 156 SLC6A8

deficiency, 155

dosage, 151 physiology, 153 accumulation, 153 clearance, 153 tissue distribution, 153 sources, 156

Index

805

therapeutic use, 156, 157 in biosynthesis disorders, 156 ergogenic aid, 156 in neuromuscular disease, 156, 157

in sports, 156 transporter, 151, 153, 155 Creatine kinase (CK), 151-153 Creatine phosphate, 541 as a phosphorus source, 537-538, 541

DADS, and garlic, 230, 231, 233, 234, 235 Daidzein, 363, 365, 366 Dam, H., and vitamin K, 771

Dang gui, 159-166 biochemistry and function, 159-162 pharmacodynamics, 159 pharmacokinetics, 159 dosages, 163 effects, 162

analgesic, 162 antioxidant, 161 on bone cells, 162 cardiovascular,

drug interactions, 173 sources and preparations, 173 therapeutic applications, 170-172 adrenal insufficiency, 170 adrenopause and age-related disorders, 170-171 potential benefits, 171-172 Depressant, valerian and, 691 Depression, SAMe and, 3, 4 DHEA. See Dehydroepiandrosterone. Diallyl disulfide. See DADS. Diallyl sulfide. See DAS. Diallyl trisulfide. See DATS. Diatoms, and EFA, 505

Dihomo-y-linolenic acid (DHLA), 494 Dihydrotestosterone (DHT), 571, 572 Docosahexaenoic acid (DHA) and omega-3 fatty acids, 493, 494, 495, 496, 497, 498, 499 and omega-6 fatty acids, 505, 507, 509, 512 Docosapentaenoic acid (DPA) * and omega-3 fatty acids, 493, 497 Dolichol synthesis, and coenzyme Qj, 121, 123, 129 Doping, and anabolic steroids, 7, 11 Du Vigneaud, V., experiments relating to SAMe, 1

159

on driving, 164 gynecological, 161 hematopoietic, 161 hemorrheological, 159 hepatoprotective, 160 hormonal, 161 immunomodulatory, 161 menopausal, 161 on smooth muscle, 161 wound-healing, 162

Echinacea, 177-187

active principles, effects, and standardization, 178-183 adverse effects, 184-185 alkamides, 181-183 caffeic acid derivatives, 180-181

clinical efficacy, 183-184 clinical particulars, 184-185 contraindications, 184-185 dosage, 184 immunomodulatory activities, 183 polysaccharides and glycoproteins, 179-180 toxicological considerations, 185 historical use, 177 preparations, 177 regulatory status, 177

medical indications, 162

regulatory status, 164 safety profile, 163 carcinogenicity, 164 contraindications, 163 interactions, 163 lactation, use in, 164 mutagenicity, 164 overdose, 164 precautions, 163 pregnancy, use in, 164

taxonomy

side effects of, 163

toxicology, 164 DAS, and garlic, 231, 233, 234, 235 DATS, and garlic, 234, 235 Dehydroepiandrosterone, 167-176

biochemistry and physiology, 167-169 adrenarche and adrenopause,

169

biosynthesis and metabolism, 167—168 mechanisms of action, 169 production, regulation of, 168

compendial/regulatory status, 173 and cortisol, 168

history, 167 pharmacokinetics, 169-170

and upper 7h respiratory tract infections, 177, 184 Echinilin (echinacea), 184 @3 EFA. See Omega-3 fatty acids. w6 EFA. See Omega-6 fatty acids. Efacal® (evening primrose), 203 Efamast~ (evening primrose), 197 Efamol® (evening primrose), 204 Eicosapentaenoic acid (EPA) and omega-3 fatty acids, 493, 494, 495, 496, 497, 498, 499 and omega-6 fatty acids, 505, 507, 513 End-stage renal disease. See ESRD. Endurance sports androstenedione and, 7 creatine and, 156

Energy storage, role of phosp horus, 539, 540, 541 Ephedra, 189-195

adverse reactions, 173 contraindications, 173

action, 190 on asthma, 190 on heart, 190 adverse events, 192 clinical trials, 191 constituents, 190 alkaloids, 190, 191, 192 contraindications, 193

dosage and administration, 173

description, botanical, 189

absorption and tissue distribution, 169 bioavailability, metabolism, and clearance, 169-170 supplementation, 170 pharmacology and toxicology, 172-173

Index

806

Ephedra (contd.) drug interactions, 193 efficacy, 191 for athletic performance, 192 for weight loss, 191 historical use, 189-190 Nagai, N., and, 189 pharmacokinetics, 191 preclinical studies, 191 toxicology, 192 in vitro, 192 in vivo, 192 Ephedra spp., 189 Epidermal growth factor (EGF), 571, 573 EPO. See Evening primrose. Epogam™® (evening primrose), 197, 199 Epogen® (iron), 361 Ergotamine, 213 Eritadenine, 658, 660 ESRD, and calcium, 66, 69, 70

Essential fatty acids (EFA), 505, 507, 509, 511 Estradiol, 7, 9, 10. See also Estrogens. Estrogens, 7, 9, 10, 61, 169 Evening primrose, 197-210

and atopic dermatitis, 199-200

flavonoids, 213 parthenolide, 211, 213, 215 sesquiterpene lactone (STL), 211, 213, 215 clinical studies, 214-215 mechanism of action, 215 safety, 215 adverse effects, 215 anti-platelet aggregation, 216 contraindications, 215 drug interactions, 215 toxicology, 215 Flavin adenine dinucleotide. See FAD. Flavin mononucleotide. See FMN. Flavonoids, 363. See also Isoflavones.

and quercetin, 577, 578, 579, 581 in saw palmetto, 638 Flavonol, and quercetin, 577 FloraGlo® (lutein), 418 FMN, 623, 624, 625, 626, 630 Folate, 219-228 active forms, 219, 220, 221 biochemistry and functions, 219-221 methionine synthesis, 221, 224 nucleotide synthesis, 220-221 serine—glycine interconversion, 220

and cancer, 202—203

and choline, 105, 107, 109, 110

and cardiovascular disease, 201-202 antioxidant, 201 description, botanical, 197 and diabetes mellitus-induced neuropathy, 205-206 essential fatty acids, 197 and inflammatory diseases, 198-199 omega-3 fatty acids, 197 omega-6 fatty acids, 197 eicosanoids, 198, 201 metabolic functions, 197

coenzymes. See active forms. compendial/regulatory status, 226 contraindications, 226 deficiency, 222-224 cancer, 223-224 megaloblastic anemia, 222-223 neural tube defects, 223 vascular disease, 223 genetic diseases involving, 223 homocysteine and, 221, 223, 224, 225 indications and usage, 224-226 alcoholism, 225 cancer prevention, 225-226 deficiency, treatment of, 224 drugs, adverse effects on folate status, 225 hyperhomocysteinemia, 223, 225 neural tube defects, prevention of, 224-225 pregnancy, 223, 224 renal conditions, 225 supplementation, 224 one-carbon metabolism, 219, 220, 222 physiology, 221-222

and rheumatoid arthritis, 200-201 and women’s health, 203-205

hot flashes, use in, 203 mastalgia, use in, 204 PMS, use in, 204

pregnancy, use in, 204 Evening primrose oil. See Evening primrose.

@3 FA. See Omega-3 fatty acids. FAD, 623, 624, 625, 626, 627, 629 Farnesyl pyrophosphate, coenzyme Qj 9 precursor, 22, WA I AAG ARS eS FastOne™ (proanthocyanidins), 563 Fatty acids (FA), 493, 495, 498, 499 B-oxidation, CoA in, 521 synthesis, ACP and CoA in, 520 transesterification of, 73, 74, 77 Feminization, androstenedione and, 10, 11

Ferritin, 357, 358, 360 Feverfew, 211-217 ancient uses, 212 emmenagogue,

use as, 212, 215

description, botanical, 211 modern uses arthritis, use in, 212, 214-215 migraine, use in, 212, 214 psoriasis, use in, 212

chemical constituents, 213 essential oil, 213

absorption, 221 bioavailability, 221-222 excretion, 222 tissue retention and turnover, 222

transport, 221-222 precautions and adverse reactions, 226 drug interactions, 226 overdosage, 226 recommended intake, 224 structure, 219 and vitamin Bj>, 221, 222, 223, 224, 225,

226, 737-738 Folic acid. See Folate. Follicle-stimulating hormone (FSH) levels, 595 Foudaril® (Pygeum africanum extract), 574 Fragile histidine triad (FHIT) gene, 107 French maritime pine bark extract. See Pycnogenol”. Funk, C., thiamin and, 677

Index

GAGs chondroitin sulfate, 113, 114, 115, 116, 117 glucosamine and, 279, 283 GAIT, 115, 116 Ganoderma lucidum. See Reishi.

Ganopoly®, reishi and, 607, 609

Ginseng, American, 259-264. See also Ginseng, Asian. adaptogenic properties, and Asian ginseng, 259, 260 chemistry, 259 P dammarane saponins, 259 ginsenosides, 259, 260, 261

phytosterols, 260

Garlic, 229-240

description, botanical, 259

adverse effects of, 229 chemistry, 229-230 allicin, 230, 231, 232, 235 alliin, 230, 231 allyl sulfur compounds, 230, 231, 232, 233, 234, 235, 236 health promotion, 231-236 antimicrobial effects, 231 apoptosis, 235 bioactivation and response to carcinogens, 234 cancer prevention, 231, 233-235 cell differentiation, 235 cell proliferation, 235 cardiovascular effects, 231 detoxification and specificity, 234-235 dietary modifiers and efficacy of, 236 nitrosamine formation and metabolism, 233 usage, 229 Genistein, 363, 364, 365, 366, 367 Gerard, J., and feverfew, 212 Ginger, 241-248 adverse effects, 245, 246 antiemetic activity, 241-244 chemotherapy-induced nausea and vomiting, 243-244 hyperemesis gravidarum (morning sickness), 242-243 motion sickness, 242 postoperative nausea and vomiting, 244 anti-inflammatory activity, 244-245 osteoarthritis, 244-245 cardiovascular effects, 245 constituents, 241 gingerols, 241, 242, 244, 245 contraindications, 246 description, 241 dose, 245-246 gastrointestinal effects, 245 -herb—drug interactions, 246 pharmacodynamics, 241-245 pharmacokinetics, 241 pregnancy, use in, 242, 246 toxicity, 246 Ginkgo biloba, 249-257 chemistry, 250-252 bilobalide, 249, 251, 254 flavonoids, 249, 250, 251, 253 formulation and analysis, 252 ginkgolides, 249, 251, 253, 255 triterpene lactones, 250 clinical studies, 254-256 on adverse effects, 255 on cerebral insufficiency, 254

effects, 260-261 on cancer, 261 on immune system, 261 on memory, 260

on memory, 254, 255

on toxicity, 255 cultivation and history of use, 250 description, botanical, 249 extract, 250 preclinical studies, 252-254 antioxidant activity, 252 gene expression, 252 protein function, 252 regulatory status, 256

on metabolism, 260 on stress and fatigue, 260-261

interactions, 261-262 with diseases or conditions, 262 with drugs, 261 with food, 262

nutritional and medicinal values, 260-261 antioxidant activity, 260

preparation of products, 259 quality control, 262 regulatory status, 262 safety, 261

Ginseng, Asian, 265-277. See also Ginseng, American. chemistry and preparation of products, 266-267 active constituents, 266 formulation and analysis, 266-267 ginsenosides, 265, 266, 268, 270, 272 source materials, 266

clinical studies, 271-274 aqueous extract, 273 immunomodulation, 273 performance, 271-273 standardized extract, 273 contraindications, 274

indications, 274 from clinical data, 274 in folk medicine, 274 in pharmacopoeias and traditional medicine, 274 interactions and side effects, 274-275 lactation, use in, 275 pregnancy, use in, 275 overdose, 275 preclinical studies, 267-271 CNS, action in, 268-269 embryo, fetal, and perinatal toxicity, 271 free radical scavenging effects, 267-268 genotoxicity, 271 immunological effects, 268 metabolic effects, 269-270 pharmacodynamic properties, 267 pharmacokinetics and metabolism, 270-271 preclinical safety data, 271 repeated dose toxicity, 271 reproduction toxicity, 271 single dose toxicity, 271 Ginsenosides in American ginseng, 259, 260, 261 in Asian ginseng, 265, 266, 268, 270, 272 Gitelman’s syndrome, 451

Glucosamine, 279-286 chemistry and physiology, 279, 280 proteoglycans, 279 structure, 279 transporter, 280 chitin (precursor), 281, 282, 284 and chondroitin, 113, 114, 115, 116, 117

Index

808

Glucosamine (contd.) clinical trials, 283-284 disease modification, 284 dose, 283, 284 glycosaminoglycans (GAGs), 279, 283 and osteoarthritis (OA), 279-284 cartilage, 279, 280, 283, 284 osteoarthritis therapy, use in, 280-281

pharmacology and pharmacokinetics, 280 preparations, 281 cocrystals and coprecipitates, 282 comparison, 283 glucosamine hydrochloride, 282 glucosamine sulfate, 281 glucosamine with sulfate, 281-282 physical mixtures, 282-283 safety, 284 adverse effects, 281, 283 Glucosamine/chondroitin Arthritis Intervention Trial. See GAIT. Glutamine, 287-296 biochemistry and functions, 287—288 biological synthesis and utilization, 287-288 metabolic functions, 288 catabolic disease, 290-291 catabolic states, 290-291

interorgan transport, 291 description, general, 287 glutamate dehydrogenase (GDH), 288, 290 glutaminase (GA), 288, 290 hepatic glutaminase (hGA), 288 kidney glutaminase (kGA), 288 glutaminolysis, 288 nutritional supplementation, 291-293 athletic performance enhancement, 292 immune system enhancement, 293 intestinal renewal and function, 292 sickle cell disease, 292 total parenteral nutrition (TPN), 291-292 in very-low-birth-weight infants, 292 physiology, 288-289 cell proliferation, 288-289 cellular functions, 288 synthesis, 288, 290, 291 systemic metabolism, 289-290 acidosis, control of, 290 cycling in brain and liver, 289-290 production by muscle and lung, 290 Glutamine synthetase (GS), 287, 289, 290 Glycyrrhiza glabra. See Licorice. Glycones, and quercetin, 577, 578 Glycosaminoglycans. See GAGs. Goldenseal, 297-308 berberine, 297, 298, 302, 303, 304, 305 chemistry, 299 alkaloids, 299 formulation and analysis, 299 clinical studies on berberine, 302 cardiovascular and circulatory disorders, 302-303 cirrhosis, 303 diarrhea, 303 digestive, hepatic, and gastrointestinal disorders, 303

parasitic disease, 303 contraindications, 303 description, 297

yellow root, 297 dosage and toxicity, 303

drug interactions, 304 history and traditional uses, 298-299 immunostimulatory effect, 298

B-hydrastine, 297, 299, 300, 302 precautions, 304-305 preclinical studies on berberine, 300-302

adrenal functions, 301 antiarrhythmic effects, 301 antiproliferative activity, 300 cancer, 300 cardiotonicity and cardioprotection, 301 cardiovascular and circulatory functions, and berberine, 301

chemopreventive activity, 300 chemotherapy adjunct activity, 300 congestive heart failure, 301 connective, tissue functions, 302 cytotoxicity, 300 diarrhea, 301 digestive, hepatic, and gastrointestinal functions, 301 endocrine and hormonal functions, 301 fungal infections, 301 hepatic functions, 301 hypertension, 301 immunology, 301 infectious diseases, 301-302 integumentary, muscular, and skeletal functions, 302 microbial infections, 301 osteoporosis, 302 parasitic infections, 302 receptor and neurotransmitter mediated functions, 302 respiratory and pulmonary functions, 302 preclinical studies on goldenseal and berberine, 299-300 antimicrobial and antifungal activity, 300 cardiovascular and circulatory functions, 299 immune functions, 299-300

respiratory and pulmonary functions, 300 pregnancy and lactation, use in, 304 toxicity, 304 Grape seed extract, 309-325 angiotensin, 316 anthocyanidin, 309, 314 catechin, 309, 310, 311, 312, 314, 315, 317, 318, 319 chemical composition, 309-311 endothelial cells and, 313, 316, 317 endothelium-dependent vasorelaxation (EDR), 313 epicatechin, 310, 313, 314, 316, 318

mechanisms of action, 311-313 anti-inflammatory, 312-313 antioxidant, 311-312 immunostimulatory, antiviral, and anticancer, 313 vasodilation, 313 oligomeric proanthocyanidins (OPC), 310, 314, 318 pharmacokinetics, 314 animal and human studies, 314 pharmacology, 314-318 anticarcinogenic and anticancer effects, 314 energy metabolism, effects on, 318 hair growth effects, 316 hepatoprotective and tissue protective effects, 316 inflammation and allergy protective effects, 316 metabolic and nutritional effects, 315-316 obesity management, 318 postoperative edema effects, 317 premenstrual syndrome effects, 316 renal and cardioprotective effects, 315-316 skin and wound healing effects, 317

Index

809

vascular effects, 317 visual effects, 317 procyanidin, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319 regulatory issues of, 318 usage, 318 Green tea polyphenols, 327-336 biotransformation and bioavailability, 331-333 enzymology, 332 pharmacokinetics, 332 cancer, effect on, 329 animal studies, 329

antioxidant/pro-oxidant activity, 320 epidemiological studies, 329 inhibitory activity, 330-331 mechanisms of prevention, 329-330 mechanisms, other, 331 cardiovascular diseases, effect on, 328 animal studies, 328 human studies, 328 catechins; 327;,3305331, 332;.333 epigallocatechin-3-gallate (EGCG), 327, 329, 330, 331, 332,, 333 diabetes, effect on, 329 neurodegenerative disorders, effect on, 328-329 prevention of diseases, 327, 329 GSE. See Grape seed extract. Gyorgy, P., and vitamin Be, 715

Haber—Weiss—Fenton reaction, 357 Hallervorden—-Spatz syndrome, 524 Haptocorrin, 736, 737 Hawthorn, 337-347 action and pharmacology, 338-341 antiarrhythmic effects, 339 cardioprotective effects, 340 coronary flow, increase of, 339 pharmacological activities, other, 340-341 pharmacokinetics, 341 positive inotropic action, 339 vasorelaxing effects, 339 adverse reactions, 342 drug interactions, 342 effects, 342 clinical studies, 339, 341, 343-344 compendial/regulatory status, 345 contraindications, 342 general description, 337-338 fruits (berries), 337, 338 leaves and flowers, 337, 338 plant, 337 preparations, 338 starting material, 337

starting material constituents, 337 indications and usage, 341-342 in heart failure, 341, 342

overdosage, 345 precautions, 342

toxicology, 341 HDL. See Lipoproteins. Hereditary hemochromatosis, 359 Highly unsaturated fatty acids (HUFAs) and omega-3 fatty acids, 493, 494 and omega-6 fatty acids, 505, 507, 508, 509 Hill, J., and feverfew, 212

Hirase, S., and ginkgo, 249 His, W., experiments relating to SAMe, | HMG-COA reductase, and coenzyme Qjo, 121, 122, 127, 129 *Hormone replacement therapy (HRT), and isoflavones, 366, 368

Host defense potentiator (HDP), and shiitake, 657, 659 Hova® (valerian), 693 5-HTP. See 5-Hydroxytryptophan. Hydrastis canadensis. See Goldenseal. 5-Hydroxytryptophan, 349-355. See also Serotonin. adverse side effects of, 353 clinical use of, 350-353

depression, 350-352 fibromyalgia, 352 headaches, 352 neurological disorders, 352-353 obesity, 350 sleep aid, 352

eosinophilia—myalgia syndrome, 349, 353 pharmacokinetics and metabolism, 349-350 regulatory issues of, 354 Hypericum perforatum. See St. John’s wort. Hyperphosphatemia, 539

IdB 1016" (milk thistle), 476 Idebenone, coenzyme Qj analog, 129 Insulin, 60 Insulinlike growth factor (IGF), 571 Intrinsic factor (IF), 736 Tron, 357-362 acute effects of, 358-359 bioavailability, 358 compendial/regulatory status, 362 dietary forms, 358 heme, 358 iron response element (IRE), 358 nonheme, 358, 359 regulation of absorption, 358 transport proteins, 358 doses, 358, 359, 361 hemoglobin, and iron, 358, 360 indications and usage, 360-361 contraindications, 361 deficiency, treatment of, 360, 361 with drugs affecting iron status, 361 recommended intakes, 360 supplementation, 360, 362 myoglobin, and iron, 358 overload, 359-360 Bantu siderosis, 360 hereditary hemochromatosis, 359 transfusion, 360 precautions and adverse reactions, 361-362 and riboflavin, 626, 628 and vitamin A, 711 and vitamin C, 747, 754 Isoflavones, 363-372 aglycones in, 363, 365 Asian intake, 365 chronic disease prevention and treatment, 366-368 cancer, 366-367 cognitive function, 367-368 coronary heart disease, 367 menopausal symptoms, 367 osteoporosis, 367

Index

810

Isoflavones (contd.) content in soy, 363-365

controversies in use, 368-369 breast cancer patients, 368 infants, 368 reproduction, 368 thyroid function, 368 B-glycosides in, 363, 364 and hormone replacement therapy (HRT), 366, 368 metabolism, 365 physiological properties, 365-366 antioxidant activity, 366 estrogenlike activity, 365, 367, 368 signal transduction, 366 recommended intakes, 369 and Rhizobium, 363

safety, 369 Isoprostanes, 511

Jarsin® (St. John’s wort), 666 Jet lag, and melatonin, 463

Kava, 373-380 awa, 373 chemistry, 374-376 kavain, 374, 376, 379

methysticin, 374, 376 clinical studies, 377-379 on anxiolytic quality, 377 on metabolism, 376, 378 Cuzent, research on, 374 description, botanical, 373

Forester, J.G., description of, 374 Gobley, research on, 374 historical background, 373 preclinical studies, 376-377

on anxiolytic quality, 376 on metabolism, 376, 377 toxicity, 378

Keratinocyte growth factor (KGF), 571 testosterone, 572 Keratomalacia, and vitamin A, 703 Keshan disease, 645, 649

Kunzle® (Pygeum africanum extract), 574

Lactobacilli, 381-389 action and pharmacology, 383 host defense, mechanisms of, 383-384 vaccine development, implications, 384 adverse effects, 386-387 bioavailability, 382-383 proprietary and probiotic strains, 382-383 description, general, 381 drug interactions/safety, 387 regulatory status, 387-388 indications and usage, 385-386 dose-range effects, 386 labeling, 385 oral vaccines, 384, 388

preparation, 385 Lactobacillus acidophilus. See Lactobacilli.

Lactobacillus casei. See Lactobacilli. Lacto-lycopene— (lycopene), 421 LAP. See Shiitake. Lapacho. See Pau d’arco. L-Arginine, 15-24 absorption, 15

adverse effects, 20 and aging, 17 biochemistry, 15-17 arginine paradox, 19 endogenous production, 16 and carcinogenesis, 20

deficiency, 15-16 endocrine actions, 17-18 ACTH secretion, 18 growth hormone (GH) secretion, 17-18 insulin secretion, 18 prolactin (PRL) secretion, 18 metabolic pathways, 16-17, 19 and nitric oxide, 15, 17, 18, 19, 20 nonendocrine actions, 18-20 on cardiovascular system, 18-19 on immune system, 19-20 ornithine, active form, 17, 20 physiology, 17—20 and somatostatin release inhibition, 17 sources, 15 toxicity, 16 L-Carnitine. See Carnitine. LDL. See Lipoproteins. Lecithin, and choline, 106, 107, 108, 109 Legalon® (milk thistle), 470, 476 Lentinacin. See Eritadenine. Lentinan, 656, 657, 658, 659, 660, 661, 662. See also Shiitake. Lentinus edodes. See Shiitake. Lentinus edodes mycelium (LEM). See Shiitake. Lentysine. See Eritadenine. Lepidium meyenii. See Maca. Lerner, A., discoverer of melatonin, 460 L-Glutamine. See Glutamine. Licorice, 391-399 chemistry, 393

flavonoids, 391, 394 glycyrrhizin, 391, 393, 394 triterpene saponins, 391 description, botanical, 391 historical uses, 392 efficacy, 396 modern uses, 393 as flavoring agent, 393 in food industry, 393 in medicine, 393 pharmacokinetics, 396 preclinical and clinical studies, 394-396 activity on sex hormones, 394 anti-Helicobacter pylori activity, 395 anti-inflammatory activity, 395 antimicrobial activity, 395 antitumoral activity, 395 antiulcer activity, 395 antiviral effects, 396 effect on oxidative stress, 396 glucocorticoid and antiglucocorticoid activity, 394 mineralocorticoid activity, 394 preparation of products, 393 analysis, 394 formulations, 393

Index

811

side effects, 397 drug interactions, 397 Ligustrum, 25

Ligustrum lucidum. See Ligustrum. Lind, J., and scurvy, 751

Ling zhi. See Reishi. Linoleic acid (LA), 505, 506, 507, 509, 512, 513 a-Linolenic acid (ALA), 493, 505, 506, 507, 513 a-Lipoic acid, 401-407 antioxidant properties of, 401 chemistry, 401 lipoate, 401, 402, 403, 404 dihydrolipoate, 401, 402 metabolism and dynamics, 401-402 and reactive oxygen species (ROS), 402, 403, 404 supplement uses, 402 glutathione, 402 vitamin C, 402 vitamin E, 402

synthesis, 401, 403 therapeutic uses, 403

burning mouth syndrome, 404 cancer, 404 hepatitis C, 404 liver disease, 404 mitochondrial enzyme defects, 404 smell dysfunction, 404 LipoKinetix® (yohimbe), 787 Lipoproteins and $-carotene, 81, 84 and coenzyme Qj, 124, 126 and garlic, 232, 233 and grape seed extract, 312, 313, 315, 319 and isoflavones, 367 and a-lipoic acid, 403 and lutein, 412, 415, 416 and lycopene, 425 and niacin, 488 and proanthocyanidins, 558 and quercetin, 580 and red clover, 592, 598 and vitamin C, 746 and vitamin E, 759, 760 and zinc, 797 Liu Wan-Tai, and ginkgo, 250 Liver disease, SAMe deficiency and, 3, 4 Lower urinary tract symptoms (LUTS), and Pygeum africanum extract, 569, 570, 572 Lutein, 409-420. See also Carotenoids. adverse effects, 418 and age-related macular degeneration, 409, 417 biochemistry and functions, 409-412 cell membrane structure, 410 interactions with vitamins, 412 and oxygen partial pressure, 412 redox balance, 411 short wavelength light filtering, 410-411 signal transduction, 412 site of action, 411-412

biophysics, 409-412 and cataract, 409, 417 compendial/regulatory status, 418 indications and usage, 416-418 benefits, 417-418

concentration vs. action, 417 sources, 416-417 photosynthesis, 409

physiology, 412-416 bioavailability, 414-416 bioavailability, factors affecting, 416 macular response, 414 plasma concentration, 413-414 potential variable factors, 416

serum response, 414

tissue distribution, 412-413 reference intakes, 418 and retinal |iinjury, 410 Lyc-O-Mato™ (lycopene), 421, 428 - Lycopene, 421-434 activity, whole food vs. supplements, 430 analysis and characterization, 423-424 bioavailability, 425-426 chemistry and biochemistry, 421-423 chronic disease, role in, 426-427 antioxidant activity, 426, 427 cancer, 427-429 cardiovascular disease, 429 distribution, in plasma and tissue, 426, 427 safety and adverse effects, 429 lycopenemia, 429 sources, dietary and supplement, 421, 422 stability and isomerization, 424-425 Lycopenemia, 429 LycoVit® (lycopene), 421

Ma huang. See Ephedra. Maca, 435-443 biological studies, 439-441 aphrodisiac activity in, 439 cytotoxic and chemopreventive activities in, 440-441 fertility-enhancing activity in, 439 activities, other, 441 chemical studies, 436-439 alkaloids, 437 essential oils, 437

glucosinolates, 437 macamides, 438 nutritional constituents, 436 secondary metabolites, 436-439 classification, 435-436

commercial preparations, 441 cosmetics, use in, 441 cultivation, 436

standardization of products, 437, 441 traditional and medicinal uses, 435, 436 AIDS, 436 anticancer, 436 fertility, 436 Macular pigment, 409, 703. See also Lutein, Vitamin A. Magnesium, 445-455 biochemistry and physiology, 445-446 enzyme interactions, 445 ion channels, 445-446 nucleic acids and membranes, structure of, 445 composition of, 446 deficiency, causes of, 450-451 diabetes mellitus, 451 gastrointestinal disorders, 450 renal disorders, 450-451 deficiency, manifestations of, 451-454 chronic latent, 453-454 moderate to severe, 451-453

Index

812

Magnesium (contd.) deficiency, prevalence of, 450 depletion, management of, 454 homeostasis, cellular, 446 homeostasis, in body, 446-448 absorption, intestinal, 446-447 bioavailability, 447 renal regulation, 447-448 sources and intake, 446 tissue sources, 448 requirements, 448-449 assessment of, 448 recommended intake, 449

status, assessment of, 449-450 analytic procedures, 449 tests, 449-450

Megaloblastic anemia and folate, 222—223

and vitamin Bj>, 737, 741 Melatonin, 457-466

adverse effects of, 464 circadian rhythms in production of, 458, 459 effects of, 461-464 antiaging action, 464 antihypertensive action, 464 as antioxidant, 463-464

circadian rhythms, phase-shifting of, 463 sleep promotion, 458, 459, 461-463 history, 457-458 Lerner, A., 460

metabolism, 458-459 pineal gland, in production of, 457, 460 plasma levels, 459-461 receptors, 458-459

regulatory status, 458, 464 response to light, 457, 458, 459, 460 and serotonin, 458 synthesis, 458

tryptophan (precursor), 458, 459 usage, 464 Menopausal symptoms. See Climacteric symptoms. Menopause, black cohosh and, 42

Metallochaperones, copper and, 133 Methylcrotonyl-CoA carboxylase, biotin and, 32 Milk alkali syndrome, 70 Milk thistle, 467-482 absorption and transport, 468-470 action and pharmacology, 470-474 anticancer effects, 471 antilipidemic effects, 471 antioxidant activity, 470 liver cells, effect of, 470-471 chemistry, 468 flavonolignans, 467, 468, 476, 477 silibinin, 468, 469, 471, 476 silymarin, 467, 468, 470, 471, 474, 477

contraindications/adverse effects, 477 current use, 468

dosages/forms, 476 drug interactions, 477 historical uses, 467

indications and usage, 474-476 alcoholic or virus-induced hepatitis, 474 cancer, 474 cirrhosis, 474 lipidemia, 474 liver disorders, 474

primary biliary cirrhosis, 474 subacute or acute liver disease, 474 overdosage, 477 regulatory status, 477 Minner, H., and pine bark, 546 Muscle mass, androstenedione and, 7, 10 Muscle protein breakdown, and glutamine, 291, 292, 293

Nagai, N., and ephedra, 189 Neo Urgenin® (Pygeum africanum extract), 574 Niacin, 483-491 adverse reactions, 488 flushing, 488 liver toxicity, 489 contraindications, 488 deficiency, treatment of, 487 Hartnup’s syndrome, effect in, 487 description, general, 483 dietary sources, 483 drugs, adverse effects of, 488 genomic stability, 486, 489 indications and usage, 486

recommended dietary allowance (RDA), 486 mono-ADP-ribosylation, 485 NAD-consuming enzymes, 486 and nicotinamide, 484, 486, 487

oxidant-induced cell injury, prevention of, 488 pharmacology, 483 precautions, 488 Sir2-like protein, 485, 486 Night blindness, and vitamin A, 703, 707 Non-insulin-dependent diabetes mellitus (NIDDM), 170 Nonsteroidal anti-inflammatory drugs. See NSAIDs. Norandrostenedione,

7

Normobrost® (Pygeum africanum extract), 574 Normoprost® (Pygeum africanum extract), 574 Novasoy™ (isoflavones), 364 NSAIDs chondroitin and, 116, 117 feverfew and, 215

Oenothera biennis. See Evening primrose. Omega-3 fatty acids, 493-504. See also Omega-6 fatty acids. arachidonic acid (AA), 494 biochemistry and function, 493-495 efficiency of conversion, 493 functions, 493 membrane ion channels, 495 synthesis, 493 tissue distribution, 493 dihomo-y-linolenic acid (DHLA), 494 and evening primrose, 197 food sources, 495, 496 a-linolenic acid (ALA), 493 potential health benefits, 496-499 asthma, 498 cancer, 498-499 coronary heart disease (CHD), 496-498 diabetes, 498 diabetes, arthritis, and inflammatory diseases, 498 human development, 499 neurological and psychological conditions, 499

Index

813

recommended intakes, 499

safety, 495-496 Omega-6 fatty acids, 505-515. See also Omega-3 fatty acids. arachidonic acid (AA), 505, 506, 507, 509, 511, 512, 513 biochemical selectivity, 512 biological actions, 512 dietary sources, 505—507 and evening primrose, 197 health consequences; 513 linoleic acid (LA), 505, 506, 507, 509, 512, 513 a-linolenic acid (ALA), 505, 506, 507, 513

biosynthesis of membrane phospholipids, 509 distribution of, 507 in membrane phospholipids, 507-509 metabolism of, 507-509 pathologies, 507 production by plants, 512-513 structures and biosynthesis of, 505 transformation of, into eicosanoids, 509-512

biosynthesis and actions of prostaglandins, 509-511 biosynthesis of leukotrienes, 511-512 P450 hydroxylase pathways, 512 related hydroxy fatty acids, 511-512 Osteoarthritis chondroitin and, 113-117 ginger and, 244-245 glucosamine and, 279-284 SAMe

and, 4

Osteoporosis calcium and, 66, 69, 70 copper and, 136 isoflavones and, 367 red clover and, 596, 597 vitamin K and, 771, 778

Panax ginseng. See Ginseng, Asian. Panax quinque folium. See Ginseng, American. Pantothenic acid (vitamin Bs), 517-525 bioavailability, 523 cofactor degradation, 518-519 cofactor synthesis, 517-518 acyl carrier protein (ACP) synthesis, 518 coenzyme A, cellular location, 518 coenzyme A synthesis, 517, 518 deficiency, 523 food sources, 523 inherited disorder of, 524 metabolic role, 519-522 carnitine, interrelations with, 522

fatty acid B-oxidation, CoA in, 521 fatty acid synthesis, ACP and CoA in, 520 ketone body metabolism, CoA in, 521 organic acid metabolism, CoA in, 522 oxidative decarboxylation, CoA in, 520-521

microbial synthesis, 517 physiology, 522-523 absorption, 522 excretion, 522-523 transport, 522 recommended intakes, 523 structure, 517, 518

supplementation, 523-524 Parathyroid hormone. See PTH. Pau d’arco, 527-535 antifungal use, 529

botanical products, 529 chemistry, 528-529 naphthaquinones, 527, 528, 530, 531, 532 clinical trials, 531-532 anticancer efficacy, 531

anticervicitis/antivaginitis, 532 description, botanical, 527 historical uses, 527 preclinical studies, in vitro, 530-531 antibacterial activity, 530

anticancer activity, 530 antifungal activity, 530 antimalarial activity, 530 antioxidant activity, 530 antipsoriatic activity, 530 antitrypanosomal activity, 531 antiviral activity, 530 neurodegenerative effects, 531 snakebites, 531 preclinical studies, in vivo, 531 anticancer activity, 531 antischistosomiasis activity, 531 cancer chemopreventive action, 531

toxicity, 531 regulatory status, 532 Pausinystalia johimbe. See Yohimbe. Pellagra, niacin and, 483, 487, 488 Pernicious anemia. See Megaloblastic anemia. Phosphates. See Phosphorus. Phosphatidylcholine. See Lecithin. Phosphatidylethanolamine N-methyltransferase (PEMT),

106, 107 Phosphocreatine (PCr), 151-153 Phosphorus, 537-543 absorption, intestinal, 538 magnesium in, 447 additives, 537, 538 blood concentration, 538 and calcium, 538, 539, 540, 541, 542

compendial/regulatory status, 542 contraindications, 541 dietary reference intake (DRI), 538 dietary sources, 537 forms, 537 homeostasis, 538-539 parathyroid hormone (PTH) in, 538, 539 indications and usage, 540-541 deficiency, 540-541 overdosage, 542 upper limit (UL), 542 precautions and adverse reactions, 541 roles of, 539-540 bone mineralization, 540 phosphorylation reactions, 540 structural functions, 539, 540 supplements, 538 urinary excretion, 539 and vitamin D, 538, 540 Phytates, copper and, 134 Pinus pinaster. See French maritime pine. Piper methysticum. See Kava. PMS calcium and, 66 Chasteberry and, 96-97 evening primrose and, 204 grape seed extract and, 316 vitamin Be and, 724-725

Index

814

Polyunsaturated fatty acids (PUFA), 505, 509, 511 Potenzia® (Pygeum africanum extract), 574 Prasterone. See DHEA. Pregnenolone, DHEA precursor, 167 Prestara (DHEA), 167, 172 Premenstrual syndrome. See PMS. Prenyltransferases, and coenzyme Qjo, 123, 127, 128, 129 Proanthocyanidins, 555-568

adverse biological effects, 563-564 biochemistry, 555 biological markers, alteration of, 557-561 animal models and human studies, 559-560 antioxidants, 557 atherosclerosis, 558

bacterial antiadhesion, 561 cancer, 557 cytotoxicity, 557 diabetes, and glucose and insulin metabolism, 561

free radicals, 557 immune function, 561

inflammatory response, inhibition of, 558-559 liposome and LDL oxidation, inhibition of, 558 mechanisms of action, 558 NO-dependent vasodilation, 560 platelet aggregation, decreased in, 559 reperfusion, 561

vasoconstriction, 560 compendial/regulatory status, 563 description, general, 555

dietary sources and intake, 561-563 foods, 561, 563 supplements, 562, 563 and grape seed extract, 309, 314, 315 physiology, 555-557 and Pycnogenol”, 559, 562 Probiotics, 382, 383, 386. See also Lactobacilli. action, 383 bifidobacteria, 382, 386 dietary supplement, 385 lactobacilli, 386 modulation, 384 strains, 382, 384, 385, 386

therapy, 384 Pro Flow® (Pygeum africanum extract), 574 Progesterone receptor (PR), 590, 591 Prolitrol® (Pygeum africanum extract), 574 Pronitol® (Pygeum africanum extract), 574 Propionyl-CoA carboxylase, biotin and, 32, 36 Proscar’ (saw palmetto), 639 Prostaglandin endoperoxide H, (PGH)>), 509, 510 Prostaglandin endoperoxide H synthase (PGHS), 509, 510, 512 Proteoglycans, and chondroitin, 113, 114 Provitamin A, 701, 702, 705, 706, 707 Provol® (Pygeum africanum extract), 574 Prunus africana. See Pygeum africanum extract. PTH and calcium, 65, 66 and magnesium, 451-452 Pursh, F., and cascara sagrada, 89

Pycnogenol”, 545-553 chemistry and production, 546 analysis, 546 composition, 546 formulations, 546 phenolic compounds, 546 stability, 546

children, use in, 551

clinical studies, 549-551 antidiabetic effects, 551 antihypertensive effect, 549 asthma symptoms, reduction of, 550

cholesterol lowering, 550 circulation, effect on, 545, 551

deep vein thrombosis, prevention of, 550 edema formation, inhibition of, 550 erectile function improvement, 550 gingival bleeding, 550 immune modulation, 550 menstrual cramps and pain, reduction of, 551 microcirculation improvement, 549 platelet aggregation, inhibition of, 549 sperms, improvement of quality of, 551 UV-induced damage, inhibition of, 551 vascular retinopathy, effects in, 550 contraindications, 551 description, botanical, 545 preclinical studies, 546-549 absorption and metabolism, 549 adhesion molecules, inhibition of, 548 adrenaline-induced vasoconstriction, inhibition of, 547 animal toxicology, 549

antiaging effects, 548 antihypertensive effect, 547

antioxidative effects in biological systems, 548 antioxidative substances, stimulation of synthesis of, 548 blood glucose lowering, 549 capillary sealing, 547 cognitive function, enhancement of, 548 e-NOS, stimulation of, 546 immune system, stimulation of, 548 inflammatory mediators, inhibition of, 548 matrix metalloproteases, inhibition of, 548 NF-«B, interaction with, 548 protein binding, 547 radical-scavenging activity, 545, 547 spasmolytic activity, 549 UV-induced damage, inhibition of, 548 pregnancy, use in, 551 observed drug interactions, 551 optimum intake, 551

regulatory status, 551 side effects, 551 Pygeum africanum extract, 569-575 adverse effects, 573 cell proliferation, 571, 573 clinical studies, 569-570 benign prostatic hyperplasia (BPH), 569-570 mechanism of function, 571-573 bladder function, effects on, 571-573 prostate growth, effects on, 571 pharmaceutical description, 569 products, 573 Pyridoxal, 715 Pyridoxamine, 715 Pyridoxine, 715

Pyruvate carboxylase, biotin and, 32, 36

Quercetin, 577-585 absorption of, 577-578 adverse effects, 581 bioavailability, 578-579

Index

815

biosynthesis and natural sources of, 577 cellular and molecular actions, 579-581 antioxidant properties of, 579 cancer, effects on, 579-580 cardiovascular diseases, effects on, 580 inflammation, effects on, 580-581 effects, other, 581 indications and usage, 581 metabolism, 578 regulatory status, 582

Red clover, 587-602

adverse effects, 598-600 botanical misidentification, 599 clinical trials, reported, 600 coumarins, presence of, 599 cytochrome P450, inhibition of, 599

heavy metal contamination, 598 pesticide residues, 598-599 thyroid function, 599 chemical constituents, 588-589 compounds used for standardization, 589 common names, 587 description, botanical, 587-588 dosage and preparation of, 598 isoflavones in, 363 pharmacokinetics and metabolism, 589 pharmacological activity, 589-591 cancer-related effects, antioxidant, 591-592 cancer-related effects, antiproliferative, 592 glucose and lipid metabolism, 592 hormonal effects, estrogen receptor dependent, 589-591 hormonal effects, other receptor dependent, 591 hormonal effects, receptor independent, 591 inflammation and immune function, 592

biological activities, other, 592-593 thyroid function, 592

regulatory status, 600 safety, 599-600 in cancer patients, 599 in pregnant women and children, 599-600 traditional and medicinal uses, 588 external, 588 internal, 588 usage, 593-598

breast cancer, 595 cardiovascular disease risk, 596-598 caveats, 593, 594 cyclical mastalgia, 595 endometrial effects, 595 menopause, 593-595 osteoporosis prevention and treatment, 596, 597 prostate cancer, 595-596 5-a-Reductase inhibitors

and Pygeum africanum extract, 570, 573 Reishi, 603-622

antibacterial and antiviral effect, 615-618 anti-HIV activity, 616 on Epstein-Barr virus, 616 on Gram-positive and Gram-negative bacteria, 615 on HBV infection, 618 on Helicobacter pylori, 615

chemo- and radiopreventive, 609 PC-SPES, 609 as single agent, 607 cardiovascular and circulatory functions, 615 cholesterol and lipid metabolism, 615 hypertension, 615 complement system, effect of, 613 cultivation, 603-604 description, general, 603

dosage forms, 618 edibility, 603 effect on immune effector cells, 611-613 macrophages, 612, 617 mast cells, 612 natural killer (NK) cells, 612, 617 splenocytes, 611

Tecells oils 617; enzyme-inhibiting activity, 609 habitat, 603 hepatoprotective activity, 613 histamine release inhibition, 613 immunomodulatory effects, 610, 613, 617 mitogenic activity, 610 nutritional components, 604-607 bioactive constituents, 605 nucleotides and nucleosides, 607 polysaccharides, 605, 607, 609, 612, 616 proteins, 605 triterpenes, 605 triterpenoids, 607, 609, 615 related species, 603 safety profile, 618 drug interactions, 618 side effects, 618 therapeutic applications, 607 preclinical and clinical studies, 607 toxicity, 618 traditional uses, 604 Remifemin® (black cohosh), 42, 48, 50 Retinal injury, and lutein, 410 Retinal pigment. See Macular pigment. Retinaldehyde, retinoic acid, retinol. See Vitamin A. Rhamnus purshiana. See Cascara sagrada. Riboflavin, 623-624 adverse effects, 640 antioxidant potential assessment of status, 628-629

biochemistry and functions, 523-524 deficiency, 624

in alcoholism, 625 anemia, 628 carcinogenesis, 628 dietary recommendations, 628-629 recommended dietary allowance (RDA), 629 fat metabolism, 627-628 flavin coenzymes, 623, 624 in HIV infection, 628 homocysteine metabolism, 627 and iron, 626, 628 safety, 630 UV light, sensitivity to, 623-624

Rokan’” (ginkgo), 250

mechanism consideration, 617 on other viruses, 616 antidiabetic effect, 614-615 antitumor effect, 607-609, 612

Sabal serrulata. See Saw palmetto. S-Adenosyl-L-methionine. See SAMe. S-Adenosylmethionine, 105, 108

Index

816

S-Allylcysteine (SAC), 231, 233, 234, 235, 236 S-Allyl mercaptocysteine. See SAMC. SAMC, and garlic, 234, SAMe adverse effects of, 4 Cantoni, G., | deficiency, 3 and depression, 3, 4 discovery of, 1-2 drug/supplement interactions, 5 Du Vigneaud, V., 1 enzymes in synthesis of SAMe, 2-3 and gene expression, 3

hepatic metabolism of, 2

regional variations in, 645, 649 sources, 648-649 supplementation, 650 toxicity, 650 Selenomethionine, 649, 650 Serenoa repens. See Saw palmetto. SERMs and calcium, 69 isoflavones, 365, 366 Serotonin and 5-HTP, 349, 350, 351, 352, 353

5-hydroxyindoleacetic acid (5-HIAA), metabolite of, 350, 351, 353 and melatonin, 458

His, W., 1

Sex-hormone-binding globulin (SHBG)

indications and usage, 3—5 liver disease and, in animals, 3

and DHEA, 170 and red clover, 591, 595, 596 Shiitake, 653-664 artificial cultivation, 655 blood serum cholesterol and, 658 edibility, 653 habitat and distribution, 653 medicinal uses, 660-662 antifungal activity, 661 toxicity and side effects, 661-662 nutritional value, 653-654 carbohydrates, 653 minerals, 654

osteoarthritis and, 4

pharmacokinetics, 4 side effects, 4

synthesis of, 2 Saw palmetto, 635-644 BPH, 638-639 current medical treatment, 638-639 efficacy in control of symptoms, 639 botanical treatments, other, 638 chemistry, 637-638

fatty acids, 637, 640, 642 flavonoids, 638

description, botanical, 635 ecological distribution, 635 historical use, 636-637 pharmacokinetics, 642

pharmacology, 640-642 antiandrogenic activity, 641 antispasmodic activity, 641 estrogenic properties, 641 urological symptoms, 640 primary use, 635-636 regulatory status, 642 safety, 639-640 lactation, use in, 639 pregnancy, use in, 639 side effects, 639 Scurvy, 746, 751 Sedative action, and valerian, 687, 691, 697

Sedonium® (valerian), 696, 697

Selective estrogen receptor modulators. See SERMs. Selenium, 645-652 assessment of status, 649 Berzelius, 645

biochemistry and function, 646-648 antioxidant properties, 647 cancer chemoprevention, 647-648 selenoproteins, and thiol redox reactions, 647

selenoproteins, and thyroid hormone metabolism, 647 selenoproteins, synthesis of, 646 transport, 647 chemical forms of, 646 and coenzyme Qjo, 127 deficiency, 645, 649

Keshan disease, 645, 649 dietary intake, 648-649 dietary reference intake (DRI), 649-650 recommended dietary allowance (RDA), 650 metabolism, 648

polysaccharides, 654, 656, 657, 660 protein, 653, 654, 656, 657, 658, 660, 661 vitamins, 653, 654, 662 preclinical studies, 655-660 AIDS, 657, 659, 661, 662 anticarcinogenic and antitumor effects, 656-657 antiparasitic effects, 659 antiviral and antibacterial effects, 659-660 cardiovascular effects, 658

hepatoprotective effects, 659 HIV, 659, 661 immune-modulating effects, 657 KS-2-a-Mannan peptide, 657 LEM and LAP extracts from, and culture media, 657 therapeutic applications, 655 preparations of, 662 dosage, 662 drug interactions, 662 sarcoma, 180, 656, 657 taxonomic classifications, 655

Siliphos® (milk thistle), 476 Silipide® (milk thistle), 476

Silybum marianum. See Milk thistle. Single nucleotide polymorphisms, 106 Skin photosensitivity, and carotenoids, 84 Sleep promotion melatonin and, 458, 459 valerian and, 688

Sleep-Qik® (valerian), 697 Soy, isoflavones in. See Isoflavones. Squalene synthase, and coenzyme Qj, 123, 127, 128, 129 Statins, and coenzyme Qo, 121, 127, 129, 130 Steroid hormones. See also Anabolic steroids. androstenedione, 7-13 DHEA, 167-176 St. John’s wort, 665-676

chemistry and preparation of products, 665 hyperforin, 666, 667

Index

817

hypericin, 666, 671 clinical studies, 667-673

adverse effects, 669-670 anxiety disorders, 669 depression, 667-669, 671 drug interactions, 671 herb-drug interactions, 665, 673

mutagenicity, 671 photosensitivity, 670 reproduction, 670 current uses, 665 Super Prostate Formula® (Pygeum africanum extract), 574 Symptomatic slow-acting drugs in osteoarthritis (SYSADOA), 115, 280

Trinovin® (red clover), 595 Triterpene glycosides, in black cohosh, 41 Tryptophan, melatonin precursor, 458, 459 TSH, 70

Ubiquinol, ubiquinone. See Coenzyme Q\}po. UDP-glucuronosyltransferases (UGT), 578, 580 UTI. See Urinary tract infection.

Vaccinium macrocarpon. See Cranberry. Valerian, 687-700 chemical constituents, 689 iridoids, 689, 693, 697 monoterpenes, 689

Tabebuia. See Pau d’arco. Tadenan® (Pygeum africanum extract), 574 Tanacetum parthenium. See Feverfew. Tanakan (ginkgo), 250 Tardive dyskinesia, 108

TCM, 25, 28, 159, 189, 250, 392, 604 Tea. See Green tea polyphenols. Tebonin (ginkgo), 250 Tebonin forte (ginkgo), 250 Testosterone, 7, 9, 10, 169, 571, 572

Tetrahydrofolates, 220, 221, 222 Thiamin, 677-686 bioavailability, 681-682 antagonists, 682 lipid-soluble analogs, 682 biochemistry and functions, 677-678 phosphates, 677 coenzymatic function, 677

congenital disorders, 684 Leigh’s disease, 684 maple syrup urine disease, 684 Roger’s syndrome, 684 deficiency, 683 beriberi, 677, 683 Funk, C., 677 general description, 677 non-coenzymatic function, 677-678 pharmacokinetics, 681 physiology, 678-681 absorption and transport, 678-679 placental transport, 680-681 renal excretion, 680 potential variable factors, 683-684 requirements and allowances, 684 recommended dietary allowance (RDA), 684 sources, 684 status, 682-683 toxicity, 684 Williams, R.R., 677 Thioctic acid. See a-Lipoic acid. Thiouracil, and coenzyme Qjo, 127

Thisilyn® (milk thistle), 476

Thisilyn Pro’ (milk thistle), 476 Thyroid stimulating hormone. See TSH. a-Tocopherol. See Vitamin E. Tocopherol-mediated peroxidation, 758 Tomato, lycopene source, 421, 430 Traditional Chinese medicine. See TCM. Transferrin, 358, 360 Trifolium pratense. See Red clover.

sesquiterpenes, 689, 693, 697 valepotriates, 689-690 volatile oil, 689 clinical studies, 693-697 sleep disorders, in, 693

description, botanical, 688 pharmacological activity, 691-693 alkaloids, 692 total extracts, 691 toxicology of valepotriates, 692 valepotriates, 692 volatile oil, 692

pharmacopeial status, 688-689 dosage, 688 dosage forms, 688

safety of, 697-698 contraindications and drug indications, 697 taxonomy, 687-688 traditional uses, 687

Valeriana Natt® (valerian), 697 Valeriana officinalis. See Valerian. Valmane™ (valerian), 689 Viagra®, and yohimbe, 784 Visual pigments. See Macular pigments. Vitamin A, 701-713

adverse effects, 710-711 and B-carotene, 81-85, 701, 702, 705, 706, 707, 708 biochemistry and function, 702-704 activation, 702 nuclear receptors, in cell differentiation and fetal development, 702-703 ocular functions, 703 and coenzyme Qjo, 127, 128 contraindications, 711 deficiency, 704, 707 dietary reference intake (DRI), 708, 709

food sources, 707—708 interactions, 711

and iron, 711 metabolism, 705-707 intestinal absorption, 705 plasma transport, 706-707 renal loss, 707 storage, 706 physiology, 703-707 transport and binding proteins, 704—707 recommended dietary allowance (RDA), 709 status, 707 units of activity, 708-709 upper intake level, 710, 711 use of, in disease, 709-710

Index

818

Vitamin B,. See Thiamin. Vitamin B3. See Niacin. Vitamin B;. See Pantothenic acid. Vitamin Be, 715-734 assessment of status, 715-719 dietary intake, 716

direct indices, 716-717 indirect indices, 718-719 biochemistry and functions, 715 in disease and toxicity, 723-726 asthma, 725 carpal tunnel syndrome, 725 coronary heart disease, 723-724 drug—vitamin Bg interactions, 725-726 HIV/AIDS, 724 overdosage, 726 premenstrual syndrome, 724-725 sickle cell anemia, 725 food sources, 723 functions, 719-722 erythrocyte function, 719-720 gluconeogenesis, 719 hormone modulation/gene expression, 721-722 immune system functions, 719 lipid metabolism, 721

nervous system functions, 720-721 niacin formation, 720 Gyorgy, P., 715 history, 716

and magnesium, 447 names, 715 physical and chemical properties, 715, 717 requirements, 722—723

recommended dietary allowance (RDA), 723 Vitamin B,>, 735-744 coenzyme function, 735-736 deficiency, 738, 740-742 causes, 740-741 hematological changes due to, 741 and immune function, 742 indicators of, 740, 741 neuropathies due to, 741-742 dietary reference intake (DRI), 738, 739

and folate, 221, 222, 223, 224, 225, 226, 737-738 food sources, 738 metabolism, 736-737 digestion and absorption, 736 excretion and storage, 737 transport proteins, 736-737 status, 738-740 in breast milk, 739-740 in the neonate and infant, 739 in pregnancy, 738-739 structure, 735 and vegetarianism, 735, 740-741

Vitamin C, 745-755 biochemistry and functions, 745-747 as antioxidant, 746 atherogenesis, effects on, 746 blood flow and endothelial function, effects on, 746 enzymatic functions, 746 iron absorption, 747, 754 nitrate tolerance, prevention of, 746-747

stomach and duodenum, effects in, 747 and coenzyme Qjo, 123, 124 and copper, 137

deficiency (scurvy), 751 food sources, 751 indications and usage, 751-753 adverse effects, 754 concentration vs. function, 751

deficiency, prevention of, 752 dietary reference intake (DRI), 752-753 disease, use in, 753

optimum intake, 753 possible benefits, 752 pregnancy, use in, 753 scurvy, treatment of, 752

and lutein, 412, 417 physiology, 747-751 bioavailability, 749-750 blood cell concentrations, 749 plasma concentrations, 748-749 potential variable factors recycling, 747-748. renal excretion, 751-752 tissue accumulation, 747 tissue distribution, 747 transport, 747 Vitamin D and calcium, 65, 66, 67 and magnesium, 447, 451-452, 454 and phosphorus, 538, 540 Vitamin E, 757-769 adverse effects, 762-763

drugs, interactions with, 763 upper tolerable limits, 762-763 biochemistry and functions, 757 antioxidant activity, 757-758 biologic activity, 758 molecular functions, 758-759 recycling, 758 and coenzyme Qo, 124, 129 deficiency, 761

excretion, 760-761 forms, 757 indications and usage, 761-762 chronic disease prevention, 761-762 deficiency, treatment of, 761

dietary reference intake (DRI), 762 food sources, 761 units, 762 and lutein, 412, 417

metabolism, 760-761 physiology, 759-761 absorption and transport, 759 tissue delivery, 760 supplements; 757 vitamin K, interaction with, 762 Vitamin K, 771-782 biochemical role of, 774-775 chemistry, 771 menaquinone, 771, 777, 778 phylloquinone, 771, 775, 777, 778 Dam, H., 771 deficiencies and requirements, 776-777 dietary sources, 772 metabolism, 775-776 osteoporosis, 771, 778 proteins, 773-774 blood coagulation, 773, 777 matrix Gla protein, 771, 774, 778 osteocalcin, 771, 774, 777

Index

819

and skeletal health, 778 vitamin E, interaction with, 762 Vitex agnus castus. See Chasteberry.

alcohol and drugs, 797 biochemistry, 791

and copper, 136, 137 deficiency of, 794 identification, 794 symptoms, 794

William, R.R., structure and synthesis of thiamin, 677

exchangeable zinc pool (EZP), 793

Western clinical herbal medicine, 28

functions of, 791-792

catalytic, 791 lipid and carbohydrate metabolism, 792 protein and nucleic acid metabolism, 792

Xangold® (lutein), 418 Xanthophylls, 409. See also Carotenoids. Xerophthalmia, and vitamin A, 703

Yohimbe, 783-789 adverse side effects, 787 anxiety and manic symptoms, 787 drug interactions, 787 chemistry, 783 yohimbine, 783, 784, 786, 787

yohimbine hydrochloride, 783, 784, 785 description, botanical, 783

efficacy, 785 as aphrodisiac, 783, 785 in erectile dysfunction, 783, 784, 785

mechanism of action, 785-787 nitric oxide, role of, 786 preclinical studies, 784-785 preparation, 783

Zeaxanthins, 409. See also Carotenoids. Zinc, 791-800 adverse interactions, 797

regulatory, 791-792 structural, 791 indications and usage, 794-797 Alzheimer’s disease, treatment of, 795-796 bioavailability, 794-795 cold, treatment of, 795 contraindications, 796 depletion, prevention of, 795, 796

depletion, treatment of, 795 HIV/AIDS, treatment of, 796 recommended intake levels, 795 sources, 794 supplement use, 795 and magnesium absorption, 447 metabolism, 792-794 absorption, 792-793 distribution, in humans, 792 excretion, 793-794 homeostatic regulation, 793 reserves, 793 turnover and transport, 793 metallothionein, 791, 792 recommended dietary allowance (RDA), 792, 793, 795 ’ synthesis, 791, 792, 796 Zingiber officinale. See Ginger. Zintona® (ginger), 242

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