The Encyclopedia of Vitamin E 9781845930752, 1845930754

Table of contents :
Contents
Contributors
Preface
Acknowledgements
Part I: Nomenclature and Biochemical, Physical and Chemical Aspects of Vitamin E-related Compounds
1. Overview of Antioxidant Activity of Vitamin E
2. Vitamin E, Metabolism and Biological Activity of Metabolic Products
3. PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds
4. Recycling of Vitamin E by Natural Products: a Dietary Perspective
5. Mössbauer Spectroscopy Studies of Tocopherol
6. Chemical Modifications of Alpha-Tocopherol
7. Functions and Activities of Tocotrienols and Tocopherols In Vitro
8. An Overview of Free Radicals and Oxidative Stress: Setting the Scene
9. Tocopherol Composition of Plants and Their Regulation
10. Efficiency of Extracting Vitamin E from Plant Sources
11. Commercial Extraction of Vitamin E from Food Sources
12. Overview of Tocopherol Composition of Oils
13. Changes in Tocopherol Composition of Edible Oils after Extreme Heat Exposure (frying)
14. Analysis of Vitamin E by HPLC
15. Capillary Electrochromatographic Analysis of Tocopherols
16. GC–MS and LC–MS/MS Techniques to Quantify Alpha-Tocopherol and Alpha-Tocopherolquinone: Applications to Biological and Food Matrices
17. Assay of Free Radical-generated Cholesterol Metabolites and Alpha-Tocopherol in Plasma and Tissues
18. Interactions of Vitamin E with Biomembrane Models: Binding to Dry and Water-containing Reversed Micelles
Part II: Dietary and Nutritional Influences and General Effects
19. Vitamin E Bioavailability
20. Circulating Concentrations of Vitamin E in Populations
21. The Relationship Between Tocotrienols and Tocopherols in the Diet
22. The Relationship Between Alpha-Tocopherol and Selenium in the Diet
23. The Relationship Between Alpha-Tocopherol, Selenium and Fish Oil in the Diet and Effects on the Heart and Liver
24. Vitamin E Status in the Elderly
Part III: Cocktails, Antioxidant Mixtures and Novel Analogues
25. Efficacy of Tocopherol Mixtures Compared with Alpha-Tocopherol in Cardioprotection
26. Hyperlipidaemia and the Use of Tocopherol in Antioxidant Cocktails in Smokers
Part IV: General Physiological Systems, Metabolism and Metabolic Stress
27. Tissue Distribution of Alpha-Tocopherol in Oxidative Stress in Rats
28. Cellular Metabolism in Vitamin E Deficiency
29. Role of Vitamin E and Vitamin C in Nuclear Factor-κB-Dependent Nitric Oxide Signalling
30. Modification of the Activation of NF-κB by Vitamin E
31. Vitamin E and Cell Signalling
32. Vitamin E Supplementation in Diabetes Mellitus
33. Serum Alpha-Tocopherol and Insulin Therapy in Diabetes: Relationship to Retinol
34. Lead-induced DNA Damage and Relationship with Vitamin E
35. Alpha-Tocopherol and Surgical Stress
36. Ischaemia–reperfusion Injury and Tocopherols
Part V: Brain, Neurological and Optical Systems
37. Vitamin E Cocktails and their Preventive Effects on the Retina in Type 1 Diabetes
38. Neuronal Damage, Cognitive Impairment and Alpha-Tocopherol
39. Vitamin E and Coenzyme Q[sub(10)] in Parkinson’s Disease
40. Protective Effects of Alpha-Tocopherol on Nicotinic Acetylcholine Receptors in Alzheimer’s Disease
41. Effects of Vitamin E on the Apoptotic Cascade: Studies in Neuronal Cells
42. Effects of Vitamin E on the Brain in Diabetes
Part VI: Reproductive Systems, Fetus and Infant
43. Alpha-Tocopherol and Male Fertility
44. Alpha-Tocopherol and Pre-eclampsia
45. Alpha-Tocopherol and Antioxidant Status in Premature Infants
46. Alpha-Tocopherol in Infants and Relationship with the Mothers
47. Transfer of Vitamin E in Milk to the Newborn
48. Improving Thin Endometrium by Combined Pentoxifylline–Tocopherol Treatment if Fibrosis is Present
Part VII: Musculo-skeletal Systems and Exercise
49. Vitamin E Uncouples Joint Destruction and Clinical Inflammation in a Transgenic Mouse Model of Rheumatoid Arthritis
50. The Effect of Vitamin E on Post-exercise Fatigue
51. Post-contraction Damage to Muscle and Vitamin E
52. Muscle Damage after Running and Effects of a Vitamin E and C Cocktail
Part VIII: Cardiovascular, Haematological and Pulmonary Systems
53. Alpha-Tocopherol and Vascular Endothelial Growth Factor
54. Alpha-Tocopherol and Adhesion Molecules in Endothelial Cells
55. Overview of Vitamin E and Human Low-density Lipoprotein
56. Alpha-Tocopherol in Lipoprotein Apheresis
57. Smooth Muscle Cells and Alpha-Tocopherol
58. Vitamin E and Atherosclerosis
59. Vitamin E and Blood Pressure
60. Vitamin E and Platelets
61. Vitamin E and the Lung: Uptake Mechanism and Function in Alveloar Type II Cells
62. Smoking and Tocopherol Status
63. Failure of Alpha-Tocopherol to Prevent Cigarette Smoke-induced Protein Oxidation: Comparison with Other Antioxidant Vitamins
64. Protective Effects of Alpha-Tocopherol in Pulmonary Fibrosis
65. Alpha-Tocopherol and Cystic Fibrosis
66. Hypoxia-induced Lung Damage and Alpha-Tocopherol
Part IX: Skin
67. Overview of Alpha-Tocopherol and the Skin
68. The Skin and Vitamin E Cocktails
69. Relationship Between Alpha-Tocopherol and Glutathione in Keratinocytes
Part X: Liver, Kidney, Intestinal and Other Organ Systems
70. Alcoholic Liver Disease, Oxidative Stress and the Role of Tocopherol
71. Liver Cirrhosis and Vitamin E Status
72. Inflammatory Bowel Disease and Oxidative Stress: Role of Vitamin E
73. Vitamin E, Gastric Pathology and Helicobacter pylori: Causes and Relationships
74. Does Oxidative Stress in Haemodialysis Patients Require an Overprotection with Vitamin E Supplementation?
Part XI: Immune and Haematological Systems
75. Natural Forms of Vitamin E as Anti-Inflammatory and Anti-Cancer Agents: their Effect on Cyclo-oxygenase and Other Membrane-associated Enzymes
76. Vitamin E and its Effects on Macrophages and Neutrophils
77. Vitamin E in Relation to Other Antioxidants in HIV Disease
78. Vitamin E and Hepatic Effects of Cyclosporins
79. Mycotoxins and Protection with Vitamin E
80. Alpha-Tocopherol and Expression of the CD95 Ligand
Part XII: Cancer
81. Overview of Tocopherols in Cancer Chemoprevention
82. Chemical and Biological Properties of Tocopherols and their Relation to Cancer Incidence and Progression
83. Cancer Progression and Alpha-Tocopherol in Relation to Other Antioxidants
84. Chemoprevention and Vitamin E With or Without Selenium
85. Effects of Dietary Vitamin E on Mutatect Tumours
86. Tocopherols and Lung Cancer
87. Gastrointestinal Cancers and Vitamin E
88. Alpha-Tocopheryl Succinate Exhibits Potent Anticancer Activity: its Interaction with Ionizing Radiation and Chemotherapeutic Effects
Index
A
B
C
D
E
F
G
H
I
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z

Citation preview

The Encyclopedia of Vitamin E

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The Encyclopedia of Vitamin E Edited by

Professor Victor R. Preedy Department of Nutrition and Dietetics, King’s College London, UK and

Professor Ronald R. Watson Arizona Cancer Center, University of Arizona, USA

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CABI is a trading name of CAB International CABI Head Office Nosworthy Way Wallingford Oxon OX10 8DE UK

CABI North American Office 875 Massachusetts Avenue 7th Floor Cambridge, MA 02139 USA

Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 E-mail: [email protected] Website: www.cabi.org

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© CAB International 2007. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. A catalogue record for this book is available from the Library of Congress, Washington, DC.

ISBN-13: 978-1-84593-075-2 The paper used for the text pages in this book is FSC certified. The FSC (Forest Stewardship Council) is an international network to promote responsible management of the world’s forests. Typeset in 9pt Melior by Columns Design Ltd, Reading Printed and bound in the UK by Cromwell Press, Trowbridge

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Contents

Contributors

xi

Preface

xvii

Acknowledgements

xviii

Part I: Nomenclature and Biochemical, Physical and Chemical Aspects of Vitamin E-related Compounds

1

1.

Overview of Antioxidant Activity of Vitamin E M. Lucarini and G.F. Pedulli

3

2.

Vitamin E, Metabolism and Biological Activity of Metabolic Products M.C. Polidori, H. Sies, W. Stahl and R. Brigelius-Flohé

11

3.

PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds T. Rosenau

21

4.

Recycling of Vitamin E by Natural Products: a Dietary Perspective G.F. Pedulli and M. Lucarini

44

5.

Mössbauer Spectroscopy Studies of Tocopherol K. Burda and O. Kruse

53

6.

Chemical Modifications of Alpha-Tocopherol T. Rosenau

69

7.

Functions and Activities of Tocotrienols and Tocopherols In Vitro B.Y. Rubin and S.L. Anderson

96

8.

An Overview of Free Radicals and Oxidative Stress: Setting the Scene M.C.Y. Wong and H. Wiseman

104

9.

Tocopherol Composition of Plants and Their Regulation H.-P. Mock

112

10. Efficiency of Extracting Vitamin E from Plant Sources G. Sacchetti and R. Bruni

122

11. Commercial Extraction of Vitamin E from Food Sources S.-Y. Quek, B.-S. Chu and B.S. Baharin

140

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vi

Contents

12. Overview of Tocopherol Composition of Oils F. Khallouki, B. Spiegelhalder and R.W. Owen

153

13. Changes in Tocopherol Composition of Edible Oils after Extreme Heat Exposure (frying) M. Battino and J.L. Quiles

162

14. Analysis of Vitamin E by HPLC B.J. Burri

172

15. Capillary Electrochromatographic Analysis of Tocopherols S. Fanali

183

16. GC–MS and LC–MS/MS Techniques to Quantify Alpha-Tocopherol and Alpha-Tocopherolquinone: Applications to Biological and Food Matrices P. Mottier and P.A. Guy

190

17. Assay of Free Radical-generated Cholesterol Metabolites and Alpha-Tocopherol in Plasma and Tissues L. Iuliano, F. Micheletta, S. Natoli and U. Diczfalusy

201

18. Interactions of Vitamin E with Biomembrane Models: Binding to Dry and Water-containing Reversed Micelles A. Ruggirello and V. Turco Liveri

210

Part II: Dietary and Nutritional Influences and General Effects

219

19. Vitamin E Bioavailability M.G. Traber

221

20. Circulating Concentrations of Vitamin E in Populations E.S. Ford

231

21. The Relationship Between Tocotrienols and Tocopherols in the Diet K. Yamashita

252

22. The Relationship Between Alpha-Tocopherol and Selenium in the Diet M. De Cerqueira Cesar

263

23. The Relationship Between Alpha-Tocopherol, Selenium and Fish Oil in the Diet and Effects on the Heart and Liver J. Poirier and S. Kubow

273

24. Vitamin E Status in the Elderly M. Dehghan and A.T. Merchant

285

Part III: Cocktails, Antioxidant Mixtures and Novel Analogues

297

25. Efficacy of Tocopherol Mixtures Compared with Alpha-Tocopherol in Cardioprotection K. Sarkar, S. Parthasarthy, T.G.P. Saldeen and J.L. Mehta

299

26. Hyperlipidaemia and the Use of Tocopherol in Antioxidant Cocktails in Smokers J.C.-J. Chao

307

Part IV: General Physiological Systems, Metabolism and Metabolic Stress

315

27. Tissue Distribution of Alpha-Tocopherol in Oxidative Stress in Rats J. Antosiewicz, R.A. Olek and W. Ziolkowski

317

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Contents

vii

28. Cellular Metabolism in Vitamin E Deficiency P. Fattoretti and C. Bertoni-Freddari

327

29. Role of Vitamin E and Vitamin C in Nuclear Factor-
B-Dependent Nitric Oxide Signalling J. Neuzil, P.K. Witting, A. Kontush and J. Headrick

339

30. Modification of the Activation of NF-
B by Vitamin E H.P. Glauert

353

31. Vitamin E and Cell Signalling F. Galli, M.C. Aisa, C. Annetti and A. Floridi

365

32. Vitamin E Supplementation in Diabetes Mellitus B. Manuel-y-Keenoy

381

33. Serum Alpha-Tocopherol and Insulin Therapy in Diabetes: Relationship to Retinol F. Granado-Lorencio and B. Olmedilla-Alonso

399

34. Lead-induced DNA Damage and Relationship with Vitamin E K. Wozniak

409

35. Alpha-Tocopherol and Surgical Stress J.J. Ochoa and A. Muñoz-Hoyos

419

36. Ischaemia–reperfusion Injury and Tocopherols G.E. Gondolesi

429

Part V: Brain, Neurological and Optical Systems

445

37. Vitamin E Cocktails and their Preventive Effects on the Retina in Type 1 Diabetes F. Franconi, M.A.S. Di Leo, G. Seghieri, P.P. Urgeghe, R. Coinu, P. Di Simplicio and G. Ghirlanda

447

38. Neuronal Damage, Cognitive Impairment and Alpha-Tocopherol K. Fukui and S. Urano

454

39. Vitamin E and Coenzyme Q10 in Parkinson’s Disease M. Ebadi and S. Sharma

461

40. Protective Effects of Alpha-Tocopherol on Nicotinic Acetylcholine Receptors in Alzheimer’s Disease Z.-Z. Guan

472

41. Effects of Vitamin E on the Apoptotic Cascade: Studies in Neuronal Cells R. W. Gracy

480

42. Effects of Vitamin E on the Brain in Diabetes G. Baydas

487

Part VI: Reproductive Systems, Fetus and Infant

495

43. Alpha-Tocopherol and Male Fertility S. Azhar

497

44. Alpha-Tocopherol and Pre-eclampsia A. Ben-Haroush and J. Bar

509

45. Alpha-Tocopherol and Antioxidant Status in Premature Infants J.J. Ochoa and M.C. Ramirez-Tortosa

515

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viii

Contents

46. Alpha-Tocopherol in Infants and Relationship with the Mothers G. Baydas

524

47. Transfer of Vitamin E in Milk to the Newborn C. Lauridsen and S.K. Jensen

530

48. Improving Thin Endometrium by Combined Pentoxifylline–Tocopherol Treatment if Fibrosis is Present H. Letur-Konirsch and S. Delanian

539

Part VII: Musculo-skeletal Systems and Exercise

549

49. Vitamin E Uncouples Joint Destruction and Clinical Inflammation in a Transgenic Mouse Model of Rheumatoid Arthritis M. De Bandt

551

50. The Effect of Vitamin E on Post-exercise Fatigue S. Asha Devi

555

51. Post-contraction Damage to Muscle and Vitamin E D.R. Moore and S.M. Phillips

564

52. Muscle Damage after Running and Effects of a Vitamin E and C Cocktail B. Dawson and C. Goodman

572

Part VIII: Cardiovascular, Haematological and Pulmonary Systems

581

53. Alpha-Tocopherol and Vascular Endothelial Growth Factor L.C. Vona-Davis and D.W. McFadden

583

54. Alpha-Tocopherol and Adhesion Molecules in Endothelial Cells G.A. Ferns and D.A.J. Lamb

592

55. Overview of Vitamin E and Human Low-density Lipoprotein S. Badiou, M.-A. Carbonneau, M. Morena, E. Mas, C.L. Leger and J.-P. Cristol

608

56. Alpha-Tocopherol in Lipoprotein Apheresis · V. Bláha, D. Solichová, M. Bláha, P Zdánsky´ and Z. Zadák

617

57. Smooth Muscle Cells and Alpha-Tocopherol J.-M. Zingg and A. Azzi

624

58. Vitamin E and Atherosclerosis T. Cyrus and D. Praticò

645

59. Vitamin E and Blood Pressure S. Vasdev, P. Singal and V. Gill

655

60. Vitamin E and Platelets S. Riondino, F.M. Pulcinelli and P.P. Gazzaniga

666

61. Vitamin E and the Lung: Uptake Mechanism and Function in Alveloar Type II Cells F. Guthmann and B. Rüstow

674

62. Smoking and Tocopherol Status R.T. Murphy

683

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Contents

ix

63. Failure of Alpha-Tocopherol to Prevent Cigarette Smoke-induced Protein Oxidation: Comparison with Other Antioxidant Vitamins K. Panda and I.B. Chatterjee

690

64. Protective Effects of Alpha-Tocopherol in Pulmonary Fibrosis J.W. Card and T.E. Massey

700

65. Alpha-Tocopherol and Cystic Fibrosis L.G. Wood, P.G. Gibson and M.L. Garg

708

66. Hypoxia-induced Lung Damage and Alpha-Tocopherol T. Minko

719

Part IX: Skin

729

67. Overview of Alpha-Tocopherol and the Skin J.M.L. White and E.M. Higgins

731

68. The Skin and Vitamin E Cocktails A.-K. Greul

739

69. Relationship Between Alpha-Tocopherol and Glutathione in Keratinocytes H. Masaki and H. Sakurai

749

Part X: Liver, Kidney, Intestinal and Other Organ Systems

763

70. Alcoholic Liver Disease, Oxidative Stress and the Role of Tocopherol C. Loguercio and A. Federico

765

71. Liver Cirrhosis and Vitamin E Status G. H. Koek, A. Bast and A. Driessen

773

72. Inflammatory Bowel Disease and Oxidative Stress: Role of Vitamin E J. P. Allard

782

73. Vitamin E, Gastric Pathology and Helicobacter pylori: Causes and Relationships B. Annibale and G. Capurso

791

74. Does Oxidative Stress in Haemodialysis Patients Require an Overprotection with Vitamin E Supplementation? J.-P. Cristol, S. Badiou, N. Terrier, A.-M. Dupuy, B. Canaud and M. Morena

796

Part XI: Immune and Haematological Systems

805

75. Natural Forms of Vitamin E as Anti-Inflammatory and Anti-Cancer Agents: their Effect on Cyclo-oxygenase and Other Membrane-associated Enzymes Q. Jiang

807

76. Vitamin E and its Effects on Macrophages and Neutrophils I. Politis and S. Fragou

819

77. Vitamin E in Relation to Other Antioxidants in HIV Disease C. Spada, G. L. Baggio and A. Treitinger

826

78. Vitamin E and Hepatic Effects of Cyclosporins S. Grub, C. Trendelenburg, J. Kapp-Schwoerer and A. Wolf

836

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x

Contents

79. Mycotoxins and Protection with Vitamin E Z. W. Jaradat

846

80. Alpha-Tocopherol and Expression of the CD95 Ligand M. Li-Weber and P. H. Krammer

855

Part XII: Cancer

863

81. Overview of Tocopherols in Cancer Chemoprevention K. Krishnan, S. Campbell and W. L. Stone

865

82. Chemical and Biological Properties of Tocopherols and their Relation to Cancer Incidence and Progression Y. Tanaka and R. V. Cooney

876

83. Cancer Progression and Alpha-Tocopherol in Relation to Other Antioxidants G. Mantovani and C. Madeddu

886

84. Chemoprevention and Vitamin E With or Without Selenium A. A. Karagözler

894

85. Effects of Dietary Vitamin E on Mutatect Tumours A. S. Haqqani, J. K. Sandhu and H. C. Birnboim

904

86. Tocopherols and Lung Cancer V. Cohen, S. Dawood and F. R. Khuri

915

87. Gastrointestinal Cancers and Vitamin E F. Kamangar

922

88. Alpha-Tocopheryl Succinate Exhibits Potent Anticancer Activity: its Interaction with Ionizing Radiation and Chemotherapeutic Effects K. N. Prasad and W. C. Cole

931

Index

945

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Contributors

Dr Maria Cristina Aisa, Department of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, University of Perugia, Via del Giochetto, 06126 Perugia, Italy. Dr Johane P. Allard, Department of Medicine, University of Toronto, Toronto General Hospital, 585 University Avenue, 9N973, Toronto, Ontario M5G 2C4, Canada. Dr Sylvia L. Anderson, Fordham University, Department of Biological Sciences, Bronx, NY 10458, USA. Dr Claudia Annetti, Department of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, University of Perugia, Via del Giochetto, 06126 Perugia, Italy. Professor Bruno Annibale, Digestive and Liver Disease Unit, University La Sapienza, Viale del Policlinico 155, 00161 Rome, Italy. Dr Jedrzej Antosiewicz, University of Pittsburgh, Hillman Cancer Center, UPCI Research Pavilion, Room 242, 5117 Center Ave, Pittsburgh, PA 15213–1863, USA. Professor S. Asha Devi, Laboratory of Gerontology, Department of Zoology, Bangalore University, Bangalore 560056, India. Dr Salman Azhar, Department of Veterans Affairs Palo Alto Health Care System, Stanford University, 3801 Miranda Avenue (182B), Palo Alto, CA 94304, USA. Dr Angelo Azzi, Institute of Biochemistry and Molecular Biology, Universität Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland. Dr Stéphanie Badiou, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, F34295 Montpellier Cedex 05, France. Dr Giovana L. Baggio, Health Sciences Center, Clinical Analysis Department, Campus Universitário-Trindade, Box 476, 88.010-970 Florianopolis, Santa Catarina, Brazil. Dr Badlishan Sham Baharin, Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang Selangor DE, Malaysia. Dr J. Bar, Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel. Dr A. Bast, Faculty of Medicine, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Dr Maurizio Battino, Institute of Biochemistry, Faculty of Medicine, Università Politecnica delle Marche, Via Ranieri 65, 60100 Ancona, Italy. Dr G. Baydas, Department of Physiology, Faculty of Medicine, Firat University, 23119 Elazig, Turkey. Dr Avi Ben-Haroush, Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel. Dr Carlo Bertoni-Freddari, Neurobiology of Aging Laboratory INRCA Research Department, Via Birarelli 8, 60121 Ancona, Italy. Dr H. Chaim Birnboim, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Room 2051, Ottawa, ON, K1A 0R6, Canada. Dr Milan Bláha, Second Department of Internal Medicine, University Hospital, Sokolská 581, 50005 Hradec Králové, Czech Republic. Professor Vladimír Bláha, Department of Metabolic Care and Gerontology, University Hospital, Sokolská 581, 50005 Hradec Králové, Czech Republic. Professor R. Brigelius-Flohé, Department of Vitamins and Atherosclerosis, German Institute of Human Nutrition, Potsdam-Rehbruecke, Nuthetal, Germany. Dr Renato Bruni, Dipartimento di Biologia Evolutiva e Funzionale, Sezione Biologia Vegetale e Orto Botanico, Università degli Studi di Parma, Parco Area delle Scienze 11A, 1–43100 Parma, Italy. Dr Kvetoslava Burda, Institute of Nuclear Physics, Polish Academy of Sciences, ul. Radzikowskiego 152, 31–342 Krakow, Poland.

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xii

Contributors

Dr Betty J. Burri, University of California, 229 Cruess Hall, One Shields Avenue, Davis, CA 95616, USA. Dr Sharon Campbell, Department of Internal Medicine, East Tennessee State University, Johnson City, TN 37614, USA. Dr Bernard Canaud, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, 34295 Montpellier, France. Dr Gabriele Capurso, Digestive and Liver Disease Unit, University La Sapienza, Viale del Policlinico 155, 00161 Rome, Italy. Dr Marie-Annette Carbonneau, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, F34295 Montpellier Cedex 05, France. Dr Jeffrey W. Card, Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada. Dr Jane C.-J. Chao, School of Nutrition and Health Sciences, Taipei Medical University, 250 Wu Hsing Street, Taipei, Taiwan 110. Dr Indu B. Chatterjee, Centre for Genetic Engineering and Biotechnology, Calcutta University, College of Science, 35 Ballygunge Circular Road, Calcutta 700019, India. Dr Boon-Seang Chu, Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor DE, Malaysia. Dr Victor Cohen, McGill University School of Medicine, Department of Oncology, 3755 Cote Ste., Suite E-177, Catherine Road, Montreal, H3T 1E2, Canada. Dr Rita Coinu, Department of Pharmacology and Centre for Biotechnology Development and Biodiversity Research, University of Sassari, Via Muroni 23a, 07100 Sassari, Italy. Dr William C. Cole, Premier Micronutrient Corporation, 14 Galli Drive, Suite 200, Novato, CA 94949, USA. Dr Robert V. Cooney, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813, USA. Professor Jean-Paul Cristol, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, 34295 Montpellier, France. Dr Tillman Cyrus, Washington University, Division of Cardiology, Saint Louis, MO 63110, USA. Dr Shaheenah Dawood, McGill University School of Medicine, Department of Oncology, 3755 Cote Ste., Suite E-177, Catherine Road, Montreal, H3T 1E2, Canada. Professor Brian Dawson, School of Human Movement and Exercise Science, University of Western Australia, Crawley, WA 6009, Australia. Dr Michel De Bandt, Centre Hospitalo-Universitaire Xavier Bichat, 46 rue Henri Huchard, Paris 75018, France. Dr Marcelo de Cerqueira Cesar, Universidade de Sao Paolo, Faculdade de Zootecnia e Engenharia de Alimentos, Departamento de Ciencias Basicas, Pirassununga SP, Brazil 13635–900. Dr Mahshid Dehghan, Population Health Research Institute, 237 Barton Street East, Hamilton, ON L8L 2X2, Canada. Dr Sylvie Delanian, Department of Oncology-Radiotherapy, Hôpital Sant-Louis, Paris, France. Dr Mauro A. S. Di Leo, Department of Emergency Medicine, Catholic University, Largo F. Vito 1, 00168 Roma, Italy. Dr Paolo Di Simplicio, Department of Neuroscience, University of Siena, Via A. Moro 4, 53100 Siena, Italy. Dr U. Diczfalusy, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge, Sweden. Dr A. Driessen, Department of Pathology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Dr Anne-Marie Dupuy, Hôpital Lapeyronie, 371 Av. Doyer Gaston Giraud, 34295 Montpellier, France. Professor Manuchair Ebadi, Center of Excellence in Neurosciences, University of North Dakota School of Medicine and Health Sciences, 501 North Columbia Road, Grand Forks, ND 58203, USA. Dr Salvatore Fanali, Istituto di Metodologie Chimiche, Consiglio Nazionale delle Richerche, Area della Ricerca di Roma I, Via Salaria Km 29, 300, 00016 Monterotondo Scalo, Rome, Italy. Dr Patrizia Fattoretti, Neurobiology of Aging Department, Research Department, INRCA, Via Birarelli 8, 60121 Ancona, Italy. Dr Alessandro Federico, Via Pansini 5, 80131 Napoli, Italy. Professor Gordon A. A. Ferns, School of Biomedical and Molecular Science, University of Surrey, Stag Hill, Guildford, Surrey GU2 7XH, UK. Dr Earl S. Ford, Division of Adult and Community Health, National Center for Chronic Disease Prevention and Health Promotion, 4770 Buford Highway, Mailstop K-66, Atlanta, GA 30341, USA. Dr Sofia Fragou, Department of Animal Science, Agricultural University of Athens, 75 Iera Odos Street, Athens 11855, Greece. Dr Flavia Franconi, Department of Pharmacology and Centre for Biotechnology, Development and Biodiversity Research, University of Sassari, Via Muroni 23a, 07100 Sassari, Italy. Dr Koji Fukui, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 108-8548, Japan. Dr Francesco Galli, Department of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, University of Perugia, Via del Giochetto, 06126 Perugia, Italy. Professor Manohar L. Garg, Faculty of Health, School of Health Sciences, University of Newcastle, Callaghan, NSW 2308, Australia. Dr Pier Paolo Gazzaniga, Department of Experimental Medicine and Pathology, Universitá La Sapienza, viale Regina Elena 324, 00161 Rome, Italy. Dr Giovanni Ghirlanda, Servizio di Diabetologia, Policlinico A. Gemelli, Largo A. Gemelli 8, 00123 Roma, Italy.

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Contributors

xiii

Professor Peter G. Gibson, Department of Respiratory and Sleep Medicine, John Hunter Hospital, Locked Bag 1 HRMC, NSW 2310, Australia. Dr Vicki Gill, Room 4310, Health Sciences Centre, Memorial University of Newfoundland, St. John’s, NL A1B 3V6, Canada. Professor Howard P. Glauert, Graduate Center for Nutritional Sciences, University of Kentucky, 222 Funkhouser Building, Lexington, KY 40506–0054, USA. Dr Gabriel E. Gondolesi, Mount Sinai Hospital, Box 1104, One Gustave Levy Place, New York, NY 10029, USA. Dr Carmél Goodman, School of Human Movement and Exercise Science, University of Western Australia, Crawley, WA 6009, Australia. Dr Robert W. Gracy, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107–2699, USA. Dr F. Granado-Lorencio, Unidad de Vitaminas, Servicio de Endocrionología y Nutricíon, Hospital Universitario Puerta de Hierro, C/San Martin de Porres 4, 28035 Madrid, Spain. Dr Anne-Katrin Greul, Department of Pathology, University of Vermont, C-232 Given Building, 89 Beaumont Avenue, Burlington, VT 05405–0068, USA. Dr Sibylle Grub, Novartis Pharma AG, CH-4002 Basel, Switzerland. Dr Zhizhong Guan, Karolinska Institutet, Neurotec Department, Division of Molecular Neuropharmacology, SE-141 86 Stockholm, Sweden. Dr Florian Guthmann, Clinic for Neonatology, Charité, Schumannstr. 20/21, D-10098 Berlin, Germany. Dr Philippe A. Guy, Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland. Dr Arsalan S. Haqqani, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Room 2051, Ottawa, ON K1A 0R6, Canada. Dr John Headrick, Heart Foundation Research Centre, School of Medical Science, Griffith University Gold Coast Campus, Southport, 9726 Queensland, Australia. Dr Elisabeth M. Higgins, Department of Dermatology, King’s College Hospital, London, SE5 9RS, UK. Dr Luigi Iuliano, Department of Internal Medicine, University La Sapienza, via del Policlinico 155, 00161 Rome, Italy. Dr Ziad W. Jaradat, Department of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, PO Box 3030, Irbid 22110, Jordan. Dr Søren Krogh Jensen, Research Centre Foulum, Danish Institute of Agricultural Sciences, Postboks 50, DK-8830 Tjele, Denmark. Dr Qing Jiang, Department of Foods and Nutrition, Purdue University, 700 W State Street, West Lafayette, IN 47906, USA. Dr Farin Kamangar, National Cancer Institute, 6116 Executive Blvd, Rm 705, Bethesda, MD 20892–8314, USA. Dr Jacqueline Kapp-Schwoerer, Novartis Pharma AG, CH-4002 Basel, Switzerland. Dr A. Alev Karagözler, Adnan Menderes University, Faculty of Arts and Sciences, Department of Chemistry, 09010 Aydin, Turkey. Dr F. Khallouki, Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Dr Fadlo R. Khuri, Winship Cancer Institute, Emory University, 1365 Clifton Road NE, Bldg C 3094, Atlanta, GA 30322, USA. Dr G. H. Koek, Department of Internal Medicine, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Dr Anatol Kontush, Heart Foundation Research Centre, School of Medical Science, Griffith University Gold Coast Campus, Southport, 9726 Queensland, Australia. Dr Peter H. Krammer, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Dr Koyamangalath Krishnan, UT MD Anderson Cancer Center, 1515 Holcombe Blvd-236, Houston, TX 77030–4009, USA. Dr Olaf Kruse, University of Bielefeld, Department of Biology, Molecular Cell Physiology, Universitaetstraase 25, 33501 Bielefeld, Germany. Dr S. Kubow, School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, 21,111 Lakeshore, Ste Anne de Bellevue, QC, Canada H9X 3V9. Dr David J. Lamb, The Royal Surrey County Hospital, Egerton Road, Guildford, Surrey GU2 7XX, UK. Dr C. Lauridsen, Research Centre Foulum, Danish Institute of Agricultural Sciences, Postboks 50, DK-8830 Tjele, Denmark. Dr Claude Louis Leger, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, F34295 Montpellier Cedex 05, France. Dr H. Letur-Konirsch, Fertility Center, Institut Mutualiste Montsouris, 42 Boulevard Jourdan, 75014 Paris, France. Dr Min Li-Weber, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Professor Carmela Loguercio, Via Pansini 5, 80131 Napoli, Italy. Professor Marco Lucarini, University of Bologna, Via San Giacomo 11, 40126 Bologna, Italy. Dr Clelia Madeddu, Policlinico Universitario Cagliari, Presidio di Monserrato, Strada Statale 554, 09042 Monserrato, Cagliari, Italy.

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xiv

Contributors

Professor Giovanni Mantovani, Policlinico Universitario Cagliari, Presidio di Monserrato, Strada Statale 554, 09042 Monserrato, Cagliari, Italy. Dr Begona Manuel-y-Keenoy, Laboratory of Endocrinology, Campus Drie Eiken, Metabolic Research Unit (AMRU) TA.37, Universiteitsplein 1, B-2610 Antwerp-Wilrijk, Belgium. Dr Emilie Mas, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, F34295 Montpellier Cedex 05, France. Dr Hitoshi Masaki, Nikkol Group Cosmos Technical Centre Co. Ltd, 3-24-3, Hasune, Itabashi-ku, Tokyo 174-0046, Japan. Professor Thomas E. Massey, Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada. Dr D. W. McFadden, Robert C. Byrd Health Sciences Center, West Virginia University, PO Box 9238, Morgantown, WV 26506-9238, USA. Dr J. L. Mehta, Department of Medicine and Physiology, University of Arkansas for Medical Sciences, Mail Slot 532, 4301 West Markham St, Little Rock, AR 72205, USA. Professor Anwar T. Merchant, Population Health Research Institute, 237 Barton Street East, Hamilton, ON L8L 2X2, Canada. Dr F. Micheletta, Department of Internal Medicine, University La Sapienza, Via del Policlinico 155, 00185 Rome, Italy. Dr Tamara Minko, Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854–8020, USA. Dr Hans-Peter Mock, Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, D-06466 Gatersleben, Germany. Dr Daniel R. Moore, Exercise Metabolism Research Group, McMaster University, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada. Dr Marion Morena, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, F34295 Montpellier Cedex 05, France. Dr Pascal Mottier, Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland. Dr Antonio Muñoz-Hoyos, Department of Pediatrics, San Cecilio University Hospital, 18012, Granada, Spain. Dr Ross T. Murphy, Department of Cardiovascular Medicine, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Dr S. Natoli, Department of Internal Medicine, University of La Sapienza, Via del Policlinico 155, 00185 Rome, Italy. Dr Jiri Neuzil, Heart Foundation Research Centre, School of Medical Science, Griffith University Gold Coast Campus, Southport, 9726 Queensland, Australia. Dr Julio J. Ochoa, Institute of Nutrition and Food Technology, University of Granada, Ramon Y. Cajal 4, 18071 Granada, Spain. Dr Robert A. Olek, Department of Bioenergetics, Jedrzej Sniadecki Academy of Physical Education and Sport, Wiejska 1, 80–336 Gdansk, Poland. Dr B. Olmedilla-Alonso, Hospital Universitario Puerta de Hierro, C/San Martin de Porres 4, 28035 Madrid, Spain. Professor Robert W. Owen, Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Dr K. Panda, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Dr Sam Parthasarthy, Department of Medicine and Physiology, University of Arkansas for Medical Sciences, Mail Slot 532, 4301 West Markham St, Little Rock, AR 72205, USA. Dr Gian Franco Pedulli, University of Bologna, Via San Giacomo 11, 40126 Bologna, Italy. Dr Stuart M. Phillips, Exercise Metabolism Research Group, McMaster University, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada. Dr Johanne Poirier, School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, 21,111 Lakeshore, Ste Anne de Bellevue, QC, H9X 3V9 Canada. Dr Maria Cristina Polidori, Institute of Biochemistry and Molecular Biology I, Heinrich-Heine University, Postfach 101007, D-40001 Dusseldorf, Germany. Professor Ioannis Politis, Department of Animal Science, Agricultural University of Athens, 75 Iera Odos Street, Athens 11855, Greece. Dr Kedar N. Prasad, Premier Micronutrient Corporation, 14 Galli Drive, Suite 200, Novato, CA 94949, USA. Dr Domenico Praticò, University of Pennsylvania, Department of Pharmacology, John Morgan Building – Room 124, 3620 Hamilton Walk, Philadelphia, PA 19104, USA. Professor Victor R. Preedy, Department of Nutrition and Dietetics, King’s College, London, UK. Dr Favio M. Pulcinelli, Department of Experimental Medicine and Pathology, Universita La Sapienza, viale Regina Elena 324, 00161 Rome, Italy. Dr S.-Y. Quek, Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor DE, Malaysia. Dr José Luis Quiles, Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, Ramon y Cajal 4, 18071 Granada, Spain. Dr Maria Carmen Ramirez-Tortosa, Department of Biochemistry and Molecular Biology, University of Granada, Ramon y Cajal 4, 18071, Granada, Spain.

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Contributors

xv

Dr Silvia Riondino, Department of Experimental Medicine and Pathology, Universita La Sapienza, viale Regina Elena 324, 00161 Rome, Italy. Professor Thomas Rosenau, Department of Chemistry, Muthgasse 18, A-1190 Vienna, Austria. Dr Berish Y. Rubin, Fordham University, Department of Biological Sciences, Bronx, NY 10458, USA. Dr A. Ruggirello, Dipartimento di Chimica-Fisica ‘F. Accascina’, Universita degli Studi di Palermo, Viale delle Scienze Parco D’Orleans II, 90128 Palermo, Italy. Professor Bernd Rüstow, Clinic for Neonatology, Charité, Schumannstr. 20/21, D-10098 Berlin, Germany. Dr G. Sacchetti, Universita degli Studi di Ferrara, C.so Porta Mare 2, I-44100 Ferrara, Italy. Professor Hiromu Sakurai, Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Dr Tom G. P. Saldeen, Department of Medicine and Physiology, University of Arkansas for Medical Sciences, Mail Slot 532, 4301 West Markham St, Little Rock, AR 72205, USA. Dr Jagdeep K. Sandhu, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Room 2051, Ottawa, ON K1A 0R6, Canada. Dr Kunal Sarkar, Department of Medicine and Physiology, University of Arkansas for Medical Sciences, Mail Slot 532, 4301 West Markham St, Little Rock, AR 72205, USA. Dr Giuseppe Seghieri, Department of Internal Medicine, Spedali Riuniti, Pistoia, Italy. Dr Sushil Sharma, Center for Excellence in Neurosciences, University of North Dakota School of Medicine and Health Sciences, 501 North Columbia Road, Grand Forks, ND 58203, USA. Professor Helmut Sies, Institute of Biochemistry and Molecular Biology I, Heinrich-Heine University, Postfach 101007, D-40001 Dusseldorf, Germany. Dr Pawan Singal, Institute of Cardiovascular Sciences, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, Canada. Dr Dagmar Solichová, Department of Medical Care and Gerontology, University Hospital, Sokolská 581, 50005 Hradec Králové, Czech Republic. Dr Celso Spada, Health Sciences Center, Clinical Analysis Department, Campus Universitário-Trindade, Box 476, 88.010970 Florianopolis, Santa Catarina, Brazil. Dr B. Spiegelhalder, Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Professor Wilhelm Stahl, Institute of Biochemistry and Molecular Biology I, Heinrich-Heine University, Postfach 101007, D-40001 Dusseldorf, Germany. Dr William Stone, James H. Quillen College of Medicine, Department of Pediatrics, PO Box 70578, Johnson City, TN 37614, USA. Dr Yuichiro Tanaka, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813, USA. Dr Nathalie Terrier, Hôpital Lapeyronie, 371 Av. Doyen Gaston Giraud, 34295 Montpellier, France. Dr Maret G. Traber, Department of Nutrition and Exercise Sciences, Linus Pauling Institute, Oregon State University, Corvallis, OR 97331–6512, USA. Dr Aricio Treitinger, Health Sciences Center, Clinical Analysis Department, Campus Universitário-Trindade, Box 476, 88.010-970 Florianopolis, Santa Catarina, Brazil. Dr Christian Trendelburg, Novartis Pharma AG, CH-4002 Basel, Switzerland. Professor V. Turco Liveri, Dipartimento di Chimica-Fisica ‘F. Accascina’, Universita degli Studi di Palermo, Viale delle Scienze Parco D’Orleans II, 90128 Palermo, Italy. Dr Shiro Urano, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 108-8548, Japan. Dr Pietro P. Urgeghe, Department of Pharmacology and Centre for Biotechnology Development and Biodiversity Research, University of Sassari, Via Muroni 23a, 07100 Sassari, Italy. Professor Sudesh Vasdev, Room 4310, Health Sciences Centre, Memorial University of Newfoundland, St. John’s, NL A1B 3V6, Canada. Dr Linda C. Vona-Davis, Robert C. Byrd Health Sciences Center, West Virginia University, PO Box 9238, Morgantown, WV 26506-9238, USA. Professor Ronald R. Watson, Arizona Cancer Center, University of Arizona, USA. Dr Jonathan M. L. White, St John’s Institute of Dermatology, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK. Dr Helen Wiseman, Department of Nutrition and Dietetics, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK. Dr Paul K. Witting, School of Medical Science, Griffith University Gold Coast Campus, Southport, 9726 Queensland, Australia. Dr Armin Wolf, Novartis Pharma AG, CH-4002 Basel, Switzerland. Dr Max C. Y. Wong, Department of Nutrition and Dietetics, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK. Dr Lisa Wood, Department of Respiratory and Sleep Medicine, Hunter Medical Research Institute, John Hunter Hospital, Newcastle 2310, NSW, Australia.

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Contributors

Dr Katarzyna Wozniak, Department of Molecular Genetics, University of Lodz, Banacha 12/16 90-237 Lodz, Poland. Dr Kanae Yamashita, Department of Food and Nutrition, School of Life Studies, Sugiyama Jogakuen University, 17-3 Hoshigaoka-Motomachi, Chikusa-ku, Nagoya, 464-8662 Japan. Dr Zdeneˇk Zadák, Department of Metabolic Care and Gerontology, University Hospital, Sokolská 581, 50005 Hradec Králové, Czech Republic. Dr Petr Zˇdánsky´, Department of Metabolic Care and Gerontology, University Hospital, Sokolská 581, 50005 Hradec Králové, Czech Republic. Dr Jean-Marc Zingg, Institute of Biochemistry and Molecular Biology, Universität Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland. Dr Wieslaw Ziolkowski, Department of Bioenergetics, Jedrzej Sniadecki Academy of Physical Education and Sport, Wiejska 1, 80-336 Gdansk, Poland.

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Preface

This book describes important biomedical features of vitamin E in one volume. Vitamin E is the collective name given to different homologues (i.e. vitamers) of tocopherols and tocotrienols. In general, the biological activity of alpha-tocopherol is greater than the other vitamers so that very often the terms ‘vitamin E’, α-tocopherol and ‘alpha-tocopherol’ are used interchangeably. The focus of vitamin E pertains to its properties as an important dietary constituent that helps in the defence against cellular damage. The absorption of vitamin E from food, and its utilization by the body embodies an intricate series of reactions. At the cellular level, the incorporation of vitamin E directly into membranes of subcellular organelles may be responsible for its potential to confer antioxidant protective properties and prevent both the initiation and propagation of lipid peroxidation. Its impact on cellular processes is also multifaceted, from its involvement in preventing DNA damage to its influence in intracellular signalling. However, it is also becoming very clear that vitamin E has great potential in being used therapeutically in a number of diseases and conditions. These range from the effects of vitamin E in treating skin damage to its actions in preventing pathological lesions in major organs such as the liver and heart. Beneficial effects of vitamin E have also been shown in metabolic disorders as well. The aim of this book is to bring together the many features of vitamin E-related research. This is no easy task. The Editors recognize, for example, the difficulty and complexity of categorizing the effects of tocopherol therapy. A chapter on a vitamin E-containing cocktail could easily be categorized into a section pertaining to organ pathology. In addition, some studies describing changes in vitamin E also report other antioxidants. In this handbook, both the negative as well as the positive effects of vitamin E are also described. This represents an all-embracing approach in describing the effects and actions of vitamin E. The book is divided into a number of subsections and is suitable for all those interested in nutrition in general, vitamins or the specific effects of vitamin E and/or oxidative stress. Victor R Preedy and Ronald Ross Watson Editors

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Acknowledgements

H. B. and Jocelyn Wallace, through the Wallace Research Foundation, have supported studies on the role of vitamin E in health promotion by Ronald Ross Watson for two decades. Their strong interest and encouragement is gratefully acknowledged. This research provided background and interest. The National Health Research Institute’s understanding of the importance of natural vitamin E is also appreciated. Finally, both groups’ encouragement to communicate research findings to the scientific and general public is appreciated, making this book ultimately possible.

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Part I Nomenclature and Biochemical, Physical and Chemical Aspects of Vitamin E-related Compounds

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1 Overview of Antioxidant Activity of Vitamin E Marco Lucarini and Gian Franco Pedulli Department of Organic Chemistry ‘A. Mangini’, University of Bologna, Via San Giacomo 11, 40126 Bologna, Italy

Abbreviations: AAPH, 2,2-azobis(2-amidinopropane)dihydrochloride; AH, phenolic antioxidants; AIBN, 2,2-azobis(isovaleronitrile); AMVN, 2,2-azobis(2,4-dimethylvaleronitrile); BDE, bond dissociation enthalpy; BHA, 3,5-di-tert-butyl-4-hydroxyanysole; BHT, 3,5-di-tert-butyl-4hydroxytoluene; EPR, electron paramagnetic resonance; ESR, electron spin resonance; LDL, low-density lipoprotein; MTMP, 4-methoxytetramethylphenol; PC, dimyristoylphosphatidylcholine; PMC, 2,2,5,7,8-pentamethyl-6-chromanol; TMP, 2,4,6-trimethylphenol; Trolox, 2-carboxy-2,5,7,8-tetramethyl-6-chromanol.

Abstract The action of vitamin E as an antioxidant has been the subject of extensive studies in the last 30 years. In this report, we summarize the results reported in numerous review articles as well as original papers. A bibliography pertinent to the topic is also reported.

Introduction Vitamin E, a generic term that includes all entities that exhibit the biological activity of natural D--tocopherol1, represents the major lipid-soluble antioxidant found in cells. The name originated in the early 1920s. Evans and Scott Bishop (1922) described a ‘substance X’ present in vegetable oil that was essential to maintain rat fertility. Pure -tocopherol was isolated from wheat-germ oil in 1936 (Evans et al., 1936). Because this compound permitted an animal to have offspring, it was named tocopherol from the Greek word tokos, meaning childbirth, and the verb phero, meaning to bring forth. To indicate the presence of an OH group in the molecule, ol was added to the ending. Its correct structure was given in 1938 (Fernholz, 1938) and its first synthesis (all-racemic form) was realized in the same year (Karrer et al., 1938). In nature, eight substances, which differ in the methylation site and side chain saturation, have been found to have vitamin E activity: -, -, - and -tocopherol and -, -, - and -tocotrienol. Tocopherols contain saturated phytyl side chains, and -, -, - and -tocotrienols have three double bonds in the side chain. The most active form of vitamin E, -tocopherol, is a 1

6-hydroxychroman derivative with methyl groups in positions 2, 5, 7 and 8 and a phytyl side chain attached at carbon 2. Notice that, due to the presence of three chiral centres at positions 2, and 4 and 8 of the phytyl tail, -tocopherol may exist under one of eight stereoisomeric forms. The most abundant isomer in nature is the RRR form. Also, the acetate and succinate derivatives of the natural tocopherols have vitamin E activity, as do synthetic tocopherols such as Trolox (2-carboxy-2,5,7,8-tetramethyl-6chromanol) and PMC (2,2,5,7,8-pentamethyl-6-chromanol) and their acetate and succinate derivatives (see Fig. 1.1). -Tocopherol is the most plentiful and the most biologically active of these compounds. The main function of vitamin E is to prevent the peroxidation of membrane phospholipids, and to avoid cell membrane damage through its antioxidant action. In the auto-oxidation of lipids (Howard, 1973), activated C–H bonds (generally those in a bisallylic position) are cleaved by peroxyl radicals, ROO
, to give a hydroperoxide molecule, ROOH, and a lipid radical, R
, which by reaction with O2 regenerates the peroxyl radical (Fig. 1.2). Phenolic antioxidants (AH), such as -tocopherol, are able to inhibit this reaction by scavenging the chain carrying peroxyl radicals, thus behaving as chain-breaking

The term ‘alpha’ and the symbol ‘’ are used interchangeably, as are the terms ‘beta’ and ‘’, ‘gamma’ and ‘’ and ‘delta’ and ‘’.

© CAB International 2007. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

3

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4

M. Lucarini and G. F. Pedulli

Fig. 1.1. Chemical structures of tocopherols, tocotrienols and some phenolic antioxidants.

antioxidants. The mechanism by which chain-breaking antioxidants inhibit the auto-oxidation of an oxidizable substrate (Niki, 1987) involves, at least in lipophilic phases, the transfer of the phenolic hydrogen to peroxyl radicals which propagate the radical chain oxidation (Fig. 1.2, Equation 5). This subtracts peroxyl radicals from the reaction medium and leads to a reduction of the rate of auto-oxidation. Since the phenoxyl radicals produced are resonance stabilized, they are usually persistent enough to capture a second peroxyl radical, so that every molecule of

antioxidant interrupts two oxidative chains (Fig. 1.2, Equation 6). In biological systems, many factors are important in determining the antioxidant activity of vitamin E: the chemical reactivity toward free radicals; its localization and concentration; its interaction with other antioxidants; and finally the fate of the antioxidant-derived radical. In this chapter, we mainly consider the first and the last aspects, while other chapters in this book will cover the remaining points.

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Overview of Antioxidant Activity of Vitamin E

Initiation Ri

R·

(1)

ROO·

(2)

ROOH + R·

(3)

Products

(4)

ROOH + A·

(5)

Products

(6)

Propagation R · + O2 ROO· + RH

kp

T Termination 2k t 2 ROO·

Inhibition steps ROO· + AH ROO· + A·

k inh

Fig. 1.2. Reaction scheme for the inhibited auto-oxidation of a hydrocarbon RH by chain-breaking antioxidants.

Chemical Reactivity Toward Radicals The effectiveness of a chain-breaking antioxidant, particularly one that must function in living organisms, depends on a number of factors including its reactivity towards peroxyl radicals. If a chain-breaking antioxidant is to be effective, a relatively small quantity must protect a much greater quantity of lipid. This implies that in Fig. 1.2 the rate constant for hydrogen atom transfer to the chainpropagating peroxyl radicals (reaction 5) must be much greater than that for propagation reaction 3, i.e. kinh >> kp (typical values of kp are generally comprised between 0.01 and 100/Ms). To compare quantitatively different chainbreaking antioxidants, it is essential to measure their kinh values under comparable conditions. This has been done more than 20 years ago by Ingold and co-workers (Burton et al., 1980, 1985; Burton and Ingold, 1986). The employed method simply involves the measurement of the rate of auto-oxidation of a pure hydrocarbon which has been inhibited with a phenolic derivative, under conditions where the rate of chain initiation (Ri) is known, the oxidation chain is not completely suppressed and the hydrocarbon is one for which kp has been determined (styrene or cumene). The auto-oxidation of a given substrate can be followed by measuring either the oxygen consumption or the formation of reaction products (hydroperoxides or their decomposition products, conjugated dienes in the case of lipid peroxidations) (RiceEvans et al., 1991). To initiate the substrate peroxidation, azo compounds (AIBN (2,2-azobis(isovaleronitrile)), AMVN (2,2-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2-azobis(2-amidinopropane)dihydrochloride)) which generate alkyl radicals at a convenient, known and

5

constant rate, are usually employed as radical initiators. Thermal decomposition of azo derivatives produces two alkyl radicals which rapidly react with oxygen (Maillard et al., 1983) to give peroxyl radicals. The latter radicals may attack lipids by abstracting an activated hydrogen atom to give a lipid radical which induces a sequence of propagation reactions to give lipid hydroperoxides. As an example, in Fig. 1.3 is reported the oxygen consumption trace observed during the auto-oxidation of 1.1 M cumene in tert-butanol in the presence of 4.6  102 M AIBN and 8.0  105 M -tocopherol, at 50°C. The two different slopes correspond to the inhibited and uninhibited autooxidation, the latter being observed when -tocopherol is completely consumed. The antioxidant disappears at a constant rate during the inhibition period and, when it is all consumed, the inhibition period is over and the oxidation proceeds as rapidly as observed in the absence of antioxidants. The time, t, at which this change is observed corresponds to the induction period which provides the value of Ri by means of the equation Ri = 2[-tocopherol]/t obtained under the assumption that every -tocopherol molecule (AH) leads to the disappearance of two peroxyl radicals (Equations 5 and 6). By measuring the rate of oxygen consumption during the inhibition period, it is possible to determine the inhibition constant, i.e. kinh, by means of the following equation d[O2]/dt = kp[RH]Ri/(n[AH]kinh) where n is the number of oxidation chains terminated per molecule of antioxidant. By using this method, Ingold and co-workers have determined the rate constants for H-atom abstraction at 37°C by peroxyl radicals from -tocopherol and a large number of structurally related phenols (Burton et al., 1985). The values of kinh for the four tocopherols and five synthetic antioxidants are reported in Table 1.1. The results summarized in Table 1.1 show that all tocopherols are exceptionally good chain-breaking antioxidants in vitro. In particular, -tocopherol has a larger rate constant for H-atom transfer to a peroxyl radical than any synthetic phenolic antioxidant, while -tocopherol and -tocopherol are only slightly less reactive. -tocopherol is about four time less reactive than -tocopherol. PMC is, within limits of error, as reactive as -tocopherol, indicating that the substitution of the phytyl chain with a methyl group does not change the reactivity of 6-hydroxychromanic derivatives in homogeneous solution (Niki et al., 1985). Also in the case of tocotrienols, the reactivities decrease in the order  >  ≈  > -tocotrienol, and the corresponding tocotrienols and tocopherols have similar reactivities toward peroxyl radicals (Suarna et al., 1993; Yoshida, 2003). Comparison of the 2,4,6trimethylphenol (TMP) and 3,5-di-tert-butyl-4-hydroxytoluene (BHT) rate constants indicates that phenols having two tert-butyl substituents in the ortho positions are less reactive than the corresponding 2,6-dimethylphenols. The factors responsible for the magnitude of the rate of inhibition, kinh, are the steric crowding about the hydroxyl group and the bond dissociation enthalpy (BDE) of the ArO–H bond cleaved in the inhibition process. As far as the first point is concerned, phenols having two tert-butyl substituents in the ortho positions are less reactive,

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M. Lucarini and G. F. Pedulli

Table 1.1. Rate constant for H-atom transfer to a peroxy radical and BDEs of different phenolic derivatives.

2.0 t

[O2 × 103 M

Phenol -Tocopherol - Tocopherol - Tocopherol - Tocopherol PMC MTMP TMP BHT BHA

1.5

1.0

0.5

kinh (1/Ms) (30°C)a

BDE ArO-H (kcal/mol)

3.2  106 1.3  106 1.4  106 4.4  105 3.8  106 3.9  105 8.5  104 1.4  104 1.1  105

78.2b 80.7c 80.7c 81.7c 78.2b 81.9b 82.7b 81.0 b 78.3 b

aFrom

Burton et al. (1985). Lucarini et al. (1996). cEstimated from additivity rules. bFrom

0

0

2000

4000

6000

Time(s) Fig. 1.3. Oxygen consumption observed during the auto-oxidation of 1.1 M cumene in tert-butanol in the presence of 4.6  102 M AIBN and 8.0  105 M -tocopherol, at 50°C. The rate of oxygen consumption has been measured in a closed tube using a method based on the variation of the EPR spectral line width of a stable nitroxide radical dissolved in solution (Pedulli et al., 1996).

because of the steric hindrance to the approach of peroxyl radicals to the hydroxyl group. Since steric hindrance to abstraction of the phenolic hydrogen by ROO
must be very similar in all tocopherols, the difference in the kinh values for these phenols must be due mainly to differences in the exothermicities of reaction 5, i.e. the O–H bond in -tocopherol must be weaker than in the other tocopherols. Thus attention has been focused in the literature on the energetic of the phenolic bond and on the effect on it of the number, nature and position of the aromatic ring substituents. Low values of BDE are important in determining a good antioxidant activity not only from a kinetic but also from a thermodynamic point of view. In fact, since the bond formed in the first step of the inhibition reaction (the O–H bond of the hydroperoxide) is characterized by a BDE of 88 kcal/mol (Lucarini et al., 1996), only phenols with oxygen–hydrogen BDE values - and -tocopherol; - and -CEHC < - and -tocopherol

Yoshida and Niki (2002)

Cell culture

LPS-stimulated nitrite production LPS-stimulated microglial nitrite efflux Microglial prostaglandin E2 production

-CEHC inhibits - and -CEHC inhibit - and -CEHC inhibit

Grammas et al. (2004)

Animal

Rats

-CEHC protects against metal-induced nephrotoxicity

Appenroth et al. (2001)

LPS = lipopolysaccharide.

exhibited similar activities toward radicals and exerted the same antioxidant activities against lipid peroxidation in organic solution as the corresponding parent compound, with the partition coefficient decreasing from -tocopherol to -tocopherol to -CEHC to -CEHC to Trolox; in accordance with their more hydrophilic nature, - and -CEHC were shown to scavenge aqueous radicals more efficiently but to inhibit membrane lipid peroxidation less efficiently than the parent compounds (Table 2.2), and to exert a minimal pro-oxidant effect in the presence of cupric ion (Yoshida and Niki, 2002).

Cell culture studies The first study investigating the metabolism of -tocopherol to carboxychroman metabolites in cell culture used HepG2 cells incubated with a medium containing fetal bovine serum enriched with RRR--tocopherol (Parker and Swanson, 2000). The analysis of -tocopherol metabolites released into the cell culture medium by gas chromatography–mass spectrometry (GC–MS) revealed the cellular secretion of -CEHC as well as of the 5-carboxychroman analogue of -CEHC, 2,7,8-trimethyl-2(-carboxymethylbutyl)-6-hydroxychroman (Parker and Swanson, 2000), now called -CMBHC or 5-carboxychromanol. As mentioned in the metabolism section, all further intermediates of vitamin E metabolism have been identified in HepG2 cells. Also, the first hints for functions of metabolites have been obtained from cell culture studies. -CEHC decreased prostaglandin E2 synthesis in lipopolysaccharide-stimulated RAW264.7 macrophages and interleukin-1 (IL-1)-treated A549 human epithelial cells due to its inhibition of cyclooxygenase-2 (COX-2) activity, suggesting an antiinflammatory activity of -CEHC at physiological concentrations (for a review, see Jiang et al., 2001) (Table 2.2). This effect did not depend on antioxidant activity. -CEHC suppressed tumour necrosis factor- (TNF-)- or bacterial lipopolysaccharide-stimulated nitrite production in rat aortic endothelial cells and mouse microglial cultures (Grammas et al., 2004) (Table 2.2). Both - and CEHC inhibited lipopolysaccharide-stimulated microglial nitrite efflux. In the same study, - and -CEHC inhibited

microglial prostaglandin E2 production (Table 2.2), but neither - nor -tocopherol was effective in inhibiting cytokine-stimulated inflammatory processes (Grammas et al., 2004), indicating that the anti-inflammatory effects of tocopherols and tocopherol metabolites are highly cell type, stimulus and end-point dependent. Animal studies In rats, after a single oral administration of 2 mg (20 Ci) of SRR- and RRR--[5-methyl-14C]tocopherol, no difference in the recovery of radioactivity until 12 h after administration was observed in the liver. In contrast, in other tissues, radioactivity derived from RRR--tocopherol was clearly higher than that derived from SRR--tocopherol after 12 h (Kaneko et al., 2000). In the same study, total faecal excretions of SRR- and RRR--tocopherol were 87.6 and 83.0%, respectively; about 1.3% of the total urinary radioactivity was found to be RRR--tocopherol-derived -CEHC. If 83% is not absorbed, this is 80 years after the discovery of vitamin E, the definition of its biological role remains a challenge.

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Kaneko, K., Kiyose, C., Ueda, T., Ichikawa, H. and Igarashi, O. (2000) Studies of the metabolism of alpha-tocopherol stereoisomers in rats using [5methyl-(14)C]SRR- and RRR-alpha-tocopherol. Journal of Lipid Research 41, 357–367. Kliewer, S.A. (2003) The nuclear pregnane X receptor regulates xenobiotic detoxification. Journal of Nutrition 133, 2444S–2447S. Kluth, D., Landes, N., Pfluger, P., Müller-Schmehl, K., Weiss, K., Bumke-Vogt, C., Ristow, M. and Brigelius-Flohé, R. (2004) Modulation of CYP3a11 expression by -tocopherol but not -tocotrienol in mice. Free Radical Biology and Medicine 38, 507–514. Landes, N., Pfluger, P., Kluth, D., Birringer, M., Rühl, R., Bol, G.F., Glatt, H. and Brigelius-Flohé, R. (2003) Vitamin E activates gene expression via the pregnane X receptor. Biochemical Pharmacology 65, 269–273. Landi, M.T., Sinha, R., Lang, N.P. and Kadlubar, F.F. (1999) Human cytochrome P4501A2. International Agency for Research on Cancer (IARC) Scientific Publications 148, 173–195. Lodge, J.K., Ridlington, J., Leonard, S., Vaule, H. and Traber, M.G. (2001) - and -tocotrienols are metabolized to carboxyethyl-hydroxychroman derivatives and excreted in human urine. Lipids 36, 43–48. Morinobu, T., Yoshikawa, S., Hamamura, K. and Tamai, H. (2003) Measurement of vitamin E metabolites by high-performance liquid chromatography during high-dose administration of alpha-tocopherol. European Journal of Clinical Nutrition 57, 410–414. Müller-Schmehl, K., Beninde, J., Finckh, B., Florian, S., Dudenhausen, J.W., Brigelius-Flohé, R. and Schuelke, M. (2004) Localization of tocopherol transfer protein in trophoblasts, fetal capillaries’ endothelium and amnion epithelium of human term placenta. Free Radical Research 38, 413–420. Ouahchi, K., Arita, M., Kayden, H., Hentati, F., Ben Hamida, M., Sokol, R., Arai, H., Inoue, K., Mandel, J.L. and Koenig, M. (1995) Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein. Nature Genetics 9, 141–145. Parker, R.S. and Swanson, J.E. (2000) A novel 5-carboxychroman metabolite of gamma-tocopherol secreted by HepG2 cells and excreted in human urine. Biochemical and Biophysical Research Communications 269, 580–583. Podda, M., Weber, C., Traber, M.G. and Packer, L. (1996) Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols, and ubiquinones. Journal of Lipid Research 37, 893–901. Polidori, M.C., Stahl, W., Eichler, O., Niestroj, I. and Sies, H. (2001) Profiles of antioxidants in human plasma. Free Radical Biology and Medicine 30, 456–462. Polidori, M.C., Mecocci, P., Stahl, W. and Sies, H. (2003) Cigarette smoking cessation increases plasma levels of several antioxidant micronutrients and improves resistance towards oxidative challenge. British Journal of Nutrition 90, 147–150. Pope, S.A., Burtin, G.E., Clayton, P.T., Madge, D.J. and Muller, D.P. (2002) Synthesis and analysis of conjugates of the major vitamin E metabolite, alpha-CEHC. Free Radical Biology and Medicine 33, 807–817. Radosavac, D., Graf, P., Polidori, M.C., Sies, H. and Stahl, W. (2002) Tocopherol metabolites 2,5,7,8-tetramethyl-2-(2-carboxyethyl)-6hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2-carboxyethyl)-6-hydroxychroman (gamma-CEHC) in human serum after a single dose of natural vitamin E. European Journal of Nutrition 41, 119–124. Raucy, J.L. (2003) Regulation of CYP3A4 expression in human hepatocytes by pharmaceuticals and natural products. Drug Metabolism and Disposition 31, 533–539. Saito, H., Kiyose, C., Yoshimura, H., Ueda, T., Kondo, K. and Igarashi, O. (2003) Gamma-tocotrienol, a vitamin E homolog, is a natriuretic hormone precursor. Journal of Lipid Research 44, 1530–1535. Schuelke, M., Elsner, A., Finckh, B., Kohlschütter, A., Hübner, C. and Brigelius-Flohé, R. (2000) Urinary alpha-tocopherol metabolites in alphatocopherol transfer protein-deficient patients. Journal of Lipid Research 41, 1543–1551. Schultz, M., Leist, M., Petrzika, M., Gassmann, B. and Brigelius-Flohé, R. (1995) Novel urinary metabolite of alpha-tocopherol, 2,5,7,8tetramethyl-2(2-carboxyethyl)-6-hydroxychroman, as an indicator of an adequate vitamin E supply? American Journal of Clinical Nutrition 62 (Suppl. 6), 1527S–1534S. Sen, C.K., Khanna, S., Roy, S. and Packer, L. (2000) Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60(cSrc) kinase activation and death of HT4 neuronal cells. Journal of Biological Chemistry 275, 13049–13055. Simon, E.J., Eisengart, A., Sundheim, L. and Milhorat, A.T. (1956) The metabolism of vitamin E II. Purification and characterization of urinary metabolites of alpha-tocopherol. Journal of Biological Chemistry 221, 807–817. Smith, K.S., Lee, C.L., Ridlington, J.W., Leonard, S.W., Devaraj, S. and Traber, M.G. (2003) Vitamin E supplementation increases circulating vitamin E metabolites tenfold in end-stage renal disease patients. Lipids 38, 813–819. Sontag, T.J. and Parker, R.S. (2002) Cytochrome P450 -hydroxylase pathway of tocopherol catabolism. Journal of Biological Chemistry 277, 25290–25296. Stahl, W., Graf, P., Brigelius-Flohé, R., Wechter, W. and Sies, H. (1999) Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7,8tetramethyl-2-(2-carboxyethyl)-6-hydroxychroman and 2,7,8-trimethyl-2-(2-carboxyethyl)-6-hydroxychroman in human serum. Analytical Biochemistry 275, 254–259. Stahl, W., van den Berg, H., Arthur, J., Bast, A., Dainty, J., Faulks, R.M., Gärtner, C., Haenen, G., Hollman, P., Holst, B., Kelly, F.J., Polidori, M.C., Rice-Evans, C., Southon, S., van Vliet, T., Viña-Ribes, J., Williamson, G. and Astley, S.B. (2002) European research on the functional effects of dietary antioxidants (EUROFEDA): Bioavailability and metabolism. Molecular Aspects of Medicine 23, 3–100. Swanson, J.E., Ben, R.N., Burton, G.W. and Parker, R.S. (1999) Urinary excretion of 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans. Journal of Lipid Research 40, 665–671. Traber, M.G. and Arai, H. (1999) Molecular mechanisms of vitamin E transport. Annual Reviews of Nutrition 19, 343–355. Wagner, K.H., Kamal-Eldin, A. and Elmadfa, I. (2004) Gamma-tocopherol – an underestimated vitamin? Annals of Nutrition and Metabolism 48, 169–188. Wechter, J.W., Kantoci, D., Murray, E.D., D’Amico, D.C., Jung, M.E. and Wang, W.-H. (1996) A new endogenous natriuretic factor: LLU-. Proceedings of the National Academy of Sciences of the USA 93, 6002–6007. Yap, S.P., Yuen, K.H. and Wong, J.W. (2001) Pharmacokinetics and bioavailability of alpha-, gamma- and delta-tocotrienols under different food status. Journal of Pharmacy and Pharmacology 53, 67–71. Yoshida, Y. and Niki, E. (2002) Antioxidant effects of alpha- and gamma-carboxyethyl-6-hydroxychromans. Biofactors 16, 93–103. Zingg, J.M. and Azzi, A. (2004) Non-antioxidant activities of vitamin E. Current Medical Chemistry 11, 1113–1133.

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3 PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds Thomas Rosenau University of Natural Resources and Applied Life Sciences Vienna, Department of Chemistry, Muthgasse 18, A-1190 Vienna, Austria

Abbreviations: ABMV, 2,2-azobis(2,4-dimethylvaleronitrile); AIBN, azobis(isobutyronitrile); DDQ, 2,3-dichloro-5,6-dicyano-parabenzoquinone; DFT, density functional theory; ED, ethano-dimer of -tocopherol, 1,2-di(5--tocopheryl)-ethane; ENDOR, electron nuclear double resonance; EPR, electron paramagnetic resonance; HOMO, highest occupied molecular orbital; IR, infrared spectroscopy; LUMO, lowest unoccupied molecular orbital; NMR, nuclear magnetic resonance; oQM, ortho-quinone methide; PMC, 2,2,5,7,8-pentamethylchroman-6-ol; pQ, para-quinone; SD, spiro-dimer of -tocopherol; SIBL, strain-induced bond localization; ST, spiro-trimer of -tocopherol; THF, tetrahydrofuran; TMC, 2,2,7,8-tetramethylchroman-6-ol; TMHQ, trimethylhydroquinone.

Abstract 2,2,5,7,8-Pentamethylchroman-6-ol (PMC, 1) is the most frequently used model compound for -tocopherol, in which the isoprenoid side chain of the tocopherol is replaced by a methyl group. The antioxidation chemistry of PMC equals that of its parent compound completely, thus PMC is mainly used for studies in vitro and for all investigations regarding reaction mechanisms, product analysis, oxidant studies or model oxidative systems. After a brief survey of the advantages of employing the model compound PMC in reaction studies instead of -tocopherol itself, the first part of this chapter focuses on the oxidation chemistry of PMC. The primary oxidation intermediates – the chromanoxyl radical as the product of a one-electron process as well as a chromanoxylium cation and an orthoquinone methide as products of two-electron oxidations – are presented, and literature reports are critically evaluated. The oxidation chemistry of PMC is strongly dependent on the oxidant and the solvent used. Products and mechanisms of oxidations under different reaction conditions are reported and discussed. Two final schemes summarize the oxidative processes proceeding in either protic or inert apolar solvents. The second part of the chapter covers PMC derivatives modified at different positions of the chroman skeleton, their syntheses and special properties. In a third part, special PMC-related derivatives are communicated. First, the oxidation behaviour of 3-oxachromanols, a class of PMC-derived antioxidants, is discussed, which is governed by the amount of co-reacting water present, and exhibits either similarities to or differences from that of PMC, depending on the chemical structure. Secondly, the ‘Siamese twin’-PMC represents a model compound that possesses two interlinked chroman units, useful for mechanistic and theoretical studies, and for the construction of supramolecular assemblies of antioxidants. Finally, PMC derivatives with a different ring strain in the annulated ring are presented as they are key compounds in studies on the regioselectivity of oxidative changes in PMC and the related tocopherol system.

PMC – General Facts The tocopherols are highly effective antioxidants thoroughly optimized by nature, and appear to be ‘ideal’ natural antioxidants. So why is a model compound

frequently used instead, which – like any model – must have a limited functionality? 2,2,5,7,8-Pentamethylchroman-6-ol (PMC, 1) is the standard model compound for -tocopherol, the main component of vitamin E. It represents a ‘shortened’ or

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T. Rosenau

‘truncated’ -tocopherol, the isoprenoid side chain of the vitamin being replaced by a methyl group. Although lacking the side chain, the antioxidation chemistry of PMC equals that of its parent compound almost completely, since the chromanol system and its substitution pattern, which are responsible for the antioxidative action, are entirely unchanged in PMC. In contrast to the vitamin itself which is used for in vivo experiments, the model is mainly used for studies in vitro and for all investigations regarding reaction mechanisms, oxidant studies or model oxidative systems. While the isoprenoid side chain appears indispensable in vivo, it is less advantageous – if not an obstacle – in many in vitro experiments, especially if it comes to analysis of the mixture or separation and identification of the reaction products. Historically – and also today – PMC has been used to study the reactions of vitamin E without having to struggle with the inherent difficulties of tocopherol analytics. In addition, PMC was easier to synthesize and thus readily available in pure form. In summary, the reason for the wide usage of PMC instead of tocopherol is not a changed chemical behaviour. It is to be seen as advantageous concerning the ‘practical’ or ‘applied’ side of the usage: benefits in availability, handling, reaction work-up and general analytics, some of which are listed in Table 3.1 in comparison with -tocopherol. PMC is generally synthesized by condensation of trimethylhydroquinone (TMHQ) with a C5 unit that provides the three carbons of the alicyclic ring and the two methyl groups in the 2-position. The synthesis must accomplish bond formation between O-1 and C-2 in a formal etherification as well as bond formation between C-4 and C-4a in a Friedel-Crafts alkylation/acylation step. Possible precursors thus offer a hydroxyl group at C-2 or a double bond extending from C-2, and a hydroxyl group,

halogen or double bond at C-4, 3-methyl-but-2-en-1-ol giving optimal results (see for instance Nilsson et al., 1968c) (Fig. 3.1). The condensation reaction is catalysed either by Bronstedt acids (H2SO4, phosphoric acid) or better by Lewis acids (FeCl3, AlCl3 or BF3). It is also possible to introduce C-4 as a keto function by using a carboxylic acid or carboxylic halide as precursor in a Friedel-Crafts acylation. The primarily formed chroman-4one is then reduced in a subsequent step, preferably with a metal/acid system (Zn/HCl, Mg/HOAc, etc.). Nowadays, PMC is a commercial product with a relatively low price which is thus widely available: an indepth discussion of synthetic approaches and their difference appears unnecessary.

Oxidation Reactions of PMC Primary oxidation products of PMC and their general reactions Oxidation chemistry of PMC (1) generally involves three key intermediates (2–4), which are formed according to the respective reaction conditions used, their intermediacy being especially dependent on the oxidant employed (twoelectron or one-electron oxidant) and the solvent chosen (polar or apolar, aprotic or protic, dry or containing traces of water), see Fig. 3.2. The chromanoxyl radical 2 is the primary homolytic (one-electron, radical) oxidation product of PMC. Its formation and occurrence are comprehensively supported and confirmed by electron paramagnetic resonance (EPR) (Boguth and Niemann, 1971; Mukai et al., 1982; Matsuo and Matsumoto, 1983; Matsuo et al., 1983; Tsuchija et al., 1983; Burton et al., 1985) and electron nuclear double resonance (ENDOR) (Mukai et al., 1981) experiments.

Table 3.1. Comparison of -tocopherol with its model compound PMC. PMC

-Tocopherol

One-step synthesis, easily possible in the lab

Complex multi-step synthesis to build up the correct stereochemistry

No stereo centres, no changes in stereochemistry or occurrence of other stereoisomers possible

Three stereo centres; changes in stereochemistry can occur

White, crystalline solid: easy to handle, to weigh and to dose

Colourless to yellow oil/wax: handling, weighing and dosage less convenient

Relatively stable to air and light

Sensitive towards air and light

Products readily separable by chromatographic techniques

Products more difficult to separate and analyse due to their high lipophilicity

Most reaction products are crystalline; crystal structure analysis can be performed

Reaction products are oils

NMR spectra of derivatives easy to analyse

Region below 1.7 p.p.m. (1H) and 30 p.p.m. (13C) crowded due to the side chain

Derivatives can be readily ionized and analysed in (hyphenated) mass spectrometry techniques

Mass spectrometric analysis more difficult (higher molecular weight, lipophilicity)

Good solubility in polar aprotic, polar protic and apolar solvents

Good solubility in apolar solvents, limited solubility in polar aprotic and protic solvents

Miscibility with other reagents good due to the good solubility characteristics

Miscibility with other (especially polar) reagents difficult, application of auxiliaries (co-solvents, solubilization in micelles)

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

23

Fig. 3.1. General synthesis approach to PMC.

Fig. 3.2. Primary key intermediates in the oxidation chemistry of PMC.

The spin density of chromanoxyl 2, a classical phenoxyl radical, is mainly concentrated at oxygen O-6, which is the major position for coupling with other C-centred radicals, leading to chromanyl ethers 5. Also at the ortho- and parapositions of the aromatic ring, the spin density is increased. Coupling with other radicals, especially O-centred ones, proceeds mainly at the para-position (C-8a) (Fig. 3.3) leading to differently 8a-substituted chromanones 6 (Fig. 3.4). The ‘disproportionation’ of the chromanoxyl radical is actually a multi-step process, but not just an alteration of oxidation states as the term suggests. It involves radical coupling of the O-centred form of 2 with its C-8a-centred resonance form to give an 8a-chromanyl-chromanone of the general structure 6, which undergoes 1,4-elimination involving C-5a, forming ortho-quinone methide (oQM) 3 and a molecule of regenerated PMC. Alternatively, one molecule of 2 abstracts an H atom at C-5a of another molecule of 1. The net outcome is the observed ‘disproportionation’ of two chromanoxyl radicals 2 into phenol 1 (reduction) and a quinoid structure 3 (oxidation). In several instances, the ‘chromanol methide radical’ 7 has been inaccurately described in the literature as being a resonance form of the primary chromanoxyl radical (Goodhue and Risley, 1964; Inglett and Mattill, 1964; Skinner, 1964; Fujimaki et al., 1970; Kamal-Eldin and Appelqvist, 1996), and as a consequence the 5a-position was presented as a position much favoured for radical coupling. However, radical 7 is rather a tautomer of chromanoxyl 2, being formed by a chemical reaction, namely a 1,4-shift of one 5a-proton to the 6-oxygen (Fig. 3.5). Even though this process would proceed via a cyclic transition state according to a concerted mechanism, computations on the density functional theory (DFT) level

(B3LYP/6–31G*) readily showed that radical 7 was energetically much less favoured (by 78.4 kJ/mol) than 2. Computations and theoretical considerations provide no evidence that in the aromatic radical 2 position 5a should be favoured over the other ortho-position 7a; the radical centred at C-5a was calculated to be only insignificantly more stable (by 1.5 kJ/mol) than the one centred at C-7a. In contrast, in quinoid structures, the 5a methyl group is clearly energetically favoured over C-7a and C-8b, cf. oQM 3 (see later). Also EPR provided no direct evidence for the presence of 7 in higher concentrations, by giving a neat spectrum of 2. Thus, radical reactions of PMC involve mainly 2; the contribution of its tautomer 7 is less important. Heterolytic (two-electron, ionic) oxidation of 1 or, alternatively, further one-electron loss from the primary radical 2, affords chromanoxylium cation 4 with its positive charge mainly localized at C-8a. Cation 4 is stabilized by resonance so that a positive partial charge also results at C-5 and C-7, where nucleophilic attack is consequently facilitated. Chromanoxylium cation 4 preferably adds nucleophiles in the 8a position producing 8a-substituted tocopherones 6, similar in structure to those obtained by radical recombination between C-8a of chromanoxyl 2 and coreacting radicals (Fig. 3.6). Addition of a hydroxyl ion to 4, for instance, results in the 8a-hydroxy derivative (30) which in a subsequent step gives the para-quinone (pQ) 8. The reactivity of 4 can be displayed by means of the lowest unoccupied molecular orbital (LUMO), i.e. the orbital which receives electrons upon reaction and which is thus large at atoms with positive partial charge where nucleophiles attack (Fig. 3.7).

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Fig. 3.3. Spin density in the chromanoxyl radical 2, displayed as grey surfaces on the tube model (left) and mapped on the bond density at 0.08 a.u. (right, shading indicates atoms of increasing spin density). Calculated by the Spartan package at DFT level (B3LYP/6–311G*+).

Fig. 3.4. Resonance forms and general reactions of chromanoxyl radical 2.

Fig. 3.5. Tautomerism leading from O-centred radical 2 to C-centred radical 7.

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

25

Fig. 3.6. Reactions and products of chromanoxyl radical 2, ortho-quinone methide 3 and chromanoxylium cation 4.

The second main two-electron oxidation product of PMC, oQM 3, is formed either from 4 by proton loss at C-5a, or by a further single-electron oxidation step from 2 with proton loss from C-5a. In aqueous or protic media, formation of 4 is preferred; in apolar media, aprotic media or media containing small amounts of water, only the oQM 3 is formed. In contrast to the aromatic system of PMC and the derived radicals, position 5a is favoured in the derived quinoid system, explainable by the model of strain-induced bond localization (SIBL), see later. Thus, the regioselective involvement of position 5a in the deprotonation of 4 is now strongly energetically favoured over position 7a, since the system is now quinoid and no longer aromatic. The resulting oQM 3 undergoes the usual subsequent reactions, which can be generally divided into three categories: (i) reduction back to the parent phenol; (ii) 1,4-addition of nucleophiles to C-5a and O-6; and (iii) hetero-Diels-Alder reaction with inverse electron demand, the oQM itself reacting as the electron-deficient diene. All three reaction types restore aromaticity, which is the driving force, starting from a cyclohexadienone structure. According to frontier orbital theory, the reactivity of oQM 3, especially in cyclic reactions, can be explained and displayed by means of its highest occupied molecular orbital (HOMO) and its LUMO (see Fig. 3.8). Thus, dimerization to spiro-dimer (SD) 11 is a typical reaction of 3. It proceeds in the case of no other co-reactant than the oQM ‘itself ’ present, the reaction being a Diels-Alder reaction with C-5a–C-5–C-6–O-6 of one oQM reacting as the hetero-analogous diene, and C-5a–C-5 of the second, co-reacting oQM as the dienophile (Fig. 3.6). Also addition of nucleophiles to 3 restores aromaticity, and leads to 5a-

Fig. 3.7. LUMO of the chromanoxylium cation 4 displayed on a tube model. The ‘size’ of the orbitals correlates with the positive partial charge and with centres being attacked by nucleophiles. Calculated by the Spartan package at DFT level (B3LYP/6–311G*+).

substituted chromanols 9 which are, however, formed in an ionic process. As the same compounds would have been formed by recombination of the C-centred radical 7 with co-reacting radical species, it becomes understandable why in the (especially older) literature – based on product analysis – position 5a has frequently been described as favoured, readily undergoing radical

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Fig. 3.8. HOMO (left) and LUMO (right) of ortho-quinone methide (3) displayed on a tube model. Position and ‘size’ of the orbitals explain the reactivity in typical reactions, e.g. Diels-Alder reactions. Calculated by the Spartan package at DFT level (B3LYP/6–311G*+).

coupling (Fig. 3.6). However, most alleged radical coupling products at C-5a are actually products formed heterolytically involving oQM 3.

Oxidation reactions of PMC – products and mechanisms PMC reacts with the radical initiator azobis(isobutyronitrile) (AIBN) in inert solvents, such as benzene or dioxane, mainly to (1-cyano-1-methyl)ethyl chroman-6yl ether (see general structure 5), the coupling product of isobutyronitrile radicals and the chromanoxyl radical (Skinner, 1964). The reaction with 2,2-azobis(2,4dimethylvaleronitrile) (ABMV) proceeds analogously. Also in most studies aimed to determine its antioxidative action in lipids, PMC is converted into PMC ethers via coupling of chromanoxyl 2 at O-6. When incubated with a linoleic acid hydroperoxide, (9Z, 11E)-13hydroperoxy-9,11-octadecadienoic acid, and Fe(II) as

inducer of the decomposition in a solvent mixture containing methanol, PMC afforded four PMC ethers of general structure 5, differing in the position of coupling to the lipid model (Fig. 3.9). The composition of the product mixture was largely independent of whether anaerobic or aerobic conditions were used (Kaneko and Matsuo, 1985). Auto-oxidation processes, mimicked by incubation of PMC with linoleic acid under aerobic conditions, also afforded different chroman-6-yl ethers as the main coupling products (Peers and Coxon, 1983; Kamal-Eldin and Appelqvist, 1996). The reaction of PMC with dibenzoyl peroxide in inert solvents was described to lead to a mixture of the 5asubstituted benzoyl derivative 10 and pQ 8 (Inglett and Mattill, 1955) (see Fig. 3.10). If the oxidation was carried out with tert-butyl hydroperoxide or di-tert-butyl diperoxyoxalate in benzene or acetonitrile as inert solvents, the SD (11), the ethanodimer (ED, 12) and the spiro-trimer (ST, 13) – the latter

Fig. 3.9. Reaction products of PMC with a linoleic acid hydroperoxide.

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

formed by reaction of the SD 11 with one molecule of oQM 3 in a hetero-Diels-Alder reaction – were obtained (Matsuo et al., 1989) (see Fig. 3.11). In addition, 8a(hydro)peroxides 14 were isolated. Also 8a(hydro)peroxide derivatives additionally epoxidized in the 4a,5-position (15) were found (Matsumoto et al., 1986b). With an increasing concentration of alkoxyl, peroxyl, hydroperoxyl or other radicals, the corresponding 8acoupling products of 2 with these radicals become more and more dominant (Suarna and Southwell-Keely, 1988, 1989; Suarna et al., 1992). In their absence, the other products derived from the primary tocopheroxyl radical and from the intermediate oQM 3, such as 11–13, dominate (Nilsson et al., 1968a, b). In the presence of radical initiators, the corresponding 6-O-derivatives as radical coupling products of the chromanoxyl radical 2 with initiator-derived radicals dominate, as discussed above. The tocopheroxyl radical was postulated to ‘disproportionate’ easily into ED 12 (Nilsson et al., 1968b; Rousseau-Richard et al., 1988). Even though, theoretically, recombination of two molecules of 7 would afford 11, this mechanism is questionable as the radical concentration of the C-centred radical 7 is far too low to account for the relatively large amounts of ED 11 formed. This product is

27

thus not the result of 5a–5a coupling, but is formed by reduction of the SD, which oxidizes one molecule of 1 to the oQM 3 in turn. The latter reacts with another SD to form the ST, so that in the overall process two molecules of SD 11 and one molecule of PMC (1) react to form the ST 13 and the ED 12 (Yamauchi et al., 1988, 1989a, b). Oxidation of 1 with tert-butyl hydroperoxide in chloroform – but in the presence of ethanol – afforded a complex product mixture containing four main components (11 and 16–18) (Suarna et al., 1988a, b; Suarna and Southwell-Keely, 1989, 1991) (see Fig. 3.12). The SD 11 was formed by dimerization of the intermediate oQM 3. The oQM intermediate also reacted with pQ 8 in an inverse hetero-Diels-Alder reaction to give the observed tetracycle 16. It should be noted that 16 is not an addition or oxidation product of the ED 12 or the SD 11, as described in the literature. Both 11 and 12 possess a C2 unit, formed by the two ‘former’ C-5a, connecting the two chromanol moieties, whereas 16 was produced by bond formation between C-5a and C-5, thus having a different carbon skeleton. 5a-Ethoxy-PMC (17) is the 1,4-addition product of ethanol to the intermediate oQM 3. 5-Formyl-PMC (18) was described as the product of further oxidation of 17. As a minor product, the addition product (19) between

Fig. 3.10. Main products of the reaction between PMC and benzoyl peroxide.

Fig. 3.11. Reaction of PMC with (hydro)peroxyl radicals in inert solvents.

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oQM 3 and PMC reacting as the alcohol was found (Fig. 3.13). Alternatively, 19 can be seen as formally formed by radical coupling of 2 and 7. Furthermore, the 5a-hydroxy derivative of PMC (20) – formed from traces of water – and its quinoid oxidation product 21 were found. In the presence of alcohols other than ethanol, ranging from methanol to cholesterol, the corresponding 5a-alkoxy-PMC derivatives 22 were formed in fair yields, and also by addition of the alcohol to the intermediate oQM 3 (Suarna and Southwell-Keely, 1988, 1989; Suarna et al., 1992). Reaction of PMC with potassium superoxide under oxygen in tetrahydrofuran (THF) afforded two monooxygenated products (23, 24) with cyclohexadienone structures (Clennan et al., 1986), the latter being formed by 1,2-migration of the 5a-methyl substituent as shown using isotopically labelled material, such as the 5a-CD3 and 7aCD3 derivatives (Matsumoto et al., 1986a) (Fig. 3.14). By means of isotopic labelling experiments with 18O2, it was demonstrated that the oxygen atom entering position 5a originates from molecular dioxygen (Matsumoto et al., 1981b). In an earlier account, the oxidation by O2 in THF in the presence of potassium tert-butoxide was described to afford the same two products 23 and 24 in addition to a bis-oxygenated cyclohexene 25 carrying an exo-methylene

group (Matsumoto et al., 1981a). In a later report, an additional trioxa-tricyclus 26 was found (Matsumoto et al., 1993). Another reaction with oxygen and solubilized superoxide radicals in aqueous media provided a similar product mixture, consisting of bis-epoxide 27 and the two mono-epoxides (28, 29) (Matsuo et al., 1987) (see Fig. 3.15). The three compounds are derived from pQ 8; the first one is the bis(epoxide) of the hemiketal para-quinone precursor 30, and the latter two are mono-epoxidation products of 8. Similar products were obtained upon photo-oxidation with visible light using sensitizers, such as proflavin or methylene blue (Grams et al., 1972). Oxidation of PMC by FeCl3 or RuCl3/H2O2 produces the corresponding pQ 8 in high yields or even quantitatively (Ito et al., 1983). This oxidation in aqueous media proceeds via the unstable 8a-hydroxy-chromanone 30 (Dürckheimer and Cohen, 1962), which rearranges into the pQ 8 (Fig. 3.16). Compound 30 is the intramolecular semi-ketal of 8. Experiments with several reductants showed that reduction back to PMC is easier from the 8a-hydroxy-derivative 30 than from the pQ 8, particularly under mildly acidic conditions (Kohar and Southwell-Keely, 2002). Reaction of PMC with nitric acid produces a red-

Fig. 3.12. Reaction of PMC with (hydro)peroxyl radicals in inert solvents in the presence of alcohols (ethanol): main products.

Fig. 3.13. Reaction of PMC with (hydro)peroxyl radicals in inert solvents in the presence of alcohols (ethanol): minor reaction products.

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

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Fig. 3.14. Oxygenated reaction products of PMC from reactions in inert solvents.

Fig. 3.15. Oxygenated reaction products of PMC from reactions in aqueous media.

Fig. 3.16. Two-electron oxidation of PMC affording para-quinone 8.

Fig. 3.17. Formation of a red-coloured ortho-quinone by HNO3 oxidation of PMC.

coloured ortho-quinone 31 (Smith et al., 1939; Kohar et al., 1993), the short-chain analogue of the tocopherol derivative -tocored (Fig. 3.17). It was shown by deuteration experiments that the reaction involved a cycohexadienone intermediate similar to 23, from which the 5a-substituent was transferred to 6-O in a [1,3]-shift to give a 5-hydroxy-6-methoxy-PMC intermediate, which was immediately oxidized to ortho-quinone 31 under the release of the C-5a-methyl as methanol (Rosenau et al., 1997). Reaction of PMC with elemental iodine under alkaline conditions caused the formation of 4-methoxy-PMC (33) via the 8a-methoxy-quinone derivative 32 according to a multi-step rearrangement mechanism (Omura, 1989). The

Fig. 3.18. Reaction of PMC with iodine in alkaline media.

4-methoxy derivative 33 eliminated methanol upon acidic treatment to give 3,4-dehydro-PMC (34), which was thus directly accessible from PMC by alkaline iodine/acid treatment (Fig. 3.18). The action of singlet oxygen on PMC was described to cause formation of the 8a-hydroperoxy-chromenone 14 as the main product (Grams, 1971; Gorma et al., 1984; Matsumoto et al. 1987a; Kamal-Eldin and Appelqvist, 1996), which reacts further to epoxidized and ring-

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contracted products. Treatment of the 8a-hydroperoxide with Pb(OAc)4 gave a mixture of the meso- and (±)peroxide 35 (Matsumoto et al., 1987), which upon heating in acetonitrile underwent a disproportionation-type decomposition into a mixture of PMC, its SD (11) and ST (13). Reduction of 14 by Me2S gave the pQ 8 (Fig. 3.19). In the presence of NO and dioxygen in 1,2dichloroethane, PMC afforded a complex product mixture, in which the ortho-quinoid -tocored analogue 31, 5aformyl-PMC (18), pQ 8 and its addition product to the intermediate oQM (16) were identified (Nagata et al., 2000) (see Fig. 3.20). Oxidation with hypochlorite gave the pQ 8 as the main product, along with minor amounts of monoand bis-chlorinated byproducts (Ho et al., 2000).

Summary of the oxidation chemistry The oxidation chemistry of PMC is strongly dependent on the oxidants and on the reaction medium, especially the solvent used. The product spectrum and the underlying pathways in protic solvents, such as alcohols, water or

their mixtures (Fig. 3.21), are different from those in inert, aprotic solvents, such as chloroform, THF or hexane (Fig. 3.22). In protic solvents, the oxidation chemistry is based on ortho-quinoid (3) and para-quinoid (4) intermediates, whereas in aprotic solvents radical coupling reactions of the chromanoxyl radical 2 dominate. In Figs 3.21. and 3.22, emphasis is placed on generalization of the pathways.

Derivatives of PMC Modified at Different Positions of the Chroman Skeleton Oxidation of PMC in different solvents, with different oxidants under different conditions provided various reaction products, as discussed above. In most cases, a product mixture is obtained, with the yield of a specific product being very poor to fair. In contrast to such lowyield conversions, reactions and syntheses will be reported in the following which give smaller amounts of byproducts, providing the target compound, a substituted PMC, in good yields or even quantitatively.

Fig. 3.19. Reaction of PMC with singlet oxygen and subsequent processes.

Fig. 3.20. Oxidation of PMC in the presence of NO and O2.

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

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Fig. 3.21. Summary: oxidation of PMC in protic (aqueous, alcoholic, acidic) media.

5-Substituted PMC derivatives Oxidation of PMC by elemental bromine afforded 5abromo-PMC (36) in quantitative yield (Adelwöhrer et al., 2003a) according to a non-radical two-step mechanism consisting of oxidation to the intermediate oQM 3 followed by 1,4-addition of HBr (Rosenau and Habicher, 1995) (see Fig. 3.23). The intermediacy of 3 was proven by trapping reactions. Reaction of PMC with sulphuryl chloride, according to a postulated radical mechanism, yielded the 5a-chloro derivative, which was used as an intermediate for the alkylation of different phenols (Murase et al., 1974). Also starting from pQ 8, which can be readily obtained from PMC by oxidation with FeCl3, neat conversions into 5a-substituted derivatives are possible: reaction with

trimethylsilyl bromide afforded the 6-trimethylsiloxy-5abromo derivative 37, while addition of acetyl chloride gave 6-O-acetyl-5a-chloro-PMC (38) (Dallacker et al., 1991) (Fig. 3.24). The reaction mechanism consisted of an initial cyclization of 8 initiated by nucleophilic attack of the hydroxyl group at the C-1 carbonyl under formation of a hemiketal, followed by 1,4-elimination of water involving the OH group at C-1 and a proton at C-5. The process is catalysed by traces of acid. The resulting oQM 3 adds trimethylsilane (TMS) bromide or acetyl chloride, respectively, in a 1,4-manner, the regioselectivity being clearly dictated from the aromatic resonance form of 3: the nucleophile attacks C-5a while the electrophile adds to O-6. The O-protected chloride 38 was further converted into 5-formyl-PMC (18) by oxidation with para-nitroso-

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Fig. 3.22. Summary: oxidation of PMC in inert (apolar or less polar) solvents.

Fig. 3.23. Synthesis of 5a-bromo-PMC (36).

N,N-dimethylaniline followed by deacetylation of the phenolic hydroxyl group. The 5-formyl derivative is distinguished by a strong intramolecular hydrogen bond from the phenolic hydroxyl to the carbonyl oxygen (Dallacker et al., 1991).

5a-Hydroxy-PMC (20) was obtained by hydroxymethylation of 2,2,7,8-tetramethylchroman-6-ol (TMC), the -tocopherol analogue, with formaldehyde (Nakamura and Kijima, 1972) (Fig. 3.25). Similar alkylation reactions of TMC have also been used to obtain the 5a-methoxymethyl-,

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

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Fig. 3.24. Synthesis of 5a-substituted PMC derivatives according to a cyclization–addition mechanism starting from para-quinone 8.

Fig. 3.25. 5a-Substituted PMC derivatives by alkoxymethylation or aminomethylation.

Fig. 3.26. PMC spiro-dimer (11) and PMC ethano-dimer (12) as a reversible redox pair.

upon radical oxidation of PMC (Nilsson et al., 1968b; Matsuo et al., 1989), but is far more likely to be formed by reduction of the SD 11 (see above). The two-electron oxidation of PMC to dimers (Nelan and Robeson, 1962; Schudel et al., 1963) and trimers (Skinner and Alaupovic, 1963; Skinner and Parkhurst, 1964) via oQM 3 has been reported in the early days of vitamin E chemistry, including the publication of standard procedures for nearquantitative preparation. The ED 12 was obtained quantitatively from SD 11 by treatment with several reductants, including ascorbate, NaBH4 or metal/acid systems. The ready reduction can be explained by the energy gain upon re-aromatization of the cyclohexadienone system. Since the reverse process, oxidation of 12 leading to 11, also proceeds quantitatively by various oxidants, SD 11 and ED 12 can be regarded as a reversible redox system (see Fig. 3.26). The 5-allyl-derivative of PMC (39) was obtained by cyclization of 5-allyl-2,3-dimethyl-hydroquinone with 3-methyl-but-2-en-1-ol according to the general pathway for PMC synthesis (Matsumoto et al., 1994) (Fig. 3.27).

7-Substituted PMC derivatives The 7-formyl derivative 40, obtained by Vilsmeier formylation, and its further reduction to 7a-hydroxy-PMC were reported in two instances (Nakamura and Kijima, 1972; Dean et al., 1981). The latter compound was further converted into the chloromethyl derivative followed by Wurtz-type coupling to the 7a,7a-ethano-dimer 41 (Fig. 3.28).

Fig. 3.27. Synthesis of 5-allyl-PMC (39).

8-Substituted PMC derivatives 5a-dimethylaminomethyl-, benzyloxymethyl- and ethoxymethyl derivatives (Skinner et al., 1967), which are similar in structure to the 5a-substituted products 22 obtained in oxidation reactions (Suarna et al., 1988). The ED of PMC (12) can also be regarded as a 5asubstituted PMC derivative. It has been described as the coupling product of two 5a-centred chromanoxyl radicals 7

Starting from 2,2,5,7-tetramethylchroman-6-ol, 8b-chloroPMC was obtained according to a Blanc chloromethylation (Omura, 1989), and was subsequently etherified with methanol to give the 8b-methoxy-PMC (42). The same compound was obtained by treatment of 8a-methoxychromanone 32 with MeOH/KOH according to a

Fig. 3.28. 7-Formyl derivative 40 and conversion into dimer 41.

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rearrangement mechanism involving oQM intermediates (Omura, 1989) (Fig. 3.29). The 8-allyl-derivative 43 was produced by cyclization of 2-allyl-5,3-dimethylhydroquinone with 3-methyl-but-2-en1-ol, in analogy to the preparation of the 5a-allyl derivative 39 (Matsumoto et al., 1994). Cyclization of 2,6-dimethylhydroquinone to 2,2,5,7tetramethylchroman-6-ol followed by Vilsmeier formylation provided the 8-formyl derivative 44, which was further converted into other 8-substituted chromanols, such as the -CN, -COOH or -CONH2 derivatives (Fujishima, 1996). A similar procedure provided the 8b-acetyl derivative 45 by Friedel-Crafts acylation of 2,2,5,7tetramethylchroman-6-ol (Fig. 3.30).

PMC derivatives modified in position 2a Probably the most important 2a-substituted PMC derivative is 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox, 46), which carries a carboxyl group in the 2-position instead of one 2a-methyl group. Through this chemical alteration, the compound is rendered readily water soluble, but remains soluble in protic and many polar aprotic solvents. This allows its oxidation chemistry to be studied directly in aqueous media – without application of co-solvents or other means of solubilization, such as micelle formation, which are frequently required in the cases of PMC and in particular -tocopherol. Through

these advantages in its handling, Trolox has become a standard compound in antioxidant research: in many studies, the effectiveness of antioxidants is given in ‘Trolox equivalents’. The fact that the hydrophilic Trolox was studied almost exclusively in homogeneous aqueous solution raises questions of comparability with the strongly lipophilic -tocopherol, which are not to be discussed here, however. In principle, the oxidation chemistry of Trolox is similar to that of PMC, but is not the subject of this review. One notable difference is the formation of intermediate 8alactones, or spiroketals, involving the 2a-carboxyl group, which are hydrolysed in a subsequent step to the corresponding para-quinone 47 (Thomas and Bielski, 1989) (Fig. 3.31). Numerous precursors in the synthesis of the -tocopherol – in particular intermediates obtained in the build-up of the isoprenoid side chain with its natural (RRR)-stereochemistry – can formally be regarded as 2amodified PMC derivatives, but are outside the scope of this discourse, so that only two recent examples from the literature are given. Derivative 48, combining both antioxidants vitamin E and vitamin C in one molecule (Fig. 3.32), showed interesting antioxidative properties (Manfredini et al., 2000). In contrast, compound 49 which unites the two 2a-methyl groups in one cyclopropyl ring was found to have inferior antioxidative properties compared with its parent compound PMC (Gilbert and Pinto, 1992).

Fig. 3.29. Syntheses of 8-methoxy-PMC (42).

Fig. 3.30. 8-Substituted PMC-derivatives.

Fig. 3.31. Oxidation of Trolox into its para-quinone.

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

PMC derivatives modified at the 3-position and/or 4-position The preparation of 4-methoxy-PMC (33) from PMC by treatment with I2/KOH has already been discussed in the section on oxidation. The methoxy derivative eliminated methanol upon acidic treatment to give 3,4-dehydro-PMC (34) (Omura, 1989), which is a valuable starting material for modifications of the alicyclic ring. Compound 34 can also be obtained in a simple preparation by treatment of acetylated PMC with 2,3-dichloro-5,6-dicyano-1,4benzoquinone (DDQ) (Burton et al., 1985).

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group at C-6, such as the hydroxyl or amino function, were ineffective as antioxidants as they cannot be converted into a stable radical, e.g. the 6-methyl derivative 52 (Parker, 1969) or the 6-bromo compound 53 (Smith and Ungnade, 1939) (Fig. 3.33). The antioxidative efficiency of different PMC derivatives and its maximization have been comprehensively studied (Burton et al., 1985; Mukai et al., 1989).

Other PMC-related Derivatives 3-Oxa-derivatives of PMC

PMC derivatives modified at the 1-position and/or 6-position Replacement of the two oxygen atoms in PMC was performed to study the specific role of these atoms in the antioxidative action. 2,2,5,7,8-Pentamethyl-thiochroman-6ol (50), the 1-thia-derivative of PMC, was obtained by Clemmensen reduction of the corresponding thiochroman4-one (Robillard et al., 1986). It proved to be a good antioxidant, as was the 6-amino-derivative, 2,2,5,7,8pentamethyl-chroman-6-ylamine (51) (Merck & Co., 1939). In contrast, all derivatives lacking a readily oxidizable

3-Oxa-chromanols of the general formula 54 – named 5,7,8trimethyl-4H-benzo[1,3]dioxin-6-ols according to IUPAC rules – were obtained by condensation of trimethylhydroquinone with the double equivalent of aldehydes in a straightforward one-pot reaction (Adelwöhrer et al., 2003b) (see Fig. 3.34). The reaction did not proceed with ketones, so that derivatives monosubstituted at C-4 and C-2 were always obtained. In addition, these two substituents were always similar. In contrast to the PMC and its derivatives described above,

Fig. 3.32. 2-Substituted PMC derivatives.

Fig. 3.33. 1-Substituted and 6-substituted PMC derivatives.

Fig. 3.34. Synthesis of 3-oxa-chromanols as a mixture of cis/trans-isomers.

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Fig. 3.35. Oxidation of 3-oxa-chromanol 55 in aqueous media.

the basic chroman skeleton is altered by replacement of C-3 by an oxygen atom. As insignificant as this modification might appear, it causes some fundamental differences in the reaction behaviour of the compounds as compared with PMC. 3-Oxa-chromanols have recently been tested for their antioxidative properties, as they represent an interesting novel class of phenolic antioxidants (Gregor et al., 2005). EPR measurements of the radicals derived from 3-oxachromanol derivatives revealed similar stabilities as compared with the PMC-derived radical 2 (Adelwöhrer et al., 2003b; Gregor et al., 2005), producing well-resolved multi-line spectra, the hyperfine coupling constants for the methyl substituents at the aromatic ring being quite similar to those measured for chromanoxyl radical 2 or the -tocopheroxyl radical. The oxidation behaviour of 3-oxa-chromanols was mainly studied by means of the 2,4-dimethyl-substituted compound 55 applied as a mixture of isomers; it showed an extreme dependence on the amount of co-reacting water present (Rosenau et al., 2002a). In aqueous media, 55 was oxidized by one oxidation equivalent to 2,5-dihydroxy-3,4,6-trimethyl-acetophenone (57) via 2-(1-hydroxyethyl)-3,5,6-trimethylbenzo-1,4quinone (56) (Fig. 3.35). Two oxidation equivalents gave directly the corresponding para-quinone 58. Upon oxidation, C-2 with the methyl substituent was always lost in the form of acetaldehyde. Oxidation of 3-oxa-chromanol 55 in the presence of just one equivalent of water produced acetophenone 57 as well, but according to a different mechanism involving orthoquinone methide intermediate 59 and styrene derivative 60, from which finally acetaldehyde was released (Fig. 3.36). By means of deuterated starting material, the selective [1,5]-sigmatropic proton shift from the C-4a methyl group to the exocyclic methylene group was demonstrated, and the occurrence of both intermediates was additionally confirmed by trapping in hetero-DielsAlder reactions (Rosenau et al., 2002a). In the absence of water, oxidation of 55 produced chromenone 62, again via the two intermediates 59 and 60, and via chromanone 61, which was formed by internal group rotation (Fig. 3.37). The further oxidation of chromanone 61 to chromenone 62 was favoured over the oxidation of the starting material 55. If the formation of an exocyclic methylene group at C-4

Fig. 3.36. Oxidation of 3-oxa-chromanol 55 in the presence of 1 equivalent of water: mechanistic study by means of selectively deuterated starting material.

Fig. 3.37. Oxidation of 3-oxa-chromanol 55 in the absence of water: mechanism and reaction intermediates.

is impossible due to structural prerequisites, as in the 2,4diphenyl-3-oxachromanol 63, oxidation in the absence of water provided spiro-dimer 64, since the primary orthoquinone methide intermediate cannot rearrange involving

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

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Fig. 3.38. Oxidation of 3-oxa-chromanols lacking protons in the 4a-position.

C-4a (Fig. 3.38). Oxidation in aqueous media produced para-quinone 65, both reactions in analogy to PMC. In summary, the oxidation behaviour of 3-oxachromanols showed both differences from and similarities to that of PMC. Paralleling the chemistry of PMC, oneelectron oxidation caused formation of the corresponding chromanoxyl radicals, which are relatively stable. In the absence of a C-4a substituent with protons, the oxidation behaviour entirely resembled that of PMC. In the presence of a C-4a substituent with protons, the oxidation behaviour changed fundamentally. The primary ortho-quinone methide of type 59 underwent different subsequent reactions depending on the water content present, the proton transfer to styrene derivatives of type 60 being largely preferred over spiro-dimerization.

The ‘Siamese twin’-PMC Tetracyclus 66 was obtained by condensation of trimethylhydroquinone with 1,1,3,3-tetramethoxypropane (Rosenau et al., 2002b). It consists of two PMC molecules connected with each other at the alicyclic pyran ring having C-2, C-3 and C-4 in common, which gave the compound its name ‘Siamese twin’-PMC. Compound 66 is the first vitamin E model which ‘locks’ the alicyclic chroman ring into a specific geometry, but without achieving this by means of sterically demanding, large substituents. Any

conformational change in one of the two chromanol moieties in 66, which would influence radical stability (Nagaoka and Mukai, 1992), is accompanied by the reverse change in the second ‘twin’ chromanol moiety, causing the opposite effect there. The chromanoxyl radical derived from 66 gave EPR spectra that resembled those of -tocopherol, but exhibit additional hyperfine structure due to the other ‘half’ of the molecule. The compound showed antioxidant properties which were superior to that of PMC in several test systems (Gregor et al., 2005; Staniek et al., 2005). Since it contained two chromanol moieties, compound 66 gave all reactions characteristic of PMC twice, such as the oxidation to the bis(para-quinone) 67, bromination to the dibromide 68 and oxidation to an oQM on both sides (Fig. 3.39). Due to the double oQM formation, each one of the two ‘twins parts’ in 66 was able to undergo a reaction similar to the spiro-dimerization of PMC. Thus, the simple spiro-dimerization of PMC eventually became a spirooligomerization/spiro-polymerization in the case of 66: after both sides of the twin molecule reacted in a spirodimerization, each of the two newly attached twin molecules again possessed an end capable of undergoing spiro-dimerization, and so on, finally affording linear molecules consisting of twin molecules connected by spiro links that were formed in sequential hetero-Diels-Alder reactions. The lengths of the spiro-polymers as well as the molecular weight distribution varied according to reaction time and reaction temperature. Different oxidants, solvents,

Fig. 3.39. ‘Siamese twin’-chromanol 66 and its reaction products.

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reaction times and reaction temperatures afforded polymers with 9–215 units depending on the conditions. After reaction of the first ‘twin side’ of 66 by spirodimerization, for instance as dienophile, the second half can theoretically react either as a dienophile or as a diene (Fig. 3.40). Thus, a pyrano/spiro pair (reaction of the ‘left twin’ as a diene and the ‘right twin’ as a dienophile), a pyrano/pyrano pair or a spiro/spiro couple (reaction of ‘both twins’ as dienes or dienophiles, respectively) is formed. However, only products were observed that contained exclusively asymmetric pairs (pyrano/spiro = spiro/pyrano pairs) as the building blocks – but no symmetric (pyrano/pyrano or spiro/spiro) couples.

The spiro-pyrano regioselectivity was rationalized in terms of frontier orbital theory. Reaction of the first oQM as a diene (pyrano structure) resulted in an increase of the HOMO energy of the neighbouring oQM. Therefore, these oQMs will react as a dienophile due to increased π-donor ability, and will thus form a spiro structure. By analogy, reaction of the first oQM as a dienophile (spiro structure) decreased the LUMO energy of the neighbouring oQM, leading to increased π-acceptor capability and subsequent reaction as a diene (pyrano structure). In both cases, spiro/pyrano couples resulted, but no spiro/spiro or pyrano/pyrano neighbours, since there was a significantly decreased HOMO–LUMO energy difference for the

Fig. 3.40. Regioselectivity of spiro-pyrano link formation.

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

asymmetric pairs as compared with the symmetric couples (Fig. 3.40). Working at low temperatures produced exclusively the cyclic tetramer 69 instead of linear products (Rosenau et al., 2002b). Also 69 contained only pyrano/spiro (=spiro/pyrano) pairs as the building blocks, but no pyrano/pyrano or spiro/spiro couples. Each spiro-link in linear or cyclic spirooligomers and spiro-polymers can be reduced in analogy to the SD of PMC (11), which is readily reduced to the ED 12. Consequently, reduction of 69 provided the tetra-ethano derivative 70 (Fig. 3.41), which shows similarities to calixarenes. In the presence of excess oxidant and reductant, respectively, 69 and 70 form a reversible redox pair, by analogy to the PMC SD and ED 11 and 12.

PMC derivatives with different ring strain According to literature accounts, oxidation chemistry of -tocopherol and PMC regioselectively involves C-5a, where the oQM 3 is formed (‘up’-oQM); the isomeric compound with the exo-methylene group at C-7a (‘down’oQM) was reportedly not observed (Machlin, 1980; Parkhurst and Skinner, 1981). Usually, the so-called ‘Mills–Nixon effect’ (Mills and Nixon, 1930), reported for the first time in 1930, is given as an explanation for the regioselectivity observed (Badger, 1951; Behan et al., 1975). Applying the Mills–Nixon explanation to vitamin E, a widely accepted postulate was derived, which was frequently repeated throughout the literature: PMC-type chromanols are regioselectively oxidized at C-5a to form ‘up’-oQMs, whereas PMC-type benzofuranols are always oxidized at C-6a to form ‘down’-oQMs. The strict regioselectivity is due to the different ring size of the alicycle and to the electronic effect of the alicyclic ring exerted on the aromatic. The issue of regioselectivity in oxidations of PMC-type (-tocopherol-type) antioxidants was recently clarified by a combined experimental and theoretical study (Rosenau et al., 2005). The approach was based on measuring the ratio

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between the ‘down’-oQM and ‘up’-oQM products obtained upon oxidation of 11 PMC-type antioxidants (1, 66 and 71–79) carrying differently sized alicycles (Fig. 3.42), thus keeping the electronic effects constant and changing only the angular strain of the systems, as seen by the ( + )values, which cover an angle range between 219° (for 71) and 246° (for 79) (see Fig. 3.43 and Table 3.2). The model compounds were oxidized to the corresponding oQMs, which were trapped by the fast reaction with ethyl vinyl ether (Fig. 3.43). Product analysis provided the ratio between the two oQM intermediates, and proved unambiguously that the regioselectivity, i.e. the ratio between ‘up’-oQM and ‘down’-oQM, was not a function of the ring size: it changed gradually, but not abruptly when going from a six-membered to a fivemembered ring system, in contrast to what has been assumed so far (Table 3.2). The ‘up’-oQMs were increasingly favoured when going from small ( + )-values to large ones, with down-oQMs showing the opposite trend. The data clearly disproved that vitamin E-related benzofuranols form only one oQM (the ‘down’-form), while chromanol-type tocopherol models give only the opposite one (‘up’-oQMs). By measuring the ratio between the trapped ‘up’-oQMs and ‘down’-oQMs at different temperatures, the relative activation energies H‡ for the formation of the two intermediates were obtained. The tetrasubstituted hydroquinones 80 and 81 represent the ‘open ring version’ of chromanol 16, having the same substituents and thus the same inductive electronic substituent effects as this chromanol, but no annulated ring (Fig. 3.44). Upon oxidation, both compounds afforded the ‘up’-oQMs and ‘down’-oQMs in a nearly perfect 50/50 ratio (Rosenau and Stranger, 2005). This proved that the regioselectivity in oQM formation from PMC-type oxidants is also not a consequence of substitution, as hitherto assumed. Regioselectivity in oxidative oQM formation is only observed if TMHQ is annulated, i.e. attached to another ring structure. It is not an intrinsic property of chain-substituted TMHQ or caused by electronic substituent effects; it is, moreover, caused by strain imposed through annulation.

Fig. 3.41. Cyclic spiro-tetramer 69 and reduction to macrocycle 70.

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Fig. 3.42. PMC derivatives with a different strain in the alicyclic ring.

Fig. 3.43. Oxidation of PMC derivatives with a different ring strain to mixtures of two possible ortho-quinone methides: the oxidation behaviour agrees with the theory of strain-induced bond localization (SIBL).

Table 3.2. Ratio between the trapped ‘up’-oQMs and ‘down’-oQMs, and kinetically determined activation enthalpy difference. Compound 71 72 73 74 75 76 66 77 1 78 79

( + )

Up-oQM (%, 373 K)

Down-oQM (%, 373 K)

H† (kcal/mol)

219 221 221 223 231 233 239 240 242 244 246

0.9 2.3 14.9 43.3 54.3 66.9 93.1 94.2 97.9 99.3 99.8

99.1 97.7 85.1 56.7 45.7 30.1 6.9 5.8 2.1 0.7 0.2

3.49 ± 0.12 2.86 ± 0.08 1.22 ± 0.04 0.18 ± 0.01 –0.109 ± 0.002 –0.458 ± 0.009 –1.61 ± 0.09 –1.96 ± 0.06 –2.768 ± 0.005 –3.24 ± 0.12 –4.77 ± 0.13

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

Fig. 3.44. Non-annulated ‘derivatives’ of PMC.

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The agreement of the experimental data for H‡ derived from the ‘up’/‘down’ ratio of the two possible oQMs with the theoretical model of SIBL (Stanger et al., 1997) was superb, showing that the observed regioselectivity in oxidations of PMC-type antioxidants is simply a function of angular strain. This peculiar oxidation selectivity was thus fully explained by the SIBL theory, which ended the decade-long Mills–Nixon controversy.

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Substitution derivatives of 5-hydroxyhydrindene. Journal of the Chemical Society, 2510–2524. Mukai, K., Okabe, K. and Hosose, H. (1989) Synthesis and stopped-flow investigation of antioxidant activity of tocopherols. Finding of new tocopherol derivatives having the highest antioxidant activity among phenolic antioxidants. Journal of Organic Chemistry 54, 557–560. Mukai, K., Tsuzuki, N., Ishizu, K., Ouchi, S. and Fukuzawa, K. (1981) Electron nuclear double resonance studies of radicals produced by the PbO2 oxidation of -tocopherol and its model compound in solution. Chemistry and Physics of Lipids 29, 129–135. Mukai, K., Tsuzuki, N., Ouchi, S. and Fukuzawa, K. (1982) Electron spin resonance studies of chromanoxyl radicals derived from tocopherols. Chemistry and Physics of Lipids 30, 337–345. Murase, K., Matsumoto, J., Tamazawa, K., Takahashi, K. and Murakami, M. (1974) Chlorinations of 5-methyl-6-chromanols and reactivities of 5-chloromethyl-6-chromanols. Yoamanouchi Seiyaku Kenkyu Hokoku 2, 66–73. Nagaoka, S. and Mukai, K. (1992) Mechanism of antioxidant reaction of vitamin E. 2. Photoelectron spectroscopy and ab initio calculation. Journal of Physical Chemistry 96, 8184–8187. Nagata, Y., Nishio, T., Matsumoto, S., Kanazawa, H., Mochizuki, M. and Matsushima, Y. (2000) Reaction of 2,2,5,7,8-pentamethyl-6-chromanol, an -tocopherol analog, with NO in the presence of oxygen. Bioorganic and Medicinal Chemistry Letters 10, 2709–2712. Nakamura, T. and Kijima, S. (1972) Studies on tocopherol derivatives. IV. Hydroxymethylation reaction of -, -tocopherol and their model compounds with boric acid. Chemical and Pharmaceutical Bulletin 20, 1681–1686. Nelan, D.R. and Robeson, C.D. (1962) The oxidation product from -tocopherol and potassium ferricyanide and its reaction with ascorbic and hydrochloric acids. Journal of the American Chemical Society 84, 2963–2965. Nilsson, J.L.G., Daves, D.G. and Folkers, K. (1968a) New tocopherol dimers. Acta Chimica Scandinavica 22, 200–206. Nilsson, J.L.G., Daves, D.G. and Folkers, K. (1968b) The oxidative dimerization of -, -, -, and -tocopherols. Acta Chimica Scandinavica 22, 207–218. Nilsson, J.L.G., Sievertsson, H. and Selander H. (1968c) Synthesis of methyl substituted 6-hydroxychromans, model compounds of tocopherols. Acta Chimica Scandinavica 22, 3160–3170. Omura, K. (1989) Iodine oxidation of -tocopherol and its model compound in alkaline methanol: unexpected isomerization of the product quinone monoketals. Journal of Organic Chemistry 54, 1987–1990. Parker, V.D. (1969) Anodic alkyl transfer from hydroquinone ethers. II. Anodic oxidation of an -tocopherol model compound. Journal of the American Chemical Society 91, 5380–5381. Parkhurst, R.M. and Skinner, W.A. (1981) Chromans and tocopherols. Chemistry of Heterocyclic Compounds 36, 59–137. Peers, K.E., Coxon, D.T. and Chan, W.S. (1981) Autoxidation of methyl linolenate and methyl linoleate: the effect of -tocopherol. Journal of the Science of Food and Agriculture 32, 898–904. Robillard, B., Hughes, L., Slaby, M., Lindsay, D.A. and Ingold, K.U. (1986) Synthesis of 2-substituted 5,7,8-trimethyl-6-hydroxythiochromans and purported syntheses of sulfur-containing analogues of vitamin E. Journal of Organic Chemistry 51, 1700–1704. Rosenau, T. and Habicher, W.D. (1995) Novel tocopherol compounds I. Bromination of -tocopherol – reaction mechanism and synthetic applications. Tetrahedron 51, 7919–7926. Rosenau, T. and Stanger, A. (2005) Novel tocopherol compounds XXI. Synthesis and oxidation of ‘non-annulated’ vitamin E-type model compounds. Tetrahedron Letters 46, 7845–7848. Rosenau, T., Gruner, M. and Habicher, W. D. (1997) Novel tocopherol compounds VIII. Reaction mechanism of the formation of -tocored. Tetrahedron 53, 3571–3576.

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PMC – 2,2,5,7,8-Pentamethyl-chroman-6-ol and Related Model Compounds

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Rosenau, T., Potthast, A., Elder, T., Lange, T., Sixta, H. and Kosma, P. (2002a) Synthesis and oxidation behaviour of 2,4,5,7,8-pentamethyl-4H-1,3benzodioxin-6-ol, a multi-functional oxa-tocopherol type antioxidant. Journal of Organic Chemistry 67, 3607–3614. Rosenau, T., Potthast, A., Hofinger, A. and Kosma, P. (2002b) Calixarene-type macrocycles by oxidation of phenols related to vitamin E. Angewandte Chemie (International Edition) 41, 1171–1173. Angewandte Chemie 114, 1219–1221. Rosenau, T., Ebner, G., Stanger, A., Perl, S. and Nuri, L. (2005) From a theoretical concept to biochemical reactions: strain induced bond localization (SIBL) in oxidation of vitamin E. Chemistry – a European Journal 11, 280–287. Rousseau-Richard, C., Richard, C. and Martin, R. (1988) Kinetics of bimolecular decay of -tocopheroxyl free radicals studied by ESR. FEBS Letters 233, 307–310. Schudel, P., Mayer, H., Metzger, J., Rüegg, R. and Isler, O. (1963) Über die Chemie des Vitamins E. 2. Mitteilung. Die Struktur des Kaliumferricyanid-Oxydationsproduktes von -Tocopherol. Helvetica Chimica Acta 46, 636. Skinner, W.A. (1964) Vitamin E oxidation with free radical initiators. Azobis-isobutyronitrile. Biochemical and Biophysical Research Communications 15, 469–472. Skinner, W.A. and Alaupovic, P. (1963) Oxidation products of vitamin E and its model, 6-hydroxy-2,2,5,7,8-pentamethyl-chroman. V. Studies of the products of alkaline ferricyanide oxidation. Journal of Organic Chemistry 28, 2854–2858. Skinner, W.A. and Parkhurst, R.M. (1964) Oxidation products of vitamin E and its model, 6-hydroxy-2,2,5,7,8-pentamethylchroman. VII. Trimer formed by alkaline ferricyanide oxidation. Journal of Organic Chemistry 29, 3601–3603. Skinner, W.A., Parkhurst, R.M., Scholler, J., Alaupovic, P., Crider, Q.E. and Schwarz, K. (1967) Structure–activity relations in the vitamin E series. I. Effects of 5-methyl substitution on 6-hydroxy-2,2,5,7,8-pentamethylchroman. Journal of Medical Chemisry 10, 657–661. Smith, L.I. and Ungnade, H.E. (1939) The chemistry of vitamin E. IV. The synthesis of tocopherols. Journal of Organic Chemistry 4, 298–340. Smith, L.I., Irwin, W.B. and Ungnade, H.E. (1939) The chemistry of vitamin E. XVII. The oxidation products of -tocopherol and of related 6-hydroxychromans. Journal of the American Chemical Society 61, 2424–2429. Stanger, A., Ashkenazi, N., Boese, R. and Stellberg, P. (1997) Evidence for metal induced bond localization in cyclobutabenzenes: the crystal and molecular structures of 6-Cr(CO)3 and 4-Fe(CO)3 complexes of cyclobutabenzene. Journal of Organometal Chemistry 542, 19. Staniek, K., Rosenau, T., Gregor, W., Nohle, H. and Gille, I. (2005) The protection of bioenergetic functions in mitochondria by new synthetic chromanols. Biochemical Pharmacology 70, 1361–1370. Suarna, C. and Southwell-Keely, P.T. (1988) New oxidation products of -tocopherol. Lipids 23, 137–139. Suarna, C. and Southwell-Keely, P.T. (1989) Effect of alcohols on the oxidation of the vitamin E model compound, 2,2,5,7,8-pentamethyl-6chromanol. Lipids 24(1), 56–60. Suarna, C. and Southwell-Keely, P.T. (1991) Antioxidant activity and oxidation products of -tocopherol and of its model compound 2,2,5,7,8pentamethyl-6-chromanol. Lipids 26, 187–190. Suarna, C., Craig, D.C., Cross, K.J. and Southwell-Keely, P.T. (1988a) Oxidations of vitamin E and its model compound, 2,2,5,7,8-pentamethyl-6hydroxychroman. A new dimer. Journal of Organic Chemistry 53, 1281–1284. Suarna, C., Nelson, D. and Southwell-Keely, P.T. (1988b) New oxidation products of 2,2,5,7,8-pentamethyl-6-chromanol. Lipids 23, 1129–1131. Suarna, C., Baca, M. and Southwell-Keely, P.T. (1992) Oxidation of the -tocopherol model compound 2,2,5,7,8-pentamethyl-6-chromanol in the presence of alcohols. Lipids 27, 447–453. Thomas, M.J. and Bielski, B.H.J. (1989) Oxidation and reaction of trolox c, a tocopherol analogue, in aqueous solution. A pulse-radiolysis study. Journal of the American Chemical Society 111, 3315–3319. Tsuchija, J., Niki, E. and Kamiya, Y. (1983) Oxidation of lipids. IV. Formation and reaction of chromanoxyl radicals as studied by electron spin resonance. Bulletin of the Chemical Society of Japan 56, 229–232. Yamauchi, R., Kato, K. and Ueno, K. (1988) Formation of trimers of -tocopherol and its model compound, 2,2,5,7,8-pentamethylchroman-6-ol, in autoxidizing methyl linoleate. Lipids 23, 779–783. Yamauchi, R., Matsui, T., Kato, K. and Ueno, K. (1989a) Reaction products of -tocopherol with a free radical initiator, 2,2-azobis(2,4dimethylvaleronitrile). Lipids 24, 204–209. Yamauchi, R., Matsui, T., Kato, K. and Ueno, K. (1989b) Reaction of -tocopherol with 2,2-azobis(2,4-dimethylvaleronitrile) in benzene. Agricultural and Biological Chemistry 53, 3257–3262.

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4 Recycling of Vitamin E by Natural Products: a Dietary Perspective Gian Franco Pedulli and Marco Lucarini Università di Bologna, Dipartimento di Chimica Organica ‘A. Mangini’, Via S. Giacomo 11, 40126 Bologna, Italy

Abbreviations: AIBN, 2,2-azobis(isobutyronitrile); AMVN, 2,2-azobis(2,4-dimethylvaleronitrile); BC, 4-tert-butylcatechol; BDE, bond dissociation enthalpy; BHA, 3,5-di-tert-butyl-4-hydroxyanysole; BHT, 3,5-di-tert-butyl-4-hydroxytoluene; GSH, glutathione; ML, methyl linoleate; MPTZ, 1,3-dimethylphenothiazine; TMP, 2,4,6-trimethoxy phenol.

Abstract A very important feature of vitamin E is its ability to be regenerated by co-antioxidants which, when used alone, are much less efficient in inhibiting the peroxidation reaction of hydrocarbons and unsaturated lipids. An overview of the various compounds showing a synergistic behaviour with vitamin E is presented and the mechanisms by which it can be recycled are discussed.

Introduction The observation that the antioxidant activity of vitamin E is enhanced by the presence in the system of vitamin C (ascorbic acid) was reported for the first time by Golumbic and Mattill (1941). Since then, several investigations have demonstrated that these two vitamins show a synergistic behaviour and that their mixtures display much better antioxidant properties than expected on the basis of the activity of the single components. Subsequent studies have shown that vitamin E can be recycled by several other natural products such as ubiquinols, flavonoids and other polyphenol derivatives.

Homogeneous Solutions Golumbic and Mattill (1941) did not propose any mechanism for the reaction between ascorbic acid and -tocopherol although they pointed out that the oxidation potential of -tocopherol is appreciably higher than that of ascorbic acid. Many years later, Mahoney (1972) reported synergism between hindered and unhindered phenols in inhibiting the auto-oxidation reaction of hydrocarbons. In order to explain these results, the overall reaction 44

scheme describing the initiated auto-oxidation of a hydrocarbon, RH, should be considered (see below). Here Equations 1 and 2 represent the initiation, 3 and 4 the propagation, and 5 the termination steps (Howard, 1973). Ri ——→ InOO
InOO
+ RH ——→ InOOH + R


(1) (2)

R
+ O2

——→ ROO


(3)

ROO
+ RH

kp ——→ ROOH + R


(4)

2ROO


2kt ——→ Products

(5)

In the presence of a chain-breaking antioxidant, AH, two more reactions should be taken into account, i.e. the trapping by AH of the chain-propagating peroxyl radicals ROO
(Equation 6), with rate constant of inhibition kinh, and the termination reaction (Equation 7). ROO
+ AH

kinh ——→ ROOH + A


(6)

ROO
+ A


——→ Products

(7)

Mixtures of certain unhindered (AH) and hindered (CoAH) phenols were found by Mahoney to have much

© CAB International 2007. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

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Recycling of Vitamin E by Natural Products: a Dietary Perspective

better antioxidant properties than either alone. In this case, the three additional reactions (Equations 8–10), involving the second antioxidant CoAH and the related phenoxyl radical CoA
, may take place. The reason for the synergistic behaviour was attributed by Mahoney to the regeneration of the unhindered and more reactive (kinh>kinh) phenol AH by the co-antioxidant CoAH, in the hydrogen atom exchange reaction (Equation 9). ROO
+ CoAH A
+ CoAH ROO
+ CoA


kinh ——→ ROOH + CoA
kr —→ AH + CoA
k–r ——→ Products

(8) (9)

45

regenerating AH. As a result, AH will be consumed at a lower rate while the co-antioxidant will disappear at a rate higher than predictable on the basis of the inhibition rate constants kinh and kinh. At the same time, the oxidation reaction will show an inhibition period longer than expected on the basis of the initial AH concentrations. The overall behaviour can be conveniently represented as in Fig. 4.1. On this basis, regeneration of AH can be an efficient process only if the equilibration reaction (Equation 9) is largely shifted to the right. This may not be true in the presence of additional reactions leading to the disappearance of the radical from the co-antioxidant, CoA
, thus making the reaction shown in Equation 9 irreversible (see above).

(10)

In the absence of hydrogen exchange (Equation 9), the two antioxidants would be consumed with rates only depending on the kinh/kinh ratio and on the relative concentrations of the two species, so that the inhibition effect of the mixture would be purely additive. If, however, hydrogen exchange takes place at a rate higher than or comparable with that of the termination reaction (Equation 7) leading to the disappearance of A
, the latter radical will abstract a hydrogen atom from the co-antioxidant thus

The case of vitamin C The structures of vitamin E and vitamin C are given in Fig. 4.2. A rationalization of the synergism between vitamin E and vitamin C, in terms of hydrogen exchange between ascorbic acid and tocopheroxyl radicals leading to recycling of tocopherol, was given by Niki et al. (1984) to explain the results obtained by studying the inhibition of oxidation of methyl linoleate (ML) by mixtures of vitamin E and vitamin

Fig. 4.1.

Fig. 4.2. Chemical structures of vitamins E and C.

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46

G. F. Pedulli and M. Lucarini

C. Evidence that the radical from vitamin E reacted with ascorbic acid to regenerate vitamin E was previously reported by Packer et al. (1979) who also determined the rate constant for this reaction as 1.55  106 Ms. Niki et al. (1984) measured the rate of oxidation of ML in alcoholic solution (tert-butanol and methanol, 3:1 by vol.) inhibited by vitamin E and vitamin C separately and by mixtures of the two. It was found that oxidation of ML was almost suppressed by the presence of vitamin E alone for a period tinh (the induction period) proportional to the vitamin E concentration. The length of tinh indicated that every -tocopherol molecule scavenged two peroxyl radicals, ROO
, i.e. one in reaction 6 and another one in reaction 7, and the rate constant kinh, in alcoholic solution, was determined from the oxidation rate during the inhibition period as 5.1  105 Ms. Similar experiments, performed in the presence of only vitamin C, showed that ascorbic acid also acted as a chainbreaking antioxidant and produced a distinct induction period. Vitamin C, however, scavenged only one peroxyl radical and reduced the rate of oxidation of ML less than vitamin E. Actually, its rate constant of inhibition, kinh, = 7.5  104 Ms, was about seven times smaller than that of -tocopherol. When both vitamin E and vitamin C were added to the oxidating solutions, the induction period was lengthened to the sum of the induction periods observed when using either vitamin E or vitamin C and the rate of oxidation was the same throughout the whole induction period and very close to the inhibited rate observed in the presence of the better antioxidant alone, i.e. vitamin E. The rate constant of inhibition for this system, kinh = 4.0  105 Ms, was much larger than that for vitamin C and only slightly smaller than that observed for vitamin E. By measuring the rate of consumption of both vitamin E and C during the oxidation reaction, vitamin C was consumed first and vitamin E remained almost constant. Only after the complete disappearance of vitamin C, did vitamin E start to be consumed. These results were interpreted (Niki et al., 1984; Mukai et al., 1989) by admitting that -tocopherol, due to an inhibition rate constant larger than that of ascorbic acid (kinh/kinh = 6.8), reacts with peroxyl radicals more quickly, but then the resulting -tocopherol radicals react with vitamin C to regenerate vitamin E (Equation 9). In the following years, synergism with -tocopherol in homogeneous solution has also been reported for ubiquinol-10 (Landi et al., 1992), and for flavonoids such as epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate and gallic acid (Jia et al., 1998), and for quercetin, catechin and epicatechin (Pedrielli and Skibsted, 2002). In all these studies, synergism between vitamin E and the various co-antioxidants was explained in terms of regeneration of tocopherol from the corresponding tocopheroxyl radical. However, no rationalization of the

structural features needed by a co-antioxidant to show synergism with vitamin E was given until recently when Amorati et al. (2002, 2003) reported a systematic investigation on the effect on the rate of oxidation of hydrocarbons by mixtures of phenols and/or polyphenols. This rationalization was possible due to the current availability of thermochemical data (O–H and N–H bond dissociation enthalpies (BDEs)) concerning the reactants involved in the regeneration reactions as well as their inhibition and hydrogen exchange rate constants. Thus, recycling of vitamin E (AH) by the co-antioxidant CoAH may occur when the hydrogen exchange reaction (Equation 9) is faster than the termination reaction (Equation 7) and it is shifted to the right, both conditions being usually satisfied when the reaction shown in Equation 9 is exothermic. This is certainly true when the co-antioxidant is vitamin C since the free energy change for the reaction of -tocopheroxyl radicals with ascorbate1 to regenerate vitamin E has been estimated as –5.0 kcal/mol, on the basis of the standard one-electron reduction potentials of the involved species (Buettner, 1993), and calculated as –7.7 kcal/mol in a recent theoretical paper (DiLabio et al., 2000). Thus the value of Kr can be estimated to be in the range 5  103–5  105 and therefore the reaction shown in Equation 9 can be considered completely shifted to the right and practically irreversible, this implying that recycling of -tocopherol by ascorbic acid should be complete and that no consumption of vitamin E should be observed until some vitamin C is present.

Synergism between vitamin E and phenolic antioxidants Vitamin E can also be recycled by co-antioxidants containing easily abstractable hydrogen atoms such as those of phenolic (O–H) or even amino (N–H) groups. Antioxidants of these classes capable of regenerating -tocopherol from the corresponding -tocopheroxyl radicals are usually sterically crowded about the reactive hydroxylic or amino function so that their reaction with peroxyl radicals is much slower than that of -tocopherol. At the same time, they are also characterized by O–H (or N–H) BDEs lower than or at most comparable with that of -tocopherol (78.2 kcal/mol) (Lucarini et al., 1994, 1996), so that the hydrogen exchange reaction (Equation 9) is exothermic or thermoneutral. Examples are represented by 2,6-di-tert-butyl-4methoxyphenol (BHA) (Amorati et al., 2003) and by 1,3dimethylphenothiazine (MPTZ) (Amorati et al., 2002) (Fig. 4.3). Figure 4.4 shows the effect of the addition of BHA, -tocopherol and of combinations of the two antioxidants on the rate of auto-oxidation of styrene at 30°C initiated by 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN). BHA (trace b), a relatively poor antioxidant able only to retard the oxidation reaction, has a rate constant for reaction with peroxyl radicals, kinh = 1.2  105 Ms, 26 times lower than

1 Although the reaction shown in Equation 9 could take place via an initial electron transfer followed by proton transfer, a recent computational study suggests the direct hydrogen transfer mechanism as more likely (DiLabio et al., 2000).

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47

Recycling of Vitamin E by Natural Products: a Dietary Perspective

Fig. 4.3. Chemical structures of common antioxidants.

1.2

3

a

2

1

b

c

d

e

[Antioxidant]  104

[O2]  103 (M)

1.0 0.8 0.6 0.4 0.2 0.0

0 0

2000

4000

6000

0

2000

Time(s)

4000

6000

8000

Time(s)

Fig. 4.4. Oxygen consumption traces recorded at 30°C during the autooxidation initiated by 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) of 7.5 M styrene in the absence of any antioxidant (a) and in the presence of (b) 5.0  10–5 M 3,5-di-tert-butyl-4-hydroxyanysole (BHA); (c) 5.0  10–5 M α-tocopherol; (d) 5.0  10–5 M -tocopherol and 5.0  10–5 M BHA; and (e) 5.0  10–5 M -tocopherol and 1.0  10–4 M BHA.

Fig. 4.5. Rate of disappearance of -tocopherol (filled circles) and 3,5di-tert-butyl-4-hydroxyanysole (BHA) (open circles), determined by HPLC–MS, observed during the thermal decomposition of 5 mM 2,2azobis(isobutyronitrile) (AIBN) at 60°C in air-saturated chlorobenzene. The dashed line represents the expected rate of disappearance of -tocopherol in the absence of BHA.

that of -tocopherol (3.2  106 Ms)2 (Burton et al., 1985) which, instead, behaves as a strong inhibitor (see Fig. 4.4, trace c). When using a 1:1 or a 1:2 mixture of -tocopherol and BHA, they behave as solutions containing twice and three times the amount of -tocopherol, respectively. Thus, similarly to what was observed with vitamin C, the mixtures behave as better antioxidants than expected from the activities of the single components, and practically as if the amount of vitamin E was equal to the sum of vitamin E and BHA. This behaviour is entirely expected on the basis of our previous considerations, since the BDE value of the 2,6-di-tert-butyl phenol (78.2 kcal/mol) (Lucarini et al., 1996) is identical to that of -tocopherol so that the hydrogen exchange reaction (Equation 9) is thermoneutral. Thus, the regeneration of vitamin E from BHA can be explained as follows: when the reaction medium contains both -tocopherol and BHA in comparable amounts, the chain-propagating peroxyl radicals react first with

-tocopherol since kinh/kinh = 26.6, with negligible formation of the phenoxyl radical from BHA. Then, equilibration of -TO
with BHA (a fast process in the present case, being kr ≈ 6  103 Ms) leads to regeneration of tocopherol. Since the O–H BDE values of -tocopherol and BHA are the same, the equilibrium constant Kr (Equation 11) is equal to ≈1 and, thus, the concentration ratio of the two antioxidants is expected to remain unchanged during their decay3. –RT lnKr = G ≈ BDE

(11)

Therefore, vitamin E will be present in solution and will continue to inhibit the oxidation until complete disappearance of BHA. By thermally decomposing 2,2azobis(isobutyronitrile) (AIBN) in air-saturated chlorobenzene at 60°C in the presence of equimolecular amounts of the two antioxidants, and by measuring their rate of disappearance by high-performance liquid chromatography

2 It should be pointed out that the rate of inhibition of -tocopherol in chlorobenzene is larger than in tert-butanol due to hydrogen bond formation between solvent and the hydroxyl group of -tocopherol in alcoholic solution. 3 Since the entropy change, S, of the hydrogen atom exchange between two phenols is negligible, Equation 11 holds.

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48

G. F. Pedulli and M. Lucarini

2

[O2]  103 (M)

a

b

c

d

1

0 0

1000

2000

3000

4000

Time(s) Fig. 4.6. Oxygen consumption traces recorded at 30°C during the autooxidation initiated by 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) of 7.5 M styrene in the absence of any antioxidant (a) and in the presence of: (b) 5.0  105 M 2,4,6-trimethoxy phenol (TMP); (c) 5.0  105 M -tocopherol; (d) 5.0  105 M TMP and 5.0  105 M -tocopherol.

(HPLC) and mass spectrometry, it was found that they are consumed at the same rate (see Fig. 4.5). This provides additional evidence that vitamin E is recycled by BHA and also indicates that the radicals from BHA and -tocopherol react with peroxyl radicals with identical rate constants kA
and kCoA
. The fact that synergism between phenolic antioxidants is essentially determined by the BDE difference of the O–H bonds and independent on other factors, such as the relative values of the rate constants for their reactions with peroxyl radicals, is supported by other experimental data. For instance, Fig. 4.6. shows the effect on the oxygen uptake traces of inhibiting the oxidation of styrene with -tocopherol, with 2,4,6-trimethoxy phenol (TMP) and with a mixture of the two. TMP has a rate constant of inhibition (2.3  105 Ms) twice as large as that of BHA, but an O–H BDE value of 80.0 kcal/mol, i.e. 1.8 kcal/mol larger than that of -tocopherol. From plot d of Fig. 4.6, it is seen that the inhibiting effect exhibited by the 1:1 mixture of the two antioxidants is purely additive, since the first part is coincident with the trace obtained in the presence of vitamin E only and the second part is parallel to the trace obtained with TMP only. A similar effect (not shown) is observed when the oxidation is inhibited by mixtures of -tocopherol and 3,5-di-tert-butyl-4-hydroxytoluene (BHT) (Amorati et al., 2003), the latter having an O–H BDE value 2.8 kcal/mol higher than that of tocopherol. The behaviour of TMP and BHT, when mixed with vitamin E, can be rationalized on the basis of the same reaction mechanism previously discussed. Peroxyl radicals react first with tocopherol, which, however, is not recycled by the co-antioxidant since the equilibrium is completely shifted to the left because of the large BDE difference. Therefore, the co-antioxidant begins to react with peroxyl radicals only after tocopherol is completely consumed and, thus, no synergism is observed.

Another interesting example is that of resveratrol, a naturally occurring phenol particularly abundant in grapes and red wine. Many reports (Fauconneau et al., 1997; Soares et al., 2003) attribute to trans-resveratrol an antioxidant activity comparable with that of vitamin E, and others claim that it acts synergistically with -tocopherol (Alonso et al., 2002). Since the O–H BDE value for transresveratrol was estimated recently (Amorati et al., 2003) as 5.5 kcal/mol higher than that of -tocopherol, its reported extraordinary antioxidant activity was surprising. Actually the oxygen uptake traces recorded while inhibiting the oxidation of styrene with vitamin E, with resveratrol and with a 1:1 mixture of the two indicate that resveratrol, in homogeneous solution, is neither an outstanding antioxidant (its inhibition rate constant is only 2  105 Ms) nor capable of effectively regenerating -tocopherol, as also confirmed by the measured rate of disappearance of tocopherol during the thermal decomposition of AIBN, which is completely unaffected by the presence of resveratrol in the reaction mixture (Amorati et al., 2003). Other phenolic derivatives present in red wine might be more likely responsible for its antioxidant activity, among them the numerous flavonoids. Indeed, important synergistic effects with vitamin E have been observed when using as co-antioxidants flavonoids containing the catechol ring (Amorati et al., 2002). These are capable of giving synergism with vitamin E despite their O–H BDE values being higher by 2 kcal/mol or more than tocopherol. Figure 4.7 (left) shows that 4-tertbutylcatechol (BC), that may be considered a model system for flavonoids, can completely regenerate tocopherol despite its estimated BDE value (80.0 kcal/mol) being about 1.8 kcal/mol larger than that of tocopherol. The regeneration effect is even more evident from the plots of Fig. 4.7 (right) showing the relative rates of disappearance of -tocopherol and 4-tert-butylcatechol observed while thermally decomposing AIBN in a chlorobenzene solution containing equimolar amounts of the two antioxidants. It is clearly seen that the concentration of -tocopherol remains unchanged from the initial value until all BC is completely consumed despite the fact that its rate constant for the reaction with peroxyl radicals, kinh = 5.6  105 Ms, is six times lower than that of -tocopherol, and that the antioxidant activity of the mixture is so effective that it can only be due to -tocopherol rather than to the catechol itself. This behaviour can be explained by a mechanism somewhat different from that we have seen previously (see Fig. 4.1), and involves two steps. In the first one, reversible hydrogen transfer takes place between BC and the tocopheroxyl radical to give a semiquinone radical and -tocopherol (Equation 12, i.e. the explicit form of Equation 9 in the present case); in the second step, the resulting semiquinone reacts with another -tocopheroxyl radical to give back tocopherol and tert-butyl-ortho-quinone (Equation 13). Thus, the latter equation should be added to the overall reactions in Fig. 4.1 previously considered. This second reaction is calculated to be highly exothermic (~25 kcal/mol) due to the very low O–H bond strength in the semiquinone radical (~55 kcal/mol) and

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Recycling of Vitamin E by Natural Products: a Dietary Perspective

1.2

2 a

b

c

d

1

0

[Phenol]  104 (M)

[O2]  103 (M)

3

0.8 d 0.4

0.0 0

2000

4000

6000

8000

Time(s)

0

5000

10000

15000

Time(s)

Fig. 4.7. Left: oxygen consumption during the 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN)-initiated auto-oxidation at 30°C of styrene (7.5 M) in chlorobenzene in the presence of 1  104 M 4-tert-butylcatechol (BC) (dashed line). Solid lines have been recorded in the presence of 5  105 M -tocopherol and of BC (a) 0 M; (b) 5  105 M; (c) 1  104 M; (d) 1.5  104 M. Right: disappearance of -tocopherol (open circles) and of BC (filled squares) observed during the thermal (60°C) decomposition under air of 2,2-azobis(isobutyronitrile) (AIBN) (0.002 M) in chlorobenzene containing both antioxidants at initial concentrations of 1  104 M.

therefore fast enough to compete favourably with the termination reactions (Amorati et al., 2002). Thus, as soon as some semiquinone radicals are formed, they are immediately subtracted from the solution in a reaction leading to irreversible regeneration of -tocopherol and to 4-tert-butyl-ortho-quinone. Several other polyphenols, such as epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate and gallic acid, have been found to slow the rate of peroxidation of linoleic acid in tert-butanol–water solution, without showing a definite inhibition period. On the other hand, when used in combination with vitamin E, they behave as much better antioxidants (Jia et al., 1998) due to the synergistic mechanism involving recycling of tocopherol. A detailed study of the recycling of -tocopherol by flavonoids (quercetin, epicatechin and catechin) both in tert-butanol and in chlorobenzene (Pedrielli and Skibsted, 2002) showed that synergism is observed both in chlorobenzene, where the polyphenolic derivatives behaved as inhibitors with a definite induction period, and also in tert-butanol where they behaved as weak retarders when used alone. This means that kinetic solvent effects (Avila et al., 1993; Snelgrove et al., 2001) which are known drastically to reduce the antioxidant activity of phenols and polyphenols do not affect the regeneration of -tocopherol by flavonoids.

Regeneration of tocopherol in homogeneous solution was also observed with polyphenols containing the hydroquinone moiety (Landi et al., 1992). However, in this case, a quantitative rationalization of the experimental results is more difficult to give since the semiquinone radicals, beside reacting with peroxyl radicals to give termination and with tocopheroxyl radicals to regenerate vitamin E, can also react with molecular oxygen to give superoxide or hydroperoxyl radicals (Loshadkin et al., 2002; Roginsky, 2003; Roginsky et al., 2003). More studies are needed in order to clarify the relative importance of the various reactions taking place in these systems. In conclusion, the results of the work carried out on the recycling of vitamin E by mono- and polyphenols in solution can be rationalized by admitting that this process takes place following one of the following two routes: equilibration with a co-antioxidant characterized by a BDE value lower than or comparable to that of tocopherol and, in the case of catechol or hydroquinone derivatives, equilibration followed by reaction of -TO
with the semiquinone radical arising from the polyphenol. In the former case, tocopherol is consumed from the beginning of the oxidation reaction, although at a reduced rate. With polyphenols, on the other hand, -tocopherol is completely preserved until all co-antioxidant has been consumed. Due to this peculiar behaviour, polyphenolic species are ideal (12)

(13)

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G. F. Pedulli and M. Lucarini

co-antioxidants to be used together with a small amount of vitamin E. Also non-phenolic derivatives, besides the obvious example of vitamin C, have been claimed to be capable of regenerating -tocopherol from the tocopheroxyl radical, some of these being -carotene and -carotenoid derivatives. This assertion was based on pulse radiolysis of hexane solutions of -tocopherol and -carotenoids with time monitoring of transients attributed to -CAR+
in the near infrared (Böhm et al., 1997). However, subsequent electron paramagnetic resonance (EPR) studies on the rate of decay of the tocopheroxyl radical in cyclohexane and benzene have shown that this is not influenced by carotene (Valgimigli et al., 1997). Other claims about the ability of -carotene to recycle vitamin E have been reported by Palozza and Kinsky (1992) and by Li et al. (1995), although a convincing explanation of the possible regeneration mechanism was not given.

Non-homogeneous Systems A large number of studies has also been reported on the recycling effect of vitamin E by co-antioxidants in nonhomogeneous systems. Doba et al. (1985) and Niki et al. (Niki et al., 1985, Niki, 1987a, b) investigated the effect of ascorbate on the antioxidant ability of -tocopherol in phospholipid liposomes, and Barclay et al. (1985) in micelles. Large differences were found in these systems when initiating the peroxidation in lipid or in aqueous phases. Thus, with lipid-soluble initiators, vitamin C behaved as a very poor antioxidant while it was an excellent synergist when used together with -tocopherol to inhibit the peroxidation of liposomes. On the other hand, with water-soluble initiators, it is itself a good antioxidant. The inability of ascorbate to trap peroxyl radicals originating in the lipid phase, in contrast to aqueous peroxyl radicals, and the synergism with -tocopherol have been interpreted as evidence that -tocopheroxyl radicals reside with their polar head group close to the membrane surface where they can interact with vitamin C (Doba et al., 1985). Similar results and conclusions were reached by Niki et al. (1985) and by Barclay et al. (1985).

After these reports on the synergistic inhibition of oxidation by a combination of vitamin E and vitamin C, several other papers appeared in the literature describing synergy between -tocopherol and other co-antioxidants including cysteine (Motoyama et al., 1989) and ubiquinol10 (Frei et al., 1990; Mukai et al., 1990; Stocker et al., 1991; Landi et al., 1992) both in dispersed phosphatidylcholine liposomes and in low-density lipoproteins. Glutathione (GSH), on the other hand, seems to be unable to regenerate vitamin E from the tocopheroxyl radical (Frei et al., 1990), this being in agreement with the much lower rate constant for the reaction between -TO
and GSH (25 M/s) in hexamethyl-trimethylammonium chloride (HTAC) micelles when compared with the similar reaction between -TO
and ascorbate (7.2  107 Ms) under the same conditions (Bisby and Parker, 1991). Also uric acid does not show synergistic inhibition of the oxidation of liposomes induced by lipid-soluble initiators when used in mixture with vitamin E (Sato et al., 1990). Synergism with vitamin E in inhibiting the peroxidation of linoleic acid in sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB) micelles has instead been observed with polyphenols containing the catechol and pyrogallol groups (Zhou et al., 2000) when the oxidation was initiated with a water-soluble azo derivative, while only additive effects were observed when initiating the peroxidation with the lipid soluble di-tert-butyl hyponitrite (Chen et al., 2001). Finally, we would like to mention a few examples of studies on the regeneration of vitamin E in biologically relevant systems by ascorbate and other co-antioxidants (Kagan et al., 1990, 1998, 2003; Stoyanovsky et al., 1995; Halpner et al., 1998a, b; Guo and Packer, 2000; James et al., 2004).

Acknowledgements Financial support from the University of Bologna and MIUR (Research project ‘Free Radical Processes in Chemistry and Biology: fundamental aspects and applications in environment and material sciences’) is gratefully acknowledged.

References Alonso, A.M., Domìnguez, C., Guillén, D.A. and Barroso, C.G. (2002) Determination of antioxidant power of red and white wines by a new electrochemical method and its correlation with polyphenolic content. Journal of Agricultural and Food Chemistry 50, 3112–3115. Amorati, R., Ferroni, F., Lucarini, M., Pedulli, G.F. and Valgimigli, L. (2002) A quantitative approach to the recycling of -tocopherol by coantioxidants. Journal of Organic Chemistry 67, 9295–9303. Amorati, R., Ferroni, F., Pedulli, G.F. and Valgimigli, L. (2003) Modeling the co-antioxidant behavior of monofunctional phenols. Applications to some relevant compounds. Journal of Organic Chemistry 68, 9654–9658. Avila, D.V., Brown, C., Ingold, K.U. and Lusztyk, J. (1993) Solvent effects on the competitive -scission and hydrogen atom abstraction reactions of the cumyloxyl radical. Resolution of a long-standing problem. Journal of the American Chemical Society 115, 466–470. Barclay, L.C.R., Locke, G.J. and MacNeil, J.M. (1985) Autoxidation in micelles. Synergism of vitamin C with lipid soluble vitamin E and water soluble Trolox. Canadian Journal of Chemistry 63, 366–374. Bisby, R.H. and Parker, A.W. (1991) Reactions of the -tocopheroxyl radical in micellar solutions studied by nanosecond laser flash photolysis. FEBS Letters 290, 205–208.

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Böhm, F., Edge, R., Land, E.J., McGarvey, D.J. and Truscott, T.G. (1997) Carotenoids enhance vitamin E antioxidant efficiency. Journal of the American Chemical Society 119, 621–622. Buettner, G.R. (1993) The pecking order of free radicals and antioxidants: lipid peroxidation, -tocopherol, and ascorbate. Archives of Biochemistry and Biophysics 300, 535–543. Burton, G.W., Doba, T., Gabe, E.J., Hughes, L., Lee, F.L., Prasad, L. and Ingold, K.U. (1985) Autoxidation of biological molecules. 4. Maximizing the antioxidant activity of phenols. Journal of the American Chemical Society 107, 7053–7065. Chen, Z.H., Zhou, B., Yang, L., Wu, L.M. and Liu, Z.L. (2001) Antioxidant activity of green tea polyphenols against lipid peroxidation initiated by lipid-soluble radicals in micelles. Journal of the Chemical Society, London, Perkin Transactions 2, 1835–1839. DiLabio, G.A. and Wright, J.S. (2000) Hemiketal formation of dehydroascorbic acid drives ascorbyl radical anion disproportionation. Free Radical Biology and Medicine 29, 480–485. Doba, T., Burton, G.W. and Ingold, K.U. (1985) Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes. Biochimica et Biophysica Acta 835, 298–303. Fauconneau, B., Waffo-Teguo, P., Huguet, F., Barrier, L., Decendit, A. and Merillon, J.-M. (1997) Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests. Life Sciences 61, 2103–2110. Frei, B., Kim, M.C. and Ames, B.N. (1990) Ubiquinol-10 is an effective lipid-soluble antioxidant at physiological concentrations. Proceedings of the National Academy of Sciences of the USA 87, 4879–4883. Golumbic, C. and Mattill, H.A. (1941) Antioxidants and the autoxidation of fats. XIII. The antioxygenic action of ascorbic acid in association with tocopherols, hydroquinones and related compounds. Journal of the American Chemical Society 63, 1279–1280. Guo, Q. and Packer, L. (2000) Ascorbate-dependent recycling of the vitamin E homologue Trolox by dihydrolipoate and glutathione in murine skin homogenates. Free Radical Biology and Medicine 29, 368–374. Halpner, A.D., Handelman G.J., Belmont, C.A., Harris J.M. and Blumberg J.B. (1998a) Protection by vitamin C of oxidant-induced loss of vitamin E in rat hepatocytes. Journal of Nutritional Biochemistry 9, 355–359. Halpner, A.D., Handelman G.J., Harris J.M., Belmont, C.A. and Blumberg J.B. (1998b) Protection by vitamin C of loss of vitamin E in cultured rat hepatocytes. Archives of Biochemistry and Biophysics 359, 305–309. Howard, J.A. (1973) Homogeneous liquid-phase autoxidations. In: Kochi, J.K. (ed.) Free Radicals. John Wiley & Sons, New York, Vol. 2, pp. 3–62. James, A.M., Smith, R.A.J. and Murphy, M.P. (2004) Antioxidant and prooxidant properties of mitochondrial coenzyme Q. Archives of Biochemistry and Biophysics 423, 47–56. Jia, Z.S., Zhou, B., Yang, L., Wu, L.M. and Liu, Z.L. (1998) Antioxidant synergism of tea polyphenols and -tocopherol against free radical induced peroxidation of linoleic acid in solution. Journal of the Chemical Society, London, Perkin Transactions 2, 911–915. Kagan, E.V., Serbinova, E.A. and Packer, L. (1990) Generation and recycling of radicals from phenolic antioxidants. Archives of Biochemistry and Biophysics 280, 33–39. Kagan, V.E., Arroyo, A., Tyurin, V., Tyurina, Y., Villalba, J.M. and Navas, P. (1998) Plasma membrane NADH-coenzyme Qo reductase generates semiquinone radicals and recycles vitamin E homologue in a superoxide-dependent reaction. FEBS Letters 428, 43–46. Kagan, V.E., Kuzmenko, A.I., Shvedova, A.A., Kisin, E.R., Lj, R., Martin, I., Quinn, P.J., Tyurin, V.A., Tyurina, Y.Y. and Yalowich, J.C. (2003) Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6hydroxychromane, by ascorbate/dihydrolipoate in living HL-60 cells. Biochimica et Biophysica Acta 1620, 72–84. Landi, L., Cabrini, L., Fiorentini, D., Stefanelli, C. and Pedulli, G.F. (1992) The antioxidant activity of ubiquinol-3 in homogeneous solution and in liposomes. Chemistry and Physics of Lipids 61, 121–130. Li, Z.L., Wu, L.M., Ma, L.P., Liu, Y.C. and Liu, Z.L. (1995) Antioxidant synergism and mutual protection of -tocopherol and -carotene in the inhibition of radical-initiated peroxidation of linoleic acid in solution. Journal of Physical Organic Chemistry 8, 774–780. Loshadkin, D., Roginsky, V. and Pliss, E. (2002) Substituted p-hydroquinones as a chain-breaking antioxidant during the oxidation of styrene. International Journal of Chemical Kinetics 34, 162–171. Lucarini, M., Pedulli, G.F. and Cipollone, M. (1994) Bond dissociation enthalpy of -tocopherol and other phenolic antioxidants. Journal of Organic Chemistry 59, 5063–5070. Lucarini, M., Pedrielli, P., Pedulli, G.F., Cabiddu, S. and Fattuoni, C. (1996) Bond-dissociation energies of O–H bonds in substituted phenols by equilibration studies. Journal of Organic Chemistry 61, 9259–9263. Mahoney, L.R. (1972) Antioxidants. Angewandte Chemie, International Edition 8, 547–555. Motoyama, T., Miki, M., Mino, M., Takahashi, M. and Niki, E. (1989) Synergistic inhibition of oxidation in dispersed phosphatidylcholine liposomes by a combination of vitamin E and cysteine. Archives of Biochemistry and Biophysics 270, 655–661. Mukai, K., Nishimura, M., Nagano, A., Tanaka, K. and Niki, E. (1989) Kinetic study of the reaction of vitamin C derivatives with tocopheroxyl (vitamin E radical) and substituted phenoxyl radicals in solution. Biochimica et Biophysica Acta 993, 168–173. Mukai, K., Kikuchi, S. and Urano S. (1990) Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radicals by reduced ubiquinone-10 in solution. Biochimica et Biophysica Acta 1035, 77–82. Niki, E. (1987a) Interaction of ascorbate and -tocopherol. Annals of the New York Academy of Sciences 498, 186–199. Niki, E. (1987b) Antioxidants in relation to lipid peroxidation. Chemistry and Physics of Lipids 44, 227–253. Niki, E., Saito, T., Kawakami, A. and Kamiya, Y. (1984) Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C. Journal of Biological Chemistry 259, 4177–4182. Niki, E., Kawakami, A., Yamamoto, Y. and Kamiya, Y. (1985) Oxidation of lipids. VIII. Synergistic inhibition of oxidation of phosphatidylcholine liposome in aqueous dispersion by vitamin E and vitamin C. Bulletin of the Chemical Society of Japan 58, 1971–1975. Packer, J.E., Slater, T.F. and Willson, R.L. (1979) Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278, 737–738. Palozza, P. and Krinsky, N.I. (1992) -Carotene and -tocopherol synergistic antioxidants. Archives of Biochemistry and Biophysics 297, 184–187. Pedrielli, P. and Skibsted L.H. (2002) Antioxidant synergy and regeneration effect of quercetin, (–)-epicatechin, and (+)-catechin on -tocopherol in homogeneous solutions of peroxidating methyl linoleate. Journal of Agricultural and Food Chemistry 50, 7138–7144.

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Roginsky, V. (2003) Chain-breaking antioxidant activity of natural polyphenols as determined during the chain oxidation of methyl linoleate in Triton X-100 micelles. Archives of Biochemistry and Biophysics 414, 261–270. Roginsky, V., Barsukova, T., Loshadkin, D. and Pliss, E. (2003) Substituted p-hydroquinones as inhibitors of lipid peroxidation. Chemistry and Physics of Lipids 125, 49–58. Sato, K., Niki, E. and Shimasaki, H. (1990) Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C. Archives of Biochemistry and Biophysics 279, 402–405. Snelgrove, D.W., Lusztyk, J., Banks, J.T., Mulder, P. and Ingold, K.U. (2001) Kinetic solvent effects on hydrogen-atom abstractions: reliable, quantitative predictions via a single empirical equation. Journal of the American Chemical Society 123, 469–477. Soares, D.G., Andreazza, A.C. and Salvador, M. (2003) Sequestering ability of butylated hydroxytoluene, propyl gallate, resveratrol, and vitamins C and E against ABTS, DPPH, and hydroxyl free radical in chemical and biological systems. Journal of Agricultural and Food Chemistry 51, 1077–1080. Stocker, R., Bowry, V. and Frei, B. (1991) Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does -tocopherol. Proceedings of the National Academy of Sciences of the USA 88, 1646–1650. Stoyanovsky, D.A., Osipov, A.N., Quinn, P.J. and Kagan V.E. (1995) Ubiquinone-dependent recycling of vitamin E radicals by superoxide. Archives of Biochemistry and Biophysics 323, 343–351. Valgimigli, L., Lucarini, M., Pedulli, G.F. and Ingold, K.U. (1997) Does -carotene really protect vitamin E from oxidation? Journal of the American Chemical Society 119, 8095–8096. Zhou, B., Jia, Z.S., Chen, Z.H., Yang, L., Wu, L.M. and Liu, Z.L. (2000) Synergistic antioxidant effect of green tea polyphenols with -tocopherol on free radical initiated peroxidation of linoleic acid in micelles. Journal of the Chemical Society, London, Perkin Transactions 2, 785–791.

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5 Mössbauer Spectroscopy Studies of Tocopherol Kvetoslava Burda1,2 and Olaf Kruse3

1Institute

of Nuclear Physics, Polish Academy of Sciences, ul. Radzikowskiego 152, 31–342 Kraków, Poland; 2Institute of Physics, Jagiellonian University, ul. Reymonta 4, 30–059 Krakow, Poland; 3University of Bielefeld, Department of Biology, Molecular Cell Physiology, Universitaetsstrasse 25, 33501 Bielefeld, Germany

Abbreviations: Chl, chlorophyll; cyt b559, cytochrome b559; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; Em,7, mid-point potential at pH 7.0; FCCP, carbonylcyanide-p-trifluoromethoxy-phenyl-hydrazone; HP, high potential; HS, high spin; LP, low potential; LS, low spin; P680, reaction centre, a primary electron donor in photosystem II; PQA, plastoquinone A, also called PQ9; PSII, photosystem II; QA, non-exchangeable plastoquinone at the acceptor side of photosystem II; QB, exchangeable plastoquinone with the external pool of plastoquinones at the acceptor side of photosystem II; QA–Fe–QB, iron–quinone complex containing the non-haem iron.

Abstract Mössbauer spectroscopy is a useful method to investigate chemical and dynamic properties of a probing atombinding site, for example of the iron isotope 57Fe. We show that using this method one can study interactions of tocopherols with redox active components of the electron transfer chain within photosystem II containing iron atoms. -Tocopherol and -tocopherol quinone, being the most abundant forms of vitamin E, quench singlet oxygen and scavenge various active forms of oxygen. Additionally, they are expected to participate in photosynthetic electron transport. Mössbauer spectroscopy allows the elucidation of the action of -tocopherol quinone on the haem iron of cytochrome b559 and the non-haem iron of the iron–quinone complex at the acceptor side of photosystem II. The results give a new insight into a possible protective role for -tocopherol quinone in the cyclic electron flow and energy dissipation in photosystem II under light stress conditions. These results are discussed in detail, and other applications of Mössbauer spectroscopy in investigations of vitamin E actions are outlined.

Introduction Nowadays, it is well established that tocopherols and tocotrienols, from the vitamin E group, play an essential role in human nutrition and health (see other chapters in this volume). They consist of the prenyl chain formed in the isoprenoid pathways (Lichtenthaler, 1999) and the chromanol head formed in the shikimate pathway (Herrmann and Weaver, 1999). The shikimate pathway leading to production of chorismate, which is the precursor of the aromatic amino acids and many aromatic secondary metabolites, is found only in some microorganisms (bacteria, fungi and algae) and plants, but not in animals (Bentley, 1990). Thus, oxygenic photosynthetic organisms,

mainly plants, are the sole source of vitamin E for animals. Although the physiological importance of tocopherols as scavengers and quenchers of reactive oxygen species as well as regulators of membrane permeability and fluidity, gene and intracellular signalling is well recognized in animal systems (for comprehensive reviews see other chapters in this volume), comparatively little is known about their functions in photosynthetic organisms. In some cases, tocotrienols show greater antioxidative activity than the corresponding tocopherols (see review by MunnéBosch and Alegre, 2002). Tocotrienols differ from tocopherols only in the degree of saturation of their hydrophobic tails. They have double bonds at the 3, 7 and 11 positions of the chain (Fig. 5.1A). In nature, four types

© CAB International 2006. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

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A R2

R1 CH 3 CH 3 H H

HO CH3 R1 CH3

O

CH 3

CH 3 3

R2 CH 3 H CH 3 H

α-Tocopherol β -Tocopherol γ -Tocopherol δ -Tocopherol

R2 HO CH3 R1 CH3

Tocotrienols

O

CH3

CH3 3

B CH 3

O2 , O2 , HO2, OH

CH3 CH3

O CH3

CH3

. .

.-

1

HO

O CH3 CH3

CH 3

CH 3

3

CH3

O

CH3

CH3 3

α-Tocopherol

OOH

Hydroperoxydienone

CH3

.O

CH3 CH3 CH3

O

CH3

CH3 3

α-Tocopheroxyl radical OH

CH 3

O

CH3 3

OH

+2e+2H +

(x2)

.O

OH

CH3

CH3

OH

CH 3

CH 3

O

+O 2

CH3

CH3

-H2 O

O

CH3 O

CH3

CH3

3

CH 3

3

O-

CH3

Epoxyquinones

α-Tocopherol hydroxyquinone

+2H +

CH3 3

O CH3

CH3

CH3 CH3

CH3

CH3

CH 3

HO

CH3

OH

CH3 O

CH3

α-Tocopherol semiquinone radical

.-

+O

OH

CH3

+O 2

CH3

O CH3

2

CH3

CH3 3

O CH3

α-Tocopherol quinone

Fig. 5.1. (A) Structures of naturally occurring tocopherol and tocotrienol isomers. Tocotrienols differ from tocopherols only in the degree of saturation of their hydrophobic tails. They have double bonds at the 3’, 7’ and 11’ positions of the chain. Their isomers, , ,  and , vary in the number and position of methyl substituents on the chromanol ring. (B) A scheme of a possible cycling between -tocopherol and -tocopherol quinone, the most abundant forms of tocopherols in oxygenic photosynthetic organisms, including a role for the -tocopherol semiquinone radical in protection of the -tocopheroxyl radical against oxidation. Scavenging of singlet oxygen or other active oxygen species by -tocopherol leads to formation of an -tocopheroxyl radical or hydroxydienone, which is decomposed further to epoxyquinones and -tocopherol quinone. -Tocopherol quinone may react with a superoxide radical, forming an -tocopherol semiquinone radical, or can be reduced and accept 2H+, forming -tocopherol hydroxyquinone. -Tocopherol hydroxyquinone has been shown to be an effective antioxidant by regenerating the -tocopheroxyl radical to -tocopherol. The reactivity of -tocopherol hydroxyquinone with the -tocopheroxyl radical has been found to be comparable with that of ascorbic acid. Thus, -tocopherol hydroxyquinone as a regenerator of -tocopherol may occur in thylakoids. In the regeneration process of -tocopherol, a dehydrotocopheroquinol radical is formed, which can be protonated 2 -TQH
– + 2 H+ and transferred to -tocopherol quinone + -tocopherol hydroxyquinone. This scheme may partially clarify the inter-relationships between -tocopherol and -tocopherol quinone in thylakoids.

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Mössbauer Spectroscopy Studies of Tocopherol

of tocopherols (and four types of tocotrienols) are synthesized: -tocopherol (-tocotrienol), -tocopherol (tocotrienol), -tocopherol (-tocotrienol) and -tocopherol (-tocotrienol). They vary in the number and position of methyl substituents on the chromanol ring (Fig. 5.1A). Tocopherols and tocotrienols together with plastoquinones (PQs) belong to a diverse group of compounds called prenylquinones and are synthesized and accumulated at the inner chloroplast envelope (Soll et al., 1985). The pathway and enzymes for prenylquinone synthesis are homologous in cyanobacteria, algae and higher plants, but cyanobacteria appear to have an alternative route for PQ (Whistance and Threlfall, 1970; Cheng et al., 2003). It is suggested that during plastid development, massive transport of prenylquinones from the inner envelope membrane to the thylakoid membranes occurs by a mechanism that has not yet been identified (Soll et al., 1985). The content of prenylquinones differs qualitatively and quantitatively in photosynthetic organisms, depending on the development stages of the species and localization within the membranes, as well as on environmental conditions, such as light intensity, temperature, drought, nutrients and pollutants (reviewed in Fryer, 1992; MunnéBosch and Alegre, 2002). In leaves, fruits, roots and flowers, -tocopherol dominates over the other isomers, but in seeds both - and -isomers are present at higher concentrations. The - and - forms are present in seeds and leaves in minor amounts (DellaPenna, 1999). Such a distribution of tocopherol forms is probably closely related to their biosynthetic pathways (Cheng et al., 2003). It has been shown that -tocopherol methyltransferase regulates the spectrum of accumulated tocopherol in plants (Koch et al., 2003). In spinach chloroplasts, the ratio of -tocopherol to chlorophyll a (Chl a) is typically 1:10 (Halliwell, 1981). One-third of the total amount of -tocopherol is accumulated in the envelope but two-thirds are in the thylakoid membranes (Wiese and Naylor, 1987). The molar ratio of -:-:-isomers of tocopherol in spinach thylakoids has been found to be 1:0.06:0.02 (Asada and Takahashi, 1987). In in vivo experiments, a relative efficiency in antioxidant protection against lipid oxidation of the tocopherol isomers with  >  >  >  has been reported. The efficiency is correlated with the electron-donating capacity of the alkyl substituents on the aromatic ring (Mukai et al., 1989) and the abundance of the various tocopherol forms in thylakoids. However, it has to be emphasized that the studied systems were modified by substrates and therefore the action of tocopherol in native systems can differ from those observed in these investigations. Moreover, recent studies provide evidence that the function of tocopherols in plants is far more diverse (Fryer, 1992; Munné-Bosch and Alegre, 2002). Plants are very sensitive to high light exposure but, under unfavourable environment conditions such as drought, temperature or nutrient stress, they are sensitive even to low light intensities (Foyer et al., 1994; Asada, 1999). As a consequence, plants developed various enzymatic and non-enzymatic protection mechanisms against generated reactive oxygen species (1O2, O2–
, HO2


55

and HO
) which could cause photoinhibition of photosynthesis and protein degradation. Ascorbate peroxidase, glutathione reductase and superoxide dismutase (Rabinovich and Fridovich, 1983; Foyer et al., 1994; Asada, 1999) belong to the enzymatic antioxidants, and tocopherols, carotenoids, ascorbate and glutathione are non-enzymatic antioxidants (Fryer, 1992; Foyer et al., 1994; Smirnoff, 1996; Cogdell and Frank, 1996).

Tocopherols in Photosystem II Photosystem II (PSII), as the site of electron and proton abstraction and of oxygen evolution by water splitting, is a multimeric protein complex, which is incorporated into the thylakoid membranes of chloroplasts. PSII is extremely sensitive to environmental changes. As a consequence, plants developed photoprotection close to PSII, whose molecular mechanisms are still not well understood. In this chapter, we will concentrate on the influence of tocopherols on the action of PSII, in particular the most abundant isomer -tocopherol and its oxidized form -tocopherol quinone. -Tocopherol and -tocopherol quinone, being natural components of the photosynthetic membranes, occur at about 25–30% and 10% of the amount of plastoquinone A (PQA, also known as PQ9), respectively (Lichtenthaler et al., 1981; Kruk and Strzałka, 1995). PQA is the main component of the prenylquinone pool in thylakoids and the only one whose role in photosynthesis is fully described (Rich and Moss, 1987; Kruk et al., 1998). The function of -tocopherol as an antioxidant is well recognized. As a single molecule, it can undergo approximately 120 1O2 quenching events before it is oxidized to -tocopherol quinone (Fahrenholtz et al., 1974; Neely et al., 1988). It can trap fatty acylperoxy radicals formed during lipid oxidation and effectively scavenge many active forms of oxygen (Fryer, 1992). The scheme of its action is shown in Fig. 5.1B. -Tocopherol quinone and/or its epoxide forms are final products of these reactions (Ham and Liebler, 1995). Thus, it is clear that the -tocopherol quinone which appears in thylakoids is not only directly synthesized but may also result from the oxidation of -tocopherol. -Tocopherol quinone has been detected in all photosynthetic algae and higher plants, except for Anacystis nidulans and Synechococcus sp. (reviewed by Kruk and Strzałka, 1995). Usually, it occurs together with its reduced form -tocopherol hydroxyquinone. Dark-stored spinach chloroplasts contain oxidized and reduced forms of -tocopherol quinone in almost equal amounts, whereas light stimulates its oxidation (Dilley and Crane, 1963). This process is reversible in darkness. It seems that both forms of -tocopherol quinone exist in a state of dynamic equilibrium, which is affected by light. Intriguingly, a similar stimulation of -tocopherol quinone accumulation (twofold) has been observed in illuminated stored chloroplasts, whereas the amount of -tocopherol was almost unchanged (Michalski and Kaniuga, 1981). There are two possibilities to explain this: (i) irreversibly inactivated superoxide dismutase during cold storage

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caused intensive formation of superoxide radicals responsible for -tocopherol oxidation to -tocopherol quinone as has been claimed by the authors; and/or (ii) the increased amount of -tocopherol quinone came from partial oxidation of -tocopherol hydroxyquinone stored in darkness and therefore the -tocopherol pool stayed unchanged. The first possibility provides no explanation as to why the -tocopherol pool should be almost stable. Probably a combination of these two hypotheses should be considered. -Tocopherol hydroxyquinone has been shown to be an effective antioxidant by scavenging the lipid peroxyl radical and by regenerating the -tocopheroxyl to -tocopherol (Mukai et al., 1992). The reactivity of -tocopherol quinone with -tocopheroxyl has been found to be comparable with that with ascorbic acid, at least in in vitro experiments. Ascorbic acid is essential for recycling of -tocopherol from tocopheroxyl radicals in chloroplasts (Fryer, 1992). A synergistic action of ascorbic acid and -tocopherol in scavenging oxygen radicals has been already suggested by Packer et al. (1979). The same effect could be expected in the case of -tocopherol hydroxyquinone which might be biologically important as its abundance in thylakoids is rather too low for it to play the role of a radical scavenger. In the regeneration process of -tocopherol, the dehydrotocopheroquinol radical (TQH
–) is formed, which can be protonated: 2 -TQH
– + 2 H+ → -tocopherol quinone + -tocopherol hydroxyquinone. Assuming that the action of -tocopherol hydroxyquinone as a regenerator of -tocopherol takes place in thylakoids during illumination, it is clear why the amount of -tocopherol quinone increases twofold without influencing the -tocopherol pool. The semiquinone form of -tocopherol quinone can be protonated, for example, in desaturation reactions of fatty acids (Infante, 1999). In Fig. 5.1B, we present the proposed cycling of -tocopherol and -tocopherol quinone, which may partially clarify the inter-relationships between these two prenylquinones in thylakoids.

-Tocopherol and 1O2 in Photosystem II PSII catalyses the light-induced water oxidation and PQ reduction (Debus, 1992). The reaction centre of PSII is composed of two homologous polypeptides D1 and D2 containing binding sites for six molecules of Chl a, two pheophytins, two plastoquinones (QA and QB), two carotenes, one non-haem iron, manganese complex and cytochrome b559 (cyt b559) (Fig. 5.2). There are many redox cofactors participating in the photosynthetic electron transport chain within PSII (Ferreira et al., 2004). The primary electron acceptor pheophytin receives an electron from the excited reaction centre Chl a species P680*, transferring it to plastoquinone, QA. The electron is transferred from the reduced QA– to a second plastoquinone, QB. In two turnovers, QB, which has accepted two electrons and taken up two protons, is replaced by a new oxidized plastoquinone. P680+ is reduced by the primary donor tyrosine 161 Yz. The water-oxidizing complex

extracts electrons from H2O transferring them to Yz. The linear transfer of electron flow in PSII, which is responsible for electron and proton delivery to subsequent components of the photosynthetic chain, is shown in Fig. 5.2B. For many years, the rapid turnover of the D1 protein during the process of oxygen evolution has been known (Kyle et al., 1984). It occurs at any light intensity but is enhanced by increasing light intensity (Keren et al., 1997; Jansen et al., 1999). When the plastoquinone pool of PSII is over-reduced, the iron–quinone complex (QA–Fe–QB) also stays reduced. This leads to a charge recombination between the primary electron acceptor Pheo– and the Chl dimer P680+, resulting in the formation of a harmful longer lived 3P680 triplet state (103 s) (Rutherford and KriegerLiszkay, 2001; Diner and Rappoport, 2002). This triplet state can be quenched by neighbouring -carotenes, which further dissipate its energy, but this reaction seems to be insufficient or even impossible due to the location, orientation and distance of -carotenes in the PSII reaction centre (Fig. 5.2A and B), which are too far away from each other for orbital overlap (Trebst, 2003). Oxygen, present in PSII, has an excited single state of low energy and therefore can quench the 3P680 triplet state efficiently (Knox and Dodge, 1985). This singlet oxygen appears to trigger the degradation of the D1 protein (Telfer and Barber, 1989; Hideg et al., 1998). 1O2 can be quenched further by carotene (Gorman and Rodgers, 1981) or -tocopherol, which has been shown to be an effective quencher (in physical processes) and scavenger (in chemical processes) of singlet oxygen (Fahrenholtz et al., 1974). The chemical 1O deactivation by tocopherol results in the formation of 2 tocopherol quinone and tocopherol quinone epoxides through intermediate hydroperoxydienones (Neely et al., 1988). It has been suggested that the protection of PSII against the singlet oxygen action by the tocopherol is the main reason why plants need tocopherols and continuously produce them (Trebst et al., 2002). The failure of PSII protection from singlet oxygen leads to the degradation of the D1 protein, disassembly of PSII and liberation of Chl responsible for photodynamic bleaching if not removed via ubiquitous Chl degradation (Matile et al., 1999). Secondary reactive oxygen species accompany the pigment bleaching, but the formed oxygen radicals may also be scavenged by -tocopherol, resulting in its oxidation (see Fig. 5.1B).

-Tocopherol and its Derivatives in Photosynthetic Electron Transport In stress conditions, cyclic electron transport around PSII has been described as a first step of protection against photoinhibition (reviewed by Stewart and Brudvig, 1998). There are some data suggesting participation of -tocopherol quinone and -tocopherol in photosynthetic electron transport (Barr and Crane, 1977; Michalski and Kaniuga, 1981; Kruk and Strzałka, 1995), but the site of their action has not been identified. The role of -tocopherol quinone as an electron acceptor in PSII has been excluded (Kruk et al., 1997, 1998). It has been shown

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Mössbauer Spectroscopy Studies of Tocopherol

57

Fig. 5.2. Photosystem II (PSII) complex structure adapted from Ferreira et al. (2004). (A) Structural model representing the PSII complex including the reaction centre proteins D1 and D2, the oxygen-evolving complex (OEC), the proximal antenna proteins CP43 and CP47 and several PSII subunits (Psb) in the symmetric dimeric complex. (B) Structural model representing the PSII reaction centre (D1, D2 and cyt b559) including cofactors involved in the electron transfer chlorophylls (D1 and D2 should be as bottom indexes in ChlD1, ChlD2, PD1, PD2 and ChlzD1), -carotene and pheophytin (D1 and D2 should be as bottom indexes in PheoD1 and PheoD2). (C) Structural model representing the binding site of the exchangeable plastoquinone QB in the iron–quinone complex in PSII.

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that -tocopherol quinone decreases oxygen evolution of tobacco thylakoids, while -tocopherol increases it (Kruk et al., 1997). In flash light experiments, both prenylquinones increase the probability of a fast mode of oxygen evolution, but they differently influence the dark distribution of the redox states of the manganese complex, which is a site of water depletion in PSII, and the probabilities of transition between those subsequent states. These results suggest some specific interactions of -tocopherol and -tocopherol quinone with the donor (the manganese complex) and acceptor side (QA–Fe–QB) of PSII (Kruk et al., 1997). Another specific site of their action is probably cyt b559, which is generally accepted to be a component participating in the cyclic electron flow around PSII (Stewart and Brudvig, 1998). This idea is supported by the fact that in two algae in which -tocopherol and -tocopherol quinone are absent, namely Synechococcus 6301 (Anacystis nidulans) (Omata and Murata, 1984) and the Scenedesmus obliquus PS28 mutant (Bishop and Wong, 1974), no high potential (HP) form of cyt b559 can be identified. The protection function of cyt b559 against photoinhibition is probably associated with the conversion between its HP and low potential (LP) forms, as it is known that it has an unusually high and variable mid-point reduction potential. Cyt b559 occurs mainly in two potential forms, an HP (Em,7 = 350–400 mV) and an LP form (Em,7 = 0–80 mV), in thylakoids but also other potential forms were described, such as intermediate potential (IP; Em,7 = 150–270 mV) and the very low potential form (VLP; Em,7 = –45 mV) (Stewart and Brudvig, 1998). However, it has been found that none of the prenylquinones present in thylakoid membranes (-tocopherol, -tocopherol quinone and PQA) were able to restore the HP form of cyt b559 (Kruk et al., 2000). PQA and -tocopherol did, however, increase the reduction level of the cyt b559 HP form and -tocopherol quinone decreased the level of this form in a Scenedesmus wild type. It appeared that -tocopherol quinone quenched fluorescence in the wild-type of Scenedesmus and its mutant PS28 as well as in tobacco thylakoids to a higher extent compared with PQA. -Tocopherol showed a small opposite effect. It has been proposed that -tocopherol may act on the HP of cyt b559 because it was most active in tobacco thylakoids containing the highest amount of this cyt b559 form among the studied systems. -Tocopherol stimulated the accumulation of a reduced form of cyt b559 HP. Most probably it prevents the oxidation of HP similarly to the way PQA acts (Cox and Bendall, 1974). It has been shown that the reduced form of HP cyt b559 is converted much more slowly to the LP form than the oxidized form of HP (Ortega et al., 1990). In particular, the interaction of -tocopherol quinone with the cytochrome seems to be interesting with respect to the cyt b559 photoprotective function, because it was shown that this prenylquinone effectively quenches PSII fluorescence by a mechanism similar to that of carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) and Cu2+, i.e. by deprotonation of the HP form of cyt b559 (Arnon and Tang, 1988; Kruk et al., 2000; Burda et al., 2003a), causing oxidation of this cyt b559 form. -Tocopherol quinone is able to oxidize directly the LP form of cyt b559 (Kruk and Strzałka, 2001). It is

worth mentioning here that -tocopherol hydroxyquinone changes the fluorescence induction kinetics similarly to the PSII QB acceptor site inhibitor 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU). However, it turned out that addition of -tocopherol hydroxyquinone in a molar ratio to Chl of 1:1 inhibited oxygen evolution only by 30%. Therefore, the action of -tocopherol hydroxyquinone in PSII is certainly much more complex than simply blocking the QB-binding site (Kruk et al., 2000). These results indicate a highly specific role for -tocopherol quinone in the process of electron and energy transport within PSII. The function of -tocopherol quinone seems to be closely related to its interaction with many redox active components of PSII, the action of some of which has not yet been explained. Cyt b559 and the non-haem iron from the quinone–iron complex on the acceptor side of PSII are the most intriguing components in the electron transfer chain within PSII. Applying Mössbauer spectroscopy in the studies of the interaction of the prenylquinone with these two ironcontaining components present in PSII gives a unique possibility to look for its specific local interaction with cyt b559 and the non-haem iron (Burda et al., 2003b).

Mössbauer Spectroscopy There are many comprehensive reviews on the basic principles of Mössbauer spectroscopy (e.g. Greenwood and Gibb, 1971). Here, only a brief description of the method summarizing the most relevant facts is given. Mössbauer spectroscopy, discovered in 1957, has already been proven to be a powerful tool in studies of biological samples containing iron as the isotope, 57Fe being one of the most suitable Mössbauer probes. The method is based on a study of the energy dependence of the resonant absorption or emission of -rays. The resonance energy spectrum is scanned by Doppler shifting of the source relative to the absorber changing the energy of the emitted -rays (E) by E = (v/c)E, where v is the source velocity and c is the light velocity (c ≈ 3  108 m/s). The radiation transmitted through the absorber is recorded as a function of velocity; for 57Fe: 1 mm/s = 4.8  108 eV = 11.6 MHz. In the recoil-free transition from an excited nuclear state to the ground state, no energy is lost to the system and the -ray carries the total energy of this transition. The Mössbauer -ray energies (E) are usually of the order of 104–105 eV and free atom recoil energies (ER) are 104–101 eV, according to the formula:

ER =

Eγ

2

, where c is 2Mc2 the light velocity and M is the mass of the atom. The transition energy between the first excited state and the ground state of 57Fe is 14.4 keV, which gives the recoil energy of about 2  103 eV. It is considerably lower than chemical and lattice (phonon) energies of solids, which are of the order of 1–10 eV. The chemical bonds prevent the atom from being freely recoiled and therefore the recoiling mass is the mass of the whole molecule, which makes ER completely negligible. The line width ( ) of the source emission or the absorption line is determined by the

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Mössbauer Spectroscopy Studies of Tocopherol

lifetime of the excited state () according to Heisenberg’s uncertainty principle. The line width is defined as the full width at half maximum and it is ≈ 5  109 eV for the first excited state of 57Fe, whose lifetime  is about 1.4  107 s. The line width is many orders of magnitude smaller than typical phonon energies. However, there are many inherent processes causing line broadening, such as, for example, diffusive motion of atoms. From the line broadening, one can derive the diffusion coefficient of large organic molecules (Craig and Sutin, 1963). The ratio of the natural line width and the photon energy /E is a measure of the accuracy in the determination of relative energy (frequency) changes. This ratio is 3  1013 for 57Fe. Taking into account that line shifts can be measured with an accuracy of 1%, the sensitivity of Mössbauer spectroscopy is, in the case of 57Fe, of the order of 1015. The ability to detect such extremely small changes in relative energy makes the Mössbauer effect unique. This method enables resolution of hyperfine interactions of the 57Fe nucleus with its electronic environment such as: (i) electric monopole interaction; (ii) electric quadrupole interaction; and (iii) magnetic dipole interaction. The electric monopole interaction affects the nuclear energy levels and thus the position of the resonance lines on the velocity scale, giving rise to an important quantity called isomer shift (). The isomer shift reflects the Coulomb interaction of the nuclear charge distribution over a finite nuclear radius (R), in the excited and ground states and the electron charge density at the nucleus. In the nonrelativistic approximation, the isomer shift is expressed by: δ =C

2 2 δR ( ψ A (0) − ψ S (0) ) R

(1)

δR R is the relative change of the nuclear radius between the excited state and ground state, |S(0)|2 and |A(0)|2 are the total electron density evaluated at the nucleus for source and absorber, respectively. The isomer shift is affected by changes in the electronic structure of the valence shell resulting, for example, from the change of charge state or change of bond properties due to electron delocalization. In the case of 57Fe, electrons from the 4s and 3d outer shells make the main contribution to the isomer shift. The changes in the s-electron population in the valence shell directly influence the electron density inside the nucleus, whereas d-electrons act indirectly via shielding of s-electrons causing the effective decrease of the s-electron density in the nucleus volume. Because R is negative for 57Fe (the nuclear radius of the excited state is smaller than the radius of the ground state), the increase of 3d electrons, lowering the total s-density at the nucleus, leads to the increase of the isomer shift. The details on the isomer shift are given in Shenoy (1978). The distribution of the nuclear charge is not uniform and spherically symmetrical in many nuclei. It may be different in each state of excitation. An electric quadrupole moment (eQ), which is a measure of the deviation from spherical symmetry, is constant for a given Mössbauer nuclide and is observable only for nuclei with where C is a constant characterizing a given isotope,

1 . A negative quadrupole moment indicates that 2 the nucleus is oblate (flattened) and a positive moment that the nucleus is prolate (elongated). For 57Fe, the 14.4 keV 3 transition occurs between the nuclear excited level and 2 1 the ground level of identical parity. Only the first excited 2 state of 57Fe has an electric quadrupole moment (Q ≈ 0.2 b), which splits into two double degenerated sublevels in a 3 3 3 1 ,± non-homogeneous electric field: and ,± 2 2 2 2 spin I >

without shifting the centre of mass. Due to the selection rule for dipole magnetic transition, M = 0, ±1, the detected spectrum consists of two absorption lines corresponding to 3 1 3 3 1 1 1 1 ,± → ,± ,± , ± and → transitions. The 2 2 2 2 2 2 2 2 twofold degeneracy of the substates can be removed only by magnetic perturbation, but we will not discuss this here. The eigenvalues of the Hamiltonian describing the electric quadrupole interaction in a coordinate system aligned along the principal axes of the potential field at the nucleus give the energy values of the split levels:

[

]

1

eQVZZ ⎛ η2 ⎞ 2 2 EQ = 3I − I ( I + 1) ⋅ ⎜ 1 + ⎟ 4I (2I − 1) z 3⎠ ⎝

(2)

where e is the charge of proton; VXX, VYY and VZZ are the second spatial derivatives of the electrostatic potential in the principal axes directions (diagonal elements of the electric field gradient tensor); = (VXX – VYY)/VZZ is the asymmetry parameter; I is the nuclear spin quantum number; and IZ = –I, –I+1, …, I–1, I is the nuclear magnetic spin quantum number. Thus the energy separation of the substates in 57Fe for its first excited state is: 1

eQVZZ ∆EQ = EQ ( ±3 / 2) − EQ ( ±1 / 2) = 2

⎛ η2 ⎞ 2 ⎜1 + ⎟ 3⎠ ⎝

(3)

EQ is called the quadrupole splitting and is generally quoted in velocity units. In an axially symmetric field = 0. The quadrupole splitting observed in Mössbauer spectra is altered by changes of the electric field gradient, which may be influenced by the valence electrons of the probe atom and by the surrounding ions. Non-cubic electron distribution in partially filled valence orbitals of the Mössbauer atom makes the so-called valence electron contribution to the electric field gradient, and the charges from surrounding atoms in non-cubic symmetry make the lattice contribution. The s-electronic wave functions make no contribution to the electric field gradient because they have spherical symmetry, but p- and d-electronic wave functions do. The valence orbitals are influenced by the crystal field and by the anisotropic bond properties, i.e. anisotropic population of molecular orbitals. Usually, the major contribution to the electric field gradient for iron ions comes from the valence molecular orbitals unless the ion has a high intrinsic symmetry as high spin (HS) Fe3+ (d5), in which case the main contribution is from lattice. A

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low spin (LS) Fe2+ cation behaves similarly to the HS ferric one, but the latter is highly sensitive to the anisotropy of covalency. The discussion of the Mössbauer results presented below will concentrate on the interpretation of the isomer shift and the quadrupole splitting, which provide the most valuable chemical information from the Mössbauer studies. They are directly related to the electronic structure of an investigated ion, its bond properties and the molecular symmetry of its ligands. It should be stressed that there is no ‘Mössbauer-silent’ iron; this means that all spin and valence states are detectable. The spectra shown consist only of doublets, thus no magnetic field at the nucleus is present in the samples. A discussion on dipole magnetic interactions is beyond the scope of this chapter, but can be found elsewhere (Greenwood and Gibb, 1971). Temperature measurements of the Mössbauer recoil-free fraction give valuable information on molecular dynamics. Mössbauer spectroscopy on the isotope 57Fe has already proved to be a useful tool to investigate the dynamics of iron-containing biological systems. Using the Mössbauer method, one can obtain knowledge about the behaviour of local modes, which is unobtainable by other techniques. Motions in proteins are known to exist in a wide range of time scales, and the dynamic transitions strongly depend on the time scale window of the technique used to observe the phenomena. Mössbauer spectroscopy enables observation of dynamic displacement of iron, which occurs on a time scale faster than the lifetime of the first excited state of 57Fe, i.e. faster than 140 ns. Essential information on dynamics is encoded in the Lamb–Mössbauer factor: f = exp(–k2 )

(4)

where k = 1/0.137Å1 is the wave number of the 14.4 KeV gamma ray for 57Fe. The Lamb–Mössbauer factor is proportional to the logarithm of the absorption area of the Mössbauer spectrum, which is determined by a least squares fit of Lorentzian lines (for a thin absorber, which is the case for biological samples). Using the Debye model, that assumes a large number of oscillator levels with frequency distribution ranging from 0 to a maximum frequency D, one obtains the following formula for the recoil-less fraction: ⎧ ⎪ 3E R f = exp⎨− ⎪⎩ 2kB θ D

2 ⎤⎫ ⎡ ⎛ ⎞ θD /T x ⎥ ⎪⎬ ⎢1 + 4 T dx ∫ ⎜θ ⎟ ⎢ ⎝ D ⎠ 0 e x − 1 ⎥⎪ ⎦⎭ ⎣

(5)

where T is the absolute temperature, D is a characteristic – = k  , k is the Debye temperature defined as h D B D B – Boltzmann constant, and h is a Planck constant normalized to 2π. The recoil-free fraction increases as the resonant nuclear transition energy decreases and the Debye temperature increases. According to the Debye model, at high temperatures (T  D), the mean square displacement of the Mössbauer atom is proportional to T. However, experimental results usually show that the linearity is disrupted by anharmonicity of the lattice vibrations. In Mössbauer spectra of biological samples, a characteristic temperature, around 200 (±30) K is observed,

above which the Lamb–Mössbauer factor decreases rapidly whereas the line width often increases. Below that temperature, the mean square displacement of iron indicates a solid-like behaviour, whereas above that temperature it is influenced by additional molecular fluctuations. One can introduce anharmonic corrections to the Debye model (Burda et al., 1994), which are significant for higher temperatures:

D = 0(1 + AT + … )

(6)

where 0 is the Debye temperature in the low temperature limit and A is an anharmonicity parameter. For low temperature (below the Debye temperature), increases linearly with temperature, with the main contribution from lattice or solid-state vibrations v (Fig. 5.5B). In an independent approach, Frauenfelder et al. (1979) has shown that the total mean square displacement t of iron can be approximated by a sum of three statistically independent terms: t = v + cf + ct

(7)

where indices v, cf and cs are related to vibrational, collective fast and collective slow (diffusional) modes. The so-called bound diffusion (slow collective motions) (Nowik et al., 1983) responsible for the appearance of a broad line can be detected at higher temperatures T > 260 K (Fig. 5.3C). The effect is associated with the fluidity of the protein matrix but we will not elaborate this point here. Within the temperature range from 78 K up to 260 K, t is the sum of vibrational and fast collective motions (Fig. 5.5B). Above a characteristic temperature, which is usually about 200 K in large biomolecules, groups of atoms can occupy different substates according to the Boltzmann distribution, but below the characteristic temperature they stay in the same substate. In the most simple case, one considers only two main substates of the resonant atom (Keller and Debrunner, 1980; Burda et al., 1994). The transition rate from the higher potential well to the lower one is given by: ⎛ Q ⎞ k H = ν0 exp⎜ − ⎟ ⎝ kBT ⎠

(8)

and from the lower potential well to the higher one by: ⎛ E + Q − T∆S ⎞ k L = ν0 exp⎜ − ⎟ kBT ⎝ ⎠

(9)

where E denotes the energetic difference between the substates and Q the separation potential barrier, S is the activation entropy, 0=1013 s1 is a typical vibrational frequency of a solid, T is the temperature and kB is the Boltzmann constant. Assuming that kH>>kL, one obtains the following expression for cf : < x 2 >cf =

d 2k L k H + τ −1

(10)

where  = 1.4  107 s is the lifetime of the excited state of Fe and d is the distance between the two substates. The model is presented in Fig. 5.5A.

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Mössbauer Spectroscopy Studies of Tocopherol

As can be seen from the above considerations, Mössbauer spectroscopy is a technique which is sensitive to the local dynamics of the part of a molecule containing iron atoms. Since iron atoms are usually located at redox active or enzymatically active sites of proteins, this is a very important method, useful in understanding the influence of the protein flexibility on its functioning. At the same time, the method allows for monitoring changes of iron states and the arrangement of its binding site. A summary of information which can be derived from Mössbauer measurements is given in Table 5.1. A disadvantage of the Mössbauer method is that it requires a solid absorber. Thus biological samples have to be studied in a frozen solution, in lyophilized form or as a single crystal. Since the natural abundance of 57Fe is only about 2%, the samples have to be enriched in this isotope. The required 57Fe concentration per iron site is approximately 0.2–1 M. The isotope can be incorporated

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into the molecule by growing the organism from which the sample is to be prepared or by chemical substitution. The latter method has to be applied very carefully because it may introduce uncontrolled modifications of the properties of the system.

Interaction of α-Tocopherol Quinone with PSII Components It is crucial for Mössbauer experiments on -tocopherol quinone interactions with cyt b559 and the non-haem iron from PSII that the samples have high purity and homogeneity and no contamination with other photosynthetic components containing iron atoms. If possible, the samples should be prepared avoiding any chemical treatment which could damage the sample. For example, this can be achieved by using a mutant organism

Table 5.1. What one can learn from Mössbauer spectroscopy measurements. Hyperfine interactions Isomer shift (IS)

The isomer shift measures the electric monopole interaction between charges within the volume of the Mössbauer nucleus and the electrostatic potential generated outside the nucleus. Electric monopole interaction shifts nuclear energy levels without altering their degeneracy, causing the shift of the position of the resonance lines in the Mössbauer spectrum called isomer shift (usually denoted by ). Isomer shift is correlated with the oxidation and spin state of the Mössbauer ion, electronegativity of ligands, covalency effects and coordination number, and thus it provides information on bond properties and electron configuration of the probing atom.

Quadrupole splitting (QS)

Quadrupole splitting results from electric quadrupole interactions. Nuclei with a charge distribution deviating from spherical symmetry have a non-zero quadrupole moment (nuclei with spin I > 
), which can interact with an inhomogeneous electric field (described by the electric field gradient) at the nucleus. The quadrupole moment for a given Mössbauer nuclide is constant. Changes in the quadrupole interactions arise only from the electric field gradient in different compounds. Non-cubic electron distribution in partially filled valence orbitals of the probing atom and aspherical charge distribution in the ligand sphere and/or surrounding lattice with non-cubic symmetry contribute to the total electron field gradient. For example, quadrupole splitting for a nucleus with spin I = 3/2, as in the case of the excited state of 57Fe, leads to its splitting into two double degenerated sublevels. Because the ground level of this iron isotope has no quadrupole moment (I = 1/2), the Mössbauer spectrum consists of two lines. The distance between them is called quadrupole splitting (the corresponding energy splitting is usually denoted by E). In the case of higher spins of ground and excited Mössbauer levels, the spectrum is characterized by larger numbers of resonant lines. Quadrupole splitting reflects the electronic structure of the probing ion, its bond properties and molecular symmetry.

Magnetic splitting

Magnetic splitting measures magnetic dipole interactions. A nucleus with spin I > 0 has a magnetic dipole moment, which can interact with a magnetic field at the nucleus. The energy level splits into 2I + 1 equally spaced sublevels, removing the original degeneracy of the state. This is called the Zeeman effect. The sublevels are characterized by the nuclear magnetic spin quantum number mI = I, I–1,…, –I. For example, the 3/2 state of 57Fe is split into four levels, while the ground state 1/2 is split into two. In this case, the Mössbauer spectrum consists of six resonant lines which can be classified according to the selection rules: I = 1 and m = 0. In practice, the six-line signal is usually perturbed by the quadrupole coupling which additionally changes the energy of sublevels. Directions of the changes depend on the sign of the quadrupole coupling constant. Therefore, the simultaneous appearance of dipole magnetic and electric quadrupole interactions allows one to determine an internal magnetic field at the Mössbauer nucleus and the sign of the electric field gradient. Lattice dynamics

Probability of recoil-less absorption by Mössbauer atom in a lattice

The Mössbauer recoil-free fraction depends on the mean inverse frequency of the lattice vibrations. In the Debye theory, for a given sample, there is a characteristic Debye temperature D, which is proportional to the inversion of the maximum frequency of the lattice D. In harmonic approximation, the recoil-less fraction is given by the Debye–Waller factor proportional to temperature T for T = 
D but in practice this factor is non-linear due to anharmonicity of the lattice vibrations. From that temperaturedependent deviation one obtains information on the dynamic features of the probing atom matrix.

Line broadening

At the melting point, the absorption decreases more rapidly than predicted by the temperature dependence of the Lamb–Mössbauer factor. This can be attributed to an increase of resonant line width, which is related to diffusion processes. The line broadening is proportional to the diffusion coefficient of the probing atom. Mössbauer spectroscopy can be used to study diffusion processes of a time range from 10–7 to 10–10 s.

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as has been done by Burda et al. (2003b). The experiments were performed on a PSI-deficient mutant of Chlamydomonas reinhardtii cultivated in TAP medium (Harris, 1989) containing Hunter’s trace solution enriched in an iron isotope 57Fe. Thylakoid membranes were isolated according to Diner and Wollman (1980) by sucrose cushion centrifugation. The PSI deficiency of the strain Xba9 was caused by the disruption of a psaA exon downstream of the psbD gene. The mutation was achieved by an in vitro mutagenesis approach, using the plasmid vector pCA1 and the aadA-selectable marker (Andronis et al., 1998) for chloroplast transformation with a gunpowderdriven DNA particle delivery instrument (Shearline-MK2, UK). In order to estimate the contamination rate of the cyt b6/f complex, immunoblottings of the isolated thylakoid membrane using an antibody raised against cyt f (PetA) and cyt b559 (PsbE) were performed. For immunoblotting with polyclonal anti-PetA (dilution 1:1000) and anti-PsbE (dilution 1:2000), nitrocellulose membranes were prepared and stained following the procedure of Kruse et al. (1997). Immunoblotting clearly demonstrated that Xba9 thylakoid membranes contain only very low amounts of cyt f (PetA) compared with wild type and no detectable amounts of the cyt b6/f complex subunit IV (PetD). From these data, an overall amount of 90% overall yield (Fig. 6.15) together with SD 7 as a byproduct. The hydrophilicity of the secondary amines produced can be utilized to separate them easily from the strongly hydrophobic products of the cleaved protecting group. Twelve example primary amines were monoalkylated according to this procedure, in addition to six amino acids which were converted into the corresponding N-alkyl-amino acids (Rosenau and Habicher, 1995). 5a-Bromo--tocopherol (14) was also used as an aminoprotecting group in the synthesis of dipeptides according to the DCC method (Bodanszky and Bodanszky, 1994), employing N-Toc-protected amino acids (23) which were coupled with amino acid esters to dipeptides, as demonstrated by means of the six examples Ala–Ala, Ala–Cys, Cys–Cys, Gly–Gly, Leu–Ala and Leu–Leu (Fig. 6.16). The overall yield of the reaction sequence was reported to be largely dependent on the coupling reaction, since both installation and removal of the protecting group from the N-Toc-protected dipeptide (24) – performed as in

Fig. 6.13. 5a--Tocopheryl esters.

Fig. 6.14. Quaternary N-(5a--tocopheryl)-ammonium bromides.

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Chemical Modifications of Alpha-Tocopherol

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Fig. 6.15. 5a-Bromo--tocopherol as auxiliary in the monoalkylation of primary amines.

the alkylation of amines – were near-quantitative steps. The Toc protecting group showed stability towards a wide range of reaction and work-up conditions as well as selective and mild removal. Its orthogonality to common aminoprotecting groups was demonstrated. After trimethylsilyl (TMS) protection of the phenolic OH group, O-trimethylsilyl-5a-bromo--tocopherol (25) was converted into O-trimethylsilyl-5a--tocopheryl mag-

Fig. 6.16. 5a-Bromo--tocopherol as auxiliary in the synthesis of dipeptides: the ‘Toc’ protecting group.

nesium bromide (26), which made the whole breadth of Grignard reactions accessible to the tocopherol structure (see Fig. 6.17) (Rosenau and Habicher, 1996b). A solvent mixture of diethyl ether and hexane provided optimum results, and the presence of trace amounts of TMS chloride used in OH protection proved to be useful in promoting the reaction with magnesium metal. The TMS group was advantageous as it was cleaved upon hydrolysis of the intermediate magnesium salts, the primary reaction products with carbonyl compounds. Rather than starting from 5a-bromo--tocopherol (14), the O-protected derivative 25 can be obtained quantitatively by reaction of the readily accessible pQ 5 with TMS bromide according to a cyclization–[1,4]addition mechanism (Rosenau and Habicher, 1997a). The reaction of tocopheryl-Grignard 26 with carbonyl compounds at –10°C produced the corresponding magnesium salts 27 as the primary intermediates, as was tested for four co-reacting aldehydes and ketones. Upon the hydrolysis of 27, a pronounced influence of the reaction medium on the formation of the respective products was observed; in all cases the TMS protecting group was removed concomitantly (Fig. 6.18). Mild hydrolysis with a

Fig. 6.17. Syntheses of ‘-tocopheryl-Grignard ’ (26).

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saturated aqueous solution of ammonium chloride yielded the alcohols 28, usage of 5 M HCl for hydrolysis produced mainly the styrene derivatives 29, exclusively in transconfiguration, whereas concentrated (85%) phosphoric acid gave the furobenzopyrans 30. All hydrolysis reactions resulted in the formation of the corresponding major products in good yields – above 70% after purification. The fact that, depending on the work-up conditions, three products can be obtained from one reaction in good yields rendered the reaction quite valuable in synthesis. In another account, the synthesis of 5a-substituted tocopherols with element-organic 5a-substituents other than oxygen or nitrogen was described (Weber et al., 1997). The silicon-containing compounds 5a-trimethylsilyl-tocopherol (31) and 5a-(tert-butyl-dimethylsilyl)-tocopherol (32) were obtained by sonochemical reaction on zinc metal dust starting from 5a-bromo--tocopherol (14) and trialkylchlorosilane (Fig. 6.19). By S-alkylation of thiourea, S-(5a--tocopheryl) thiouronium was obtained and precipitated as the picrate, but the compound was only stable at low temperatures. In contrast, the reaction with N-acyl-thioureas (Klayman et al., 1972) gave stable compounds, precipitated as bromides from acetonitrile/n-hexane: N-acetyl-S-(5a--tocopheryl) thiouronium bromide (33) and N-benzoyl-S-(5a--tocopheryl)

thiouronium bromide (34). Alkylation of the thiourea derivatives proceeded exclusively at the sulphur (Fig. 6.19). (5a--Tocopheryl)triphenylphosphonium bromide (35) was obtained by reaction of 5a-bromo--tocopherol (14) with triphenylphosphine as a white, non-filterable, voluminous solid that had to be used immediately for subsequent conversions. Reaction of the phosphonium salt with acyl chlorides in the presence of an excess of auxiliary base provided furotocopheryl derivatives 36 in fair to good yields (Hercouet and LeCorre, 1979; Adelwöhrer et al., 2003b). The reaction proceeded according to a two-step process consisting of esterification of the phenolic hydroxyl group followed by an intramolecular Wittig-type reaction (Fig. 6.20). The auxiliary base facilitated both the acylation reaction and the intramolecular cyclization. By employing different acyl chlorides, the 2-substituents of the stable 5-membered furan ring were varied (Fig. 6.20). While reaction with phthaloyl dichloride proceeded only once (Weber et al., 1997), oxalyl chloride reacted twice, affording the bis(furotocopherol) 37 in good yield (Adelwöhrer et al., 2003b). The coupling of 35 to an acyl chloride-modified Merrifield resin provided a furotocopherol-loaded polymer (38) (see Fig. 6.20). The presence of tocopheryl moieties on

Fig. 6.18. Reaction of -tocopheryl-Grignard with carbonyl compounds to three different product classes according to the hydrolysis conditions.

Fig. 6.19. 5a-Substituted tocopherols with Si-, S- and P-substituents.

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Chemical Modifications of Alpha-Tocopherol

the beads was confirmed by high-resolution solid state 13C NMR spectroscopy. Due to attachment of the tocopherol structure by stable, non-hydrolysable carbon–carbon bonds extending from C-5a and due to protection of the phenolic hydroxyl group, the furotocopherols proved to be much more stable than most tocopherol derivatives and conventional tocopheryl esters; compounds such as 38 were interesting candidates for polymer stabilizers with long-term efficiency, possibly applicable either as additives or in polymer-bound form. 5-Tocopherylacetonitrile (39) was obtained by reaction of 5a-bromo--tocopherol (14) with potassium cyanide in dimethylsulphoxide (DMSO). On hydrolysis of the nitrile in aqueous dioxane with gaseous HCl, the corresponding acid, 5-tocopherylacetic acid (40), was obtained, which showed a low, but noticeable solubility in warm water and aqueous solvent mixtures. Treatment of the nitrile 39 in concentrated formic acid with gaseous HBr provided the 5-tocopherylacetamide (41). 5-Tocopherylacetic acid lactone (42) was prepared by treatment of acid 40 with polyphosphoric acid (Fig. 6.21). The 6-O-methyl derivatives of 39–41 were obtained by etherification of 39 and subsequent hydrolysis into the respective derivatives (Rosenau et al., 1996). Several 5-tocopherylacetic acid alkyl esters (43) were prepared according to a two-step approach. First, nitrile 39 was alcoholysed in the presence of equimolar amounts of

81

an alkyl alcohol in n-hexane or ether into which dry HBr was passed at low temperatures. Secondly, the obtained imidate hydrobromides were hydrolysed in aqueous acidic media to afford the corresponding alkyl esters in >90% overall yields. An interesting feature of 5-tocopherylacetic acid and its derivatives was their appreciable thermal stability up to 200°C (Rosenau et al., 1996). In contrast to 5a-substituted tocopherols carrying an electronegative substituent at C-5a, the homopolar C–C bond in the C2-unit at the 5-position was shown to be very stable. Thermal decomposition of 40 at temperatures above 250°C caused a complete breakdown of the chroman structure, the C3-unit consisting of C-2, C-2a and C-3 being eliminated as propyne, and the side chain as 4,8,12-trimethyltridec-1-ene (Fig. 6.22). Fragmentation occurred with formation of an intermediate oQM involving C-4 and O-1, which was stabilized immediately in subsequent reactions, either by [1,4]addition or by dimerization. 5-Tocopherylacetic acid underwent ready oxidation to its corresponding pQ, present as lactono-semiketal 44 in aqueous media; 40 and 44 formed a reversible redox pair (Fig. 6.22). Apparently, the electronic effects exerted by the carboxylic acid function in the -position to C-5a changed the oxidation chemistry of the tocopherol system in such a way that oQM formation was largely disfavoured so that only pQ oxidation products were formed.

Fig. 6.20. (5a--Tocopheryl)triphenylphosphonium bromide (35) and its reaction to furotocopherols 36 and bis(furotocopherol) 37 and polymerbound furotocopherol 38.

Fig. 6.21. 5-Tocopherylacetic acid (40) and its derivatives.

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Fig. 6.22. Redox behaviour and thermal degradation of 5-tocopherylacetic acid (40).

In contrast to 5-tocopherylacetic acid (40), its higher C1homologue, 3-(5-tocopheryl)-propionic acid (45), showed the common redox behaviour of tocopherol derivatives, i.e. formation of both ortho-quinoid and para-quinoid oxidation intermediates and products depending on the respective reaction conditions. Evidently, the electronic substituent effects that changed the reactivity and oxidation behaviour in 40 and its derivatives were neutralized by homologation, so that the system returned to its ‘normal’ behaviour. Compound 45 was prepared by a ZnCl2-catalysed, inverse hetero-Diels-Alder reaction between oQM 3 and an excess of O-methyl-C,O-bis(trimethylsilyl)ketene acetal (Fig. 6.23). The former reagent was prepared in situ by thermal degradation of 5a-bromo-tocopherol (14). The primary cyclization product, an orthoester derivative, was not isolated, but immediately hydrolysed to methyl 3-(5-tocopheryl)-2-trimethylsilylpropionate. Desilylation with tetrabutylammonium fluoride

(TBAF) and acidic hydrolysis finally produced 45 in 72% overall yield relative to 14 (Rosenau et al., 1999). All three oxidation reactions typical of -tocopherol – bromination of 45 with elemental bromine to the 5a-bromo derivative 46, oxidation with Ag2O to an intermediate oQM which dimerized to spiro-dimer 47, and oxidation in aqueous media to the para-quinone 48 – proceeded as expected for -tocopherol itself, demonstrating the analogous chemical behaviour of the two compounds (Rosenau et al., 1999) (Fig. 6.23).

5a-Nitro-α-tocopherol 5a-Nitro derivatives of -tocopherol are peculiar due to both their unusual synthesis and their chemical behaviour. Direct nitration of -tocopheryl acetate (49) by nitric acid produced 6-O-acetyl-5-nitro--tocopherol (50) in good

Fig. 6.23. Synthesis and typical reaction products of 3-(5-tocopheryl)-propionic acid (45).

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Chemical Modifications of Alpha-Tocopherol

yields (Witkowski and Markowska, 1996). The acetyl group was not cleaved during the reaction, but was removed afterwards in a separate step by treatment with HCl in methanol to give 5a-nitro--tocopherol (51). Even though it is a strongly electronegative substituent, the nitro group in 50 and 51 is not eliminated upon thermal stress or alkali treatment as are most other electronegative 5a-substituents, possibly because of its ability to form the corresponding aci-nitro form which is distinguished by a double bond between C-5a and the nitrogen (see also Fig. 6.26). The mechanism of the unusual formation reaction was studied in more detail, and a non-radical, heterolytic course was established. A deacetylation–oxidation– reacylation mechanism was ruled out by performing the reaction in propionic acid as the solvent and confirming the presence of an acetyl group – but not a propionyl moiety – in the product. Supported by experiments and computational results, the nitration was shown to proceed via a 1,3,8-trioxa-phenanthrylium cation intermediate, which eventually added nitrite to afford 50 (Adelwöhrer et al., 2003a). Through spatial interaction of the partially negative acyl oxygen with the positive benzylic position, an effective charge delocalization over four atoms was effected, resulting in strong resonance stabilization (Fig. 6.24). The benzylic methylene group is arranged perpendicular to the aromatic plane, so that the compound possesses four aromatic resonance hybrids involving the acetyl group, but no quinoid canonic forms. Generally, Oacyl substituents were shown to be crucial for the nitration reaction to proceed as they stabilize the cationic intermediate by resonance. The occurrence of similar outof-plane benzylic intermediates was also reported for other

83

oxidations of -tocopherol, as observed by NMR at low temperatures (Rosenau et al., 2002b). It should be noted that in the case of free -tocopherol – without the O-protecting group – the nitration takes a completely different path, leading to an ortho-quinone, -tocored (6), according to a multi-step mechanism (Fig. 6.25). After oxidation, nucleophilic attack of water or solvent at C-5 produces a cyclohexadienone intermediate which is stabilized by rearomatization under release of C-5a as methanol, which was demonstrated by means of selectively deuterated starting material. The intermediately formed catechol is easily further oxidized into -tocored (6) as the final product (Rosenau et al., 1997). 6-O-Acetyl-5-nitro--tocopherol (50) found an interesting application as starting material in the synthesis of tocopheryl-substituted isoxazolines by [2+3]-cycloadditions with olefinic compounds (Rosenau et al., 2004) (see Fig. 6.26). Catalysed by an organic base, the aci-nitro form of 50 was formed, from which water was eliminated, which in turn was bound by reaction with Ph-NCO. The resulting nitrile oxide intermediate 52 – stable enough to be isolated in crude form – was the actual co-reactant for the excess alkene component in a stereospecific 1,3-dipolar cycloaddition to give the desired cis-isoxazolines (53) from cis-alkenes and trans-isoxazolines (54) from trans-alkenes. Interestingly, under the cycloaddition conditions used, diethyl maleate afforded exclusively the thermodynamically more stable trans-product 56, which was due to isomerization of the primarily formed cis-product 55 (Fig. 6.27). The basic TEA catalyst present induced isomerization at C-4 of the isoxazoline according to a deprotonation–reprotonation mechanism. To prove that

Fig. 6.24. Synthesis of 6-O-acetyl-5-nitro--tocopherol (50) and four resonance forms of the cationic intermediate.

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Fig. 6.25. Synthesis and formation mechanism of -tocored (6) from -tocopherol.

Fig. 6.26. Synthesis of tocopherylisoxazolines starting from 6-O-acetyl-5-nitro--tocopherol.

Fig. 6.27. Base-catalysed isomerization of tocopheryl-isoxazoline (55).

mechanism, reaction of the isolated nitrile oxide intermediate 52 with diethyl maleate under neutral conditions gave no trans-product, but exclusively cisproduct, which was isomerized by TEA in a subsequent step into trans-configured product. The NMR spectra of the isoxazolines 53–56 indicated restricted mobility of some protons, such as H-4’’ in the

isoxazoline moiety, 4-CH2 in the pyran ring and the acetyl group. This was due to the spatial proximity and related steric effects of the bulky substituents in the isoxazoline ring. This was also supported by crystal structure analysis of model compounds without an isoprenoid side chain.

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Chemical Modifications of Alpha-Tocopherol

Special 5a-substituted tocopherols This section will focus on 5a-substituted tocopherols, which deserve attention due to their special reactivity and an unexpected reaction behaviour, rather than because of their synthesis. For N-phenyl-acetamide (Acetaminophen) carrying an N-Toc group, a reactivity similar to that of other 5asubstituted tocopherols with an electronegative 5asubstituent should be expected, namely release of the 5a-substituent upon treatment with alkali, along with formation of SD 7 and pQ 5 as by-products formed from the primary intermediate, the oQM 3. However, treatment of the Toc-prodrug of acetaminophen (57) with aqueous base yielded 4-hydroxy-3-(6-O--tocopheryl)acetanilide (58) as the main product (Rosenau and Kosma, 2001). The novel reaction was regarded as an intramolecular rearrangement involving [1,4]-sigmatropic and [1,3]-sigmatropic shifts, or as an intramolecular redox process. Alternative pathways, such as intermolecular reactions, radical processes or a multi-step elimination–redox–addition sequence were ruled out experimentally. Deuteration experiments demonstrated the selective transfer of one aromatic proton from the position meta to the acetamido function in the acetaminophen moiety to C-5a in the tocopheryl moiety (Fig. 6.28). The N-acyl structure in the 4-position was

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crucial for the reaction to proceed, with an N-acetyl group giving the highest yield of rearrangement product. In the case of other positions of N-acyl substituents, or N-alkyl groups, or completely different substituents, such as alkoxy or halogen groups, no rearrangement occurred, except for the usual elimination of the 5a-substituent. 5a-Tocopheryl ascorbate (59) was obtained by reaction of 5a-bromo--tocopherol (14) with sodium ascorbate in the presence of excess ascorbic acid in the solvent DMSO. Upon thermal elimination or treatment with alkali, ascorbate was eliminated with concomitant production of oQM 3. The separated moieties of ascorbate and oQM 3 now joined in a redox process in which ascorbate was oxidized to dehydroascorbate while the oQM was reduced to -tocopherol, or the -tocopherolate anion at a higher pH value, respectively (Rosenau and Habicher, 1997c) (see Fig. 6.29). The yield of the reduction reaction was brought close to quantitative by adding excess ascorbate, one additional equivalent giving 96% reconversion of -tocopherol. The portion of oQM 3 which was not reduced underwent spirodimerization to 7 or rearrangement to pQ 5. Kinetic experiments showed the main reaction to proceed in the pH range of 8–11 under simulated physiological conditions, and the tocopherol to be generated in a finely dispersed and thus readily absorbable manner. Another interesting 5a-modified tocopherol derivative is

Fig. 6.28. Base-induced rearrangement of 57, showing the transfer of one proton from the acetanilide to the position of 5a in tocopherol during the process by means of isotopic labelling.

Fig. 6.29. Regeneration of -tocopherol by base-catalysed fragmentation of 5a-tocopheryl ascorbate (59) followed by a redox process.

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5-tocopherolcarboxylic acid (60), which was obtained by benzylic oxidation of 6-O-acetyl-protected 5a-bromo-tocopherol under phase-transfer conditions followed by deacetylation of the phenolic OH group (Fig. 6.30). Compound 60 exhibited interesting photochemical reactivity: the compound was completely consumed upon irradiation at 337 nm for 2 h in the presence of sensitizers, such as iron complexes or activated titanium dioxide (Rosenau and Habicher, 1997b). Besides -tocopherol (61) as the main product, the two coupling products 62 and 63 (Ha and Igarashi, 1990; Yamauchi et al., 1990) were isolated, which were indicative of a homolytic (radical) process (Fig. 6.30). The reaction mechanism was postulated to start with H abstraction and formation of a photochemically excited radical, since chemically generated radicals of 60 did not decarboxylate. The primary radical intermediate then

underwent decarboxylation to form the -tocopheroxyl radical (Boguth and Niemann, 1971; Mukai et al., 1984) that in turn produced -tocopherol (61) by H abstraction from the solvent. The reaction was used on a laboratory scale for preparation of the costly -tocopherol from the cheap -congener in a yield of 72%. The ‘methano-dimer’ of -tocopherol, bis(5tocopheryl)methane (9), was previously synthesized in fair yields by boric acid-assisted hydroxymethylation of tocopherol (61) followed by HCl-catalysed reaction of the product with another molecule of -tocopherol (Nakamura and Kijima, 1972), in 59% overall yield. In a recent approach, 5a-bromo--tocopherol (14) reacted with tocopherol under mild conditions, catalysed by BF3–acetic acid complex, to afford bis(5-tocopheryl)methane (9) in 94% yield (Rosenau et al., 2005b) (see Fig. 6.31).

Fig. 6.30. Synthesis of 5-tocopherolcarboxylic acid (60) and its photochemical decarboxylation to -tocopherol.

Fig. 6.31. Oxidation and bromination behaviour of the ‘methano-dimer’ of -tocopherol (bis(5-tocopheryl)methane, 9).

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Chemical Modifications of Alpha-Tocopherol

Bromination of methano-dimer 9 revealed a remarkable reactivity: at low temperatures, it proceeded quantitatively to the furano-SD 10, by analogy with the ED 8 giving SD 7 upon oxidation. With increasing temperatures, the reaction mechanism changed, however, now affording a mixture of 5-bromo--tocopherol (64) and SD 7 (Fig. 6.31). Thus, upon bromine treatment at higher temperatures, the methanodimer 9 fragmented into an ‘-tocopherol part’, in the form of oQM 3 that dimerized into 7, and a ‘-tocopherol part’, which was present as the 5-bromo derivative 64 after the reaction. Thus, the reaction was seen as oxidative dealkylation. A bromination of the central methylene group did not occur (Rosenau et al., 2006). Also 6-O-acetyl-5a-azido--tocopherol (65), obtained by O-acetyl protection of 5a-bromo--tocopherol followed by reaction with sodium azide, is remarkable because of its increased stability with regard to elimination of its 5asubstituent (Adelwöhrer et al., 2006). The compound was readily reduced to 5a-amino--tocopherol (66) which was otherwise difficult to obtain, and underwent neat [2+3]cycloadditions with alkynes to give O-acetyl protected tocopheryl-triazoles (67) and – after deacetylation – tocopheryl-triazoles (68) (see Fig. 6.32). By hydrogenative or base-induced removal of the tocopheryl moiety as -tocopherol, 4,5-disubstituted triazols were obtained, which are usually rather inaccessible. The tocopheryl moiety thus acted as a temporary protecting group during the synthetic sequence.

87

Miscellaneous 5a-substituted tocopherols 5a-Hydroxy--tocopherol (69) was obtained by hydroxymethylation of -tocopherol (61) employing formaldehyde and boric acid as an auxiliary that acted by anchoring the product in six-membered borate ring structures to prevent the product from side reactions, such as condensations (Nakamura and Kijima, 1972). Similar alkylation reactions of 2,2,7,8-tetramethylchroman-6-ol have been used to obtain several other derivatives, such as 5a-methoxy-, 5adimethylamino-, 5a-benzyloxy- and 5a-ethoxy--tocopherol (Skinner et al., 1967). Using [14C]formaldehyde, the hydroxymethyl derivative radioactively labelled at C-5a was prepared (Nakamura et al., 2000). Compound 69 occurred in two forms distinguishable by NMR depending on which of the two OH groups was involved in H-bond formation (Dallacker et al., 1991a). It was also described as a byproduct in the synthesis of the 5-formyl--tocopherol (71), starting from 6-O-acetyl-5a-chloro--tocopherol (70), which was obtained by cyclization of pQ 5 with acetyl chloride. Oxidation of the O-protected chloride 70 with pnitroso-N,N-dimethylaniline followed by deacetylation of the phenolic hydroxyl group afforded 5-formyl-tocopherol (71), which was distinguished by a strong intramolecular hydrogen bond from the phenolic hydroxyl to the carbonyl oxygen (Fig. 6.33). Also the reaction of -tocopherol with trimethylamine-N-oxide provided 5-formyl--tocopherol (71) together with the 7-formyl

Fig. 6.32. Reactions of 6-O-acetyl-5a-azido--tocopherol (65).

Fig. 6.33. 5a-Hydroxy--tocopherol (69) and synthesis of 5-formyl--tocopherol (71).

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Fig. 6.34. Alternative synthesis approach to 5-formyl--tocopherol (71).

Fig. 6.35. 5a-Amino-substituted and 5a-thio-substituted -tocopherols.

product and minor aldehyde byproducts (Ishikawa, 1974; Ishikawa and Yuki, 1975). 5-Formyl--tocopherol was also synthesized in fair yields according to a one-step procedure by treatment of -tocopherol with dioxane dibromide (Witkowski and Poplawski, 1985, 1996) (see Fig. 6.34). The reaction was postulated to involve a bromoquinone intermediate, which underwent cyclization to a brominated oQM. [1,4]Addition of water and elimination of HBr finally generated aldehyde 71. Oxidation of -tocopherol (1) under different conditions provided 5a-substituted tocopherols in low yields. As these reactions were of no preparative use (their great value lies in studying and understanding the oxidation chemistry of the vitamin), they are not covered in detail here. The Obenzoyl derivative 17 was obtained by reaction of -tocopherol with benzoyl peroxide in inert solvents (Inglett and Mattill, 1955). Similarly, low yields of 5aalkoxy--tocopherols were obtained by oxidation of 1 with tert-butyl hydroperoxide or other peroxides in inert solvents containing various alcohols ranging from methanol to cholesterol (Suarna and Southwell-Keely, 1989, 1998; Suarna et al., 1992). The products were formed by addition of the respective alcohol to the intermediately formed oQM 3. Also oxidation of -tocopherol in the presence of methyl linoleate in methanol provided the 5amethoxy derivative (Yamauchi et al., 1988). The amino-substituted derivatives 5a-dimethylamino-tocopherol (72) and 5a-morpholino--tocopherol (73) were intermediates in conversion of -tocopherol – or more

generally non--tocopherols – into -tocopherol both on a laboratory scale and on a bulk scale (Fig. 6.35). The derivatives were obtained by Mannich-type reaction with secondary amine and formaldehyde, and were subsequently reduced by hydrogenolysis (Nakamura and Kijima, 1971). 5a-Diethylamino--tocopherol (74) and 5aethylthio--tocopherol (75) were synthesized in a similar way, and tested for their antioxidative efficiency (Skinner and Parkhurst, 1970). 5-Ethyl--tocopherol (76), the higher C1 homologue of -tocopherol, was obtained either by alkylation of tocopherol (Skinner and Parkhurst, 1970) or according to the classical -tocopherol synthesis, starting from 5-ethyl2,3-dimethyl-hydroquinone instead of TMHQ (Smith and Renfrow, 1942) (Fig. 6.36). In contrast to the 5a-bromoderivative 14, the 5a-iodo--tocopherol (77) cannot be obtained by direct reaction of -tocopherol with the halogen (Knapp and Tappel, 1961). The compound was produced by addition of HI to the intermediate oQM 3, and was found to be rather unstable.

Fig. 6.36. 5-Ethyl--tocopherol and 5a-iodo--tocopherol.

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Claisen rearrangement of different -tocopheryl allyl ethers opened the way to differently substituted 5-allyl derivatives in good yields (Fig. 6.37), as demonstrated for 5-allyl--tocopherol (78), 5-(but-2-en-1-yl)--tocopherol (79) and 5-(3-methyl-but-2-en-1-yl)--tocopherol (80) (Dallacker et al., 1991b).

hydroxymethyl--tocopherol, 84) was obtained by boric acid-assisted hydroxymethylation of -tocopherol, the phenol with the corresponding free aromatic position (Nakamura and Kijima, 1972).

8-Substituted derivatives

Tocopherols Modified at Positions Other than C-5a 7-Substituted tocopherols Friedel-Crafts alkylation of -tocopherol with allyl bromide produced 7-allyl--tocopherol (81) (Karrer et al., 1938). In an analogous reaction with trans-1-bromo-but-2-ene, 7-(but-2-en-1-yl)--tocopherol (82) was obtained (Fig. 6.38). The 7-ethyl derivative 83 was synthesized starting from 3-ethyl-2,5-dimethyl-hydroquinone, analogous to the synthesis of the 5-ethyl derivative 76 from 5-ethyl-2,3dimethyl-hydroquinone, by condensation with phytol derivatives according to the classical -tocopherol synthesis (Smith and Renfrow, 1942). Despite the seemingly small change in structure, derivative 83 had only about half the activity of -tocopherol in biological tests (Karrer et al., 1938). 7a-Hydroxy--tocopherol (7-

Starting from 2-ethyl-3,5-dimethyl-hydroquinone, 8-ethyl-tocopherol (85) was obtained by analogy to the 5-ethyl (76) and 7-ethyl derivatives (83) described above (Smith and Renfrow, 1942). Also this compound showed a drastically decreased activity in biological tests as compared with -tocopherol (Karrer et al., 1938), as did those ethyl derivatives. Cyclization of 2,6-dimethylhydroquinone with isophytol followed by Friedel-Crafts acylation provided the acetophenone 86; Vilsmeier formylation of the cyclization product afforded the corresponding 8-formyl derivative 87 (Fig. 6.39). The latter was converted into derivatives, both reductively by sodium borohydride to the 8-hydroxymethyl compound 88, and oxidatively into the 8-carboxylic acid (89), nitrile 90 and amide 91 (Fujishima et al., 1996). Starting from the free aromatic position, 8b-chloro-tocopherol was obtained according to a Blanc

Fig. 6.37. Synthesis of differently substituted 5-(prop-2-en-1-yl)--tocopherols by Claisen rearrangement.

Fig. 6.38. 7-Substituted -tocopherols.

Fig. 6.39. 8-Substituted -tocopherols.

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chloromethylation (Omura, 1989), and was subsequently etherified with methanol to give 8b-methoxy--tocopherol (92) (see Fig. 6.40). The same compound was obtained by treatment of the 8a-methoxy-tocopherone (93) with MeOH/KOH according to a rearrangement mechanism involving oQM intermediates, together with 4-methoxy-tocopherol (95) (Omura, 1989). The 5a-8b-ethano-dimer of -tocopherol (94) was obtained as a minor byproduct upon UV irradiation of -tocopherol under different aerobic conditions (Fig. 6.41), e.g. in acetonitrile or in ethanol containing soy phosphatidylcholine liposomes, along with 8a-hydroperoxytocopherones which were partly epoxidized, as the main products (Kramer and Liebler, 1997).

Derivatives modified in position 3 and/or 4 Reaction of -tocopherol with elemental iodine under alkaline conditions produced 8a-methoxy-tocopherone (93), which rearranged into a mixture of 8b-methoxy--tocopherol (92) and 4-methoxy--tocopherol (95) according to a multistep mechanism. Upon treatment with acid, methanol was eliminated from 95 to afford 3,4-dehydro--tocopherol (96) (see Fig. 6.42). This compound proved to be a valuable starting material for further modifications of the alicyclic ring. It can also be obtained in a facile one-pot preparation by treatment of -tocopheryl acetate (49) with 2,3-dichloro5,6-dicyano-para-benzoquinone (DDQ) followed by deacetylation (Burton et al., 1985), or by condensation of TMHQ with the corresponding substituted propynol (Karrer et al., 1940). Starting from the O-acetyl-3,4-dehydro--tocopherol (97), which was obtained from -tocopheryl acetate (49) by

DDQ treatment, the synthesis of photoaffinity labels was achieved (Fig. 6.43). After exchange of the O-acetyl group for tert-butyl-dimethylsilyl (TBDMS), the double bond was converted into a diastereomeric bromohydrin mixture (98) by treatment with N-bromo-succinimide (NBS) in the presence of water, and then further transformed into the O-TBDMS-protected 3,4-epoxide 99. Reduction with the LiAlH4/AlCl3 reagent couple provided a mixture of the two diastereomeric, O-TBDMS-protected 3-hydroxy-tocopherols (100), one of which was finally converted into 3-(-tocopheryl) diazoacetate (101) (Lei et al., 1998; Lei and Atkinson, 2000). The configuration of the hydroxyl groups in the 3-hydroxy--tocopherol intermediates (100) was determined by nuclear Overhauser effect experiments and by comparison with authentic samples obtained by treatment of the 3,4-dehydro compound (97) with (RR)salen and (SS)-salen, respectively. Upon treatment with ZnO, the racemic 6-O-acetylbromohydrin 102 produced 4-oxo--tocopherol (103) in 74% yield (Fig. 6.44). The ZnO effected simultaneous deacetylation, dehydrobromination and tautomerization of the resulting enol intermediate. Benzofuran 104 was formed as the main byproduct (8%), by ring contraction according to an elimination–addition mechanism. 4-Oxo--tocopherol (103) rearranged under simulated physiological conditions into hydroquinone 105 in yields of about 10% (Rosenau et al., 2002a). In the presence of oxidants, the naphthalenetrione 106 was formed directly, possessing a carbon skeleton completely different from that of 103. The mechanism was shown to involve opening of the alicyclic ring, followed by formation of different tautomers, bond rotation, and electrocyclic ring closure as the key step (Fig. 6.45). The incorporation of C-5a into the alicyclic ring in 106 was demonstrated by means of isotopic

Fig. 6.40. Syntheses of 8a-methoxy--tocopherol (92).

Fig. 6.41. 5a-8b-Ethano-dimer of -tocopherol (94).

Fig. 6.42. Synthesis of 3,4-dehydro--tocopherol (96).

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Chemical Modifications of Alpha-Tocopherol

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Fig. 6.43. Synthesis of photoaffinity labels starting from 3,4-dehydro--tocopherol, according to Lei and Atkinson (2000).

Fig. 6.44. Synthesis of 4-oxo--tocopherol (103).

Fig. 6.45. Oxidative rearrangement of 4-oxo--tocopherol (103) into naphthalenetrione (106).

labelling: 4-oxo--tocopherol trideuterated at C-5a produced 106 bisdeuterated at C-4, the ‘former’ C-5a position. O-Acetyl-3,4-dehydro--tocopherol (97) was employed as a trapping agent to confirm the presence of N,Ndimethylketeniminium cations in the pure cellulose solvent DMAc/LiCl (0.5%) and corresponding cellulose solutions at temperatures above 80°C (Potthast et al., 2002).

The trapping agent was used because of its high lipophilicity resulting in good extractability even from complex matrices. The trapping reaction afforded cyclobutanone 107 (Fig. 6.46) in a thermal, suprafacial– antarafacial, [2+2]-cycloaddition followed by aqueous hydrolysis of the primarily formed iminium intermediate, which was not isolated.

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Fig. 6.46. Reaction of 6-O-acetyl-3,4-dehydro--tocopherol with N,N-dimethylketeniminium cations and formation of cyclobutanone derivative 107.

Derivatives modified in position 2 The 2-ethyl (108) and 2-propyl derivatives (109) were obtained by condensation of trimethylhydroquinone with 1-bromo-3-ethyl-7,11,15-trimethyl-hexadec-2-ene and 1-bromo-3-propyl-7,11,15-trimethyl-hexadec-2-ene, respectively, analogous to the synthesis of -tocopherol (Fig. 6.47). Also, by replacement of the 2-methyl group by ethyl – similar to the replacement of 5-methyl, 7-methyl or 8-methyl by ethyl – the biological activity was reduced significantly (Karrer and Stähelin, 1945).

Derivatives modified in position 1 or 6 Replacement of the hydroxyl group or the ring oxygen in -tocopherol was performed to study the specific role of this functionality in the antioxidative action (Smith et al.,

1942; Rüegg et al., 1967; Mayer and Isler, 1971). Naturally, all derivatives lacking the deprotonable hydroxyl group at C-6 (or groups of similar reactivity, such as mercapto or amino groups) are ineffective as an antioxidant as they cannot be converted into a stable radical. The antioxidative efficiency and its maximization was comprehensively studied (Burton et al., 1985; Mukai et al., 1989). The tocopheramines 110 and 111 were synthesized by condensation of N-formyl-protected 4-hydroxy-2,3,6trimethylaniline with isophytol followed by deprotection in aqueous HCl, and N-methylation in the case of 111 (Smith et al., 1942) (see Fig. 6.48). An alternative synthesis started from 2,3,5-trimethyl-4-nitro-phenol (Merck & Co., 1941), the cyclization product, with isophytol being subsequently hydrogenated on palladium/carbon. Both compounds, especially the N-methyl derivative, showed excellent antioxidative properties. The same applies to 1-aza-tocopherol 112 and 1-aza-tocopheramine 113, even

Fig. 6.47. 2-Substituted -tocopherols.

Fig. 6.48. -Tocopheramines and -tocopherthiols.

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Chemical Modifications of Alpha-Tocopherol

though they exhibited very low biological tocopherol activity (Mayer and Isler, 1971). 1-Thia--tocopherol (114) was obtained by Clemmensen reduction of the corresponding thiochroman-4-one (Robillard et al., 1986), or alternatively by condensation starting from 4-mercapto-2,3,6-trimethyl-phenol (Valaschek et al., 1982). A similar reaction with 4-mercapto-2,3,5trimethyl-phenol afforded tocopherthiol (115) (see Fig. 6.48). Both sulphur-containing derivatives proved to be good antioxidants – albeit inferior to -tocopherol. The thiol derivative 115 showed the strong tendency to form disulphide bridges upon oxidation.

Rearrangements Oxidation of -tocopherol with NO in oxygen-saturated inert solvents, such as cyclohexane, produced a multi-

93

Fig. 6.49. Far-reaching rearrangement in a nitrosation product of -tocopherol.

component mixture, from which a ring-contracted product (116) was isolated and identified, showing a far-reaching deconstruction of the tocopherol skeleton and additional nitrosation (see Fig. 6.49) (d’Ischia, 1995; d’Ischia and Novellino, 1995).

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Stanger, A., Ashkenazi, N., Boese, R. and Stellberg, P. (1997a) Evidence for metal induced bond localization in cyclobutabenzenes: the crystal and molecular structures of 6-Cr(CO)3 and 4-Fe(CO)3 complexes of cyclobutabenzene. Journal of Organometallic Chemistry 542, 19. Stanger, A., Ashkenazi, N., Boese, R. and Stellberg, P. (1997b) Erratum to ‘Evidence for metal induced bond localization in cyclobutabenzenes: the crystal and molecular structures of 6-Cr(CO)3 and 4-Fe(CO)3 complexes of cyclobutabenzene’. Journal of Organometallic Chemistry 548, 113. Stanger, A., Ashkenazi, N., Boese, R. and Stellberg, P. (1998) Erratum to ‘Evidence for metal induced bond localization in cyclobutabenzenes: the crystal and molecular structures of 6-Cr(CO)3 and 4-Fe(CO)3 complexes of cyclobutabenzene’. Journal of Organometallic Chemistry 556, 249–250. Suarna, C. and Southwell-Keely, P.T. (1989) Effect of alcohols on the oxidation of the vitamin E model compound, 2,2,5,7,8-pentamethyl-6chromanol. 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7 Functions and Activities of Tocotrienols and Tocopherols In Vitro Berish Y. Rubin and Sylvia L. Anderson Fordham University, Department of Biological Sciences, Bronx, NY 10458, USA

Abbreviations: apoB, apolipoprotein B-100; CTGF, connective tissue growth factor; COX-2, cyclo-oxygenase-2; CYP3A4, cytochrome P450 3A4; CYP3A5, cytochrome P450 3A5; FLIP, FLICE-inhibitory protein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; ICAM-1, intercellular adhesion molecule-1; IKAP, I
B kinase complex-associated protein; IL, interleukin; JNK, Jun N-terminal kinase; LPS, lipopolysaccharide; MMP-19, matrix metalloproteinase 19; PGE2, prostaglandin E2; PKB, protein kinase B; PKC-; protein kinase C-; PPAR-, peroxisome proliferator activated receptor-; PXR, pregnane X receptor; SR, scavenger receptor; TGF-, transforming growth factor-; VCAM-1, vascular cell adhesion molecule-1; VLA-4, very late antigen 4.

Abstract The in vivo actions of the tocopherols and tocotrienols have prompted in depth analyses of the in vitro biological responses to these forms of vitamin E. Differential cell growth responsiveness and gene expression observed in cells treated with the different forms of vitamin E reveal the complexity of this responsiveness. Elucidation of the mechanisms responsible for the in vitro biological actions of the tocopherols and tocotrienols will allow for their more efficacious use in man.

Introduction

Vitamin E-mediated Cell Growth Inhibition

The natural vitamin E family is comprised of -, -, - and -tocopherols and -, -, - and -tocotrienols. These molecules all contain a six-chromanol ring structure and an isoprenoid side chain. The tocopherols and tocotrienols differ in their side chains, and the designations , ,  and  reflect differences in the number and positions of methyl groups on the chromanol ring (Kamal-Eldin and Appelqvist, 1996). Tocopherols and tocotrienols are present in a variety of food substances. The tocopherols are found primarily in polyunsaturated vegetable oils and in the germ of cereal seeds, while the tocotrienols are found in palm oil and the aleurone and subaleurone layers of cereal seeds (Yoshida et al., 2003). Vitamin E was initially identified by Evans and Bishop (1922) as a dietary factor essential for reproduction in female rats. Early studies also revealed that vitamin E plays an essential role in neuronal development (Sokol, 1989). The biological activities of the vitamin E family members continue to be elucidated through a study of both their in vivo and in vitro activities.

The reports that demonstrated a role for vitamin E in preventing atherosclerosis (Gey, 1990; Rimm et al., 1993; Stampfer et al., 1993) and that atherosclerosis begins with the proliferation and migration of vascular smooth muscle cells (Raines and Ross, 1993) prompted extensive studies characterizing the effect of tocopherols on the growth of vascular smooth muscle cells, revealing that -tocopherol, but not -tocopherol, inhibits the in vitro proliferation of smooth muscle cells (Azzi et al., 1993; Chatelain et al., 1993; Tasinato et al., 1995). This effect appears to occur through an antioxidant-independent mechanism, as -tocopherol, which has antioxidant activity comparable with that of -tocopherol, fails to mediate a growth-inhibitory response. Vitamin E-mediated growth inhibition has subsequently been observed in a variety of cell types (Table 7.1); the observed responses to the various tocopherols and tocotrienols, irrespective of their antioxidant properties, support the notion that vitamin E-mediated growth inhibition can occur through an antioxidant-independent means.

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Table 7.1. Antiproliferative activity of tocopherols and tocotrienols. Antiproliferative activityb Cell typea

Tocopherols

Balb c/3T3 fibroblast (M), retinal neuroepithelial (H), neuroblastoma (M) Vascular smooth muscle (R, H) Retinal pigment epithelial (H) Tenon’s capsule fibroblast (H) MDA-MB-435 breast cancer (H) MCF-7 breast cancer (H) B16 melanoma (M) MCF-7 breast cancer (H), CRL-1740 prostate cancer (H), HEL erythroleukaemia (H), OCIM-1 erythroleukaemia (H) MCF-7 breast cancer (H) MDA-MB-231 breast cancer (H) IHGK oral epithelial (H) B16 melanoma (M), MCF-7 breast adenocarcinoma (H), HL-60 leukaemia (H) ALVA-101 (H), LNCaP prostate cancer (H) CL-S1, -SA and +SA mammary epithelial (M) Primary epithelial cells (M) Mammary epithelial (M) DU-145 prostate carcinoma (H), LNCaP prostate cancer (H), CaCo-2 colon adenocarcinoma (H) Aortic endothelial (B) +SA mammary cancer (M) PC-3 prostate cancer (H) HMC-1 mastocytoma (H) dRLh-84 hepatoma (R)

 (+)  (+),  (–)  (+)  (+)  (–)  (+)  (–)  (+)  (–)  (–)  (+)  (+)  (+), ,  (–)  (+), ,  (–)  (+), ,  (–) , ,  (+) , , ,  (–)  (–)  (+) , , ,  (+) ,  (–)

Tocotrienols

Reference

, ,  (+) , ,  (+) , ,  (+)

Azzi et al. (1993) Azzi et al, (1993) Mojon et al. (1994) Haas et al. (1996) Guthrie et al. (1997) Guthrie et al. (1997) He et al. (1997)

,  (+),  (–)  (+), ,  (–)  (+) , ,  (+) , ,  (+) , ,  (+) , , ,  (+)  (+)  (+)

Sigounas et al. (1997) Nesaretnam et al. (1998) Nesaretnam et al. (1998) Mason et al. (1999) Mo et al. (1999) Gunawardena et al. (2000) McIntyre et al. (2000a) McIntyre et al. (2000b) Sylvester et al. (2001) Gysin et al. (2002) Inokuchi et al. (2003) Shah et al. (2003) Galli et al. (2004) Kempna et al. (2004) Sakai et al. (2004)

aH

= human; M = murine; R = rat; B = bovine. = has antiproliferative activity; ‘–’ = does not have antiproliferative activity; if no result is shown for a particular tocopherol or tocotrienol, no result was reported in the reference. Extensive studies of cells treated with vitamin E reveal both the breadth of the antiproliferative response as well as specific responses to the different forms of tocopherols and tocotrienols. b‘+’

The mechanisms by which the tocopherols and tocotrienols mediate their antiproliferative responses are varied (Table 7.2). The inhibition of smooth muscle cell proliferation by -tocopherol occurs as a result of an activation of protein phosphatase 2A, which dephosphorylates and thereby inactivates protein kinase C- (PKC-) which is required for smooth muscle cell proliferation (Dempsey et al., 1991; Chatelain et al., 1993; Tasinato et al., 1995; Azzi et al., 1997, 1999; Ricciarelli et al., 1998). A reduction in PKC- activity in -tocopheroltreated cells may also be due to the tocopherol-mediated activation of diacylglycerol kinase (Lee et al., 1999). The

growth-inhibitory response observed in -tocopherol- and -, - and -tocotrienol-treated mouse mammary epithelial cells also appears to be mediated through a reduction in PKC- activity (Sylvester et al., 2001). Phosphorylation of protein kinase B (PKB) is reduced in HMC-1 mast cells responding to the antiproliferative action of -, -, - and -tocopherols (Kempna et al., 2004). A correlation between the reduction in phosphorylation and growth-inhibitory response suggests that the tocopherols may be mediating their effect through the reduced phosphorylation of PKB. The reduction in the cellular levels of the G1→S

Table 7.2. Mechanisms of vitamin E-mediated antiproliferative response. Mechanism

Reference

Dephosphorylation of PKC- Degradation of HMG-CoA reductase Reduction in levels of cyclins D1 and E Increase in levels/activity of caspases 3 and 8 Upregulation of PPAR- Reduced phosphorylation of PKB Activation of TGF-/Fas/JNK pathways

Chatelain et al. (1993); Tasinato et al. (1995); Ricciarelli et al. (1998) Elson (1995) Gysin et al. (2002) Shah et al. (2003); Sakai et al. (2004) Campbell et al. (2003) Kempna et al. (2004) Shun et al. (2004)

The antiproliferative response of cells following treatment with vitamin E can be mediated through a variety of different mechanisms. (HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; PKB, protein kinase B; PKC-, protein kinase C-; PPAR-, peroxisome proliferator-activated receptor-; TGF-, transforming growth factor-; JNK, Jun N-terminal kinase).

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transition-specific proteins, cyclin D1 and cyclin E, in growth-inhibited -tocopherol-treated DU-145 prostate cancer cells suggests that it is the reduced presence of these proteins that is mediating the growth-inhibitory response (Gysin et al., 2002). Treatment of cells with -tocotrienol results in the cellular degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Elson, 1995). The reduced presence of this enzyme, which is required to maintain pools of farnesyl diphosphate and other phosphorylated products of the mevalonate pathway that are needed for post-translational processing of small G proteins, nuclear lamins and growth factor receptors, probably contributes to the antiproliferative response of cells to -tocotrienol. The significant role peroxisome proliferator activated receptor- (PPAR-) plays in the control of gene expression and cell growth (Kitamura et al., 2001) prompted Campbell et al. (2003) to examine PPAR- levels in human SW 480 colon cancer cells. The upregulation by -tocopherol of both PPAR- mRNA and protein suggests that the tocopherol-mediated growth inhibition in some cells may be mediated through the elevation of PPAR- (Campbell et al., 2003). The apoptotic pathways activated in cells responding to tocotrienol-mediated apoptosis have been extensively characterized. In malignant +SA mouse mammary epithelial and rat hepatoma dRLh-84 cells responding to the apoptotic effect of -tocotrienol, increased intracellular activity and levels of processed caspase-8 and -3 are detected (Shah et al., 2003; Sakai et al., 2004). Activation of caspase-8 is probably due to the -tocotrienol-mediated reduction in the intracellular presence of the FLICE-

inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation which blocks the processing of procaspase-8 to its active form (Shah and Sylvester, 2004). In human MDA-MB-435 breast cancer cells, -tocotrienol appears to mediate its effect through the activation of the transforming growth factor- (TGF-), Fas (CD95)- and Jun N-terminal kinase (JNK) signalling pathways (Shun et al., 2004).

Vitamin E-mediated Transcriptional Regulation To elucidate the molecular mechanisms by which the tocopherols and tocotrienols mediate their effects, much research has focused on the impact of vitamin E on gene expression (Table 7.3). Because of the apparent role vitamin E plays in protecting against the development of atherosclerosis, much of this effort has focused on the study of genes involved in the inhibition of atherosclerosis. The binding and internalization of oxidized low-density lipoprotein to scavenger receptors (SRs) found on the surface of macrophages and smooth muscle cells lead to the development of foam cells within atherosclerotic plaques. The establishment of these foam cells accelerates the progression of arteriosclerosis. To study the role of vitamin E on the development of foam cells, Teupser et al. (1999) examined SR class A (SR-A) expression in macrophages and observed an -tocopherol-mediated downregulation of SR-A activity as well as SR-A mRNA. Downregulation of SR-B (CD36) has also been observed in -tocopherol-treated macrophages and aortic smooth muscle cells (Ricciarelli et al., 2000; Devaraj et al., 2001). These findings suggest that the reduced risk of athero-

Table 7.3. Vitamin E-mediated transcriptional regulation. Gene product

Reference

IL-1 (↓) E-selectin (↓) ICAM (↓) CD11b (↓) VCAM-1 (↓) Collagen 1 (↓) VLA-4 (↓) -Tropomyosin (↑) Collagenase (↓) IL-8 (↓) Scavenger receptors class A and B (↓) IL-4 (↓) MMP-19 (↓) COX-2 (↓) CTGF (↑) CYP3A4 and CYP3A5 (↑) IKAP (↑) MDR1 (↑) UDP glucuronosyltransferase 1A1 (↑)

Akeson et al. (1991) Faruqi et al. (1994) Martin et al. (1997) Yoshikawa et al. (1998) Yoshikawa et al. (1998) Chojkier et al. (1998) Islam et al. (1998) Aratri et al. (1999) Ricciarelli et al. (1999) Wu et al. (1999); Tang and Meydani, (2001); Nobato et al. (2002) Teupser et al. (1999); Ricciarelli et al. (2000); Devaraj et al. (2001) Li-Weber et al. (2002) Mauch et al. (2002) Egger et al. (2003) Villacorta et al. (2003) Landes et al. (2003); Zhou et al. (2004) Anderson et al. (2003) Zhou et al. (2004) Zhou et al. (2004)

(↑) = upregulation; (↓) = downregulation A variety of genes are transcriptionally regulated by treatment with vitamin E. (CTGF, connective tissue growth factor; COX2, cyclo-oxygenase-2; CYP3A4, cytochrome P450 3A4; CYP3A5, cytochrome P450 3A5; ICAM-1, intercellular adhesion molecule-1; IKAP, I
B kinase complex-associated protein; IL-1, interleukin-1; IL-4, interleukin-4; IL-8, interleukin-8; MDR1, multi-drug resistance protein-1; MMP-19, matrix metalloproteinase 19; UDP, uridine diphosphate; VCAM-1, vascular cell adhesion molecule-1; VLA-4, very late antigen 4).

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Functions and Activities of Tocotrienols and Tocopherols In Vitro

sclerosis associated with vitamin E consumption may in part be due to the reduced uptake of oxidized lipoprotein by SRs. The adhesion of monocytes to endothelial cells contributes to the development of atherosclerotic plaques. Specific adhesion molecules involved in monocyte– endothelial adhesion include E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells, and members of the -2 integrin family (CD11a, CD11b and CD11c/18) and the -1 integrin VLA-4 (very late antigen 4) on monocytes (Bevilacqua et al., 1994). Research on the effect of -tocopherol on monocyte–endothelial adhesion and the gene regulation involved in this process has revealed its ability to inhibit monocyte–endothelial adhesion, decrease E-selectin protein and mRNA levels (Faruqi et al., 1994) and decrease expression of ICAM (Martin et al., 1997), VCAM-1, CD11b (Yoshikawa et al., 1998) and VLA-4 (Islam et al., 1998). The adhesion-dependent expression of the matrix metalloproteinase-19 (MMP-19) in myeloid cells has also been found to be downregulated in response to -tocopherol (Mauch et al., 2002). The ability of -tocopherol to reduce the level of expression of an MMP may contribute to its anti-atherosclerotic activity. The inflammatory cytokine interleukin-1 (IL-1), through its ability to induce procoagulant activity in human vascular endothelial cells (Bevilacqua et al., 1984), to increase monocyte–endothelial adhesion (Bevilacqua et al., 1985), to activate VCAM-1 gene expression on vascular endothelial cells (Marui et al., 1993), to stimulate cholesterol esterification and deposition in macrophages (Maziere et al., 1996), and to promote smooth muscle cell growth (Raines et al., 1989), contributes to atherogenesis. The observations that IL-1 mRNA levels are greatly increased in atherosclerotic lesions (Wang et al., 1989) and that the levels of IL-1 protein levels present in coronary arteries correlate with the severity of atherosclerosis (Galea et al., 1996) further support the role of IL-1 in the development of atherosclerosis. To study the impact of -tocopherol on IL-1 production, Akeson et al. (1991) monitored the effect of -tocopherol treatment on IL-1 production by phorbol ester-stimulated THP-1 cells. A clear inhibition of IL-1 gene expression was observed. This effect on IL-1 gene expression differs from that reported by Devaraj and Jialal (1999), who observed that -tocopherol mediated a post-transcriptional downregulation of IL-1 protein levels. They reported that, in activated human monocytes, -tocopherol treatment resulted in an inhibition of IL-1 protein production without affecting the levels or stability of the IL-1 mRNA (Devaraj and Jialal, 1999). The differences observed in these two studies may reflect the cell types used. Study of the effect of vitamin E on the production of other members of the interleukin family involved in the inflammatory process has revealed that -tocopherol suppresses IL-1-, H2O2-, high glucose- and thrombininduced production of IL-8 in endothelial cells (Wu et al., 1999; Tang and Meydani, 2001; Nobata et al., 2002) as well as IL-4 production in peripheral blood T cells (Li-Weber et al., 2002).

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Differential display and gene expression array analyses have been performed to gain further insight into the biological effects of tocopherol on vascular smooth muscle cells. - but not -tocopherol has been found to upregulate expression of -tropomyosin and connective tissue growth factor (CTGF) mRNAs and protein levels (Aratri et al., 1999; Villacorta et al., 2003). Increased expression of -tropomyosin may, through its ability to modulate actin–myosin interactions, influence the size of vascular smooth muscle cells and thereby modulate arterial blood pressure. The upregulation of CTGF in smooth muscle cells may increase the resistance of vascular cells to vessel injuries and thereby serve to prevent atherosclerosis (Aratri et al., 1999; Villacorta et al., 2003). Tocopherol and tocotrienol treatment also have been observed to modulate gene expression in a variety of cell types not associated with the atherosclerotic process. In hepatic stellate cells, exposure to -tocopherol results in an inhibition of collagen 1 gene expression (Chojkier et al., 1998). This tocopherol-mediated effect suggests the possible use of -tocopherol in controlling liver fibrogenesis. The observed corresponding increase with age of PKC- and collagenase gene expression in human skin cells and in human fibroblast cultures suggested that it may be the increased PKC- that induces collagenase expression. The ability of -tocopherol to reduce PKC- activity prompted an examination of the impact of this treatment on collagenase gene expression. A clear -tocopherolmediated inhibition of collagenase gene transcription has been observed (Ricciarelli et al., 1999). In a study designed to identify therapeutic modalities for individuals with the recessive genetic disorder, familial dysautonomia, which is manifested by decreased expression of the I
B kinase complex-associated protein (IKAP) from its mutated gene, Anderson et al. (2003) assessed the ability of various compounds to upregulate the expression of IKAP. They demonstrated that the tocotrienols, but not the tocopherols, stimulate transcription of the IKAP-encoding gene. The role of the upregulation of this gene product in the biological activities of the tocotrienols awaits further study. As the tocopherols and tocotrienols are metabolized by -oxidation catalysed by cytochrome P450 enzymes and as expression of these enzymes is often induced by their substrates through the activation of the pregnane X receptor (PXR), also known as the steroid and xenobiotic receptor (SXR) (Kliewer et al., 2002), Landes et al. (2003) examined the ability of members of the vitamin E family to induce PXR-activated genes. Using the human hepatic HepG2 cell line, they observed induction of the PXRregulated CYP3A4 and CYP3A5 in -tocotrienol-treated cells. Expanding on this study, Zhou et al. (2004) demonstrated the ability of all four of the tocotrienols, but not any of the tocopherols, to bind and activate the PXR in HepG2 and human intestinal epithelial LS180 cell lines. They further demonstrated the ability of tocotrienols to upregulate CYP3A4 gene expression in primary hepatocytes, and UDP-glucuronosyltransferase 1A1 and multidrug resistance protein-1 (MDR1) gene expression in LS180

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Table 7.4. Vitamin E-mediated post-transcriptional regulation. Protein activity

Reference

Lipooxygenases (↓) Protein phosphatase 2A (↑) PKC- (↓) Diacycglycerol kinase (↑) IL-1 (↓) ApoB (↓) COX-2 (↓)

Grossman and Waksman (1984); Reddanna et al. (1985) Tasinato et al. (1998) Tasinato et al. (1998); Lee et al. (1999) Lee et al. (1999) Devaraj and Jialal (1999) Theriault et al. (1999) Jiang et al. (2000)

(↑) =increased; (↓) = decreased Treatment of cells with vitamin E results in the post-transcriptional regulation of the activities of a variety of gene products. (apoB, apolipoprotein B-100; COX-2, cyclo-oxygenase-2; IL-1, interleukin-1; PKC-, protein kinase C-).

cells. The tocotrienols’ activation of the PXR suggests that their in vivo administration may induce drug metabolism and reduce the efficacy of certain drugs.

Post-transcriptional Gene Regulation by Vitamin E Regulation of gene expression by members of the vitamin E family has been observed to occur post-transcriptionally (Table 7.4). As mentioned above, the post-translational activation of protein phosphatase 2A and the activation of the diacylglycerol kinase in -tocopherol-treated cells reduce PKC- activity (Tasinato et al., 1995; Lee et al., 1999). Additional studies have revealed that the -tocopherol-mediated inactivation of PKC-, which reduces p47(phox) membrane translocation and impairs the assembly of the NADPH oxidase, inhibits superoxide anion release from human monocytes (Cachia et al., 1998; Venugopal et al., 2002). The above-mentioned tocotrienol-mediated degradation of the HMG-CoA reductase is believed to contribute to the hypocholesterolaemic activity of the tocotrienols (Parker et al., 1993). The reduced presence of plasma apolipoprotein B-100 (apoB) in subjects responding to the hypocholesterolaemic effects of tocotrienols prompted a study of the effect of tocotrienol treatment on apoB levels in HepG2 cells. A clear reduction in apoB levels was observed and found to be due to a -tocotrienol-mediated inhibition of apoB secretion (Theriault et al., 1999). Cyclo-oxygenase-2 (COX-2)-catalysed prostaglandin E2 (PGE2) synthesis plays a key role in the generation of inflammation. To assess the impact of the tocopherols on this aspect of the inflammatory process, Jiang et al. (2000)

studied the effect of - and -tocopherol on PGE2 synthesis in lipopolysaccharide (LPS)-stimulated macrophages and IL-1-treated epithelial cells. In both cell types, tocopherol inhibited COX-2-catalysed PGE2 synthesis. COX-2 protein and mRNA levels, however, were observed to be unaffected by -tocopherol treatment. These findings suggest that, in these cells, -tocopherol mediates its effect by the direct inhibition of COX-2 activity (Jiang et al., 2000). The findings reported by Jiang et al. contrast with a recent study reporting the -tocopherol-mediated transcriptional downregulation of COX-2 gene expression in LPS-stimulated BV-2 microglia (Egger et al., 2003). Study of the vitamin E-mediated inhibition of the lipoxygenases has revealed that the inhibition is the result of the direct binding of vitamin E to these enzymes (Grossman and Waksman, 1984; Reddanna et al., 1985). The vitamin E-mediated reduction of lipoxygenase levels appears to be the mechanism by which -tocopherol blocks the release of IL-1 by activated monocytes (Devaraj and Jialal, 1999) and -tocotrienol mediates the resistance of neuronal cells to glutamate-induced cell death (Khanna et al., 2003).

Conclusions Members of the vitamin E family modulate a plethora of biological activities. In vitro studies performed to date have helped elucidate the mechanisms by which these molecules mediate their effects. The continued study of the actions of the tocopherols and tocotrienols is certain to generate new understandings and therapeutic uses for these molecules.

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8 An Overview of Free Radicals and Oxidative Stress: Setting the Scene Max C. Y. Wong and Helen Wiseman Department of Nutrition and Dietetics, King’s College, University of London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK

Abbreviations: CoQ, coenzyme Q; Cu/Zn-SOD, copper/zinc superoxide dismutase; CYP2E1, cytochrome P450 2E1; EC-SOD, extracellular superoxide dismutase; GSH, reduced glutathione; GSSG, oxidized glutathione; GPx, glutathione peroxidase; MnSOD, manganese superoxide dismutase; PUFA, polyunsaturated fatty acid; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; UV, ultraviolet.

Abstract It has been noted that animals with a lower metabolic rate, such as turtles, usually have a longer lifespan. Humans are warm-blooded animals that respire and produce heat energy continuously throughout their lifespan. It is generally accepted that free radical production and the development of oxidative stress are related to ageing. Given that the production of free radicals is unavoidable in normal metabolism, this chapter intends to give an insight into the biochemistry of free radical formation and antioxidant defence systems. The formation of free radicals such as the superoxide and hydroxyl radical will be illustrated, and antioxidant defences, such as superoxide dismutase, glutathione and catalase, will also be considered. Moreover, the general concept of oxidative-induced pathophysiology will be highlighted.

Introduction Life on earth relies on atmospheric oxygen to generate energy. Under normal physiological conditions, oxygen molecules will be transported to aerobic cells by haemoglobin and eventually will be used to generate ATP. Mitochondria are the major site for ATP synthesis, and 98% of the oxygen is metabolized in the mitochondria by cytochrome oxidase, while the rest of the oxygen, about 2%, will be converted into reactive oxygen species (ROS) including oxygen-centred radicals (Chance et al., 1979). Theoretically, a higher metabolic rate will increase the rate of ATP synthesis in the mitochondria, which in turn increases the production of free radicals. It has been suggested that free radical production increases with age and thus contributes to the ageing process (Sohal and Weindruch, 1996; Biesalski, 2002). Longevity is, to a certain extent, dependent on the rate of ROS production, which is determined partly by the basal metabolic rate (Ku et al., 104

1993). Overproduction of ROS can result in oxidative stress and cause damage to biological molecules, such as proteins, lipoproteins, polyunsaturated fatty acids (PUFAs) and DNA.

Free Radicals in Biological Systems Back to the basics By definition, a free radical refers to any atom or molecule capable of existing independently that contains one or more unpaired electrons at its outermost orbit, and a free radical is denoted by a superscripted dot (Halliwell et al., 1995). Free radicals are very reactive because of their unpaired electron(s). They attempt to ‘steal’ an electron or pair up with another atom or molecule, to create a stable compound with a lower energy level. Four different types of chemical reactions are used primarily by the free radical to achieve a more stable state (Wu and Cederbaum, 2003):

© CAB International 2007. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

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● Hydrogen abstraction – accept a hydrogen atom from

another molecule ● Addition – combine with another stable compound to become a new radical ● Termination – combine with another radical to become a stable compound ● Disproportionation – reaction between two identical radicals with formation of two different stable molecules. Some potentially damaging oxygen species are not actually free radicals, for example hydrogen peroxide, and can be described by the term ROS. Oxygen-centred free radicals and other ROS are the main focus of this chapter. Reactive nitrogen species (RNS) include radical (e.g. nitric oxide) and non-radical derivatives of nitrogen. ROS/RNS can exist freely in the biological system in different forms under different conditions and/or metabolism (Rice-Evans et al., 1991). See Table 8.1 for a selection of ROS and RNS. Free radicals are generated continuously as a by-product, or as an intermediate species, of many biochemical redox processes. The mitochondrial electron transport, peroxisomal fatty acid metabolism, cytochrome P450 reactions and phagocytic cells are suggested to be the main sources of ROS production (Biesalski, 2002).

Production of reactive oxygen species including oxygencentred free radicals The primary mechanism of ROS generation

The mitochondrion is the major organelle in the cell, which is responsible for ATP synthesis through a series of electron transport chains. Under normal physiological conditions, the body converts oxygen molecules into ATP and water, via oxidative phosphorylation in the mitochondria, in order to yield large amounts of ATP. The electron transport system consists of four different complexes, and each of them contributes to the process of ATP synthesis. Briefly, the NADH + H+ and the FADH2 from the Krebs cycle transport high-energy electrons into the first stage of the electron transport system. FMN accepts the high-energy electrons and coenzyme Q (CoQ) will transfer the electrons to complex II. The electrons will be further transferred to complex III by cytochrome c, the site where the oxygen molecules accept four electrons and thus are converted into water molecules. Superoxide radicals (O2
–) are mainly generated in two sites of the electron

transport chain, namely NADH dehydrogenase (complex I) and the ubiquinone–cytochrome c reductase (complex III) (Turrens, 1997). Superoxide radicals are generated due to the ‘leakage’ of electrons from the electron transport chain to the oxygen molecules such that fewer than four electrons are transferred to the oxygen molecules. These superoxide radicals are chemically unstable and cause damage to the cells. The superoxide radicals generated by these complexes in the inner membrane of the mitochondria are difficult to quantify as they are rapidly converted into a more stable form, hydrogen peroxide, by superoxide dismutase (SOD). SOD acts as the primary defence mechanisms against superoxide radicals. SOD converts superoxide to hydrogen peroxide, which can then be converted to oxygen and water by catalase and glutathione peroxidase. The toxicity of hydrogen peroxide is due mainly to its reactivity with superoxide and/or Fe2+ to generate the hydroxyl radical, which is an extremely reactive and damaging radical. In the presence of ferrous iron ions, hydrogen peroxide can be converted to the hydroxyl radical, and this series of reactions is usually referred to as the Fenton reaction.

Generation of superoxide will also generate hydrogen peroxide at the same time by dismutation. Superoxide and hydrogen peroxide can react with each other and result in formation of the hydroxyl radical by the Haber–Weiss reaction. O2
– + H2O2→H2O + O2 + OH


Formation of ROS by enzymatic reactions

In addition to the generation of superoxide radicals from the mitochondria, enzymatic reactions are also able to generate superoxide radicals and/or hydrogen peroxide. Xanthine oxidase is one of the enzymes that can generate ROS. Under normal physiological conditions, a hydrogen atom is transferred from hypoxanthine or xanthine to the NAD and thus generates NADH. Xanthine oxidase also catalyses the reaction of hypoxanthine with water to generate hydrogen peroxide. However, under certain pathological conditions, such as ischaemic injury, xanthine

Table 8.1. Reactive oxygen species and reactive nitrogen species found in biological systems. Reactive oxygen species Radicals

Reactive nitrogen species

Non-radicals

Superoxide (O2• Hydroxyl (OH•) Hydroperoxyl (HO2•) Alkoxyl (RO•) Peroxyl (ROO•) –)

(1O2)

Singlet oxygen Hydrogen peroxide (H2O2) Ozone (O3) Hypochlorous acid (HOCl)

Radicals

Non-radicals

Nitric oxide (NO•) Nitrogen dioxide (NO2•)

Nitrous acid (HNO2) Peroxynitrite (ONOO–)

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oxidase will be converted from the dehydrogenase to the oxidase form that leads to the production of superoxide radicals (Granger et al., 1981). Generation of ROS by other biological activities

The immune system is another major source of free radical production in biological systems. Immune cells, such as macrophages and neutrophils, are responsible for defending the body against invading microorganisms. In order to fight and destroy foreign pathogens, phagocytes have the ability to produce ROS (Rosen et al., 1995). The activation of macrophages and neutrophils generates ROS, including hydrogen peroxide, superoxide and singlet oxygen. Superoxide can react with hydrogen peroxide to produce the hydroxyl radical, while the hydrogen peroxide can be converted into the strong oxidant hypochlorite (OCl–) in the presence of chloride ions in the cells. Hydrogen peroxide can be converted into OCl– directly by myeloperoxidase in neutrophils. Studies have also shown that NADPH oxidase is involved in the body’s defence mechanism, by converting oxygen into superoxide (Shepherd, 1986; Karlsson and Dahlgren, 2002). Moreover, the production of peroxynitrite (ONOO–) is essential for killing bacteria, but the overproduction of these species can also damage the normal cell by oxidation and nitration reactions (Schulz et al., 1995). O2
–→NO→ONOO– Transition metal ions play an important catalytic role in the generation of free radicals. Iron and copper ions are suggested as two of the key metal ions (Rice-Evans et al., 1991). Usually, transition metals donate an electron to hydrogen peroxide to produce the hydroxyl radical. It is therefore suggested that most hydroxyl radical formation involves transition metals. Fortunately, in healthy individuals, most of these metal ions are bound to transport proteins and storage proteins, and only trace amounts are free in the system. ROS formation under pathological conditions and due to the environment

In addition to normal physiological metabolism, some pathological conditions such as alcohol metabolism can also induce oxidative stress. Alcohol metabolism has been studied extensively and it is believed that many different medical complications are related to it, such as cirrhosis of the liver (Lieber, 1998). Alcohol dehydrogenase is a cytosolic enzyme that catalyses the conversion of ethanol to acetaldehyde. This metabolism changes the NAD+/NADH ratio which results in a higher concentration of NADH. An increase in cytochrome P450 2E1 (CYP2E1) activity following alcohol consumption also accounts for another major route of free radical production (Albano et al., 1995). Moreover, the production of acetaldehyde can also induce the formation of free radicals as intermediates or byproducts of alcohol metabolism (for a detailed review, see Mantle and Preedy, 1999).

In addition to the internal production of ROS as part of normal physiology, there is also unavoidable exposure to ROS-generating sources in the environment: ● ● ● ● ●

Ultraviolet X- and -radiation Ozone Cigarette smoke Pesticides

It is generally believed that exposure to these environmental agents can lead to the generation of ROS. Many studies have shown that there is a strong correlation between exposure to cigarette smoke and lung cancer (e.g. Therriault et al., 2003). Skin cancer can be induced by long-term exposure to UV light (Saladi and Persaud, 2005). Studies suggest that exposure to UV light can initiate ionizing radiation and generate free radicals, such as singlet oxygen, which eventually causes damage to the skin tissue and DNA.

Damage to Biological Components by ROS Free radicals are highly reactive substances. It is undesirable that ROS exist in excess because they can react with biological molecules and cause damage. Lipid peroxidation, protein adduction and DNA cleavage will be the focus point of this chapter, but other reactions, such as the formation of oxysterols from cholesterol, can also result from reactions with ROS (Guardiola et al., 1996).

Lipid peroxidation Lipids are biological molecules that serve several important biological roles including cell membrane composition, structure and function. Lipids are major targets for ROS (Huang et al., 2000). PUFAs are easily attacked by free radicals due to the presence of double bonds. Free radicals such as the hydroxyl radical can initiate lipid peroxidation by abstraction of a hydrogen atom from a methylene group. The carbon radical formed tends to be stabilized by rearrangement to form a conjugated diene, which can react with oxygen to form a peroxyl radical. Peroxyl radicals are also capable of abstracting a hydrogen atom from another lipid molecule such as an adjacent fatty acid side chain (known as stimulation of lipid peroxidation rather than initiation), and this is the propagation stage of lipid peroxidation. The peroxyl radical combines with the hydrogen atom that it abstracts to form a lipid hydroperoxide (lipid peroxide) (see Fig. 8.1). A reduced iron complex can also stimulate lipid peroxidation by decomposing pre-formed lipid peroxides already present in the membrane, resulting in the formation of alkoxyl and peroxyl radicals. Products of lipid peroxidation (lipid peroxide breakdown products) include aldehydes, such as malondialdehyde and 4-hydroxynonenal,

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which can in turn damage other cell components such as proteins and DNA (see Fig. 8.2).

Oxidative damage to proteins Proteins are very important in biological systems as they are involved in many crucial functions of the cell. Amino acids such as cysteine, methionine and histidine are highly sensitive to free radical attack. Hydroxyl radical-dependent abstraction of a hydrogen atom from the amino acid and the peptide backbone are the major causes of protein damage which will result in the cross-linkage, peptide fragmentation, alteration of protein tertiary structures and/or deamidation (for a review, see Stadtman and Levine, 2003). The formation of protein adducts, originally found in alcoholic liver (Niemela, 1999), is also attracting attention. Various types of protein adducts are covalently formed by reaction with various products of lipid peroxidation such as malondialdehyde and 4-hydroxynonenal. The ethanol metabolite acetaldehyde binds to the reactive lysine residue of certain proteins to form acetaldehyde–protein adducts (Niemela et al., 1994). Studies have shown that the formation of acetaldehyde–protein adducts in vivo will alter the cellular and protein functions, as well as trigger the antigen-driven humoral and cellular immune response (Niemela et al., 1987). Malondialdehyde is one of the aldehydic products of lipid peroxidation which form Schiff ’s base adducts with different protein residues (Niemela et al., 1995; Tsukamoto et al., 1995) (Fig. 8.3). Malondialdehyde modifies different proteins, such as lowdensity lipoprotein and collagen, and 4-hydroxynonenal binds to the sulphydryl groups of proteins to form protein adducts (Niemela et al., 1995). These adducted proteins are resistant to degradation and also act as neoantigens, which can trigger the immune system to attack normal cells.

Fig. 8.1. A typical example of how the lipid peroxidation chain reaction can be initiated by the hydroxyl radical. The other hydrocarbon radicals generated from the initiation step also participate in the propagation stage to cause the chain reaction of lipid peroxidation.

DNA damage by ROS ROS and RNS can cause damage to DNA, including singleand double-strand breaks and structural alterations in DNA, e.g. base pair mutations, rearrangements, deletions, insertions and sequence amplification (Wiseman and Halliwell, 1996; Halliwell and Gutteridge, 1999). The hydroxyl radical is especially damaging, but other ROS/RNS such as singlet oxygen, peroxynitrite and the decomposition products of peroxynitrite are also effective damaging agents. Different ROS affect DNA in different ways. The hydroxyl radical generates oxidatively modified products from all four bases, including 8-hydroxyguanine, the most widely measured biomarker of oxidative DNA damage. ROS/RNS can affect cytoplasmic and nuclear signal transduction pathways. They can also modulate the activity of the proteins and genes that respond to stress and which act to regulate the genes that are related to cell proliferation, differentiation and apoptosis (Wiseman and Halliwell, 1996). These changes are likely to increase the incidence of cancer as well as age-related pathologies.

Fig. 8.2. The intermediates and products of lipid peroxidation. Different intermediates and by-products are formed during lipid peroxidation, and the types of products are dependent on the chemical structure of the precursor. These intermediates and the final products will react further with other biological components, such as proteins, to cause further damage.

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Fig. 8.3. Malondialdehyde–protein adduct formation. The N-terminal amino group and the lysine residue of the polypeptide chain are common sites for adduction. Malondialdehyde (MDA) can covalently bind to these sites, resulting in a change in protein conformation.

Oxidative Stress-induced Diseases Oxidative stress results from an imbalance of ROS production and antioxidant concentration. Many pathologies are suggested to be related to oxidative stress (Fig. 8.4) (Halliwell and Gutteridge, 1999). Although the actual mechanisms of how ROS cause disease is not well

defined, it is generally proposed that ROS damage the cell components, such as protein, lipid and DNA. It is therefore suggested that cumulative damage to the cell progressively decreases the cellular function and thus results in the incidence of disease (de Groot, 1994; Toyokuni, 1999). Specific inborn metabolic disorders and/or genetic defects may promote oxidative stress. It is suggested that an

Fig. 8.4. Different pathological conditions associated with free radicals. Overproduction of ROS can result in oxidative stress. Different studies have shown that oxidative stress is positively correlated with the incidence of different diseases.

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An Overview of Free Radicals and Oxidative Stress

abnormal metabolic rate or the underexpression of antioxidant enzymes are two of the major causes of oxidative stress due to genetic defects. Overproduction of free radicals under pathophysiological conditions, such as inflammation, could also result in oxidative stress. Dietary habit is an important factor that influences the development of oxidative stress. Many thousands of dietary antioxidants have been identified, of which carotenoids, vitamin C and vitamin E are the best known antioxidants and they are essential to maintain a normal metabolism. Atherosclerosis is one of the diseases that has been studied extensively in both animal and human models, which indicate the role of oxidative stress in disease progression (Maxwell and Lip, 1997). Several essential minerals, such as copper, zinc, manganese and selenium, act as cofactors that are important for the formation and activation of certain critical antioxidant enzymes. Malnutrition or an imbalanced diet will therefore impair the antioxidant defence system and hence result in oxidative stress as well as other diseases.

Defence Systems Against Oxidative Stress In general, defence systems against oxidative stress include both enzymatic and non-enzymatic mechanisms to eliminate and convert ROS to stable and non-toxic substances, such as water. SOD, catalase and glutathione peroxidase (GPx) are the key members of the enzymatic antioxidant defence system, while albumin, reduced glutathione (GSH), ascorbic acid, -tocopherol, uric acid and bilirubin are regarded as the key non-enzymatic antioxidants (Yu, 1994). These antioxidants form a complex and interactive network to protect the organism against oxidative damage. An antioxidant is a substance that, ‘when present at low concentrations, compared to those of the oxidizable substrate, significantly delays, or inhibits, oxidation of the substrate’ (Diplock et al., 1993). It acts in several ways: ● Remove oxygen or alleviate the oxygen concentration ● Remove catalytic metal ions ● Scavenge initiating radicals such as
OH

Antioxidants are very important in the biological system because of their ability to donate a hydrogen molecule to the free radical to make it stable and unreactive with other biological components. The antioxidant will become an oxidized form or a weak radical, but remains inert. A typical example is that of lipid peroxidation chain termination by vitamin E. -Tocopherol is the most biologically active form of vitamin E and protects the cell membrane by donating its phenolic hydrogen atom to the peroxyl radicals and hence terminates the chain reaction of lipid peroxidation. Vitamin E is also able to modulate the cellular response to oxidative stress through cell signalling pathways (Peus et al., 2001; or, for a review, see Packer et al., 2001). The vitamin E radical can be regenerated by GSH and vitamin C (ascorbate). Vitamin C is the major antioxidant in aqueous environments because of its hydrophilic properties and it reacts with hydroxyl and

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superoxide radicals directly as a radical scavenger (Frei et al., 1990; Frei, 1991). Antioxidant enzymes include SODs, which rapidly convert the superoxide radical into hydrogen peroxide, while both catalase and GPx will convert hydrogen peroxide into water and oxygen. Furthermore, in order to limit production of the hydroxyl radical, SOD, catalase and GPx work together to scavenge the superoxide radical and hydrogen peroxide and thus minimize the Haber–Weiss reaction. In humans, there are three different types of SOD, encoded by three different genes. Manganese SOD (Mn)-SOD is located in the matrix of the mitochondria, while the copper/zinc (Cu/Zn)SOD is located in the cytosol. The dismutation reaction of SODs catalyses the conversion of superoxide radicals into hydrogen peroxide. Mn-SOD is the first antioxidant enzyme that deals with superoxide radicals produced from the electron transport chain in the mitochondria (Halliwell and Gutteridge, 1986; Halliwell, 1989). Apart from the SODs which are localized in the cytosol and the mitochondria, it is necessary to remove the superoxide radical released into the extracellular spaces. Marklund et al. (1982) discovered the third SOD isozyme that is localized in the extracellular spaces and this is now termed extracellular SOD (EC-SOD). EC-SOD is a slightly hydrophobic glycoprotein containing one copper and one zinc atom per subunit, which are essential for its enzymatic activity. Although the mitochondrial electron transport system is the main source of superoxide generation, other cell types such as leukocytes release superoxide into the extracellular spaces, and studies have suggested that EC-SOD is important in ameliorating certain pathophysiological conditions, such as neurological disorders and arthritis (Fattman et al., 2003). SODs O2
– + O2
– + 2H+ ——→ H2O2 + O2 Catalase is a haemoprotein mainly located in peroxisomes that catalyses the conversion of hydrogen peroxide into water and oxygen. Catalase works as the primary antioxidant enzyme to remove hydrogen peroxide, while GPx shares this function in order to maximize the conversion of hydrogen peroxide into water. Catalase H2O2 + H2O2 ————→ O2 + 2H2O The GPx system is a more complicated system that consists of GPx itself, glutathione reductase and other cofactors. The primary antioxidant enzyme function is mainly that of GPx, which catalyses the reduction of hydrogen peroxide and organic hydroperoxides into water and oxygen. GPx 2GSH + H2O2 ——→ GSSG + 2H2O GPx 2GSH + ROOH ——→ GSSG + ROH + H2O GPx is mainly localized in the cytosol and mitochondrial matrix (Wendel and Cikryt, 1980), and two types of GPx, namely selenium-dependent and selenium-independent, have been identified. The Se-dependent GPx mainly removes hydrogen peroxide, while the Se-independent GPx

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Fig. 8.5. A schematic diagram showing the production of different ROS and the relevant defence mechanism. The superoxide radical, which is mainly generated from the electron transport chain of the mitochondria, is the primary ROS formed. Different substances react with the superoxide radical to generate further ROS, while several antioxidants cooperate to eliminate ROS accumulation. SOD, superoxide dismutase; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; PUFA, polyunsaturated fatty acid; G-6-P, glucose-6-phosphate; ETC, electron transport chain; e–, free electron; ROO
, lipid peroxyl radical; ROOH, lipid peroxide; Se-Px, Selenium-dependent GPx.

mainly removes organic hydroperoxides. GSH is a tripeptide with a reactive thiol group and resistance to peptidase attack, which serves as an effective reductant to reduce hydrogen peroxide, while itself becoming oxidized glutathione (GSSG) by donating hydrogen from its sulphydryl group. Due to the importance of GSH in a variety of detoxification processes, the presence of glutathione reductase is also important to regenerate the reduced form of GSH by reducing the GSSG using NADPH as substrate.

Last but not Least There is growing evidence showing that there is a strong correlation between oxidative stress and ageing, an issue that affects the future of society. This chapter highlights the basic concepts of ROS formation and its toxicity. Several major pathways of ROS production and antioxidant defence mechanisms are also described (Fig. 8.5 illustrates the complexity of ROS metabolism).

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Halliwell, B. and Gutteridge, J.M. (1986) Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Archives of Biochemistry and Biophysics 246, 501–514. Halliwell, B. and Gutteridge, J.M.C. (1999) Free Radicals in Biology and Medicine, 3rd edn. Oxford University Press, Oxford. Halliwell, B., Murcia, M.A., Chirico, S. and Aruoma, O.I. (1995) Free radicals and antioxidants in food and in vivo: what they do and how they work. Critical Reviews in Food Science and Nutrition 35, 7–20. Huang, Y., Liu, D. and Sun, S. (2000) Mechanism of free radicals on the molecular fluidity and chemical structure of the red cell membrane damage. Clinical Hemorheology and Microcirculation 23, 287–290. Karlsson, A. and Dahlgren, C. (2002) Assembly and activation of the neutrophil NADPH oxidase in granule membranes. Antioxidants and Redox Signaling 4, 49–60. Ku, H.H., Brunk, U.T. and Sohal, R.S. (1993) Relationship between mitochondrial superoxide and hydrogen peroxide production and longevity of mammalian species. Free Radical Biology and Medicine 15, 621–627. Lieber, C.S. (1998) Hepatic and other medical disorders of alcoholism: from pathogenesis to treatment. Journal of Studies on Alcohol 59, 9–25. Mantle, D. and Preedy, V.R. (1999) Free radicals as mediators of alcohol toxicity. Adverse Drug Reactions and Toxicological Reviews 18, 235–252. Marklund, S.L., Holme, E. and Hellner, L. (1982) Superoxide dismutase in extracellular fluids. Clinica Chimica Acta 126, 41–51. Maxwell, S.R. and Lip, G.Y. (1997) Free radicals and antioxidants in cardiovascular disease. British Journal of Clinical Pharmacology 44, 307–317. Niemela, O. (1999) Aldehyde–protein adducts in the liver as a result of ethanol-induced oxidative stress. Frontiers in Bioscience 4, D506–D513. Niemela, O., Klajner, F., Orrego, H., Vidins, E., Blendis, L. and Israel, Y. (1987) Antibodies against acetaldehyde-modified protein epitopes in human alcoholics. Hepatology 7, 1210–1214. Niemela, O., Parkkila, S., Yla-Herttuala, S., Halsted, C., Witztum, J.L., Lanca, A. and Israel, Y. (1994) Covalent protein adducts in the liver as a result of ethanol metabolism and lipid peroxidation. Laboratory Investigation 70, 537–546. Niemela, O., Parkkila, S., Yla-Herttuala, S., Villanueva, J., Ruebner, B. and Halsted, C.H. (1995) Sequential acetaldehyde production, lipid peroxidation, and fibrogenesis in micropig model of alcohol-induced liver disease. Hepatology 22, 1208–1214. Packer, L., Weber, S.U. and Rimbach, G. (2001) Molecular aspects of alpha-tocotrienol antioxidant action and cell signalling. Journal of Nutrition 131, 369S–373S. Peus, D., Meves, A., Pott, M., Beyerle, A. and Pittelkow, M.R. (2001) Vitamin E analog modulates UVB-induced signaling pathway activation and enhances cell survival. Free Radical Biology and Medicine 30, 425–432. Rice-Evans, C.A., Diplock, A.T. and Symons, M.C.R. (1991) Laboratory Techniques in Biochemistry and Molecular Biology – Techniques in Free Radical Research. Elsevier Science Publishers, Amsterdam. Rosen, G.M., Pou, S., Ramos, C.L., Cohen, M.S. and Britigan, B.E. (1995) Free radicals and phagocytic cells. FASEB Journal 9, 200–209. Saladi, R.N. and Persaud, A.N. (2005) The causes of skin cancer: a comprehensive review. Drugs Today (Barcelona) 41, 37–53. Schulz, J.B., Matthews, R.T. and Beal, M.F. (1995) Role of nitric oxide in neurodegenerative diseases. Current Opinion in Neurology 8, 480–486. Shepherd, V.L. (1986) The role of the respiratory burst of phagocytes in host defense. Seminars in Respiratory Infections 1, 99–106. Sohal, R.S. and Weindruch, R. (1996) Oxidative stress, caloric restriction, and aging. Science 273, 59–63. Stadtman, E.R. and Levine, R.L. (2003) Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25, 207–218. Therriault, M.J., Proulx, L.I., Castonguay, A., and Bissonnette, E.Y. (2003) Immunomodulatory effects of the tobacco-specific carcinogen, NNK, on alveolar macrophages. Clinical and Experimental Immunology 132, 232–238. Toyokuni, S. (1999) Reactive oxygen species-induced molecular damage and its application in pathology. Pathology International 49, 91–102. Tsukamoto, H., Horne, W., Kamimura, S., Niemela, O., Parkkila, S., Yla-Herttuala, S. and Brittenham, G.M. (1995) Experimental liver cirrhosis induced by alcohol and iron. Journal of Clinical Investigation 96, 620–630. Turrens, J.F. (1997) Superoxide production by the mitochondrial respiratory chain. Bioscience Reports 17, 3–8. Wendel, A. and Cikryt, P. (1980) The level and half-life of glutathione in human plasma. FEBS Letters 120, 209–211. Wiseman, H. and Halliwell, B. (1996) Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochemical Journal 313, 17–29. Wu, D. and Cederbaum, A.I. (2003) Alcohol, oxidative stress, and free radical damage. Alcohol Research and Health 27, 277–284. Yu, B.P. (1994) Cellular defenses against damage from reactive oxygen species. Physiological Reviews 74, 139–162.

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9 Tocopherol Composition of Plants and Their Regulation Hans-Peter Mock Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, D-06466 Gatersleben, Germany

Abbreviations: Fr wt, fresh weight; GGPP, geranylgeranyl pyrophosphate; HGGT, homogentisate geranylgeranyl transferase; HPPD, hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyltransferase; MMT, 2-methyl-6-phytylbenzoquinol methyltransferase; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinol; PPP, phytyl pyrophosphate; TMT, tocopherol methyltransferase.

Abstract The following contribution will cover the roles and occurrence of vitamin E compounds in plants. The spectrum of tocopherols and tocotrienols varies with the plant tissue analysed. In plant leaves, -tocopherol has been observed as the dominant form, whereas in many seeds -tocopherol and tocotrienols are accumulated. The content of tocopherols in plant tissues is influenced by environmental conditions, e.g. by light, and by developmental processes such as senescence. Genes encoding enzymes for all the biosynthetic steps of tocopherol and tocotrienol biosynthesis have been cloned in recent years making frequent use of the resources available for the model plant Arabidopsis thaliana. Mutants and transgenic lines affected in one or several steps of tocopherol biosynthesis have been generated to study the significance of individual steps in regulating tocopherol accumulation with the aim of modifying the overall levels and the spectrum of vitamin E compounds in plants. As an example, it has been shown that tocopherol methyltransferase catalysing the final methylation reaction is not limiting for overall tocopherol accumulation, but can be used to alter the spectrum in seeds. These and other examples demonstrate the feasibility of approaches to generate plants with altered composition of tocopherols and tocotrienols to improve the quality of crops as part of the human diet.

spectrum of vitamin E with respect to the nutritional quality of plants.

Introduction The biosynthetic pathway of vitamin E synthesis in plants (Fig. 9.1) has attracted considerable research efforts in recent years mainly due to the relevance of this group of compounds for human nutrition. In the following contribution, tissue-specific accumulation of vitamin E compounds in plants will be summarized. Next, the influence of environmental and endogenous factors on the levels of vitamin E compounds in plants will be addressed. In the third section, individual steps of tocopherol and tocotrienol biosynthesis will be discussed. The focus will be on results obtained with transgenic plant lines and mutants with modified expression of genes involved in vitamin E biosynthesis. These mutants and transgenic lines are valuable resources to elucidate the regulation of the pathway, but also indicate the potential to alter the 112

Tocopherol and Tocotrienol Composition of Plant Tissues The group of plant lipophilic vitamin E antioxidants comprises eight naturally occurring forms of tocopherols and tocotrienols. Their biosynthesis is restricted to photosynthetically active organisms such as plants, cyanobacteria and algae (Lichtenthaler, 1968), suggesting a protective function of the photosynthetic apparatus. The distribution of tocopherol and tocotrienol biosynthesis in photosynthetically active organisms, but also the localization of enzymes involved in biosynthesis as well as the accumulation of end-products in plastid membranes

© CAB International 2007. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

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[Non-mevalonate pathway]

Hydroxyphenylpyruvate Hydroxyphenylpyruvate dioxygenase (HPPD)

Phytylpyrophosphate

Homogentisate

Homogentisate phytyltransferase (HPT) 2-Methyl-6-phytyl-1,4-benzoquinol 2-Methyl-6-phytyl-1,4-benzoquinol methyltransferase (MMT) 2,3-Dimethyl-5-phytyl-hydrochinone

Tocopherol cyclase (TC)

γ-Tocopherol Tocopherol methyltransferase (TMT)

α-Tocopherol

Tocopherol cyclase (TC)

δ-Tocopherol Tocopherol methyltransferase (TMT)

β-Tocopherol

Fig. 9.1. Biosynthetic pathway of tocopherol synthesis.

support the endosymbiotic origin of the pathway, and it is assumed that most nuclear-encoded enzymes of tocopherol biosynthesis were acquired from endosymbiotic cyanobacteria. The spectrum of tocopherols and tocotrienols varies with the plant tissue and is dependent on environmental and developmental conditions (see Tables 9.1 and 9.2 for selected data). High concentrations of -tocopherol are associated with photosynthetically active tissues (Hess, 1993) and, due to the lipophilic nature of the molecule, its presence in the plastid membranes is assumed, but a detailed analysis of the actual distribution of -tocopherol or other members of the vitamin E family in the membrane systems of plant cells has not been published. However, in a study on barley organelles isolated from a protoplast preparation of seedlings, 18% of the accumulated tocopherol was found in the vacuolar fraction, and approximately 50% in plastids (Rautenkranz et al., 1994). Accumulation of -tocopherol has been measured in different plant tissues including roots, seeds and etiolated leaf segments. The contents reported range from as low as 150 ng/g fresh weight (fr wt) in potato tubers up to several mg/g fr wt in oil palm leaflets (see Table 1 in Hess, 1993). The content of tocopherols in leaves has been measured in

many plant species and is comparatively low, ranging from 10 to 50 g/g fr wt (Hess, 1993). Accumulation of -tocopherol has also been observed in non-photosynthetic cell culture systems such as safflower (Furuya et al., 1987), indicating that the capacity for tocopherol biosynthesis is not dependent on photosynthesis. The highest levels of tocopherols are found in seeds when comparing different plant tissues, and oils of different plants are therefore a rich source of tocopherols in the human diet. In contrast to leaf tissues, -tocopherol is not the major component in the spectrum of vitamin E compounds (Hess, 1993; Grusak and DellaPenna, 1999). In the model plant Arabidopsis, the spectrum in leaf tissue is composed of 90% -tocopherol and 10% -tocopherol, whereas in seeds -tocopherol is the major form (95%), with minor amounts of -tocopherol (4%) and -tocopherol (1%) (Grusak and DellaPenna, 1999). Seed oils of crops show considerable variation in the spectrum of vitamin E components, with 96% of -tocopherol in sunflower seed oil, but only 7% in soybean oil which contains high levels of -tocopherol (70%) and -tocopherol (22%). In palm seed oil, -tocotrienol (30%) and -tocotrienol (40%) are the main vitamin E compounds (Grusak and DellaPenna, 1999, and references therein). Tocotrienol accumulation has been mostly reported for

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Table 9.1. Seed and fruit tocopherol composition of selected crop species. ( g/g dry weight) -Tocopherol -Tocopherol

Species Barley (Hordeum vulgare) Barley (Hordeum vulgare) Pea (Pisum sativum) Maize (Zea mays) Wheat (Triticum vulgare) Pepper fruits Green Yellow Red

13 9.4 6.5 5.6 14 61 76 171

Reference

41 (tocotrienal) 19 (tocotrienal) 158 95 19 (tocotrienol)

Ref. [11] in Hess (1993) Ref. [10] in Hess (1993) Ref. [10] in Hess (1993) Ref. [10] in Hess (1993) Ref. [10] in Hess (1993)

Up to 20% - and 3% -tocopherol

Koch et al. (2002)

Table 9.2. Leaf tocopherol composition of selected plant species. Species

( g/g fresh weight) -Tocopherol -Tocopherol

Reference

Zea mays (maize) Arabidopsis thaliana Hordeum vulgare (barley) Picea abies, primary needles Melia azedarach, green Melia azedarach, senescing

2.1 12.5 15.7 43 204 510

Ref. [2] in Hess (1993) Cheng et al. (2003) Ref. [2] in Hess (1993) Ref. [18] in Hess (1993) Ref. [25] in Hess (1993) Ref. [25] in Hess (1993)

seeds, but was also found in the latex of Hevea (Dunphy et al., 1965; Hess, 1993). The levels of tocopherols might vary during developmental processes of the plant. In pepper (Capsicum annuum) fruits, which are a rich source for dietary vitamin E, the level of -tocopherol increases during fruit ripening. Red pepper fruits contain approximately threefold higher levels of -tocopherol than green fruits, in parallel to the enrichment of triacylglycerols during ripening (Koch et al., 2002). During storage of barley seeds for 10 months, the contents of vitamin E decreased from 92 to 20 g/g dry weight at 23% moisture. A higher loss of vitamin E was observed when the moisture was 28% during storage. The effect of storage was different for individual compounds and the percentage of vitamin E increased with respect to other tocols. When storage was performed under supply of additional carbon dioxide, the loss of vitamin E was decreased (Hakkarainen et al., 1983). Tocopherol levels are also influenced during senescence of plant leaves, which is a controlled process with similarities to programmed cell death. The progression of senescence is influenced by a number of endogenous and exogenous factors, e.g. by the cytokinin class of plant hormones. Nutrient recycling from the senescing leaves is one of the important aspects of this process requiring coordinated expression of genes, but also regulation on the protein and metabolite level (Buchanan-Wollaston et al., 2003; Wiedemuth et al., 2004). In Festuca pratensis leaves, -tocopherol showed an initial increase followed by a decline upon onset of senescence (Peisker et al., 1989). An early event in leaf senescence is the breakdown of chloroplasts. Based on its capacity to counteract lipid peroxidation, it has been suggested that regulation of -tocopherol accumulation is involved in controlling the breakdown of plastidal membranes during this process (Munne-Bosch and Alegre, 2002).

2.1 0.79 8.3 – – –

Tocopherol Levels are Regulated in Response to Environmental Stresses Plants are exposed to a number of environmental stresses such as high light, UV, drought, high as well as low temperature conditions, among others. In response to many of these stresses, reactive oxygen species are generated, thereby altering the pro/antioxidant balance of the plant cells. As a consequence of elevated levels of reactive oxygen species, many changes in the enzymic and lowmolecular weight components of the antioxidative defence system are observed. The tocopherol content of plastids is affected by light conditions. Chloroplasts themselves are important intracellular generators of reactive oxygen species, and the flow of electrons during photosynthesis has to be tightly controlled to avoid oxidative stress. In addition, plastids contain an elaborate system of protective mechanisms against radical damage (reviewed by Foyer et al., 2002). Many environmental stresses perturb the electron transport chain of plastids thereby leading to increased formation of reactive oxygen species. Exposure to high light conditions might severely affect the photosynthetic apparatus of plastids, and plants have evolved strategies to cope with such conditions. Beech leaves more exposed to sunlight accumulated three times higher levels of -tocopherol than shaded leaves, relative to the contents of chlorophyll (Lichtenthaler, 1979). A tenfold increase in -tocopherol was reported for Arabidopsis during photoacclimation of leaves when transferred from 100 to 1000 E photon flux density. A further increase of the light intensity to 1500 E, however, led to a depletion of -tocopherol by approximately 40%, indicating that the capacity for photoacclimation and tolerance to oxidative stress had been exceeded (Havaux et al., 2000). Elevated levels of -tocopherol in response to increasing light intensities

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have also been reported for Euglena gracilis (Shigeoka et al., 1986) and tobacco (Tanaka et al., 1999). In high alpine plant species, the highest concentrations of -tocopherol have been observed at midday, and the lowest during the night (Wildi and Lutz, 1996). The content of vitamin E in dry barley seeds was reported to be dependent on the seasonal conditions during growth of plants, with higher levels in better seasons (Hakkarainen et al., 1983). It has to be established whether the lower levels in seeds after a poor season are due to enhanced consumption of vitamin E during seed maturation or whether this shift is a consequence of altered carbon partitioning into its biosynthesis. Drought is another environmental factor influencing the content of tocopherol in plants. Drought-tolerant plants frequently have higher tocopherol contents than susceptible accessions (Price and Hendry, 1989; MunneBosch and Alegre, 2000), consistent with the finding that oxidative stress in chloroplasts is provoked by water deficit (Smirnoff, 1993). The content of -tocopherol as a consequence of water deficit increases in a number of plant species including pea, spinach, wheat, rosemary, lavender and beech seedlings (see Munne-Bosch and Alegre, 2002, and references therein). In contrast, when young rice seedlings were subjected to water stress, a loss of -tocopherol was observed (Boo and Jung, 1999). Higher resistance to salt stress correlated with severalfold increased levels of -tocopherol in cotton when comparing tolerant and susceptible cultivars (Gossett et al., 1994). Upon salt stress, the -tocopherol levels remained constant in the tolerant cultivars, but decreased further in the susceptible ones. In addition to -tocopherol, other components of the antioxidative defence system showed differences without or upon application of salt stress. Catalase activity was higher in both tolerant cultivars. Glutathione reductase activity was slightly lower in the tolerant cultivars than in the susceptible cultivars prior to stress treatment. However, glutathione reductase activity was stimulated by salt stress in the tolerant, but not in the susceptible ones. Both susceptible cultivars were also more strongly affected in the levels of reduced ascorbate and glutathione than the tolerant ones upon salt stress treatment (Gossett et al., 1994). These findings clearly indicate that the correlation between -tocopherol contents and salt stress tolerance in the cotton lines will also be affected by the network of other components operating in stress defence. Contradictory results have been obtained when low temperature stress was applied to different plant species. For maize, increasing but also decreasing levels of tocopherol upon low temperature stress treatments have been reported (Fryer et al., 1998; Leipner et al., 1999). The level of -tocopherol in tomato leaves declined upon chilling of whole plants (Walker and McKersie, 1993). In a leaf disc experiment, -tocopherol declined in cucumber but remained constant in pea upon exposure to low temperature in the dark (Wise and Naylor, 1987). In conclusion, it is obvious that the accumulation of tocopherols is regulated in response to environmental factors most probably acting as a component within the

network of cellular stress defence in plants. Higher levels of tocopherol are frequently associated with increased protection against abiotic stresses, but the interaction with the overall system in stress defence appears to be important. Upon exposure to various abiotic stresses, levels of tocopherols are modulated, but the results are dependent on the plant species used, its developmental stage and the strength of the stress treatment. To clarify further the functions of tocopherols within plants, transgenic lines and mutants with modified expression of biosynthetic genes and consequently with an altered spectrum of tocopherols and tocotrienols have become an important tool for basic studies, but also with respect to biotechnological applications. In the following, individual steps in tocopherol biosynthesis in plants will be described, summarizing molecular work and biochemical characterization of enzymes. Studies with transgenic lines for each of the biosynthetic steps will be mentioned, and the consequences of modulating the activity of a particular step with respect to the overall regulation of the pathway will be of specific interest.

Biosynthesis of Tocopherols and Tocotrienols in Plants Overview The elucidation of the pathway of tocopherol and tocotrienol biosynthesis (Fig. 9.1) using classical biochemical methods has been hampered by the difficulties frequently associated with the purification of low abundant, labile membrane-bound enzymes. In plants, plastids are the only site for the synthesis of a group of prenylated lipids, including tocopherols, tocotrienols, chlorophylls and phyllochinones. The biosynthetic pathway of tocopherol biosynthesis has been elucidated initially by precursor/ product studies with radiolabelled intermediates demonstrating homogentisate as an intermediate (Whistance and Threlfall, 1970). Homogentisate is the precursor for tocopherols as well as for plastoquinones and is synthesized from the shikimate pathway (Herrmann and Weaver, 1999). The formation of homogentisate is catalysed by p-hydroxyphenylpyruvate dioxygenase (HPPD). Synthesis of 2-methyl-6-phytylplastoquinol, the first true tocopherol intermediate, requires the phytylation of homogentisate catalysed by the phytyltransferase using phytylpyrophosphate (PPP) as substrate. The reduction of geranylgeranyl pyrophosphate (GGPP) to PPP has been shown to occur in the envelope and thylakoid membranes of plastids starting with a plastid preparation from spinach leaves (Soll and Schultz, 1981; Soll et al., 1983). Labelling studies with early precursors elegantly demonstrated that the biosynthesis of plastidic isoprenoids including GGPP and PPP occurs via 1-deoxy-D-xylulose-5-phosphate and not via the classical acetate/mevalonate pathway; for further information on the elucidation of the novel DOXP/MEP pathway, the reader is referred to recent reviews (Lichtenthaler, 1999; Dubey et al., 2003). In the following, the current knowledge on individual

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Table 9.3. Arabidopsis mutants affected in the biosynthesis of tocopherols. Mutant

Biosynthetic step affected

Phenotype

Conclusion

Reference

Vte2

Homogentisate phytyltransferase

Reduced seed longevity

Significance of tocopherols for seed viability

Sattler et al. (2004)

Vte3

2-Methyl-6-phytylbenzquinol methyltransferase

Increased accumulation of -tocopherol, reduced level of -tocopherol in seeds

Altered tocopherol composition, implications for food quality

Van Eenennaam et al. (2003)

Vte1

Tocopherol cyclase

Accumulation of dimethylphytylbenzoquinone, reduced seed longevity

Significance of tocopherols for seed viability

Porfirova et al. (2002); Sattler et al. (2003, 2004)

Vte4

Tocopherol methyltransferase

Accumulation of -tocopherol in leaves instead of -tocopherol

Altered composition had no major Bergmüller et al. (2003) impact on stress tolerance. Tocopherol methyltransferase activity not limiting for overall tocopherol accumulation

Mutants of the model plant Arabidopsis are a valuable resource for elucidation of biosynthetic pathways and their regulation in plants. Molecular and morphological phenotypes observed in mutants allow determination of the significance of individual steps in the pathway and possible cross-talk with other pathways.

enzymic steps of tocopherol (Fig. 9.1) and tocotrienol biosynthesis will be summarized. As important tools to elucidate the biosynthesis of vitamin E compounds and their regulation in plants, mutants and transgenic lines modified in one single or several biosynthetic steps have been used. A summary of selected mutants and transgenic lines is given in Tables 9.3 and 9.4.

Hydroxyphenylpyruvate dioxygenase (HPPD) This enzyme catalyses the biosynthesis of homogentisate from 4-hydroxyphenylpryuvate and molecular oxygen, a reaction which is also part of the catabolism of tyrosine and phenylalanine in many organisms. A plastidal localization of HPPD has been found in spinach (Fiedler et al., 1982), but in a carrot cell suspension culture, compartmentalization in the cytosol was claimed (Garcia et al., 1997). Additional evidence for the cytosolic localization of the enzyme was obtained by expression of Arabidopsis HPPD in tobacco plants and analysis of the transgenic lines by immunological methods (Garcia et al., 1999). HPPD has now been cloned from a number of plant species including Arabidopsis (Norris et al., 1998), barley (Dahnhardt et al., 2002) and Coleus blumei (Kim and Petersen, 2002), and recently crystal structures were obtained for HPPDs from Zea mays and Arabidopsis (Fritze et al., 2004). Functional proof for the identity of the isolated Arabidopsis clone was obtained by demonstrating HPPD activity of the recombinant protein expressed in Escherichia coli and by complementation of the Arabidopsis pds1 mutant previously isolated in a screening for mutants in the carotenoid pathway, indicating a role for quinone compounds in the synthesis of these pigments (Norris et al., 1995, 1998). The carotenoid deficiency of the psd1 mutant could be rescued by feeding the plants with the product of HPPD, homogentisic acid. Molecular analysis revealed a 17 bp deletion in the psd1 allele

resulting in the loss of the 26 C-terminal amino acids of the HPPD protein. Sequence comparison with HPPDs of other organisms also showed the presence of highly conserved amino acid residues in the C-terminal end of the protein (Norris et al., 1998). The Arabidopsis clone encodes a protein of 50 kDa and shares high homology to other known plant HPPDs. Like other eukaryotic HPPDs, the plant enzymes form a dimer in the native state (Fritze et al., 2004). As HPPD is also a herbicide target, structural analysis of plant HPPDs will be helpful in developing new herbicides. Southern blot analysis indicated the presence of a single gene for HPPD in Arabidopsis (Garcia et al., 1999) and barley (Dahnhardt et al., 2002). HPPD transcript levels in barley were found during senescence, and levels were also increased after treatment of plants with herbicides or hydrogen peroxide causing oxidative stress, but also in response to the plant hormones methyl jasmonate and ethylene (Falk et al., 2002). The control of HPPD transcript levels in response to ageing and to oxidative stress is consistent with the role of the end-products of the pathway as outlined in previous chapters.

Homogentisate phytyltransferase (HPT) and homogentisate geranylgeranyl transferase (HGGT) HPT catalyses the condensation of homogentisate and phytyl diphosphate to 2-methyl-6-phytyl-1,4-benzoquinol (MPBQ), the committed step in tocopherol synthesis (Soll and Schultz, 1980; Kaiping et al., 1984; Collakova and DellaPenna, 2001; Savidge et al., 2002). HPT has been cloned recently from Arabidopsis and Synechocystis (Collakova and DellaPenna, 2001; Schledz et al., 2001; Savidge et al., 2002). The recombinant enzyme proteins from both organisms used phytyl diphosphate as a substrate in vitro, but only the Synechocystis HPT also accepted geranylgeranyl diphosphate as a substrate in the prenylation reaction, although with lower activity. No product

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Table 9.4. Overexpression of genes in a transgenic model of crop plants as a tool to modify levels and composition of vitamin E components. Effects on Gene donor

Host plant

Tissue analysed

Target enzyme expression/activity

Tocopherol/tocotrienol level

Tocopherol/tocotrienol composition

Conclusions

Reference

HPT

Arabidopsis

Arabidopsis

Leaves and seeds (35S promoter)

HPT activity: tenfold in leaves, fourfold in seeds

Fourfold in leaves 40% higher in seeds

Mainly -tocopherol in seeds

HPT activity limiting for tocopherol accumulation

Collakova and DellaPenna (2003)

HPT + TMT

Arabidopsis

Arabidopsis

Leaves and seeds (35S promoter for HPT, seed-specific promoter for TMT)

No further increase in overall levels when compared with HPT-overexpressing plants

Nearly complete conversion of - to -tocopherol and of - to - tocopherol

TMT activity limits formation of - and -tocopherol, but not the overall tocopherol contents

HGGT

Barley

Arabidopsis

Leaves

Expression of transgene Similar tocopherol verified by Northern blot levels as in control lines, but 10- to 15-fold increase in total vitamin E antioxidants

Additional accumulation of tocotrienols in leaves, -tocotrienol primary form of vitamin E

Redirection of metabolic flux enabled the vitamin E contents in seeds of a major crop plant to be increased

Cahoon et al. (2003)

Maize

Seeds (embryospecific promoter)

Data not presented

Overall tocopherol and tocotrienol levels fourto sixfold increased; tocotrienol levels 20-fold

-Tocotrienol primary form of vitamin E

Soybean

Seeds (seedspecific promoter)

Data not presented

Unaltered

Mainly -tocopherol; -tocopherol reduced from 20 to 2%, reduced -tocopherol levels

Utility of genes from the model plant Arabidopsis to alter seed oil composition; useful co-expression of two genes for separate steps

Van Eenennaam et al. (2003)

Unaltered

Mainly -tocopherol; -tocopherol reduced

Overall accumulation similar

Shift from - to -tocopherol and from - to -tocopherol (see Fig. 9.2)

TMT activity limits formation of - and -tocopherol, but not overall tocopherol contents

Shintani and DellaPenna (1998)

2-Methyl-6Arabidopsis phytylbenzoquinol methyltransferase (vte3) MMT + TMT TMT

Arabidopis

Arabidopis

Seeds (seedspecific promoter)

TMT activity only present in seeds of transgenic lines

Tocopherol Composition of Plants and Their Regulation

Gene(s) overexpressed

A number of papers dealing with the overexpression of one or several genes involved in the biosynthesis of tocopherol or its precursors have recently been published following the identification of biosynthetic steps in recent years. Selected data from some of these papers are shown and clearly demonstrate the feasibility of the transgenic approach to modify the overall accumulation of tocopherol and tocotrienols and the spectrum of individual components. HPT, homogentisate phytyltransferase;TMT, tocopherol methyltransferase; HGGT, homogentisate geranylgeranyl transferase; MMT, 2-methyl-b-phytylbenzoquinol methyltransferase.

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formation was observed with solanesyl diphosphate, the prenyl substrate involved in plastoquinone-9 biosynthesis (Collakova and DellaPenna, 2001). Constitutive overexpression of HPT using the Arabidopsis HPT1 gene under the control of the cauliflower mosaic virus 35S rRNA promoter provided further evidence that the HPT activity is a limiting step in tocopherol biosynthesis (Collakova and DellaPenna, 2003). Transgenic Arabidopsis plants showed a tenfold increase of HPT activity and up to a 4.4-fold increase of tocopherol content in leaves, when comparing them with controls. As described above, wild-type Arabidopsis seeds contain much higher levels of tocopherols than leaves. Overexpression of HPT activity resulted in a fourfold higher enzyme activity, and the tocopherol content was further increased by 40% in seeds of transgenic lines. Leaves of wild-type and transgenic plants mostly accumulated -tocopherol, but a considerable fraction of -tocopherol was also observed in the transgenic lines. The increase of tocopherol in seeds of transgenic lines was mostly due to additional -tocopherol. No accumulation of prenylquinone intermediates was observed in leaves of transgenic lines, indicating that the cyclase and methylaton reactions subsequent to HPT are not limiting steps in tocopherol biosynthesis (Collakova and DellaPenna, 2003). HPT overexpression had no significant effect on the accumulation of chlorophylls, carotenoids and plastoquinone-9 levels, the end-products of pathways competing for common precursors. Additional overexpression of -tocopherol methyltransferase in selected lines overexpressing HPT1 resulted in the almost complete conversion of -tocopherol to -tocopherol in leaves. Similarly, - and -tocopherol were almost fully converted to - and -tocopherol in seeds, indicating that both transgenes contributed in an additive way (Collakova and DellaPenna, 2003). When using the strong, seed-specific napin promoter for overexpression of HPT in Arabidopsis, tocopherol levels were increased by 75% (Savidge et al., 2002). Tocotrienols are the major forms of vitamin E in seeds of monocotyledonous plants, and their formation requires the presence of a prenyltransferase accepting geranylgeranyl diphosphate as a substrate. HPT-related sequences were recently obtained from developing seeds of barley, wheat and rice (Cahoon et al., 2003). Overexpression of the barley clone in a tobacco tissue culture resulted in the substantial accumulation of all four tocotrienol species without influencing the levels of tocopherols, giving functional proof for HGGT activity. Similarly, overexpression of this barley HGGT in Arabidopsis led to the accumulation of large amounts of tocotrienols in leaves. When the barley HGGT gene was introduced in maize under the control of a strong embryo-specific promoter, the tocotrienol content of whole seeds was increased 20-fold relative to wild-type controls, and the total vitamin E content was increased four- to sixfold. Additional tocotrienol formation in maize seeds did not affect tocopherol levels. Accumulation of -tocotrienol as the main compound in transgenic maize seeds indicated the limiting capacity of the final methylation reaction (Cahoon et al., 2003).

Tocopherol content (ng/mg seed)

118

Wild type 400

TMT-overexpressing lines

300

200

100

0 





Seed tocopherol composition Fig. 9.2. Seed tocopherol profile of wild-type and transgenic Arabidopsis plants overexpressing tocopherol methyltransferase. In seeds of wild-type plants, the tocopherol spectrum is dominated by tocopherol. Seed-specific expression of tocopherol methyltransferase alters the spectrum towards -tocopherol, without influencing the overall tocopherol contents. Fig. 9.2 was prepared with data from Shintani and DellaPenna (1998).

2-Methyl-6-phytylbenzoquinol methyltransferase (MMT) Screening the Arabidopsis mutant population led to the identification of vte3 (vte for vitamin E) lines with reduced levels of -tocopherol but elevated amounts of -tocopherol in seeds (Van Eenennaam et al., 2003). Independently, a similar screen based on the analysis of the leaf tocopherol spectrum resulted in the isolation of additional Arabidopsis vte3 mutants with reduced - and -tocopherol, but increased - and -tocopherol levels (Cheng et al., 2003). These alterations of the seed and leaf tocopherol compositions are consistent with an impaired MMT activity. The VTE3 locus was isolated and functional proof for the identity with MMT was obtained by enzyme assays using recombinant VTE3 protein (Cheng et al., 2003; Van Eenennaam et al., 2003). The phenotype of the vte3–1 mutant line was only slightly affected, but the homozygous vte3–2 line representing a null mutation of the gene was lethal on soil, and seedlings grown on media supplemented with sucrose were pale. Biochemical analysis demonstrated that the mutants, in addition to the lack of tocopherols, were also affected in the synthesis of plastoquinone-9, consistent with a severe impairment of the photosynthetic apparatus. This observation indicates that Arabidopsis does not contain functionally redundant methyltransferase activities for plastoquinone biosynthesis. The recombinant MMT also accepted 2-methyl-6-solanyl-1,4-benzoquinone as a substrate (Cheng et al., 2003). Seed-specific expression of the VTE3 gene almost completely reduced the -tocopherol fraction in maize, and with additional ectopic expression of -tocopherol methyltransferase the percentage of -tocopherol increased from 10 to 95% of all tocopherols (Van Eenennaam et al., 2003).

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Tocopherol/tocotrienol cyclase Screening for tocopherol-deficient Arabidopsis mutants identified the vte1 mutant lacking all four tocopherols and showing reduced tocopherol activity. The VTE1 gene was isolated and the heterologously expressed protein showed tocopherol and tocotrienol cyclase activity (Porfirova et al., 2002). The lack of tocopherols had no effect on growth and photosynthesis under standard growth conditions, but high light led to a small decrease in chlorophyll contents and photosynthetic quantum yield, supporting a protective role for tocopherols under photo-oxidative stress (Porfirova et al., 2002). Further vte1 mutants were independently isolated in another laboratory (Sattler et al., 2003). The Arabidopsis VTE1 gene encoding tocopherol cyclase showed high homology to the SDX1 gene from maize, initially isolated in the context of carbohydrate metabolism (Provencher et al., 2001). Heterologous expression of the protein encoded by SDX1 demonstrated tocopherol cyclase activity (Sattler et al., 2003). In contrast to the Arabidopsis vte1 mutant line, the maize sdx1 mutant was severely affected in growth, and accumulated anthocyanins as well as high levels of carbohydrates in source leaves, consistent with a sucrose export deficiency (Provencher et al., 2001). The discrepancy between the Arabidopsis and maize mutant phenotypes is not fully understood, but results indicate additional functional roles for tocopherols or intermediates apart from acting as lipophilic antioxidants, e.g. by modulating signalling in plants (Hofius and Sonnewald, 2003; Sattler et al., 2003). The Arabidopsis vte1 mutant also lacks tocopherols in seeds, and it has been shown recently that this mutation can severely affect seed longevity, pointing to an essential role for tocopherols during seed quiescence (Sattler et al., 2004).

Tocopherol methyltransferase (TMT) -Tocopherol methyltransferase activity has been purified by standard chromatography methods from Capsicum fruits (Dharlingue and Camara, 1985; Koch et al., 2003); however, cloning of the gene was not achieved. Based on a genomics approach, TMT clones were first isolated from the cyanobacterium Synechocystis and from Arabidopsis

(Shintani and DellaPenna, 1998). Biochemical characterization of the heterologously expressed Arabidopsis protein and the purified Capsicum TMT showed that both - and -tocopherol were accepted as substrates in the TMT reaction, but not -tocopherol (Shintani and DellaPenna, 1998; Koch et al., 2003). A detailed kinetic analysis of the Arabidopsis enzyme indicated an iso-ordered bi–bi-type reaction mechanism (Koch et al., 2003). Overexpression of TMT in Arabidopsis shifted the spectrum of seed tocopherols from - to -tocopherol without altering the overall tocopherol contents (Shintani and DellaPenna, 1998). Arabidopsis mutant lines affected in TMT activity accumulated - instead of -tocopherol in leaves (Bergmuller et al., 2003). A comparative analysis of mutant lines and wild type under oxidative stress conditions showed that the shift in tocopherol composition had no impact on the stress tolerance (Bergmuller et al., 2003).

Conclusions Elucidation of the tocopherol and tocotrienol biosynthetic pathway and its regulation in plants has made considerable progress in recent years due to the availability of nucleotide sequence information of model organisms such as Arabidopsis and Synechocystis. For all the steps involved in tocopherol biosynthesis, genes have now been cloned and used to generate transgenic lines with a modified spectrum of vitamin E compounds in different tissues, e.g. in seeds or leaves. The analysis of transgenic lines indicated the feasibility of obtaining novel plant lines with an enhanced level and a modified spectrum of tocopherols. The transgenic lines obtained will also provide valuable information on the roles of tocopherols and tocotrienols in different plant tissues, e.g. in stress defence or a putative function in signalling events. Future research is required to understand better the regulation of vitamin E biosynthesis, such as the control of biosynthesis through the action of transcription factors in response to environmental or endogenous factors. The identification of regulatory factors in vitamin E biosynthesis and a deeper understanding of the cross-talk with related pathways will enable new targeted approaches in biotechnology and plant breeding to modify the vitamin E spectrum in plants.

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(2003) Characterization of gamma-tocopherol methyltransferases from Capsicum annuum L and Arabidopsis thaliana. European Journal of Biochemistry, 270, 84–92. Leipner, J., Fracheboud, Y. and Stamp, P. (1999) Effect of growing season on the photosynthetic apparatus and leaf antioxidative defenses in two maize genotypes of different chilling tolerance. Environmental and Experimental Botany 42, 129–139. Lichtenthaler, H.K. (1968) Distribution and relative concentrations of lipophilic plastid quinones in green plants. Planta 81, 140–152. Lichtenthaler, H.K. (1979) Occurrence and function of prenyl lipids in the photosynthetic membrane. In: Appelqvist, L.A., Liljenberg, C. (eds), Advances in the Biochemistry and Physiology of Plant Lipids. Elsevier/North Holland Biomedical Press, Amsterdam, pp. 57–78. Lichtenthaler, H.K. (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular Biology 50, 47–65. Munne-Bosch, S. and Alegre, L. (2000) Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants. Planta 210, 925–931. Munne-Bosch, S. and Alegre, L. (2002) The function of tocopherols and tocotrienols in plants. Critical Reviews in Plant Sciences, 21, 31–57. Norris, S.R., Barrette, T.R. and DellaPenna, D. (1995) Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. Plant Cell 7, 2139–2149. Norris, S.R., Shen, X.H. and DellaPenna, D. (1998) Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. Plant Physiology 117, 1317–1323. Peisker, C., Duggelin, T., Rentsch, D. and Matile, P. (1989) Phytol and the breakdown of chlorophyll in senescent leaves. Journal of Plant Physiology 135, 428–432. 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10 Efficiency of Extracting Vitamin E from Plant Sources Gianni Sacchetti1 and Renato Bruni2 1Dipartimento

delle Risorse Naturali e Culturali, Laboratorio di Biologia Farmaceutica e Biotrasformazioni (BFB Lab), Università degli Studi di Ferrara, Corso Ercole l d’Este 32, I-44100 Ferrara, Italy; 2Dipartimento di Biologia Evolutiva e Funzionale, Sezione Biologia Vegetale e Orto Botanico, Università degli Studi di Parma, Parco Area delle Scienze 11A, I-43100 Parma, Italy

Abbreviations: BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; HPLC high-pressure liquid chromatography; SFE, supercritical fluid extraction; TBHQ, tert-butylhydroquinone; GRAS, generally recognized as safe.

Abstract Alimentary and pharmaceutical dietary research is increasingly focusing on nutraceuticals. In fact, from cosmetics to functional foods, from the herbal market to the food preservation industries, a wide spectrum of applications involves the use of antioxidants of natural origin. A sector where a noticeable amount of data have already been generated is linked to the optimization of extractive processes from antioxidant-rich plant sources. Among them, vitamin E plays a pivotal role, both for its efficacy and for its relatively common presence in many natural sources. Tocopherols and tocotrienols (vitamin E isomers) are well-known natural lipophilic antioxidants, and besides their known activity as free radical scavengers they are also deemed to be able to counteract the onset of pathologies related to fat and cholesterol consumption in the diet. During the last decades, a huge number of matrices and extraction methods have been optimized to set up adequate, faster, cheaper and healthier techniques for vitamin E isolation. With the aim of providing an overview of the most common methods available for both analytical and preparative purposes, the most significant and recent bibliography was summarized, devoting particular attention to methods with better perspectives for the health and herbal market, such as supercritical fluid extraction. Original data regarding the development and optimization of a supercritical fluid extraction process on renewable resources from agroindustrial byproducts are provided.

Introduction Since the discovery of the fat-soluble vitamin E by Evans and Bishop (1922), a plethora of research projects on several aspects concerning tocols have been performed, ranging from their characterization, to analytical protocols and biological relapses. The main reason for their success lies in the fact that they act as natural antioxidants in a very efficient manner and can, thus, play an important role as functional additives in different productive areas. Furthermore, tocopherols and tocotrienols may also exert direct beneficial effects against the onset of various pathologies in different areas and tissues, ranging from atherosclerosis and hypercholesterolaemia to cataract

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formation and the prevention of diabetic complications (Bruni et al., 2002). The market for healthy and functional products – i.e. cosmetics, alimentary and dietetic products, and food preservatives, constantly addresses its attention to new and underutilized plant sources, to check their antioxidant properties and to evaluate their industrial use as a natural ingredient (Ohlsson and Bengtsson, 2002). Such attention is strictly related to the restriction in use of antioxidant synthetic additives such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertbutylhydroquinone (TBHQ), widely used as antioxidants in cosmetics, food lipids and health products. In fact, as a result of a major concern about possible toxic and geno-

© CAB International 2007. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

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Efficiency of Extracting Vitamin E from Plant Sources

toxic side effects, the Food and Drug Administration (FDA) in the USA and the European Community Commissions are planning the removal of BHA from the GRAS (generally recognized as safe) list, and the safety of BHT is under careful scrutiny. Furthermore, TBHQ has not been approved for food use in Europe, Japan and Canada. Thus, natural antioxidants such as vitamin E have gained popularity in recent years and their use and positive image among consumers is spreading. For these reasons, nowadays many studies focus on optimizing extraction, and analyses have been performed to evaluate the possibility of exploiting new, underutilized plants or renewable resources from agro-industrial waste as industrial sources of vitamin E. For the optimization of extraction of vitamin E from plant sources, sample treatment is a critical step, both for extraction efficiency and for the subsequent analytical processes. In fact, manipulation is time consuming, expensive and the main source of errors and, therefore, it should be kept at a minimum. Prior to extraction, it is of crucial importance to consider the nature of plant samples (leaves, fruits, stems, woody parts and seeds) and their water content. It must also be taken into account that, as a consequence of their biological role, tocols are unstable chemicals prone to oxidation and degradation. Thus, exposure to excessive light and heat could be negative factors, affecting the extraction yield and the quality of the final product. To prevent losses derived from these steps, it is advisable to collect plant samples whilst trying to maintain their integrity and to process the samples to dryness promptly. Generally, the drying process does not represent a crucial point in the perspective of a good vitamin E recovery, especially if the plant source already has a low water content (i.e. seeds). As a rule of thumb, freeze drying is the preferred method due to its efficiency and rapidity, even if it represents an expensive process for industrial-scale preparation. Subsequently, after dryness, another crucial step is the grinding. It is important, at this stage, to prevent oxidation phenomena due to the high temperature reached with mechanical friction, that could severely affect the abundance of vitamin E. The shape and, above all, the size of sample particles may be critical to extraction efficiency. Once the dried and ground matrix has been obtained, many extraction methods are available from the literature, according to the plant source, the methodological approach, the solvent used and the final purpose of the extract (analytical or preparative). In some cases, a saponification is performed with or without addition of antioxidants such as pyrogallol, BHT, ascorbic acid or EDTA (ethylenediaminetetraacetic acid); extractions are performed with organic solvents achieved by means of vigorous shaking, sonication or recourse to a Soxhlet apparatus to improve the penetration of the extractant into the plant tissues and thus maximize matrix–solvent interactions. However, sonication, if too intense, could represent a possible source of free radicals and spoil the abundance of vitamin E (Rupérez et al., 2001). Because of the usually low amount of tocopherols in plant samples, vitamin E isomers must be concentrated after extraction in many cases. Moreover, if adequate extracting conditions are

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not adopted, vitamin E recoveries will be poor as a consequence of its availability. In fact, even if tocols are not chemically bound to proteins, lipids or carbohydrates, they could be associated with other matrix components and must be isolated in the ensuing sample preparation steps. To achieve the goal of a sharp separation between tocols and undesired lipid chemicals, saponification of the entire matrix or of the sole raw extract is often performed. However, such steps may cause vitamin degradation due to the hard reaction conditions. Saponification, a severe, timeconsuming and tedious process, especially if performed prior to extraction, is classically conducted by heating the sample with potassium hydroxide in ethanol or methanol solution for a period of time (e.g. 24 h). This step involves the unsaponifiable components – including vitamin E – being extracted into the organic solvent, while other saponifiable components (fatty acids salts, glycerols and other minority components which may interfere in the analytical process) remain in the alkaline aqueous phase. However, from the saponification on, many other factors could interfere with the extraction of tocols and, among these, the kind of organic solvent, solvent concentration and the lipid/solvent concentration ratio are included (Bunnell, 1971; Mordret and Laurent, 1978; Rupérez et al., 2001). Various organic solvents have been used for vitamin E extraction, and those more frequently used are acetone, nhexane, diethyl ether, chloroform and methanol. Due to the non-polar nature of tocols and of compounds with which they are associated in plant sources, n-hexane alone or more frequently mixed with minor amounts of polar solvents such as ethanol, methanol, isopropanol, ethyl acetate or diisopropyl ether is the most employed strategy for vitamin E extraction, with good recoveries (Rupérez et al., 2001). On the other hand, supercritical fluid extraction (SFE) represents an interesting alternative to organic solvent extraction. The advantages of supercritical fluids in vitamin E extraction from plant sources are mainly related to the lower time consumption, the reduction of heat and oxygen exposure, the minimal consumption of organic solvents and often a potentially higher selectivity and increased yield due to the availability of automated instruments (Lesellier, 2001; Turner et al., 2001). On the other hand, the operating costs and the technical skill needed for a productive and competitive industrial extraction plant are higher.

The Extraction of Vitamin E: Focusing on Methods The extraction of vitamin E from plant sources is traditionally performed by means of organic solvents under a number of temperature, pressure and plant matrix– solvent interaction conditions, and numerous past and recent research efforts have described this (Tables 10.1 and 10.2). Industrial processing focused on production of edible oils rich in vitamin E is also mainly based on traditional extraction methods, even if the application of supercritical fluids in pilot and processing plants is steadily increasing.

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The data and the comments made here refer to the most significant and recent research on vitamin E extraction from plants and agro-industrial waste material. We consider, as a good way to provide an overview about the problems relating to extraction efficacy, to explain the data acquired in terms of those regarding traditional extraction strategies – consolidated on a productive industrial scale – and those about supercritical fluids – an extraction strategy which presents many difficulties in terms of extraction optimization but generally offers higher yields. With the aim of providing all the positive and negative aspects concerning both extraction strategies, we consider particularly useful to provide annotated tables (Tables 10.1 and 10.2) as a synopsis of the present status of the research in vitamin E extraction.

Solvent extraction using traditional strategies As previously stressed, the extraction of vitamin E from the majority of plant sources is traditionally performed by means of organic solvents (Table 10.1). In relation to the different sources, particular attention is devoted to sample handling as a key factor to obtain good extraction efficiency. In fact, plant sources such as fruits, leaves and tubers are frequently dried through different methods, and all of them can be easily scaled-up to productive levels. The drying methods can be either very simple and inexpensive but time-consuming, or requiring more expensive laboratory equipment, but being generally faster, i.e. in the open air, or employing traditional ovens (temperature not over 40°C) or microwave ovens (not over 900 W) (Frega et al., 1984; Zlatanov, 1999; GòmezCoronado and Barbas, 2003). However, it must be pointed out that some other authors reported solvent extractions without prior drying mainly because – as experimentally proved – the aqueous phase was not significant or because the vitamin E fraction was easily extractable with an appropriate organic solvent (Burns et al., 2003). In fact, in the case of seeds and nuts, the drying process is often not necessary because of the naturally very low water content of the plant matrices (Cocallemen et al., 1988; Sensidoni et al., 1996; Delgado-Zamarreño et al., 2001; Yoshida et al., 2002). Another critical factor for vitamin E yield prior to solvent extraction is the grinding of the dried sample and the particle size, directly related to the optimization of plant matrix–solvent interaction. As generally expected for traditional solvent extraction, the smallest particle size is a determinant to obtain the best yield of tocols, independently of the different solvents employed (GòmezCoronado and Barbas, 2003). In some cases, the use of amber-coloured glass laboratory equipment, or of synthetic antioxidants such as BHA or BHT is suggested to prevent vitamin E oxidation during extraction and this seems to be a good strategy to obtain better tocol yields from different plant sources in a laboratory-scale extraction (Gimeno et al., 2000; Yoshida et al., 2002). However, the use of synthetic antioxidants is inadvisable in a productive-scale

extraction mainly because of unfavourable health problems due to their possible genotoxic side effects. In some cases, especially in the presence of oleaginous sources (seeds and nuts) and as a pre-analytical step, saponification is suggested in order to detect the tocols with better resolution. However, for an industrial scale-up, solvent extraction processing without prior hydrolysis provided better yields (Delgado-Zamarreño et al., 2001). Extraction using organic solvents includes mostly traditional strategies (e.g. maceration or employing a Soxhlet apparatus) with n-hexane as the preferred extractant (Frega et al., 1984; Cocallemen et al., 1988; Sensidoni et al., 1996; Lechner et al., 1999; Zlatanov, 1999). n-Hexane is also reported to be the most efficient extractant with commercial vegetable oils checked for their quality and vitamin E isomer content (Lechner et al., 1999; Gimeno et al., 2000). In some situations, with the object of finding a more efficient extractant, other different solvents were tested for their extracting capacity for vitamin E isomers; solvents such as acetone, tetrahydrofuran, 2-propanol, sodium dodecyl sulphate and sometimes acetone gave better results than the traditionally employed n-hexane (Gòmez-Coronado and Barbas, 2003). In some other cases, it could be necessary to process the plant extracts through a second extraction with a more polar solvent (i.e. methanol or ethanol); this operation provides the opportunity to have pre-concentrated samples and, above all, to obtain the best conditions for the analytical steps (Bruni et al., 2002). An interesting method, but with poor perspectives of industrial scale-up notwithstanding its good vitamin E recovery when applied to vegetable oils, seems to be molecular distillation, set up for different sources at the best temperature and pressure conditions (Shimada et al., 2000). At present, the high costs which this technique may involve for industrial processing limit its use at laboratory or pilot levels of application. Furthermore, an important development in organic solvent extraction methodologies is the use of a silicone non-porous membrane for continuous and discontinuous extraction (Sànchez-Pérez et al., 2000; Hadolin et al., 2001). As Sànchez-Pérez et al. (2000) reported, these methods reduce the time required for analysis and provide more reproducible results. Even if such an approach is slower than other traditional methods, it seems to present advantages outweighing the disadvantages of faster techniques. In fact, it couples high tocol recovery and rapidity of obtaining analytical data because generally the extraction cells are directly coupled to the analytical process. For these reasons, the method is particularly suggested to be applied to routine analysis in the food industry for vitamin E quality control. Moreover, it must be underlined that Hadolin et al. (2001) suggested a mathematical model which couples theoretical operating parameters with extraction efficacy, showing a good correspondence with experimental data acquired for Sylibum marianum. This represents a good approach to the research, mainly because it allows a reduction in working time and costs.

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Table 10.1. Solvent extractions with traditional strategies. Plant material

Extraction methods

Analytical methods

Comments

References

Various fruits and vegetables

10 mg of samples were placed in a methanol– Tris–HCl (50 mM, pH 7.5) solution after being vortexed, and then left on ice for 10 min. CHCl3 was added and the organic phase was separated by centrifugation at 3000 r.p.m. and removed. The aqueous phase was then re-extracted with CHCl3 and the organic phase dried under nitrogen.

RP-HPLC; Gradient mode: methanol (A), 20% aqueous methanol containing 0.2% ammonium acetate (B), tert-butyl-methyl-ether (C); Detection: diode array set to monitor between 200 and 600 nm

- and -tocopherols were detected and quantified; -tocopherol was quantified as -tocopherol equivalents and detected at 290 nm.

Burns et al. (2003)

Aubergine (Solanum Soxhlet extraction (16 h) with n-hexane as extracting melongena) seeds solvent.

The classical extraction method followed by HPLC determination pointed to a total tocopherols content of about 325 mg/kg seeds. -Tocopherol accounted for 3.1%, -tocopherol for 1.8% and -tocopherol for 95.1%.

Cocallemen et al. (1988)

The detected quantity of total tocopherols, and of -tocopherol in particular, suggests an important preservability of the oil and of the mixtures made with it. Various seeds and nuts

Solvent extraction: 1. Alkaline hydrolysis (saponification) with a potassium hydroxide–ascorbic acid solution, coupled with hexane extraction (discontinuous method). The extracts were washed with water. The organic phase was removed by evaporation and the residue dissolved in methanol and filtered. 2. The samples were mixed with Triton X-114 in the presence of methanol and acetonitrile. Water was added and the mixture was stirred for 30 min; the solutions were centrifuged and filtered prior to injection (continuous method). A scheme showing the extraction cell with donor and acceptor (acetonitrile) solution was given.

RP-HPLC; isocratic mode: acetic acid–sodium acetate in methanol–water (97:3 v/v) Detection: coulimetric

The discontinuous method always leads to determination in all the seeds and nuts analysed (raw, fried and roasted material) of -tocopherol (range detected 0.61–22.7 mg/100 g); - and -isomers were quantified together (range detected 0.71–12.9 mg/100 g) while -tocopherol was rarely checked (0.14–1.0 mg/100 g).

Delgado-Zamarreño et al. (2001)

The continuous method presented different problems related to the optimization of different variables (donor and acceptor phase, extraction time, amount of sample, Triton X-114 concentration). The quantification was also determined chromatographically with or without previous hydrolysis.

Efficiency of Extracting Vitamin E from Plant Sources

HPLC (the authors do not report specific chromatographic conditions but refer to a reference reported therein)

The best -tocopherol yield was detected for red and yellow pepper (0.6 µg/g dry weight) while the highest quantity of -tocopherol was reached in lettuce (0.3 µg/g dry weight).

With hydrolysis: -tocopherol was always detected (range detected 0.76–25.8 mg/100 g); - and -isomers were quantified together (range detected 0.84–20.2 mg/100 g) while -tocopherol was checked in three samples (0.41–3.74 mg/100 g). Without hydrolysis: -tocopherol was always detected (range detected 0.79–26.9 mg/100 g); - and -isomers were quantified together (range detected 1.11–20.7 mg/100 g) while -tocopherol was checked in four samples (0.45–3.01 mg/100 g).

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Continued

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Table 10.1. Solvent extractions with traditional strategies – Continued Plant material

Extraction methods

Analytical methods

Comments

References

The suggested better extraction was that which was continuous without prior hydrolysis both for the tocopherol yield and for extraction time (40 min). The tubers were air dried at 40°C and then ground in a water-cooled mill (operating temperature 20 times higher than in the other plant material examined. Interestingly, with respect to other plant material employed commercially for vitamin E isomer extraction, pepper seeds contained -tocopherol in amounts similar to rapeseed and palm oil. Analogous conclusions could be made for tomato seeds + pulp extracts with respect to wheat-germ oil, even if - and tocopherol are much more abundant in the latter (Beliz and Grosch, 1999). Seeds of apricot and peach, in both the varieties examined, did not provide results sufficiently interesting to investigate in terms of a large-scale application.

Conclusions Vitamin E extraction from many and varied plant materials, both in the recent past and nowadays is a productive field related to the research, development and production of healthy products. The optimization of the extraction methods became critical in recent years, mainly because of the need to reduce the utilization of noxious solvents without an extraction yield decrease. Therefore, much attention has been devoted to find strategies for the better efficacy of solvent extraction with traditional methods. Above all, they have been developed with the aim of optimizing supercritical solvent strategies as the new frontier of vitamin E extraction from both plant material and agro-industrial waste material. Original data reported here support the importance of by-products as a natural source of vitamin E isomers.

Table 10.5. Total extraction yield (%) and qualitative and quantitative (mg/kg) tocopherol characterization of supercritical CO2 extracts obtained from ground seeds + pulp and peel samples of Solanum lycopersicon (tomato).

Seeds + pulp SFE200 SFE400 Peels SFE200 SFE400

Extraction yield









Total tocopherols

0.39 ± 0.05 0.95 ± 0.13

81.2 ± 10.7 189.7 ± 24.9

— 125.4 ± 16.5

— —

— —

81.2 ± 10.7 315.1 ± 41.5

0.32 ± 0.04 0.67 ± 0.09

41.50 ± 5.5 87.72 ± 11.5

— —

— —

— —

41.50 ± 5.5 87.72 ± 11.5

The results are the average of three determinations ± SD. SFE200, supercritical fluid extraction at 200 atm; SFE400, supercritical fluid extraction at 400 atm. Table 10.6. Total extraction yield (%) and qualitative and quantitative (mg/kg) tocopherol characterization of supercritical CO2 extracts obtained from ground seeds of Capsicum annuum (pepper).

SFE200 SFE400

Extraction yield









Total tocopherols

7.36 ± 0.97 15.12 ± 1.99

— —

— —

73.4 ± 22.8 358.37 ± 47.1

— —

173.7 ± 22.8 358.3 ± 47.1

The results are the average of three determinations ± SD. SFE200, supercritical fluid extraction at 200 atm; SFE400, supercritical fluid extraction at 400 atm.

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G. Sacchetti and R. Bruni

In fact, many papers regarding vitamin E extraction with supercritical fluids were published in the last few years, and all of them show extraction efficacies always equal to or greater than those achievable with traditional solvent extraction employing n-hexane, chloroform or chloroform/methanol. Therefore, the main advantages of the supercritical extractions are evident: an equal or even higher extraction efficacy than traditional strategies, the reduction of organic solvent use and, as a consequence, the total reduction of their disposal. Meaningful in this regard is the fact that all these researches report industrial scaleup of the laboratory instrumentation set up. However, the main hurdle related to the start-up of industrial extraction is the specific set up of the instrumentation, which is often exclusive for a given plant material.

In light of the bibliographic and experimental evidence provided, it can be argued that the trend of the research and development in vitamin E extraction strategies from different plant material is towards increasing supercritical fluid application at industrial levels, minimizing the related problems (operating costs) and optimizing the extraction plant, achieving even more efficacy and competitiveness than traditional methods.

Acknowledgements Thanks are due to the MURST (Ministero dell’Università e della Ricerca Scientifica e Tecnologica) and CNR (Consiglio Nazionale delle Ricerche) of Italy.

References Beliz, H. and Grosch, W. (1999) Food Chemistry, 2nd edn. Springer-Verlag, Berlin, Germany. Blanco Muñoz, M.A., Molero Gòmez, A. and Martônez de la Ossa, E. (1999) Optimizaciòn del proceso de extracciòn de tocoferol de germen de trigo con diòxido de carbonio liquido y supercrôtico. Grasas y Aceites 50, 275–279. Bruni, R., Guerrini, A., Scalia, S., Romagnoli, C. and Sacchetti, G. (2002) Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction. Phytochemical Analysis 13, 257–261. Bunnell, R.H. (1971) Modern procedures for the analysis of tocopherols. Lipids 6, 245–253. Burns, J., Fraser, P.D. and Bramley, P.M. (2003) Identification and quantification of carotenoids, tocopherols and chlorophylls in commonly consumed fruits and vegetables. Phytochemistry 62, 939–947. Carlucci, G., Mazzeo, P., Del Governatore, S., Di Giacomo, G. and Del Re, G. (2001) Liquid chromatographic method for the analysis of tocopherols in malt sprouts with supercritical fluid extraction. Journal of Chromatography A 935, 87–91. Cocallemen, S., Farines, M., Faill, H., Soulier, J. and Morin, O. (1988) Etude de l’huile de graines d’aubergine Solanum melongena (L.) Solanaceae. Revue Francaise des Corps Gras 35, 105–110. Colombo, M.L., Corsini, A., Mossa, A., Sala, L. and Stanca, M. (1998) Supercritical carbon dioxide extraction, fluorimetric and electrochemical high performance liquid chromatographic detection of vitamin E from Hordeum vulgare L. Phytochemical Analysis 9, 192–195. Delgado-Zamarreño, M.M., Bustamante-Rangel, M., Sànchez-Pérez, A. and Hernàndez-Méndez, J. (2001) Analysis of vitamin E isomers in seeds and nuts with and without coupled hydrolysis by liquid chromatography and coulometric detection. Journal of Chromatography A 935, 77–86. De Lucas, A., Martinez de la Ossa, E., Rincòn, J., Blanco, M.A. and Gracia, I. (2002) Supercritical fluid extraction of tocopherol concentrates from olive tree leaves. Journal of Supercritical Fluids 22, 221–228. Evans, H.M. and Bishop, K.S. (1922). On the existence of a hitherto unrecognized dietary factor essential for reproduction. Science 56, 650–651. Frega, N., Conte, I.S., Lercker, G. and Carnacini, A. (1984) Composizione dei tubercoli di Cyperus esculentus. Rivista della Società Italiana di Scienza dell’Alimentazione 13, 211–214. Ge, Y., Ni, Y., Yan, H., Chen, Y. and Cai, T. (2002a) Optimization of the supercritical fluid extraction of natural vitamin E from wheat germ using response surface methodology. Journal of Food Science 67, 239–243. Ge, Y., Yan, H., Hui, B., Ni, Y., Wang, S. and Cai, T. (2002b) Extraction of natural vitamin E from wheat germ by supercritical carbon dioxide. Journal of Agricultural and Food Chemistry 50, 685–689. Gimeno, E., Castellote, A.I., Lamuela-Raventòs, R.M., de la Torre, M.C. and Lòpez-Sabater, M.C. (2000) Rapid determination of vitamin E in vegetable oils by reversed-phase high-performance liquid chromatography. Journal of Chromatography A, 881, 251–254. Gnayfeed, M.H., Daood, H.G., Illés, V. and Biacs, P.A. (2001) Supercritical CO2 and subcritical propane extraction of pungent paprika and quantification of carotenoids tocopherols and capsacinoids. Journal of Agricultural and Food Chemistry 49, 2761–2766. Gòmez-Coronado, D.J.M. and Barbas, C. (2003) Optimized and validated HPLC method for - and - tocopherol measurement in Laurus nobilis leaves. New data on tocopherol content. Journal of Agricultural and Food Chemistry 51, 5196–5201. Hadolin, M., Škerget, M., Knez, Ž. and Bauman, D. (2001) High pressure extraction of vitamin E-rich oil from Silybum marianum. Food Chemistry 74, 355–364. King, J.W., Favati, F. and Taylor, S.L. (1996) Production of tocopherol concentrates by supercritical fluid extraction and chromatography. Separation Science and Technology 31, 1843–1857. Lechner, M., Reiter, B. and Lorbeer, E. (1999) Determination of tocopherols and sterols in vegetable oils by solid-phase extraction and subsequent capillary gas chromatographic analysis. Journal of Chromatography A 857, 231–238. Lesellier, E. (2001) Analysis of non-saponifiable lipids by super-/subcritical-fluid chromatography. Journal of Chromatography A 936, 201–214. Mendes, M.F., Pessoa, F.L.P. and Uller, A.M.C. (2002) An economic evaluation based on an experimental study of the vitamin E concentration present in deodorizer distillate of soybean oil using supercritical CO2. Journal of Supercritical Fluids 23, 257–265. Mordret, F. and Laurent, A.M. (1978) Application de la chromatographie en phase gazeuse sur colonne capillaire de verre à l’analyse des tocophérols. Revue Francaise des Corps Gras 25, 245–250.

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Ohlsson, T. and Bengtsson, N. (2002) Minimal Processing Technologies in the Food Industry. Woodhead Publishing, Cambridge, UK. Perretti, G., Miniati, E., Montanari, L. and Fantozzi, P. (2003) Improving the value of rice by-products by SFE. Journal of Supercritical Fluids 26, 63–71. Rozzi, N.L., Singh, R.K., Vierling, R.A. and Watkins, B.A. (2002) Supercritical fluid extraction of lycopene from tomato processing byproducts. Journal of Agricultural and Food Chemistry 50, 2638–2643. Rupérez, F.J., Martôn, D., Herrera, E. and Barbas, C. (2001) Chromatographic analysis of -tocopherol and related compounds in various matrices. Journal of Chromatography A 935, 45–69. Sànchez-Pérez, A., Delgado-Zamarreño, M.M., Bustamante-Rangel, M. and Hernàndez-Méndez, J. (2000) Automated analysis of vitamin E isomers in vegetable oils by continuous membrane extraction and liquid chromatography–electrochemical detection. Journal of Chromatography A 881, 229–241. Sensidoni, A., Bortolussi, G., Orlando, C. and Fantozzi, P. (1996) Olio di Borragine, importante fonte di acido -linolenico. Noa 2: contenuto di tocoferoli, clorofilla ed analisi sensoriale di oli di borragine ottenuti con diverse tecniche estrattive e miscelati con olio extra vergine di oliva. Industrie Alimentari 35, 664–669. Shimada, Y., Nakai, S., Suenaga, M., Sugihara, A., Kitano, M. and Tominaga, Y. (2000) Facile purification of tocopherols from soybean oil deodorizer distillate in high yield using lipase. Journal of the American Oil Chemists Society 77, 1009–1013. Turner, C., King, J.W. and Mathiasson L. (2001) Supercritical fluid extraction and chromatography for fat-soluble vitamin analysis. Journal of Chromatography A 936, 215–237. Yoshida, H., Hirakawa, Y., Abe, S. and Mizushina, Y. (2002) The content of tocopherols and oxidative quality of oils prepared from sunflower (Helianthus annuus L.) seeds roasted in a microwave oven. European Journal of Lipid Science and Technology 104, 116–122. Zlatanov, M.D. (1999) Lipid composition of Bulgarian chokeberry, black currant and rose hip seed oils. Journal of the Science of Food and Agriculture 79, 1620–1624.

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11 Commercial Extraction of Vitamin E from Food Sources Siew-Young Quek1, Boon-Seang Chu2 and Badlishah Sham Baharin2 1Food

Science, Department of Chemistry, The University of Auckland, Private Bag 92019, Auckland, New Zealand; 2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor DE, Malaysia

Abbreviations: FAD, fatty acid distillate; FFA, free fatty acid; SCF, supercritical fluid.

Abstract Tocopherols and tocotrienols are valuable compounds because of their activity as vitamin E and their capacity as an antioxidizing agent. The most important commercial source of vitamin E is the fatty acid distillate (FAD) of the vegetable oils obtained during the deodorization of the oils. Vitamin E is co-distilled with the free fatty acids (FFAs) and concentrated in a fatty sludge. Although the vitamin is obtained in concentrated form, it is mixed with various extraneous materials in the FAD and needs to be purified. There are many commercial methods used for extraction of vitamin E from food sources, particularly from the FAD, as discussed in this chapter.

Introduction Vitamin E is a generic name describing the bioactivities of both tocopherol and tocotrienol derivatives. Vitamin E activity is widely found in plant food groups, including seeds, grains, fruits and vegetables, where the vitamin occurs mainly in unesterified form. Seeds and grains are the sources of high potency oils and, hence, important plant sources of vitamin E. Other plant sources of vitamin E include palm leaves, lettuce, lucerne and rubber latex. The distribution of vitamin E in plant foods varies according to the species, variety, stage of maturity, season, time and manner of harvesting, processing and storage time. As more evidence of the potential benefits associated with the use of vitamin E accumulates, demand for it increases, as does the demand for purer forms thereof. Until the early 1940s, the main commercial sources of vitamin E were the vegetable oils. However, even soybean oil and wheat-germ oil, which are considered to be the best sources of vitamin E, contain only minute amounts of vitamin E. Kenneth C. D. Hickman is probably the first person who extracted vitamin E from a much cheaper and richer source, the fatty acid distillate (FAD) of vegetable oils, in 1940 (Hickman, 1944). The vitamin E levels in FAD are many times greater than that of the original crude oils

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from which they are derived. Today, almost all manufacturers concentrate vitamin E from the FAD, even though other potential commercial sources have been identified. For example, vitamin E is extracted from the leaves and stalks of some plants of the families Gramineae and Palmae (Shibue and Tamura, 1984). Kato et al. (1983) patented a method to extract vitamin E from the leaves of palm, banana, pineapple, sugarcane, rice, wheat, barley, maize and rye using organic solvents, while Lane et al. (1999) patented a method to extract the vitamin from rice bran. High purity vitamin E concentrate is also prepared from a green algae using liquid chromatography (Kajiwara, 1982). FAD is a high-melting, yellowish solid fatty substance discharged from deodorization in vegetable oil refining. It is also called deodorizer sludge, deodorizer distillate, hotwell scum, lighter-than-water scum, clabber stock, condenser oil, deodorizer trap oil and catch basin scum. Deodorization is essentially a steam-stripping process whereby the extraneous material in crude vegetable oils that affect their flavour, odour, colour and stability are evaporated off at 200–275°C under partial vacuum (80%) with only small amounts of unsaponifiables (5–10%). As compared with chemical refining, FFAs are neutralized by a caustic solution and washed off before deodorization. FAD from a chemical refining obviously has a lower FFA content (30–50%) and a higher level of unsaponifiables (25–33%) (Verleyen et al., 2001). A reason for low vitamin E content in FAD of palm oil is because the oil is physically refined. Meanwhile, the operating conditions of the deodorization process also have a great effect on vitamin E levels in FAD. The deodorization temperature, typically ranging between 240 and 275°C, is the most important variable that determines the vitamin E recovery in FAD (Walsh et al., 1998). For optimal vitamin E recovery, temperatures should be above 260°C.

Commercial Methods for Extraction of Vitamin E A brief overview The vitamin E concentrate isolated varies depending on the sources and extraction techniques applied. The ease of recovery is dependent on the similarity of the properties of the impurities to those of vitamin E (Willging and Swanson, 1986). The extraction processes include esterification, saponification, liquid–liquid extraction, crystallization, distillation, ion-exchange and adsorption chromatography. They are normally used in combination to yield vitamin Erich fractions of higher purity. Some of the processes require drastic exposure to heat, oxidizing agents and mineral acids/alkalis, resulting in some degradation of the vitamin. While some of these processes have been commercialized, none has been completely satisfactory for a number of reasons, as listed in Table 11.1. Table 11.2 shows some of the patented methods for extraction of vitamin E. Removal of the fatty components

from the starting materials – FFAs for FAD and triacylglycerols for vegetable oils, is the first step in concentrating the vitamin. Such an attempt is a good approach as the FFAs and triacylglycerols are the major components in the starting materials, and removal of them will result in a several-fold increase of vitamin E. Removal of the fatty components is commonly achieved through esterification and saponification. Their respective followon step is usually distillation of the alkyl esters and removal of the fatty acid salts. Liquid–liquid extraction and crystallization are also used to remove the fatty components, but to a lesser extent. The fraction obtained after the removal of fatty components contains, normally, the non-saponifiable matter such as the tocopherols, sterols, squalene, hydrocarbons and waxes. The fatty component residues are also commonly found, together with some solvent and salt residues. The subsequent extraction steps focus on the separation of vitamin E from the non-saponifiable matter. Chromatographic methods, crystallization and molecular distillation are commonly used for this purpose. When a single homologue of vitamin E is needed, the extraction process will usually end with a chromatographic separation of the homologues. Figure 11.1 shows the summary of the flow diagram of the vitamin E extraction methods. For a non-FAD or non-oil-based vitamin E source, such as plant leaves, stalks or seeds, extraction usually begins with grinding the material into small pieces, followed by drying in some cases, to facilitate the subsequent solvent extraction. Various solvents, such as alcohol, ether, ketone, hydrocarbons, halogenated hydrocarbons, carboxylic acid esters, or their mixtures are used. Physical extraction such as mechanical force is seldom applied unless the oil content in the plant is high. The oily fraction obtained may or may not be subjected to saponification and other refining processes as mentioned above, to remove the fatty components, prior to chromatographic extraction of the vitamin. The industrial application of an extraction method largely depends on the economics of the operation, type of vitamin E source and the purity desired in the end-product. There is a great interest, of course, in the search for a direct extraction application that results in high vitamin E concentration. While this is difficult, if not impossible, to achieve with the current separation technology, combinations of various methods are continuously used to obtained high purity products. In any case, the use of the extraction methods has to be well justified in terms of the production cost and the vitamin recovery.

Esterification Esterification is one of the most common methods used for extraction of vitamin E (Top et al., 1993; Ong et al., 1994; Barnicki et al., 1997; Hunt and Schwarzer, 1997; Hunt et al., 2000; He et al., 2003). It normally appears as the first step of a series of steps for a vitamin E extraction process from FAD. The FFAs in FAD are esterified with a lower alkyl alcohol to form alkyl esters, with the presence of an acid as the catalyst. In the esterification, alkyl alcohol is

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Table 11.1. Comparison of some vitamin E extraction methods. Extraction method

Advantages

Disadvantages

Esterification

An effective method for the removal of fatty components

Loss of tocopherols due to esterification of tocopherols Alkaline catalyst (for transesterification) degrades tocopherols and increases boiling point during distillation May need high temperature and reduced pressure

Saponification

An effective method for the removal of fatty components

Loss of tocopherols due to the use of alkali In cases where saponification is followed by solvent extraction, the formation of lumpy soap mass makes the extraction difficult May need powdering agent or chemicals for metathesis

Distillation

Quite efficient Multi-stage distillation can be employed to obtain product with high purity High product recovery is possible

Operates at high temperature and reduced pressure; high cost Need chelating agent to minimize the loss of tocopherols Nearly impossible to obtain high purity fractions in a single pass

Chromatographic methods

High product purity and recovery is possible High selectivity, possible for separation of the homologues

Extraneous materials may bind to the stationary phase irreversibly The life of the adsorbent is generally short and it needs frequent replacement Non-uniform elution profile may occur; low reproducibility Automation is difficult Sterols in the feeding material may cause the column to clog due to crystallization Solvent intensive Relatively low product throughput

Liquid–liquid extraction

Can be carried out in a single contact extraction, simple multi-stage contact extraction, countercurrent extraction, etc.

Very solvent intensive Low recovery

Crystallization

Suitable for separation of sterols from tocopherols Repeated crystallization can be carried out to maximize the purity

Solvent intensive Low recovery

Enzymatic methods

Milder reaction temperature at atmospheric pressure Lipases can be used repeatedly

Require longer reaction time compared with chemical methods Lipases may be costly

Supercritical fluid extraction High product purity and recovery High selectivity

continuously introduced into the reaction vessel and the water produced from the reaction continuously removed to prevent back-reactions such as hydrolysis. The reaction vessel is pressurized to maintain the liquid reaction phase at the reaction temperature ranging from 65 to 130°C. Transesterification is applied if the starting material is a vegetable oil. In this case, an alkaline catalyst such as potassium hydroxide, sodium hydroxide, sodium methoxide or zinc oxide is used (Top et al., 1993; Ong et al., 1994; Hunt and Schwarzer, 1997). When the reaction is completed, the catalyst is removed and the reactant mixture cooled, water washed, nitrogen purged and dried. The alkyl esters in the esterified FAD are then removed by distillation (Top et al., 1993; Barnicki et al., 1997; Hunt and Schwarzer, 1997; Hunt et al., 2000), adsorption chromatography (Ong et al., 1994), liquid–liquid extraction (Brown and Smith, 1964; Tan and Saleh, 1992) or other means. The FFAs can also be esterified by the existing sterols in FAD (4–20% depending on the type of FAD) at 150–250°C as described in a patent granted to Fizet (1996). The

High operating cost

remaining unesterified FFAs are distilled off at 120–150°C. This is followed by another distillation at 200–220°C to distil off the tocopherols into a concentrate of 15–40%. Some vitamin E loss occurs since the tocopherols can also form esters with the FFA although with a much slower reaction rate than that of the sterols. It is noted that the and -tocopherols tend to participate more in the esterification than -tocopherol, which leads to a correspondingly differential loss of vitamin E. The addition of a strong acid accelerates esterification. However, according to Fizet (1996), it is unnecessary to use any catalyst for esterification because the FAD is already weakly acidic (pH 5–6) due to its FFA content. However, a high reaction temperature (150–250°C) and a long reaction time (up to 12 h) are needed. Esterification can also be carried out to esterify the tocopherols as described by Willging (1986). Tocopherols are esterified with boric acid or a boric acid source such as alkoxy boroxine, alkoxy borate, phenoxyboroxine or phenoxyborate to form a borate tocopherol ester. The

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Table 11.2. Some commercial methods for extraction of vitamin E. Starting material

Vitamin E concentrationa

Extraction step

Concentrate

% Vitamin E recovery

Reference

Initial Ion-exchange chromatography → evaporation (removal of mobile phase)

14.3–27.5%

68–87%

89–97.3%

Kijima (1964)

FAD of soybean oil

Saponification → metathesis → solvent extraction → crystallization → evaporation

12.3%

51.0%

85.6–88.3%

Eastman Kodak Co. (1965)

FAD of soybean oil

Chemical esterification → liquid–liquid extraction → phase separation → evaporation (removal of solvent) )

9.8–10.9%

21.0–32.2%

69.0–79.5%

Brown and Smith (1964)

Pre-concentrated vitamin E source

Liquid–liquid extraction (with an alkali) → neutralization → phase separation → evaporation (removal of solvent)

58.4–60.6%

59.6–89.25%

NI

Willging (1985)

Pre-concentrated vitamin E source

Esterification (borate tocopherol ester) → distillation (removal of impurities) → releasing the tocopherols → washing (removal of boron) → distillation

72.0%

92.0–96.0%

90%

Willging (1986)

Unspecified FAD

Urea complex formation → cooling/precipitation → filtration → evaporation (removal of solvent)

6.0–13.0%

24.0–55.0%

75–97%

Sampathkumar (1986)

Fresh oil palm leaves (Palmeae sp.)

Wet grinding → solid–liquid extraction → filtration → ion-exchange chromatography → evaporation (removal of mobile phase)

Trace

70.0%

NI

Shibue and Tamura (1986)

FAD of palm oil

Crystallization → filtration → evaporation (removal of solvent) → adsorption chromatography → evaporation (removal of mobile phase)

0.7–0.75%

9.6–16.0%

65–88%

Goh et al. (1992)

Various FADs

Saponification → evaporation (removal of solvent) → crystallization → filtration → evaporation (removal of solvent)

1.1–9.0%

10.6–31.0%

86.0–95%

Fizet (1993)

FAD of palm oil

Esterification → distillation → crystallization → filtration → ion-exchange chromatography → evaporation (removal of mobile phase) → molecular distillation → deodorization

0.4%

95.2–97.9%

70.0–75.0%

Top et al. (1993)

FAD of sunflower oil

Enzymatic hydrolysis → enzymatic esterification → distillation

4.8%

30.1%

70.2%

Ghosh and Bhattacharyya (1996)

Various FADs

Esterification → two-stage distillation

10.2–23.5%

17.1–34.6%

55.2–96%

Fizet (1996)

Pre-concentrated vitamin E source

Esterification (orthoborate ester of tocopherol) → distillation → solvolyzation/hydrolysis → distillation

55–56%

92.7%

NI

McCurry et al. (2002)

Crude palm oil

Esterification → multi-stage distillation → saponification → crystallization → filtration → liquid–liquid extraction → phase separation → evaporation (removal of solvent)

0.1%

12.5–79.3%

34.9%

Lik et al. (2004)

FAD of soybean oil

Esterification → multi-stage distillation → liquid–liquid extraction → phase separation → evaporation (removal of solvent)

~12%b

43.7–62.6%

NI

Sumner (2004)

Commercial Extraction of Vitamin E from Food Sources

Pre-concentrated vitamin E source

a

Range of concentration of vitamin E in the concentrate varies depending on the conditions of extraction applied. is not provided, but typical vitamin E in FAD of soybean oil is about 12%. FAD, fatty acid distillate; NI, no information provided. b Information

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Vegetable oils

Starting material: FAD

Esterification

Transesterification

Saponification

Saponification

Crystallization or

Crystallization or

Liquid–liquid extraction

Liquid–liquid extraction

Chromatographic methods

Molecular distillation

Crystallization

Liquid–liquid extraction

Supercritical fluid extraction, etc.

Removal of the unsaponifiable matter

Separation method

Removal of fatty components

Removal of acylglycerols

Removal of FFAs

Vitamin E concentrate Fig. 11.1. Summary of flow diagram of vitamin E extraction methods.

reactant is then distilled off to leave the borate tocopherol esters in the residue. The tocopherols are then released from the esters by reacting with methanol or ethanol in water to form a boron compound and the tocopherols. The esterification was carried out preferably at 160–200°C for about 2 h under a vacuum of 270–400 Pa. Orthoborate esters will be produced if the mole ratio of the total tocopherols, sterols and alcohol to boron in the boroncontaining compound is 2.5:1 and above, and with the presence of an alcohol having 10–30 carbons at a temperature of 110–130°C and pressure under 130–670 Pa (McCurry et al., 2002). The by-products such as water, alcohol and phenol formed during the esterification are removed to prevent back-reactions. Tocopherols are recovered by solvolyzation or hydrolysis of the esters. Figure 11.2 summarizes the esterification methods used for extraction of tocopherol from its sources.

Extracting vitamin E by esterification has its disadvantages. Although adding a strong acid as the catalyst greatly accelerates esterification of FFAs, some undesired esterification of tocopherols is also promoted (Fizet, 1996). Tocopherols, which are phenolic compounds, can react with methoxide ions to form tocopherol esters and reduce the recovery of vitamin E. The use of alkaline catalysts for transesterification will lower the yield of vitamin E somewhat as the vitamin is unstable under alkaline conditions. The alkali catalyst largely present in the form of salts must be removed, otherwise it can be problematic during distillation; for example, it can lead to an increase of the boiling point. Esterification may also require multiple reactor systems and processing steps. Other drawbacks include unsatisfactory product purity and low yield and throughput (Hunt and Schwarzer, 1997).

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Esterification

Target substrate

Acylglycerols (transesterification)

Free fatty acids

Reactant

Lower alkyl alcohol

Lower alkyl alcohol

Sterols

A boric acid source

Catalyst

Alkali

Acid

Free fatty acids

Not required

Operating conditions

150–240°C for 1–3 h under reduced pressure

65–130°C for 5% moisture, the powdering agent will not work properly due to preferential wetting of the powdering agent by water. Introduction of the powdering agent to the soap mass is accomplished by admixture until the powdering agent is uniformly dispersed throughout the mass to a concentration of 6–10% (Eastman Kodak Co., 1965). Rindone and Huang (1994) improved the saponification method by adding a zinc halide, preferably the chloride, after saponification, for metathesis of the mixture. The fatty acids precipitate in fine particulate form as zinc salts, leaving tocopherols in the alcoholic solution. The zinc salts occur in granular form with uniform particle size compared with their calcium counterparts. This allows them to be

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filtered off easily. This method results in a higher vitamin E recovery without the need for mechanical granulation or the addition of powering agents. Figure 11.3 summarizes the subsequent extraction steps commonly used after saponification.

Distillation Distillation is normally the follow-on step after esterification to remove the lower alkyl esters, leaving a vitamin E-rich fraction. There are various distillers, such as short path evaporator (Fizet, 1996), high heat-transfer rate falling film vacuum distiller (Top et al., 1993), molecular distiller (Top et al., 1993) and wiped film evaporator with packed column (Willging, 1986; Hunt and Schwarzer, 1997; Hunt et al., 2000). Distillation should be accomplished without unacceptable degradation of the vitamin E. Therefore, it is preferable to treat the feeding material with a chelating agent such as ascorbic acid, phosphoric acid, malic acid, citric acid or tartaric acid before drying and distillation. It is noted that the temperature and vacuum used usually differ according to the type of distillation. For example, alkyl esters are distilled in a high vacuum of 1.3 kPa and at a temperature between 100 and 200°C using a falling film vacuum distiller (Top et al., 1993), while a wiped film distiller normally operates at 260°C and a reduced pressure of 0.3–1.2 kPa (Hunt and Schwarzer,

1997). The distilled alkyl esters are collected by condensation and discharged as by-product. The retention time of tocopherols in the distillation column must be short so that its deterioration is minimal. Since the tocopherols and the extraneous materials have different distillation points, multi-stage distillation is sometimes carried out. For example, Fizet (1996) distilled off the remaining FFAs at 120–150°C, after the esterification of the FFAs with sterols. The sterol esters were then removed in another distillation process at temperatures of 200–220°C. Lik et al. (2004) subjected the esterified oil to three stages of short path distillation at different operating conditions. The first distillation served the purpose of distilling the alkyl esters at 70–120°C and pressure 1.3–6.7 kPa; the second was to remove the impurities such as hydrocarbons, waxes and glycolipids at 130–200°C, . Generally, reacting the mixed tocopheryl acetates with cyclic amines, such as pyrolidine, in a suitable solvent at room temperature leads to rapid deacylation of -tocopheryl acetate (~15 min). The acetates of - and

-tocopherols react more slowly (~2 h) and -tocopheryl acetate does not react. These rate differences are sufficient to allow selective deacylation of -, - and -acetates in the presence of -acetate. Since the polarity of the acetate is significantly different from that of the free tocopherol, this allows an efficient chromatographic separation of the homologues (Fig. 11.4). The cyclic amines, which are fairly expensive and require recovery and ultimate disposal, can be replaced with methanol, ethanol or propanol. However, the reaction requires higher reaction temperatures, preferably 190–210°C, in a closed pressure vessel due to the low boiling point of alcohols (Foster, 1986a). Foster (1986b) then improved the method by using a basic catalyst such as potassium carbonate, potassium hydroxide or sodium hydroxide. This enables the deacylation to be carried out at temperatures 20% of the vitamin E in the US diet (in Eitenmiller and Landen, 1999). Up until 1980, the RDA for vitamin E was expressed in

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Shikimate pathway

Non-mevalonate pathway

CH CH2

NH2 COOH OPP

OPP HO

DMPP

Tyrosine

IPP

Hydroxyphenylpyruvate GGPP HO OH HOOC Homogentisic acid

PPO Phytyl-PP

HPT HO OH CH3 2-Methyl-6-phytylhydroquinone MT HO H 3C

Cyclase

OH CH3 2,3-Dimethyl-5-phytylhdroquinone Cyclase

HO H 3C

HO O CH3

O CH3

-Tocopherol

-Tocopherol

-TMT

-TMT

CH3

CH3

HO H3C

HO O CH3

O -Tocopherol

CH3

-Tocopherol

Fig. 12.2. Vitamin E biosynthetic pathway according to Hofius and Sonnewald (2003). DMPP, dimethylallyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; phytyl-PP, phytyl pyrophosphate; HPT, homogentisate phytyltransferase; IPP, isopentenyl pyrophosphate; MT, 2-methyl-6phytylhydroquinone methyltransferase; -TMT, -tocopherol methyltransferase.

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Table 12.3. Vitamin E content of selected foods (based on -tocopherol content) taken from Bauernfeind (1980). Vitamin Ea

Food (100 g portion)

Wheat-germ oil Sunflower oil Safflower oil Groundnut oil Margarine, soft Soybean oil Butter Wheat-germ, stabilized Oatmeal, rolled, cereal Sunflower seeds, raw Almonds Groundnut, dry roasted Peanut butter Cashews a1

mg

IU

119 49 40 19 14 8.1 2.2 11 1.3 50 27 7.4 6.2 0.2

178 73 59 28 21 12 3.2 17 2.2 74 41 11 9.2 0.3

mg (1TE) of -tocopherol is equivalent to 1.49 IU.

IU. However, in 1980, the term tocopherol equivalents (TEs) was used to express the RDA for vitamin E: 1 mg of D--tocopherol is equivalent to 1 TE. Other tocopherols and -tocotrienol in the diet were assigned the following values: 1 mg of -tocopherol = 0.5 TE; 1 mg of -tocopherol = 0.1 TE, and 1 mg of -tocotrienol = 0.3 TE.

Role of Vitamin E in Oils Tocols prevent the rancidity of oils during storage and thus prolong their shelf-life (Blekas et al., 1995). Extensive research work concerning the occurrence and levels of tocopherol has been carried out mainly for Italian olive oils and, to a lesser extent, for olive oils from other producing countries (Conte et al., 1993; Cert et al., 1996; Esti et al., 1996; Ranalli and Angerosa, 1996; Manzi et al., 1998; Salvador et al., 1998). During a period of three production years of Greek olive oils, the effect of the conditions of milling on -tocopherol content was studied and high concentrations of -tocopherol were observed in most of the samples selected from various Greek regions. Values ranging between 98 and 370 mg/kg were found (>200 mg/kg in 60% of samples) (Psomiadou et al., 2000). Stabilization of edible oils depends on the different chemicals they contain, among them tocopherols and tocotrienols. For example, the high instability of fish oils is due to their high degree of unsaturation, therefore the choice of antioxidant to stabilize fish oil for human consumption is restricted to a few substances, with tocopherols being among the most frequently used (see for example Tappel, 1962; Olcott and vanderVeen, 1968; Yi et al., 1991; Hamilton et al., 1998). Tocopherol is very beneficial in the case of fish oil. This is true of hydrogenated fish oils. The characteristic odour of fish oil is exacerbated by oxidation. Colouring problems are caused by oxidation of fats and oils. By adding tocopherol in

processed fish products, e.g. dried, salted and canned, these problems are prevented. Antioxidants such as tocopherols can be challenging to analyse, because they readily oxidize; when exposed to the presence of oxidizing agents or UV light, this vitamin undergoes degradation and isomerization and yields four major vitamers (i.e. -, -, - and -tocopherols). On the other hand, long exposure to alkaline conditions significantly decreases the level of -tocopherol (Lietz and Henry, 1997). The evaluation of tocopherol mixtures is from a practical point of view as important as the evaluation of the antioxidant potential of single tocopherols. Wagner and Elmadfa (2000) have evaluated the antioxidative effectiveness of -, -, -tocopherol separately or in a mixture by observing lipid oxidation expressed as the induction period (IP) in a methron Rancimat. The test systems were olive, rich in monounsaturated fatty acids, and highly unsaturated linseed. The stabilizing effect of added tocopherols depends on their mixture, their concentration and the systems used, and is, respectively (100 mg/100 g oil each), in olive oil, -/-tocopherol > tocopherol > -tocopherol > -/-tocopherol > -/tocopherol > -tocopherol; and in linseed oil, -tocopherol > -/-tocopherol > -tocopherol > -/-tocopherol > tocopherol > -/-tocopherol (Wagner and Elmadfa, 2000). More recently, in a multi-phase system, the degradation of -, - and -tocopherol in different concentrations indicates that the - and -tocopherols are more effective than tocopherol in the auto-oxidation of a 10% rapeseed oil–triacylglycerol-in-water emulsion with and without a free-radical initiator (Wagner et al., 2004).

Analytical Methods Due to the assessment of the impact of genetic modifications of oil seeds on their distribution, the separation and characterization of the various forms of tocopherols they contain is, therefore, suitable for routine analyses of large amounts of samples including oils, that avoid in addition saponification. A short time of analysis is also required. High-performance liquid chromatography (HPLC) methods are now preferred because of their speed, ease of operation and reliability (Van Niekerk and Macrae, 1988). However, the tocopherol content of oils can be established by a wide range of analytical techniques; in addition to thin-layer chromatography (TLC) and capillary gas chromatography (cGC), supercritical fluid chromatography (SFC) is also conducted (Gast et al., 2005). In HPLC, the most used technique is normal-phase (NP) HPLC with UV or fluorescence detection which gives higher resolution (Abidi and Mounts, 1994). Several authors have recommended, in addition to HPLC, a GC flame ionization detection (FID). These two techniques together produce the most sensitive method for analysis of tocopherols in oils and foods (Carpenter, 1979; Thompson and Hatina, 1979; Barnes and Taylor, 1980; Hakansson et al., 1987; Ball, 1988; Abidi, 2000). The GC analysis of tocopherols normally implies warm saponification, TLC separation and

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formation of the trimethylsilyl derivatives prior to the chromatographic injection. Lechner et al. (1999) isolated the silyl derivatives by solid-phase extraction and quantified them by GC-FID. Derivatization has been done by heating a purified extract with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (Lechner et al., 1999); N,O-(bistrimethylsilyl)trifluoroacetamide (BTSFA)-trimethylchlorosilane (TMCS) also gives reasonable data (Khallouki et al., 2003). HPLC separations of tocopherols using different packing material columns effectively provide a fast, simple, sensitive, selective and more robust technique than GC. As reported by Kamal-Eldin et al. (2000), the separations of tocopherols are performed on both NP and reversed-phase (RP) columns. Although RP columns are generally known to have the advantages of better stability and longer durability than NP columns, most RP columns are not able to separate the - and -isomers of tocopherols and tocotrienols (Tan and Brzuskiewicz, 1989; Andrikopoulos et al., 1991; Kramer et al., 1997). It was, however, possible to separate - and -tocopherol isomers on octadecylpolyvinyl alcohol (Abidi and Mounts, 1997). NP systems show elution of homologues in order of increasing polarity with separation based on methyl substituents on the chromanol moiety. Another advantage of NP columns is their ability to operate with organic solvents allowing a high solubility for lipids and tolerance of high loads of lipids which are easy to wash out by non-polar solvents. NP-HPLC is very suitable for the direct analysis of oils, fats and biological samples, as reported by Kamal-Eldin et al. (2000). In fact, the authors studied and interpreted the resolution provided by aminopropylsilica and diol-bonded silica with binary solvent systems comprising a hydrocarbon and an alcohol, an ether or an ester. Six silica, three amino and one diol column were tested for the separation of vitamin E compounds in a prepared mixture containing oat extracts and palm oil as matrix. The best separation was obtained on three silica columns and two amino columns using 5% dioxane in hexane as mobile phase, as well as on a diol column using 4% tert-butyl methyl ether in hexane as mobile phase (Kamal-Eldin et al., 2000). Silica columns with binary isocratic or gradient mobile phases of hexane-2-propanol have already been widely applied (Kramer et al., 1997; Psomiadou and Tsimidou, 1998; Qureshi et al., 2000). Anyway, poor reproducibility, prolonged equilibration times, low stability and the employment of hazardous volatile organic solvents have long been discussed. RP systems show separation based on the saturation of the phytyl side chain. The more saturated isomers are retained longer. C18 RP systems do not completely resolve - and -tocopherols. Nevertheless, when separation of - and -tocopherols is not a critical point, C18 RP systems are preferred, because equilibration times are shorter and better reproducibility is achieved. Moreover, RP-HPLC solvent systems preserve the environment more than those used in NP-HPLC (Abidi and Mounts, 1997). Isocratically, methanol with small amounts of water or other solvents is the most frequently employed mobile phase (Takeda et al., 1996; Albala-Hurtado et al., 1997; Leray et al., 1997; Ruperez et al., 1999; Weinmann et

al., 1999; Gimeno et al., 2000; Khallouki et al., 2003) and is enough for tocopherol quantification. If other analytes are to be measured, more complicated mobile phase gradients need to be developed (Podda et al., 1996; Steghens et al., 1997). Moreover, if an electrochemical detector is used, the mobile phase must be modified with an electrolyte, usually perchlorate salts (Podda et al., 1996; Takeda et al., 1996; Weinmann et al., 1999). In contrast, NP, unlike RP, resolves - and -tocopherols (Chase et al., 1994). Saponification prior to extraction is classically performed by heating with KOH, frequently in ethanol or methanol, and it has been shown that prolonged exposure to alkaline conditions resulted in significant losses of -tocopherol (Craft and Granado-Lorreencio, 1992). Therefore, in order to measure tocopherols, some studies have performed a direct analysis after only diluting the oil in an organic solvent which provides higher column stability, reproducibility of retention times, quicker equilibration and shorter analysis time. Direct analysis of tocopherols after diluting the oil in an organic solvent has been reported (Carpenter, 1979; Tan and Brzuskiewicz, 1989; Andrikopoulos et al., 1991; Shin and Godber, 1994; Abidi, 2000; Khallouki et al., 2003) because even the use of mild conditions such as darkness and high nitrogen can often not avoid pronounced losses of tocopherols after saponification (Ruperez et al., 1999). However, direct analysis after dilution, unlike saponification and extraction, simplifies the procedure and shortens the analysis. Moreover, many oil samples can be analysed several times without altering the chromatographic efficiency or the column efficiency, which remains high in liquid chromatographs more particularly. Recoveries can be a major problem in vitamin E analysis. Tocopherol acetate was selected as the internal standard owing to its availability and its structural similarity to the compounds assayed. Standard linearity was tested in each case using linear regressions (Gonzalez-Corbella et al., 1994; Leray et al., 1997; Sobczak et al., 1999; Weinmann et al., 1999; Gimeno et al., 2000). Hexane, alone or with small amounts of more polar solvents such as ethanol or ethyl acetate, or diisopropylether (never more than 5%) is the most frequently used extractant. Working with an internal standard partially corrects this non-effectiveness, and recoveries are not affected, but in some cases sensitivity could be augmented if working with solvents such as acetone (Torre et al., 2001). If vitamins have been extracted with hexane or heptane, and chromatography is going to be performed by RP-HPLC, the solvent must be evaporated and replaced by another solvent more similar to the mobile phase, or by the mobile phase itself. To avoid this evaporation–redissolution process, other organic solvents in which vitamins are soluble and which do not interfere with separation of analytes in HPLC have been investigated (Torre et al., 2001). Therefore, with these solvents, sample pre-treatment is attained in one step only, just by adding this solvent to the sample. Extraction has also been carried out with butanol–ethyl acetate, with direct injection of an aliquot of the upper layer (Sobczak et al., 1999). The chemical and physical properties of the analyte and internal standard must be as similar as possible for this to succeed. 5,7-

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Dimethyltocol (Kramer et al., 1997; Lechner et al., 1999) and tocol are among the other internal standards employed that fulfil these characteristics. Tocol (the demethylatedchromanol form of tocopherols) is one of the internal standards most used historically, but currently is not easily found commercially and can only be obtained as a generous gift or if synthesized in the same laboratory (Bortolotti et al., 1993). A treatise of the existing chromatographic methods for the analysis of tocol-derived lipid antioxidants in various sample matrices is given by Abidi (2000). It focuses on various techniques for the isolation, purification, chromatographic separation and detection of tocols. The relative merits of the two HPLC methods are assessed. NP and RP elution characteristics are delineated to aid in the identification of antioxidant components. The use of solid-phase extraction has proven to be an efficient technique for simplifying sample clean-up prior to HPLC analysis (Lechner et al., 1999). A method for the supercritical fluid chromatographic (SFC) determination of tocopherols in vegetable oils was also investigated using an octadecylsilane (ODS)-silica gel column with carbon dioxide as the mobile phase. The retention of tocopherols was affected by the density of the mobile phase and the addition of methanol as a modifier. The addition of low concentrations of methanol produced a satisfactory separation of tocopherol homologues, including the positional isomers - and -tocopherol. The results of the determination of tocopherols in vegetable oils by SFC agreed well with those obtained by NP-HPLC (Yarita et al., 1994). The use of supercritical fluids to perform extractions is a relatively new technology with a great potential for the future (Gast et al., 2005). This technique, called supercritical fluid extraction (SFE), is rapid and generates little or no hazardous reagent and solvent waste. SFE and SFC are ideally suited for analysis of medium to non-polar labile compounds, and several works have been published with one or both techniques. Nevertheless, nowadays SFE has progressed rapidly and many applications have continued to arise, but SFC has not kept pace, probably due to the complexity and cost of the system. Yarita et al. (1994) used a laboratory-built SFC system for the determination of tocopherols. Finally, it is interesting to mention that in the USA there is a Micronutrients Measurement Quality Assurance Program for these analytes run by the National Institute of Standards and Technology (NIST), and in the UK there is an equivalent scheme. With these programmes, the quality of tocopherol measurement

in the laboratory can be evaluated. For tocols, electrochemical detection, fluorescence, UV absorbance detection and light-scattering detection have been generally employed for determining tocopherols and related substances, with sensitivities decreasing in the mentioned order. For the determination of vitamin E, fluorescence detectors are generally used (Piironen et al., 1984; Podda et al., 1996) because of their higher sensitivity and specificity compared with UV and evaporative light-scattering detectors (Chase et al., 1994). Vitamin E compounds can also be quantified after sensitive and specific coulometric detection (Takeda et al., 1996). HPLC with electrochemical detection is the most sensitive and specific detection method for tocopherols by virtue of their low oxidative potentials. Both amperometric (Podda et al., 1996) and coulometric (Takeda et al., 1996) detection have been employed. The voltammetric behaviour of vitamin E in the presence of olive oil is studied with glassy carbon electrodes, in a hexane–ethanol medium, with various techniques. The influence of such variables as the proportion of hexane–ethanol, sulphuric acid concentration and instrumental parameters has been studied. Separate voltammetric peaks are obtained for -tocopherol and tocopherol, but the peaks for -tocopherol and tocopherol overlap. The proposed method is applied to the determination of the tocopherols in different vegetable oil samples. The olive oil samples needed a prior cleaning stage by solid-phase extraction on silica cartridges. The results are very acceptable (Galeano Diaz et al., 2004).

Conclusion Humans and animals are unable to synthesize vitamin E, and they must obtain it from plant sources. Whereas various homologues are found in plants, only -tocopherol and a lower proportion of -tocopherol are present in human and animal tissues including blood. The antioxidative capacity and ability to act as a free radical scavenger of tocols can reduce the risk of cancer and delay the progression of pre-cancerous lesions. Accordingly, control of vitamin E levels in foods is of great importance to ensure that daily ingestion will be optimal as an essential factor in human health. Whereas -tocopherol (5,7,8-trimethyltocol) is the most active form of vitamin E in vivo, -tocopherol (7,8-dimethyltocol) is the most active in vitro (Cooney et al., 1993; Christen et al., 1997).

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(1999) Determination of -tocopherol and -tocopheryl acetate in diets of experimental animals: study of stability in the diets. Journal of Chromatography A 839, 93–99. Ruperez, F.J., Martin, D., Herrerra, E. and Barbas, C. (2001) Chromatographic analysis of -tocopherol and related compounds in various matrices. Journal of Chromatography A 935, 45–69. Salvador, M.D., Aranda, F. and Fregapane, G. (1998) Chemical composition of commercial cornicabra virgin olive oil from 1995/96 and 1996/97 crops. Journal of the American Oil Chemists Society 75, 1305–1311. Shin, T. and Godber, J.S. (1994) Isolation of four tocopherols and four tocotrienols from a variety of natural sources by semi-preparative highperformance liquid chromatography. Journal of Chromatography A 678, 49–58. Shintani, D. and DellaPenna, D. (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282, 2098–2100. Sobczak, A., Skop, B. and Kula, B. 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13 Changes in Tocopherol Composition of Edible Oils after Extreme Heat Exposure (Frying) Maurizio Battino1 and José L. Quiles2 1Institute

of Biochemistry, Faculty of Medicine, Polytechnic University of Marche, Via Ranieri, 65, 60100 Ancona, Italy; 2Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, Ramón y Cajal 4, 18071 Granada, Spain

Abbreviations: ESR, electron spin resonance; PUFA, polyunsaturated fatty acid.

Abstract Frying is one of the most popular food preparation methods because it produces a desirable fried food flavour, golden brown colour and crisp texture. However, this culinary method has several disadvantages including the production, through several reactions, of cytotoxic aldehydes that may be harmful to health. These reactions may reduce the antioxidant content in the oils, decreasing its stability and producing new products which are responsible for a loss in the nutritional value and quality of the oils. Edible vegetable oils contain natural antioxidants such as -tocopherol for preventing oxidation and they are the main source of natural tocopherols for humans. Tocopherols are lost along with the oxidation of unsaturated fatty acids during frying, as has been separately reported for a wide variety of oils. Pan-frying is a more stressful procedure concerning -tocopherol losses than deep-fat frying, and discontinuous frying leads to greater decreases in -tocopherol content than the continuous process. Heating (a different procedure from frying since it is performed in the absence of food) also leads to a fall in tocopherol. When -tocopherol was added to an antioxidant-poor refined olive oil, the total antioxidant capacity of this oil was improved, more -tocopherol was retained in the fried oil and lower oil degradation was observed, reaching similar levels to those found in antioxidant-rich extra virgin olive oil.

Introduction Oils and fats constitute one of the three major classes of food nutrients besides proteins and carbohydrates (Takeoka et al., 1997). Fats and oils are heated at high temperatures during baking, grilling and pan-frying, although deep-fat frying is the most common method for the high temperature treatment of foods (Warner, 1999). Deep-fat frying is a popular food preparation method because it produces a desirable fried food flavour, golden brown colour and crisp texture. Among the advantages of that procedure, frying has little or no impact on the protein or mineral content of fried food, whereas the dietary fibre content of potatoes is increased after frying due to the formation of resistant starch. Moreover, the high tempera-

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ture and short transit time of the frying process cause less loss of heat-labile vitamins than other types of cooking. For example, the vitamin C concentration of French fried potatoes is as high as in raw potatoes, and thiamine is well retained in fried potato products as well as in fried pork and, although some unsaturated fatty acids and antioxidant vitamins are lost due to oxidation, fried foods are generally a good source of vitamin E (Fillion and Henry, 1998). On the side of disadvantages of the frying procedure, it has been reported how thermal stressing of polyunsaturated fatty acid (PUFA)-rich culinary oils according to routine frying generates high levels of cytotoxic aldehydic products, arising from the fragmentation of conjugated hydroperoxydiene precursors. Several compounds produced from the thermally induced autoxidation of PUFAs

© CAB International 2007. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

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Changes in Tocopherol Composition of Edible Oils after Extreme Heat Exposure (Frying)

are readily absorbed from the gut into the systemic circulation in vivo (Grootveld et al., 1998). Since such aldehydic products are damaging to human health, it has been indicated that the dietary ingestion of thermally, autoxidatively stressed PUFA-rich culinary oils promotes the induction, development and progression of cardiovascular diseases (Grootveld et al., 1998) and several alterations at the subcellular level (Battino et al., 2002; Quiles et al., 2002a). All chemical changes of fats and oils and their natural contaminants at elevated temperatures have their origin in oxidation, hydrolysis, polymerization, isomerization or cyclization reactions. All these complex reactions may affect different aspects of the heated and fried oils and may be promoted by oxygen, moisture, traces of metal and free radicals (Gertz, 1996). These processes may reduce the amount of antioxidants in the oils, decrease its stability and produce new products which are responsible for a loss in the nutritional value and quality of the oils (odour, flavour, absorption, etc.). Edible vegetable oils contain natural antioxidants such as vitamin E, for preventing PUFA oxidation. In fact, vegetable oils are the main source of natural tocopherols for humans (Andrikopoulos et al., 2002). Vitamin E is lost along with the oxidation of unsaturated fatty acids during heating of different types of edible oil, as has been separately reported for virgin and refined olive oils (Dobarganes et al., 1985; Ruiz-López et al., 1995; Pellegrini et al., 2001; Andrikopoulos et al., 2002; Battino et al., 2002; Brenes et al., 2002; Quiles et al., 2002b), rapeseed oil (Gordon and Kourimska, 1995), maize oil (Carlson and Tabacch, 1986), sunflower oil (Dobarganes et al., 1985; Andrikopoulos et al., 2002; Battino et al., 2002; Quiles et al., 2002b) and for a frying oil mixture of soybean and rapeseed oils (Miyagawa et al., 1991). However, there are no comprehensive reviews about the particular effects of frying on the changes in tocopherol composition of oils after extreme heat exposure (frying), which indeed represents the aim of this chapter.

Frying Procedures Frying must be differentiated from heating, since heating is performed in the absence of food. There are two main frying procedures: shallow or pan-frying, and deep-fat frying. In pan-frying, the food is not entirely immersed in the oil. This type of frying is carried out in pans or flat pots containing small amounts of oil. The immersed portion of the food is fried while the food not immersed is boiled, by the steam resulting from the heated oil (Cuesta and Sánchez-Muniz, 2001). The most widely used method, and for which most studies have been performed, is deep-fat frying. In this method, performed in domestic or industrial fryers at 180–190°C, the food placed into the hot fat is heated quickly and the cooking process takes place throughout the entire product simultaneously. Frying may differ depending on whether oil cooling is allowed between frying batches (discontinuous frying) or cooling is not allowed (continuous frying). The degradation

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level reached in discontinuous frying has been reported to be higher (Jorge et al., 1996). On the other hand, frying may also be classified as frying with or without replenishment of fresh unused oil during the procedure. In that sense, Cuesta and Sánchez-Muniz (1998) have reported very interesting data in which the frequent addition of fresh oil minimizes oil alteration.

Chemical and Physical Changes in Oil Composition During Frying There are numerous studies that report changes in fats and oils after heating or frying procedures (Gertz, 1996; Takeoka et al., 1997; Che Man and Jaswir, 2000). Most of them conclude that there are many parameters involved in these changes such as the presence or absence of food, the temperature, the heating cycles, the surface/volume and food/oil ratios, the fatty acid and the antioxidant composition of the oils (Melton et al., 1994). On the other hand, the wide number of methods developed to assess the effects of frying on oils are not always consistent, making it difficult in some cases to interpret the phenomena (BarreraArellano et al., 1997). All the above-mentioned are good reasons for saying that studies about thermal oxidation of oils are far from being completed. As has been stated above, all chemical changes of fats and oils and their natural contaminants at elevated temperatures have their origin in oxidation, hydrolysis, polymerization, isomerization or cyclization reactions (Gertz, 1996). Concerning hydrolysis, which is the reaction of water with a substance, this reaction in fried oils leads to the formation of free fatty acids, mono- and diglycerides and glycerol. Glycerol is the precursor of the mucosal tissue irritant acrolein, which causes irritation to the eyes and respiratory trouble at concentrations of 1800 Da are non-volatile products, and those with molecular weights 99% of unused frying oil, have molecular weights in the range of 900–100 Da and are not very volatile at normal frying temperatures. Besides the degradative processes in the oil, solubilization of components from food, including coloured compounds and food lipids, also contributes to the heterogeneity of the

components found in used frying oils and thus to increased rates of degradation. The above-described changes are summarized in Fig. 13.1 (adapted from Fritsch, 1981; Gertz, 1996; Cuesta and Sánchez-Muniz, 2001). Polymerization and isomerization are the predominant reactions when frying in the absence of air hydrolysis, although when frying oils and fats are heated in the presence of oxygen, oxidation is the main reaction. Oxygen bridges link dimers and trimers formed by oxidation at low temperatures, whereas carbon–carbon bonds link those that are formed at high temperatures. Especially dimeric reaction products may contribute to the increase of viscosity, flavour changes and the darkening of the fried oil (Gertz, 1996). Isomerization is not very important in frying, since apart from hydrogenation, food processing does not substantially alter the fatty acid composition. Margarines made of partially hardened sunflower oil contain 15–30% transisomers and abused frying fats often reach 15% on average (Gertz, 1996). Cyclization is mainly associated with increasing unsaturation of fatty acids and the length of exposure to high temperature. Oils with a high linoleic acid content such as sunflower oil form cyclic monomeric fatty acids Steam Volatiles Antioxidants

Absorption

Vaporization

Aeration Steam Oxygen+ metals Food Hydrolysis

Oxidation

Hydroperoxides (conjugated dienes) Solubilization Coloured compounds Food lipids Fission

Dehydration

Alcohols Aldehydes

Ketones

Acids

Hydrocarbons

Heating

Free fatty acids Diglycerides Glycerine Monoglycerides

Free radicals Dimers Trimers Epoxides Alcohols Hydrocarbons Dimers Cyclic compounds

Fig. 13.1. Main chemical reactions produced during frying (adapted from Fritsch, 1981; Gertz, 1996; Cuesta and Sánchez-Muniz, 2001).

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Changes in Tocopherol Composition of Edible Oils after Extreme Heat Exposure (Frying)

with 5-membered carbon rings, whereas those with a high level of linolenic acid such as linseed oil form higher levels of cyclic fatty acids with a 6-membered carbon ring (Sebedio et al., 1987). Typical levels of these compounds in frying fats and oils range from 0.01 to 0.66%. The chemical changes in the frying fats also result in changes in the physical characteristics. The colour of a fat/oil darkens and, with increasing content of polymers, viscosity increases, and greater foaming of the oil occurs. Some of the more volatile components, such as free fatty acids, accumulate to the point where the smoke point is decreased. The aroma and the flavour of the oil also change with increased frying time, as do the colour and flavour of the food fried in the oil (Melton et al., 1994).

Changes in Tocopherol Content in Oils After Frying It is very important to assess the oxidative degradation of fats and oils in the food industry, because free radicalinitiated oxidation is one of the main causes of rancidity of these foods. In fact, free radicals are known to be responsible for the oxidation of food components resulting in alteration of the major quality-control parameters such as colour, flavour, aroma and nutritional value of foodstuffs (Donelly and Robinson, 1995). As stated in the previous section, frying has a very high oxidative component, and edible oils have their own components to protect unsaturated fatty acids against oxidation, including those produced by frying or in general by thermal exposition, light, etc. (Beddows et al., 2000; Andrikopoulos et al., 2002). Among these protecting components, tocopherol is traditionally considered as the major antioxidant present in edible oils (Blekas et al., 1995), as edible oils are the main source of dietary vitamin E (Andrikopoulos et al., 2002). There are several papers reporting the response of

-tocopherol content in different edible oils against the thermal stress produced by heating or frying (Dobarganes et al., 1985; Ruiz-López et al., 1995; Pellegrini et al., 2001; Andrikopoulos et al., 2002; Brenes et al., 2002; Quiles et al., 2002b). Overall, it has been reported that -tocopherol decreases in oils with heating or frying time. Tables 13.1, 13.2 and 13.3 show results from different studies regarding changes in tocopherol content in oils after heating, pan-frying or deep-fat frying. Although it is quite difficult to make comparisons between studies in relation to the tocopherol losses because of differences in the experimental approaches, methodologies, the type of oil or the starting amount of tocopherol in the oils, it seems that pan-frying is the most stressful procedure concerning -tocopherol losses. In that way, Andrikopoulos et al. (2002) reported that only 35% (for virgin olive oil) and 18% (for sunflower oil) of the initial amount of -tocopherol in the oils was retained after 24 min of a discontinuous process of pan-frying (performed in sets of 8 min each). However, for a similar period of time, under deep-fat frying conditions, higher levels of -tocopherol were retained in the oils (Andrikopoulos et al., 2002; Quiles et al., 2002b). In fact, tocopherols retained in oils after deep-fat frying for around 30 min (the conditions most comparable with those of pan-frying) showed a 50% retention for virgin olive oil with discontinuous frying or a 97% retention with continuous frying; and for sunflower oil a 65% retention with discontinuous frying or an 88% retention with continuous frying. It seems that the higher oxidation in pan-frying comes from the fact that oil antioxidants and the other oil constituents are exposed to oxidation for longer since the air:food surface ratio is higher than in the deep-fat frying process (Cuesta and Sánchez-Muniz, 2001; Andrikopoulos et al., 2002). On the other hand, it has been reported previously that discontinuous frying leads to higher oil degradation than a continuous process (Jorge et al., 1996). Thus, according to

Table 13.1 Studies on -tocopherol changes following heating of different oil types. Tocopherol Initial (mg/kg)

Final (duration) (mg/kg)

% Retention

Virgin olive oil (Picual variety)

172

Virgin olive oil (Arbequina variety)

141

111 (5 h) 72 (10 h) 39 (15 h) 13 (20 h) Trace (25 h) 38 (5 h) 3 (10 h) Trace (15 h) Trace (20 h) Trace (25 h)

64.5 41.8 22.7 7.6 0 26.9 2.1 0 0 0

Reference

Treatment

Oil type

Brenes et al. (2002)

Heating at 180°C

Pellegrini et al. (2001)

Heating at 190°C

Virgin olive oil

308

284 (60 min)

92

Dobarganes et al. (1985)

Heating at 190°C

Virgin olive oil Sunflower oil Virgin olive oil Sunflower oil

297 350 297 350

86 (25 h) 152 (25 h) Trace (100 h) Trace (100 h)

29 43 0 0

Heating at 190°C

Only data by Dobarganes et al. (1985) considered the sum of all tocopherols.

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M. Battino and J.L. Quiles

Table 13.2. Studies on -tocopherol changes following pan-frying with different oil types. Tocopherol Initial (mg/kg)

Final (duration) (mg/kg)

Reference

Treatment

Oil type

Ruiz-López et al. (1995)

Pan-frying

Virgin olive oil

221

83 (8 min)

Andrikopoulos et al. (2002)

Pan-frying (discontinuous) at 170°C

Virgin olive oil

278

Vegetable shorteninga

535

Sunflower oil

435

222 (6 min) 144 (12 min) 103 (18 min) 97 (24 min) 428 (6 min) 332 (12 min) 273 (18 min) 241 (24 min) 317 (6 min) 165 (12 min) 87 (18 min) 78 (24 min)

aVegetable

% Retention 37.6 80 52 37 35 80 62 51 45 73 38 20 18

shortening: a mixture of sunflower oil, cottonseed oil and palm oil.

Table 13.3. Studies on -tocopherol changes following deep-fat frying with different oil types. Tocopherol Initial (mg/kg)

Reference

Treatment

Oil type

Andrikopoulos et al. (2002)

Deep-fat frying (discontinuous) at 170°C

Virgin olive oil

278

Vegetable shorteninga

535

Sunflower oil

435

Virgin olive oil

398

Sunflower oil

1460

Refined olive oil

218

Quiles et al. (2002b)

aVegetable

Deep-fat frying (continuous) at 180°C

Final (duration) % Retention (mg/kg) 250 (10 min) 166 (20 min) 139 (30 min) 111 (40 min) 444 (10 min) 428 (20 min) 283 (30 min) 267 (40 min) 391 (10 min) 369 (20 min) 183 (30 min) 265 (40 min)

90 60 50 40 83 80 53 50 90 85 65 61

402 (15 min) 389 (30 min) 378 (45 min) 327 (60 min) 1284 (15 min) 1294 (30 min) 1278 (45 min) 1239 (60 min) 212 (15 min) 196 (30 min) 153 (45 min) 140 (60 min)

101 97 94 82 87 88 87 84 97 89 70 64

shortening: a mixture of sunflower oil, cottonseed oil and palm oil.

data from studies shown in Table 13.3, this also accounts for -tocopherol degradation after frying. It is considered that the oil heating increases the speed of autoxidation (Nawar, 1984). However, in relation to the effect of heating oils on tocopherol content (Table 13.1), the disparity of conditions found in the literature, mainly those related to the oil type (and the initial amount of -tocopherols in these oils) and the time applied, makes conclusions as well as comparisons with frying almost impossible.

Effects of the Addition of ␣-Tocopherol on the Composition, Stability and Total Antioxidant Capacity of Refined Olive Oil at Different Deep-fat Frying Periods As stated above, frying influences the concentration of tocopherols in oils, and supplementation with antioxidants is a reasonable strategy to improve the stability and the

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Changes in Tocopherol Composition of Edible Oils after Extreme Heat Exposure (Frying)

nutritional value of processed food (Diplock et al., 1998). Consequently, we recently performed a study to investigate if the supplementation of refined olive oil with different amounts of -tocopherol (100 or 200 mg/kg), which effectively is the only supplementation allowed under current legislation, affects the response of this oil in a short-time deep-fat frying model in terms of oil degradation, tocopherol changes and total antioxidant capacity (measured by electron spin resonance (ESR)) of the oils. We describe and discuss below some of the most relevant unpublished results found in this study. The experiment was performed comparing extra virgin olive oil, the most pure and antioxidant-rich olive oil, versus ‘olive oil’. Briefly, extra virgin olive oil, which is obtained from the first pressing of the ripe fruit, has a high content of unsaponifiable matter rich in tocopherols, and phenolic derivatives, i.e. tirosol and hydroxytyrosol, which exhibit antioxidant properties (Kiritsakis and Markakis, 1987; Gunstone et al., 1994; Visioli and Galli, 1998). This oil conserves all lipidic and antioxidant qualities of the olives (Kiritsakis and Markakis, 1987). However, refined olive oil loses most of those antioxidants during refining procedures, although it has the same fatty acid composition as extra virgin olive oil (Petroni et al., 1995; Cinquanta et al., 1997). Virgin olive oil is the product obtained from ripened olives exclusively by physical procedures including cleaning of fruits with water, milling, cold pressing and centrifugation. The product from unfermented olives gives a low free acidity oil, usually lower than 1%, named and classified according to the European Union standards as ‘extra virgin olive oil’ (EU Directives 2568/91 and 656/95). A product with acidity higher than 1% but lower than 2% is named ‘fine virgin olive oil’ and one with acidity higher than 2% but lower than 3.3% is named ‘ordinary virgin olive oil’. Virgin olive oils contain relatively high amounts of unsaponifiable materials, mainly phenolic compounds (i.e. tirosol derivatives), free sterols and their precursors (i.e. squalene), and tocopherols (i.e. -tocopherol) and other compounds responsible for flavour (Huertas et al., 1991; Di Giovacchino et al., 1994; Ranalli et al., 1999). Low quality and high acidity virgin olive oils are usually refined by physical and chemical procedures (as in sunflower and other seed oils). Basically, they are neutralized with sodium hydroxide and the fatty acid soaps formed are eliminated by cleansing with water and centrifugation. In addition, they are passed through diatomeae or charcoal filters and extracted with hexane at low temperature and under a vacuum. The resulting oils are mostly colourless and aroma free; their fatty acid compositions are close to those of virgin olive oils but they lack the majority of unsaponifiable components, particularly phenolic compounds and tocopherols. Thus, refined olive oils have a lower stability than virgin olive oils. Laws do not allow the commercialization of refined olive oil as it is. Thus, the category ‘olive oil’ is a mixture of the refined oil with minor amounts of virgin olive oils, which results in a typical yellow to green colour and a flavour close to that of the virgin oils. Nevertheless, for the purpose of this chapter, we will refer below to ‘olive oil’ as refined olive oil for a better understanding.

167

For our experiment, domestic deep-fat fryers comprising a 3.5 l aluminium vessel (260  192  348 mm capacity) were used for each frying oil. For every experimental oil, i.e. virgin olive oil, refined olive oil, refined olive oil plus 100 mg/kg of -tocopherol, or refined olive oil plus 200 mg/kg of -tocopherol, the fryers were filled with 3 l of the same oil and heated to 180°C. Then, 300 g of potato slices (cut in regular pieces of ~40–50 mm  10 mm  10 mm) were added to each deep-fat fryer. After 30 min, fryer number one was switched off and its oil was cooled to room temperature (30 min fried oil). Thirty minutes later (60 min from the beginning of the frying period), fryer number two was switched off and the oil allowed to cool to room temperature (60 min fried oil). At the end of the experimental process, there were three different types of each oil according to the frying process: non-fried oil (0 min fried oil), 30 min fried oil and 60 min fried oil. Potato batches were added and removed every 7.5 min while fryers were working. The proportion of food to frying oil in the repeated frying was kept at 500 g/3 l. This procedure was carried out to avoid differences related to changes in the oil volume, to the exposure time of the potatoes to the oil, and to changes in the surface/volume ratio. All the oil samples were filtered and stored in a freezer at –20°C before analysis. Frying procedures were repeated six times for each of the five experimental oils. Concentrations of oils in -tocopherol, total phenolics, as well as total antioxidant capacity measured by ESR and total polar materials were measured as previously described (Quiles et al., 2002b). No differences in fatty acid content were found for any of the studied oils (data not shown), which is in agreement with the proposed model of a short-time deep-fat frying model (Quiles et al., 2002b). Concerning -tocopherol contents (Fig. 13.2), for the non-fried oils virgin olive oil contained 403.9 ± 9.2 mg/kg and the non-supplemented refined olive oil reached 212.9 ± 8.1 mg/kg. Supplementation with 100 and 200 mg/kg of pure -tocopherol led to increased values of this molecule in the respective oils (326.5 ± 18.4 mg/kg for the refined oil plus 100 mg of -tocopherol and 407.9 ± 15.8 mg/kg for the refined oil plus 200 mg of -tocopherol). These results are very interesting since on the one hand, external supplementation was properly carried out and, on the other hand, we were able to obtain refined olive oil with the same amount of -tocopherol as the virgin olive oil used for the experiment. The values of vitamin E reported in our study for non-supplemented oils are higher than those found by other authors (Dobarganes et al., 1985; Chase et al., 1994; Barrera-Arellano et al., 1997; Severini et al., 1997; Manzi et al., 1998). However, Cinquata et al. (1997) have reported values of 320 mg vitamin E/kg, and Baldioli et al. (1996) values close to 300 mg vitamin E/kg in virgin olive oil. Our group has also previously described similar levels to those reported here (Quiles et al., 2002b). The high variability in the amount of vitamin E in vegetable oils has been widely reported and depends on several factors such as genetic, agronomic, environmental, extraction procedure and others (Cimato, 1990; Mousa et al., 1996). Frying significantly decreased -tocopherol levels in all the oils after 60 min of frying and only in supplemented groups also after 30 min. These results are in

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a 400

a

VO RO RO+100mg/kg vitE RO+200mg/kg vitE

-Tocopherol (mg/kg)

a 350

b b*

300 250

a†‡

c* c d

200

b†‡ c†‡

150 d†‡

100 0

30

60

Frying time (min)

a 500 Total phenolic compounds (mg/kg)

450

a*

450

VO RO RO+100mg/kg vitE RO+200mg/kg vitE

400 a †‡ 350 b 50

b b b*

40

b* b*

b †‡ b †‡ b †‡

30 0

30

60

Frying time (min)

Fig. 13.2. -Tocopherol content in virgin olive oil (VO), refined olive oil (RO), refined olive oil plus 100 mg/kg of -tocopherol (RO+100mg/kg vitE) and refined olive oil plus 200 mg/kg of -tocopherol (RO+200mg/kg vitE) before (time = 0) and after 30 and 60 min of frying. Statistical differences (P < 0.05) with respect to frying time for each oil: * = 0 versus 30 min; † = 0 versus 60 min; ‡ = 30 versus 60 min. Statistical differences between different oils for each frying time are marked by letters; different letters mean significant differences (P < 0.05).

Fig. 13.3. Total content of phenolic compounds in virgin olive oil (VO), refined olive oil (RO), refined olive oil plus 100 mg/kg of -tocopherol (RO+100mg/kg vitE) and refined olive oil plus 200 mg/kg of -tocopherol (RO+200mg/kg vitE) before (time = 0) and after 30 and 60 min of frying. Statistical differences (P < 0.05) with respect to frying time for each oil: * = 0 versus 30 min; † = 0 versus 60 min; ‡ = 30 versus 60 min. Statistical differences between different oils for each frying time are marked by letters; different letters mean significant differences (P < 0.05).

agreement with other studies (Barrera-Arellano et al., 1997) and suggest that vitamin E is used to protect the oils against thermal damage. The importance of tocopherol as an antioxidant will be discussed below. Phenolic compounds are part of the so-called ‘minor constituents’ of olive oil. These compounds have a great importance for several characteristics of the olive oil, such as flavour, shelf-life and resistance against oxidation (Vázquez Roncero et al., 1973; Gutfinger, 1981; Tsimidou et al., 1996). However, the levels of phenolic compounds differ widely among varieties, locations, maturity and other factors (Amiot et al., 1986; Cinquanta et al., 1997). Figure 13.3 shows the total concentration of phenolics (expressed as mg of caffeic acid per kg of oil) in the experimental oils. The highest amount was found in non-fried virgin olive oil (500.1 ± 11.9 mg/kg), which is in agreement with previously reported data (Gutfinger, 1981; Cinquanta et al., 1997). Despite the refining process of virgin olive oil which eliminates almost all the phenolic compounds of olive oil (Akasbi et al., 1993), we found phenolics in the olive oil group (49.7 ± 1.6 mg/kg for non-fried refined olive oil). No differences were found among the three groups of refined olive oil concerning total content of phenolics. The frying procedure reduced the content of phenolics probably as the result of thermal destruction of these molecules or because of their use in the protection of the oils against oxidative insult. A hint of the susceptibility of phenols comes from studies where authors reported that these compounds are lost in the oil merely by the effect of time, as a consequence of hydrolytic or oxidative processes during storage (Cortesi et al., 1995; Cinquanta et al., 1997). Nevertheless, at the

end of the experimental time (i.e. 60 min frying time), differences between virgin and refined olive oil were maintained. Many studies have been carried out to elucidate the oxidative stability of edible oils as a function of their chemical composition. In general, it is assumed that high levels of PUFAs together with low concentrations of antioxidants are good factors for an increased susceptibility of oils to oxidation. Many methods have been used to evaluate the oxidative stability of oils and to compare the antioxidant activity of several compounds (Baldioli et al., 1996). We have previously described the use of ESR to study the total antioxidant activity of edible oils and their stability after frying procedures (Quiles et al., 2002b). It is a very sensitive technique that allows detection at the submicromolar level and can be used on turbid or highly coloured solutions (Gardener et al., 1997). Although frying decreased the antioxidant capacity in all the studied oils, we found important differences in these parameters between oils. Thus, the oil with the maximal percentage of total antioxidant capacity (Fig. 13.4), before frying, was virgin olive oil (53.6 ± 2.1%) and the lowest antioxidant capacity was found for refined olive oil (14.1 ± 0.8%). There was a proportional increase in total antioxidant capacity for refined olive oils (20.8 ± 1.4% for refined olive oil plus 100 mg of -tocopherol and 27.7 ± 1.8% for refined olive oil plus 200 mg of -tocopherol). It is obvious from these data and from those of -tocopherol and total phenolic compounds that both vitamin E and phenolics provide the total antioxidant capacity to the oils. Additionally, it is very important that despite the fact that

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a

50

a*

a †‡

40

30

b b c c

20 d

6.0

VO RO RO+100mg/kg vitE RO+200mg/kg vitE

d

b†‡

a †‡

5.0 4.5

b †‡

4.0

c †‡

3.5

10

VO RO RO+100mg/kg vitE RO+200mg/kg vitE

5.5 % total polar material

ESR total antioxidant potential (%)

60

a

c

b

a a a

d†

a

b b

c

30

60

3.0 0

30

60

Frying time (min)

0

Frying time (min)

Fig. 13.4. ESR total antioxidant capacity in virgin olive oil (VO), refined olive oil (RO), refined olive oil plus 100 mg/kg of -tocopherol (RO+100mg/kg vitE) and refined olive oil plus 200 mg/kg of -tocopherol (RO+200mg/kg vitE) before (time = 0) and after 30 and 60 min of frying. Statistical differences (P < 0.05) with respect to frying time for each oil: * = 0 versus 30 min; † = 0 versus 60 min; ‡ = 30 versus 60 min. Statistical differences between different oils for each frying time are marked by letters; different letters mean significant differences (P < 0.05).

Fig. 13.5. Total polar components in virgin olive oil (VO), refined olive oil (RO), refined olive oil plus 100 mg/kg of -tocopherol (RO+100mg/kg vitE) and refined olive oil plus 200 mg/kg of -tocopherol (RO+200mg/kg vitE) before (time = 0) and after 30 and 60 min of frying. Statistical differences (P < 0.05) with respect to frying time for each oil: * = 0 versus 30 min; † = 0 versus 60 min; ‡ = 30 versus 60 min. Statistical differences between different oils for each frying time are marked by letters; different letters mean significant differences (P < 0.05).

fatty acids were not modified by frying, this process decreased the antioxidant capacity of the oils due to a loss of vitamin E levels and phenolic compounds. It is interesting to distinguish between oxidative degradation after the frying process and other changes in the oils related to physico-chemical properties. Total polar components (Fig. 13.5) are a representative measurement of the total alteration of the oil. In fried oils, they are the breakdown products from the frying process (Melton et al., 1994). For this reason, they are generally considered as a good index of the alterations caused by this process. In the present study, total polar components were similar at the start of the study, and we found increased proportions in all the experimental groups after 60 min frying. The highest level was found for refined olive oil and it is noteworthy that this enhancement was not detected in refined oils supplemented with -tocopherol. The reason for this is the differences in the unsaponifiable fraction between both types of oils, as described above. The effectiveness of antioxidants in delaying oil degradation has been reported in many papers (e.g. Gordon and Kourimska, 1995). BarreraArellano et al. (1997) found a different production of total polar components between two different sunflower oils with a different composition of tocopherol, as we found in our study between refined olive and virgin olive oils.

because of differences in the experimental conditions. However, according to existing evidence, -tocopherol decreases in oils after heating or frying. Pan-frying is a more stressful procedure concerning -tocopherol losses than deep-fat frying, and discontinuous frying leads to greater decreases in -tocopherol content than the continuous process. Heating (a different procedure from frying since it is performed in the absence of food) also provides us with evidence about the decrease in tocopherol after high-temperature exposure of edible oils. When -tocopherol was added to refined olive oil in order to investigate the role of this antioxidant in the stability and antioxidant capacity of the oil during frying in comparison with extra virgin olive oil, we found that the frying procedure decreased the antioxidant capacity in all tested oils and increased chemical changes in the oils. These changes were greater in refined olive than in virgin olive oil, which was correlated with the -tocopherol content of both edible oils. That was strongly supported by the fact that supplementation of refined olive oil with tocopherol led to a higher protection and a lower degradation of these oils.

Conclusions It is quite difficult to make comparisons between studies in relation to tocopherol losses after extreme heat exposure

Acknowledgements J.L.Q. is supported by a ‘Ramón y Cajal’ contract from the Spanish Ministry of Science and Technology and the University of Granada. The helpful assistance of Ms M. Glebocki in editing the manuscript is gratefully acknowledged.

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14 Analysis of Vitamin E by HPLC Betty Jane Burri Western Human Nutrition Research Center, USDA, ARS, PWA and University of California, 475 West Health Sciences Drive, Davis, CA 95616, USA

Abbreviations: HPLC, high-performance liquid chromatography; ODS, octadecylsilane; UV, ultraviolet; MS, mass spectrometry.

Abstract HPLC (high-performance liquid chromatography) is the most common technique for identifying and measuring vitamin E concentrations. A variety of good HPLC methods are available for vitamin E analysis. Reliable and sensitive methods have been developed using reversed-phase and normal-phase HPLC columns, isocratic and gradient elutions, and fluorescent, electrochemical and ultraviolet detectors. Satisfactory internal standards include tocol, tocopheryl acetate and retinyl acetate. Current methods development focuses on analysing vitamin E in difficult sample matrices, analysing vitamin E metabolites and most commonly on combining vitamin E analysis with the analysis of other micronutrients such as vitamin A and the carotenoids.

Introduction

Characteristics of HPLC Separation Methods

High-performance liquid chromatography (HPLC) is the separation and measurement of compounds, based on their partition between a mobile (liquid) phase and a stationary (solid column) phase (Table 14.1). The most common method for analysing vitamin E is HPLC, which has displaced paper (Brown, 1952), thin-layer (Skinner and Pankhurst, 1964) and gas–liquid chromatography (Wilson et al., 1962). HPLC has become the workhorse method for determining vitamin E concentrations because it is a straightforward process that presents few technical difficulties to the trained scientist. That does not mean that HPLC analysis of vitamin E is easy, fast or simple – it is not. It does mean that a scientist working in a small laboratory with an old chromatograph and a low cost single wavelength ultraviolet (UV) detector can obtain excellent results. It also means that if a scientist wants to measure just the major isoforms of vitamin E (- and -tocopherol) in blood, older literature methods generally work as well as the newest ones and produce satisfactory data.

A variety of good HPLC separation methods have been developed for vitamin E. These methods can be grouped and characterized by differences in their stationary phase and their mobile phase. Normal-phase HPLC uses polar stationary phases (such as silica-based columns) and less polar solvents, so that hydrophobic organic compounds elute more quickly than hydrophilic compounds (described in Table 14.1 and compared in Table 14.2). Reversed-phase HPLC (Tables 14.1 and 14.2) typically uses silica-based columns to which hydrophobic alkyl chains (-CH2-CH2CH2-CH3) are attached. Reversed-phase columns elute hydrophilic compounds more quickly than hydrophobic ones. There are three common chain lengths, C18, C8 and C4. Reversed-phase chromatography using C18 columns (also called octadecyl or ODS columns) is the most common method used by chromatographers for any type of separation, and is usually the first column chosen for methods development. Not surprisingly, it appears to be the most common separation mode for vitamin E. Novice

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Table 14.1. Description of common HPLC phases. Mobile phase Solvents used to elute the sample, such as methanol, water or acetonitrile. The sample is dissolved and carried through the column by the mobile phase. Stationary phase Typically, the HPLC column. Solvents (the mobile phase) flow through the stationary phase. HPLC stationary phase types Size exclusion: separates on the basis of molecular size. Normal phase: uses a polar stationary phase (such as silica) and a less polar mobile phase. Hydrophobic compounds elute more quickly than hydrophilic compounds. Reversed-phase: uses a non-polar stationary phase (usually silica) to which n-alkyl organic chains are covalently bound. The most common types are C18 (an octadecyl carbon chain) and C8 (an octyl chain). Hydrophilic compounds elute more quickly than hydrophobic compounds. Ion exchange: the stationary phase contains charged groups (anions or cations) attached to a polymer matrix. The sample is retained when it replaces the counterions of the column with its own ions, and is usually eluted by changing the pH of the column. HPLC mobile phase types Isocratic: isocratic elutions are done using a constant mobile phase composition. It can be a single solvent, such as methanol, but is more commonly a mixture such as 95% methanol, 5% water. Gradient: the mobile phase composition changes during elution. Typically, the sample is injected while a weaker mobile phase is applied to the column, then the strength of the mobile phase is increased by raising the amount of organic solvent in the mix.

Table 14.2. Comparison of chromatography methods used in vitamin E analysis. Reversed-phase

Most common method C18, C8, C4 Stationary phase Organic solvents such as methanol or acetonitrile mobile phase Hydrophilic compounds elute first

Bieri et al. (1979); Catignani (1986); Burri et al. (2003);

Normal phase

Silica column Stationary phase Organic solvents as mobile phase Hydrophilic compounds elute last

Ha and Csallany (1988); Panfili et al. (1994)

chromatographers usually learn reversed-phase chromatography methods first, since these methods are so common and versatile. There is no compelling reason to use any other method for vitamin E analyses. However, there are also many fine normal-phase analysis methods for vitamin E. There is no compelling reason for a chromatographer to switch from a normal-phase method to a reversed-phase method to assay vitamin E. Another common type of chromatography – size exclusion – is never used to measure vitamin E because of its low molecular weight. Specialized columns, such as chiral columns, are occasionally used to separate vitamin E isomers for basic research.

HPLC column characteristics HPLC columns are characterized by their stationary phase, their dimensions, their particle size and their particle pore

size. Common analytical HPLC columns have internal diameters (i.d.) of 2.1 or 4.6 mm, and common lengths ranging from 5 to 25 cm. Larger i.d. columns require, and allow, greater sample volume. Standard sample volumes range from 20–30 l for the 4.6 m columns to 5 l for 2.1 mm i.d. columns. A larger i.d. also allows greater maximum and standard flow rates. Standard flow rates are 1–2 ml/min for 4.6 mm columns and 0.2 ml/min for 2.1 mm columns. Shorter columns have less resolution than longer columns of identical particle size, but also are cheaper, allow shorter run times and generate less backpressure. (High back-pressures decrease column life.) Particle sizes commonly range from 3 to 50 m. Columns with small particle sizes have higher resolution (Table 14.3) than columns of the same length with larger particle sizes, but also are generally more expensive and generate higher back-pressures (which decrease column life). Pore sizes range from 100 to 1000 Å, with 100 Å the most popular size for vitamin E analysis.

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Table 14.3. Terms used to describe chromatogram peak identification and measurement. Chromatogram: a plot of the detector signal versus time (or volume of mobile phase). Elution: the transport of a compound through the stationary phase column by the continuous flow of mobile phase. Sensitivity: minimum limit of detection of a given compound. Determined by the smallest ratio of ‘peak height-to-baseline noise’ (signal-to-noise ratio) that allows accurate and reproducible determination of peak height or area. Retention time, t: the time taken by the peak of interest to reach the detector is called the retention time. A better measure is derived by subtracting the time taken for an unretained solute to emerge from the column (i.e. the dead time, t0) from this retention time, resulting in an adjusted retention time, t’. Typically, compounds are identified from their retention times. Resolution: measures the degree of separation between two successively eluting components in a chromatographic run (two adjacent peaks in a chromatogram). The resolution between species A and B is: R = 2 [tB – tA]/ (WA + WB), where t and W are the retention time and peak width at baseline, respectively. A resolution value of 1.5 is a complete separation between the compounds.

Isocratic versus gradient chromatography Vitamin E can be separated from other fat-soluble vitamins by isocratic or gradient chromatography (Tables 14.1 and 14.4). Isocratic elution usually uses a single chromatography solvent or solvent mixture (such as 100% methanol or 95% methanol:5% water) throughout its separation, so it can be done with a simple, inexpensive one-pump system. Isocratic methods also tend to be more stable than gradient methods, since small changes in pressure and pump flow rate have lesser impact on a single solvent method. Gradient methods require two pumps to set up a solvent gradient; for example using 100% methanol with the first pump and 70% methanol:30% water with the second pump; then creating a gradient that changes the percentage of water throughout the run. These changing pump flows make gradient methods inherently more complex, more expensive and less stable than isocratic methods. However, they allow the chromatographer to speed up or slow down peak separations to optimize resolution. They are often used when vitamin E is measured concurrently with other fat- or water-soluble vitamins.

Comparison of HPLC detection methods Most HPLC methods for vitamin E use fluorescent, UV or electrochemical detectors (Table 14.5), though a few have used chemiluminescent (Yamauchi et al., 2000) or amperometric (Huang et al., 1986) methods. Typically,

electrochemical detectors are used for measuring vitamin E metabolites because of their high sensitivity, while diode array detectors are used to measure vitamin E in conjunction with other vitamins such as vitamin A, D and the carotenoids because of their versatility. Single and multi-wavelength UV detectors have generally been superseded by the more versatile (but more expensive) diode array detector, but give very satisfactory results when used. Fluorescent detection is more sensitive than UV but requires a less common instrument, and is less versatile since many compounds cannot be detected by fluorescence. Most UV detection of vitamin E is done at its peak absorption wavelength of 292 nm, but a few methods use the less specific and sensitive wavelength of 280 nm (Risner and Nelson, 1998).

Internal standards HPLC columns age, and can differ significantly with time, between manufacturers, and from lot to lot. The novice chromatographer should be warned that the most common HPLC columns used – C18 columns – vary notoriously between manufacturers and from lot to lot, even when they appear to be well matched in length, particle and pore size. They can give very different results for complex separations, and give somewhat different results for common vitamin E analysis methods. Other conditions that affect HPLC – such as ambient temperature, slight differences in chromatography solvents and pump rates, and chromatography software changes – are also difficult to

Table 14.4. Comparison of isocratic and gradient chromatography methods. Elution method

Advantages

Disadvantages

Isocratic

Simple Low cost equipment Stable Versatile Higher resolution Sometimes faster

Less versatile Lower resolution Can be slower Complex Higher cost Less stable

Gradient

References Finckh et al. (1995) Gueguen et al. (2002) Burri et al. (2003); Taibi and Nicotra (2002)

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Table 14.5. HPLC detectors used for vitamin E identification and analysis. HPLC detector A detector is chosen to emit a response when sample molecules of interest are eluted. Its signals are translated into the peaks seen on a chromatogram. There are many types of detectors. A partial list of detectors that have been used extensively to detect vitamin E are described below. HPLC detector types Ultraviolet: ultraviolet (UV) detectors measure the ability of a sample to absorb light at a specific wavelength. There are three common UV detectors. UV detectors have a sensitivity of about 10–11–10–12 g/l. Fixed wavelength, or single wavelength: measures at one wavelength, such as 292 nm for vitamin E. They are inexpensive, and can be sensitive and specific. They are not much used any more because of their lack of versatility. Variable wavelength: measures one wavelength at a time, but has the capacity to detect a wide range of wavelengths. For example, an aliquot of the sample can be run through the column at 292 nm to detect vitamin E, then a second aliquot can be run through at 330 nm to detect retinol. These have mostly been superseded by the diode array detector. Diode array detector: measures a spectrum of wavelengths simultaneously. For example, a single chromatography run can provide data at 292 nm for vitamin E, 330 nm for vitamin A, and 452 nm for carotenoids. Fluorescent: measures the ability of the sample compounds to absorb and then re-emit light at given wavelengths. Fluorescent detectors have a sensitivity of about 1012 to 1014 g/l. Electrochemical: measures compounds that gain or lose electrons as they pass between electrodes at a given difference in electric potential. Electrochemical detectors have a sensitivity of 10–14–10–16 g/l.

Peak identification -Tocopherol and -tocopherol are usually identified from their retention times (defined in Table 14.3) compared with standards of purified - and -tocopherol. Peaks are identified most accurately when the peaks of interest are entirely separate and distinct from other peaks (Fig. 14.1). It is important to achieve good separation only of the peaks of interest, not all of the peaks on the chromatogram. If you are only interested in measuring - and -tocopherol, it is important that these peaks are completely separated from other compounds, but it is not important what the rest of the chromatogram looks like.

half-height) and symmetrical. A good example of a chromatography peak is shown in Fig. 14.1. Peak concentrations can still be estimated by cutting out and weighing chromatogram peaks, or derived by manual calculations of width at half-height. However, they are almost always done now with commercially available chromatography software. It is important to make sure that this software identifies the correct peak, and measures it correctly. Chromatography software has improved greatly over the years, and now usually gives reliable and accurate peak area estimates. However, it is still necessary to print out, or at least look at, the chromatogram. It is not uncommon for the software to identify the wrong peak, and even more common to measure it incorrectly. The entire chromatography peak should be measured to its natural baseline, and no extra baseline or peaks should be included.

Chromatography peak 95 75 Absorption

control. For these reasons, chromatographers typically add an internal standard to their chromatography samples. Ideal internal standards are chemicals that do not exist in the matrix being tested, but are similar to the compounds being analysed. Satisfactory internal standards for fluorescent, electrochemical or simple UV detectors include tocol (Finckh et al., 1995; Kock et al., 1997) and tocopheryl acetate (Bieri et al., 1979; Gimeno et al., 2001). When diode array detection is used, molecules with UV maxima that differ from vitamin E such as retinyl acetate (Menke et al., 2000; Burri et al., 2003) are often used.

55 35 15 –5

Estimating peak concentrations Vitamin E concentrations in blood or tissues are estimated by comparison with a standard curve of purified -tocopherol of known concentration, after adjusting for differences in extraction efficiency by comparison with the internal standard. Compounds are measured most accurately when the peaks that represent them are sharp (i.e. the peak height is much greater than the peak width at

0

5

10 Minutes

15

20

Fig. 14.1. Chromatogram of -tocopherol illustrating ‘ideal’ peak characteristics. An ‘ideal’ HPLC peak is at least baseline separated from all other compounds of interest, is symmetrical, and has no leading or tailing edge. This generates the most accurate peak areas, and thus the most accurate concentration estimates. The baseline used to calculate peak area does not have to be flat, but it should be straight and should measure the entire peak from edge to edge.

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-Tocopherol concentrations measured in our laboratory have ranged from 11 to 37 mol/l, with a mean of 30.4

mol/l (n = 265). Our results are comparable with US national data, where the mean -tocopherol concentration is 29.6 mol/l (Ford and Giles, 2000). -Tocopherol concentrations are much lower, averaging 4.2 mol/l in our laboratory.

Methods for Analysing ␣- and ␥-Tocopherol in Blood There does not appear to be a universally used ‘goldstandard’ HPLC method for measuring even - and tocopherol in blood. We have used at least three different reversed-phase chromatography methods to measure vitamin E in blood; changing methods to take advantage of improvements in HPLC technology (such as the development of diode array detection) or to integrate it into other assays. The different methods produce somewhat different results even though internal standards are used, but these small differences in analytical data can be corrected for mathematically. A step-by-step isocratic HPLC method formerly used in our laboratory and a typical chromatogram (Fig. 14.2) follow. The method evolved from Catignani (1986), but uses UV detection at 292 nm instead of 280 nm. Because of a variety of difficult to control factors explained above, chromatographers should not expect to get results identical to those shown below or reported in the literature, even if they purchase an identical column and follow procedures exactly. These differences are essentially solved by adding the internal standard and other quality control procedures. However, the retention times and concentration estimates should be highly reproducible within each laboratory when conditions are stable, with variations of 5% or less. Thus, the chromatographer seeking to duplicate this method should not worry if their retention time differs somewhat from the chromatogram shown, but they should achieve internal consistency in their laboratory.

HPLC apparatus The minimum HPLC apparatus required is a single pump system with a single wavelength detector set at 292 nm for vitamin E (or 280 nm if that is necessary) with an integrating recorder and a good quality syringe for injecting samples. We use a more typical HPLC apparatus (Agilent 1100 chromatograph) that includes a gradient (two-pump) system, a diode array detector set at 292 nm for vitamin E and at 330 nm for vitamin A, a refrigerated autosampler that allows us to set up multiple chromatography runs, and a computer with chromatography data handling software for peak identification, analysis and data storage (Chemstation for LC 3D revision A.08.03 (847) running on an HP Kayak XM600 computer with Windows NT (Hewlett-Packard, Waldbronn, Germany)). HPLC column We now use a Prodigy ODS (Phenomenex, Torrance, CA, USA) 4.6 i.d. by 25 cm long, 5 m particle size and 100 Å pore size column. Over the years, we have used a variety of columns based on price and availability, including 2.1 i.d columns; 10, 25 and 30 cm long columns; columns containing 10 and 20 m particle size beads; and a 300 Å pore size. They all work for vitamin E analysis, with minor modifications of the method described herein. A larger i.d. increases the size of the sample you can apply to the column. Peak resolution is increased by longer column length and smaller particle size. Attaching a short C18 reversed-phase guard column just before the main C18 column increases column lifetime. Other equipment necessary ● Vortex mixer ● Laboratory centrifuge (preferably refrigerated) ● Laboratory (organic solvent) hood

● Use of a spectrophotometer, for checking purity and concentrations of standards Supplies

Step by Step Isocratic HPLC Method for Simultaneously Determining Vitamins E and A in Serum Equipment and supplies

● Covered ice bucket ● Vacutainers. We use 7 ml ‘red-topped’ serum tubes. In

● ●

Laboratory room Vitamin E is somewhat sensitive to heat, light and air. It is not necessary to work in a dark room or to prepare, store and run samples in an oxygen-free atmosphere, but it will increase reproducibility, reliability and accuracy to minimize or regulate heat, light and oxygen. Our laboratory uses plastic-sleeved fluorescent lights to minimize UV exposure, stores samples under nitrogen in a –70°C freezer, and uses a column heater set at 35°C for temperature control.

● ● ●

our experience, smaller sizes give accurate results but are a little more difficult to work with. You may also use plasma preserved with EDTA or heparin. Plastic Pasteur pipettes for transferring samples Analytical automatic pipettes for measuring sample and solvent volumes 1.5 ml centrifuge vials 2 ml amber glass autosampler vials, or small test tubes Cryovials if samples will be stored – at least 1 ml in size Reagents

● HPLC-grade hexane, methanol and water. ● Reagent-grade diethyl ether, absolute ethanol, -toco-

pherol, -tocopherol acetate and retinol.

● Nitrogen (or argon) gas.

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Preparing standards and mobile phase Standards Prepare the following concentrated standard solutions. Store at –20°C and prepare monthly when needed. ● ● ● ●

-Tocopherol: 50 mg/10 ml absolute ethanol -Tocopheryl acetate: 50 mg/10 ml absolute ethanol Retinol: 1 mg/10 ml absolute ethanol Working standard solutions: dilute 0.5 ml standard solution to 50 ml with absolute ethanol. Prepare fresh before each sequence of chromatography runs.

with standards that have concentrations that are one- to twofold higher (and lower) than the concentrations of your samples. If you do not know what concentrations to expect in your sample, you can prepare a preliminary standard curve by making a series of 1:10 dilutions covering a wide range of concentrations, then run a final standard curve using concentrations of standards that are close to the concentrations in your samples. Do not run the samples until you get a good standard curve (relative standard deviation (RSD) 30 years (Dupont Liquid Chromatography Methods Bulletin, 1972). Over the past decades, a few methods have been developed to take advantage of advances in technology, such as the invention of diode array (Miller and Yang, 1985) or electrochemical detection (Chou et al., 1985). Others have been developed to use plastic versus steel connectors to increase the recovery of metabolites (Menke et al., 2000), or antioxidants to increase stability (Chow and Omaye, 1983). Other methods have been developed to speed up and simplify analysis by direct extraction (Kramer et al., 1997; Taibi and Nicotra, 2002), or by avoiding time-consuming steps such as evaporation (Nierenberg and Nann, 1992; Julianto et al., 1999). Typically, these methods trade off sensitivity for speed, and are appropriate to use when sample size is not an issue or vitamin E concentrations can be depended upon to be high enough not to require the most sensitive detection methods.

Chromatography of major isoforms in tissues and other matrices Vitamin E extractions from blood or plasma are often simply fat extractions, not very different from any classic quantitative fat extraction method. Most tissues present more difficult matrices than blood, and require more laborious or more specialized extraction methods. Many methods have been developed to measure the major isoforms of vitamin E in tissues or sample matrices that present difficulties for vitamin E extraction (compiled in Table 14.6). Typically, the HPLC method itself is not especially different; it is the extraction of vitamin E from the matrix that is the problem, and the focus of the manuscript.

Sample matrix

Reference

Vitamin supplements Pharmaceuticals

Moreno and Salvado (2000) Iwase (2000)

Foods Milk Dairy products Infant formula Beverages Cheese Cereals Whole diets Fish food

Gomis et al. (2000) Blanco et al. (2000) Albala-Hurtado et al. (1997) Holler et al. (2003) Panfili et al. (1994) Panfili et al. (2003) McGeachin and Bailey (1995); Kramer et al. (1997) Huo et al. (1999)

Plants Macroalgae Malt sprouts Seeds and nuts Leaves Tissues Red blood cells White blood cells Lipoproteins Adipose Brain Lens Faeces

Sanchez-Machado et al. (2002) Carlucci et al. (2003) Delgado-Zamarrero et al. (2003) Torre et al. (2001) Bieri et al. (1979); Solichova et al. (2003) Vatassery and Smith (1987) Gimeno et al. (2001) Casal et al. (2001) Vatassery and Hagen (1977) Mitton and Trevithick (1994) Nierenberg et al. (1987)

Measuring minor isoforms and metabolites Most methods for vitamin E analysis focus on identifying and measuring just the major isoforms of vitamin E in human blood and tissues (- and -tocopherol). These methods are sufficient for assessing vitamin E status for nutrition interventions and for health surveys. However, more sensitive and specific methods needed to be developed for studies of vitamin E metabolism. Vitamin E metabolites are present in many forms, are in low concentrations and can be unstable. Electrochemical detection methods are the methods of choice for measuring metabolites. Methods have been developed for measuring minor isoforms of vitamin E (- and -tocopherol; Melchert and Pabel, 2000) and stereoisomers (Ueda et al., 1993; Kiiyose et al., 1999). Successful methods for metabolites (Delgado-Zamarrero et al., 2001; Cardenosa et al., 2002); addition products (Yamauchi et al., 2000); oxidation products (Ha and Csallany, 1988); tocotrienols (Kramer et al., 1997; Panfili et al., 2003) and tocopheryl quinones (Vatassery and Smith, 1987; Ha and Csallany, 1988) have been developed.

Combined analysis of vitamin E and other micronutrients Most of the newer methods published for the HPLC analyses of vitamin E were developed to combine the analysis of vitamin E with other nutrients. The first examples of this were methods that combined the analysis of vitamin E with its fat-soluble vitamin cousin, vitamin A

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(Bieri et al., 1979; Chow and Omaye, 1983; Catignani, 1986). There have also been methods developed to measure vitamin E, A and D (Savolainen et al., 1988; Alvarez and De Mazancourt, 2001); for vitamin E with ubiquinols (Finckh et al., 1995; Menke et al., 2000); with phytosterols and cholesterol (Huo et al., 1999); with coenzyme Q (Lang and Packer, 1987); with glutathione and ascorbate (Mitton and Trevithick, 1994); and even fat- and water-soluble vitamins (Moreno and Salvado, 2000). Most methods have been developed to measure vitamin E simultaneously with vitamin A and the major carotenoids in blood (-carotene, -carotene, lutein, lycopene, zeaxanthin and cryptoxanthin; Gueguen et al., 2002; Burri et al., 2003). The majority of these methods were developed not to improve vitamin E detection and measurement, but instead to focus on the extraction and analysis of micronutrients which are less stable or in lower concentrations. For example, the difficulty in most methods that measure vitamin E with carotenoids is the separation of lutein and zeaxanthin (Gueguen et al., 2002; Burri et al., 2003).

Stable Gradient Method for Measuring ␣- and ␥-Tocopherol, Vitamin A and Major Carotenoids in Human Serum This is the current HPLC method we use for vitamin E analysis (Burri et al., 2003). Details of the gradient method described below are identical to those described under the isocratic method, unless otherwise stated.

Equipment and supplies HPLC apparatus The minimum HPLC apparatus required is a gradient (twopump) system with a diode array detector set at 292 nm for vitamin E, 330 nm for vitamin A and 452 nm for carotenoids. We use an Agilent 1100 chromatograph with a gradient (two-pump) system, diode array detector, a column heater and a refrigerated autosampler. Peak identification, analysis and data storage is with a Chemstation for LC 3D revision A.08.03 (847) running on an HP Kayak XM600 computer with Windows NT (Hewlett-Packard, Waldbronn, Germany). The set up for the laboratory room, HPLC column, other equipment and supplies, and sample collection and storage were the same as given above.

Reagents ● HPLC-grade acetonitrile, hexane, methanol, tetrahydro-

furan and water. ● Reagent-grade toluene, absolute ethanol, ammonium

sulphate, -hydroxytoluene (BHT), -tocopherol, -tocopherol acetate, retinol, retinyl acetate and -carotene. ● Nitrogen (or argon) gas.

Preparing standards and mobile phase Standards Prepare the following concentrated standard solutions. Store at –20°C and prepare monthly when needed. Prepare -tocopherol and -tocopheryl acetate standard solutions (5 mg compound/10 ml 99:1% (v/v) toluene:absolute ethanol). Prepare retinol and -carotene standards (1 mg compound/10 ml 99:1% (v/v) toluene:absolute ethanol). Working standard solutions: dilute 0.5 ml standard solution to 5 ml with 99:1% (v/v) toluene:absolute ethanol. Mobile phases Prepare mobile phase A (85:5:5:5% (by vol.) acetonitrile:tetrahydrofuran:methanol:ammonium sulphate) and mobile phase B (55:35:5:5% (by vol.) acetonitrile: tetrahydrofuran:methanol:ammonium sulphate). Mix acetonitrile, tetrahydrofuran and methanol together, then add 10 g/l ammonium sulphate and 5 mg/l BHT in HPLC-grade water. Preparing samples Thaw samples. Pipette 100 l of tocopheryl acetate and 100 l of retinyl acetate working standards and 500 l of sample into a 12  75 mm borosilicate glass test tube and mix with 1 ml of 95% ethanol containing 5 mg/l BHT. Work under a laboratory hood under low light. Vortex the sample solution intermittently for 60 s. Add 1.7 ml of hexane. Vortex for 60 s. Remove the hexane layer into a labelled 12  75 borosilicate glass test tube with a Pasteur pipette. Repeat this extraction once, and combine the hexane layers. Evaporate to dryness under a nitrogen stream. Resuspend the sample into 100 l of solvent B. Vortex for 60 s. Transfer quantitatively to amber glass autosampler vials, and place in an HPLC autosampler.

Standard curve preparation Prepare standard curves for vitamin E with dilutions of tocopherol, for vitamin A with dilutions of retinol, and for carotenoids with dilutions of -carotene. Running chromatograms Set up a gradient at 1.0 ml/min. The total run time is 48 min. The gradient changes from 95% solvent A and 5% solvent B to 95% solvent B and 5% solvent A through the run. For the first 10 min, the mobile phase is 95% solvent A and 5% solvent B; from 10 to 29 min the percentage of solvent A decreases linearly to 5% solvent A:95% solvent B. From 29 to 35.9 min, the gradient is maintained at 5% solvent A:95% solvent B. From 35.9 to 36 min, the gradient changes abruptly to 40% solvent A:60% solvent B. This ratio is maintained from 36 to 45 min, then abruptly decreases to 95% solvent A:5% solvent B at 45 min. The

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column is re-equilibrated with 95% solvent A:5% solvent B. Vitamins E and A, and carotenoids are measured simultaneously by diode array detector set to view 292, 330 and 452 nm, respectively. Samples should be run in duplicate. Peak area reproducibility should be very good. Our RSDs (n = 14) for -tocopherol and -tocopherol were 1.8 and 4.2%, respectively. Concentration curves should be linear; our r = 0.99. A typical chromatogram of -tocopherol, retinol and -carotene standards is shown in Fig. 14.3. A typical micronutrient analysis that measures vitamin E as well as

vitamin A and carotenoids is shown in Fig. 14.4. Although this method provides good separation and analysis of - and -tocopherol, it was developed to improve the separation and analysis of the carotenoids measured, not vitamin E. The gradient elution method shown in Fig. 14.4 provides much more information on nutrient status than the simple isocratic method shown in Fig. 14.2. However, it does not improve the reproducibility, reliability, speed, cost-effectiveness or accuracy of vitamin E analysis. When scientists are sure that they only want to measure the major forms of vitamin E, they should use the isocratic method.

A UV absorption 292 nm

UV absorption 292 nm

A 245 195 145 95 45 –5

0

10

20

30

Minutes

20 Minutes

30 20 10 0 10

30

40

20

30

40

30

40

Minutes

B 35 30 25 20 15 10 5 0 –5 0

10

20 Minutes

C UV absorption 452 nm

C UV absorption 452 nm

40

0

UV absorption 330 nm

UV absorption 330 nm

10

50

40

B 35 30 25 20 15 10 5 0 –5 0

60

2.5 2 1.5 1 0.5 0 –0.5

0

10

20

30

40

Minutes

Fig. 14.3. Typical chromatogram of - and -tocopherol, vitamin A and major carotenoid standards measured with gradient reversed-phase chromatography and diode array detection. The chromatographic method (Burri et al., 2003) is the current method used in our laboratory, and is described in detail in the text. (A) -Tocopherol at 292 nm; the smaller peak in front is the retinol standard. (B) Retinol at 330 nm. (C) -Carotene at 452 nm.

10 8 6 4 2 0 0

10

20

30

40

Minutes Fig. 14.4. Typical chromatogram of the concentrations of - and tocopherol, vitamin A and major carotenoids in normal human serum measured with gradient reversed-phase chromatography and diode array detection at 292 nm. The chromatographic method (Burri et al., 2003) is the current method used in our laboratory, and is described in detail in the text. (A) -Tocopherol (first peak) and -tocopherol (second peak) at 292 nm. (B) Retinol at 330 nm. (C) Carotenoids at 452 nm. The major carotenoid peaks, in order, are lutein, cryptoxanthin, lycopene, -carotene, -carotene and cis--carotene.

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Analysis of Vitamin E by HPLC

Conclusion The most common methods for assaying the major metabolites of vitamin E use liquid–liquid extraction, reversed-phase columns and diode array UV detection at 292 nm. However, many other successful methods of chromatography are available using a variety of detectors, columns and solvents. Measuring minor metabolites is more

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difficult, and usually requires more sensitive electrochemical detection. However, many of the recent chromatographic methods reported for vitamin E were developed to assay vitamin E along with other nutrients such as vitamin A and the carotenoids. Most of these more complex methods do not give improved vitamin E detection or measurement, since they were modified primarily to improve the detection and measurement of the other micronutrients measured.

References Albala-Hurtado, S., Novella-Rodriguez, S., Veciana-Nogues, M.T. and Marine-Font, A. (1997) Determination of vitamins A and E in infant milk formulae by high-performance liquid chromatography. Journal of Chromatography A 778, 243–246. Alvarez, J.C. and De Mazancourt, P. (2001) Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, alpha-tocopherol, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human plasma with photodiode-array ultraviolet detection. Journal of Chromatography B 755, 129–135. Bieri, J.G., Tolliver, T.J. and Catignani, G.L. (1979) Simultaneous determination of alpha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. American Journal of Clinical Nutrition 32, 2143–2149. Blanco, D., Fernandez, M.P. and Gutierrez, M.D. (2000) Simultaneous determination of fat-soluble vitamins and provitamins in dairy products by liquid chromatography with a narrow-bore column. 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(1986) Sensitive high-performance liquid chromatographic analysis of plasma vitamin E and vitamin A using amperometric and ultraviolet detection. Journal of Chromatography 380, 331–338. Huo, J.Z., Nelis, H.J., Lavens, P., Sorgeloos, P. and De Leenheer, A.P. (1999) Simultaneous determination of alpha-tocopheryl acetate and tocopherols in aquatic organisms and fish feed. Journal of Chromatography B 724, 249–255.

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Julianto, T., Yuen, K.H. and Noor, A.M. (1999) Simple high-performance liquid chromatographic method for determination of alpha-tocopherol in human plasma. Journal of Chromatography B 732, 227–231. Kiyose, C., Kaneko, K., Muramatsu, R., Ueda, T. and Igarashi, O. (1999) Simultaneous determination of RRR- and SRR-alpha-tocopherols and their quinones in rat plasma and tissues by using chiral high-performance liquid chromatography. Lipids 34, 415–422. Kock, R., Seitz, S., Delvoux, B. and Greiling, H. (1997) Two high performance liquid chromatographic methods for the determination of alphatocopherol in serum compared to isotope dilution-gas chromatography–mass spectrometry. European Journal of Clinical Chemistry and Clinical Biochemistry 35, 371–379. Kramer, J.K., Blais, L., Fouchard, R.C., Melnyk, R.A. and Kallury, K.M. (1997) A rapid method for the determination of vitamin E forms in tissues and diet by high-performance liquid chromatography using a normal-phase diol column. 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(2000) Simultaneous detection of ubiquinol-10, ubiquinone-10, and tocopherols in human plasma microsamples and macrosamples as a marker of oxidative damage in neonates and infants. Analytical Biochemistry 282, 209–217 Miller, K.W. and Yang, C.S. (1985) An isocratic high-performance liquid chromatography method for the simultaneous analysis of plasma retinol, alpha-tocopherol, and various carotenoids. Analytical Biochemistry 145, 21–26. Mitton, K.P. and Trevithick, J.R. (1994) High-performance liquid chromatography–electrochemical detection of antioxidants in vertebrate lens, glutathione, tocopherol, and ascorbate. Methods in Enzymology 233, 523–539. Moreno, P. and Salvado, V. (2000) Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by highperformance liquid chromatography. Journal of Chromatography A 870, 207–215. Nierenberg, D.W. and Nann, S.L. (1992) A method for determining concentrations of retinol, tocopherol, and five carotenoids in human plasma and tissue samples. American Journal Clinical Nutrition 56, 417–426. Nierenberg, D.W., Lester, D.C. and Colacchio, T.A. (1987) Determination of tocopherol and tocopherol acetate concentrations in human feces using high-performance liquid chromatography. Journal of Chromatography 413, 79–89. Panfili, G., Fratianni, A. and Irano, M. (2003) Normal phase high-performance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals. Journal of Agricultural and Food Chemistry 51, 3940–3944. Panfili, G., Manzi, P. and Pizzoferrato, L. (1994) High-performance liquid chromatographic method for the simultaneous determination of tocopherols, carotenes, and retinol and its geometric isomers in Italian cheeses. Analyst 119, 1161–1165. Pearson, C.K., Davies, D.R. and Barnes, M.M. (1970) Separation of alpha-tocotrienol from alpha-tocopherol by polyethylene-celite column chromatography. Chemical Industry 21, 275–-276. Risner, C.H. and Nelson, P.R. (1998) Determination of (+) alpha-tocopherol in environmental tobacco smoke. Journal of Chromatographic Science 36, 80–84. Sanchez-Machado, D.I., Lopez-Hernandez, J. and Paseiro-Losada, P. (2002) High-performance liquid chromatographic determination of alphatocopherol in macroalgae. Journal of Chromatography A 976, 277–284. Savolainen, K.E., Pynnonen, K.M., Lapinjoki, S.P. and Vidgren, M.T. (1988) Determination of fat-soluble vitamins in a pharmaceutical dosage form by solid-phase extraction and reversed-phase liquid chromatography. Journal of Pharmaceutical Science 77, 802–803. Skinner, W.A. and Parkhurst, R.M. (1964) The thin layer chromatographic characterization of some oxidation products of vitamin E. Journal of Chromatography 13, 69–73. Solichova, D., Korecka, L., Svobodova, I., Musil, F., Blaha, V., Zdansky, P. and Zadak, Z. (2003) Development and validation of HPLC method for the determination of alpha-tocopherol in human erythrocytes for clinical applications. Analytical Biological Chemistry 376, 444–447. Taibi, G. and Nicotra, C.M. (2002) Development and validation of a fast and sensitive chromatographic assay for all-trans retinol and tocopherols in human serum and plasma using liquid–liquid extraction. Journal of Chromatography B 780, 261–267. Torre, J., Lorenzo, M.P., Martinez-Alcazar, M.P. and Barbas, C. (2001) Simple high-performance liquid chromatography method for alphatocopherol measurement in Rosmarinus officinalis leaves. New data on alpha-tocopherol content. Journal of Chromatography A 919, 305–311. Ueda, T., Ichikawa, H. and Igarashi, O. (1993) Determination of alpha-tocopherol stereoisomers in biological specimens using chiral phase highperformance liquid chromatography. Nutrition Science and Vitaminology (Tokyo) 39, 207–219. Vatassery, G.T. and Hagen, D.F. (1977) A liquid chromatographic method for quantitative determination of alpha-tocopherol in rat brain. Analytical Biochemistry 79, 129–134. Vatassery, G.T. and Smith, W.E. (1987) 7 Determination of alpha-tocopherolquinone (vitamin E quinone) in human serum, platelets, and red cell membrane samples. Analytical Biochemistry 167, 411–417. Wilson, P.W., Kodicek, E., and Booth, V.H. (1962) Separation of tocopherol by gas–liquid chromatography. Biochemistry Journal 84, 524–531. Yamauchi, R., Hara, Y., Murase, H. and Kato, K. (2000) Analysis of the addition products of alpha-tocopherol with phosphatidylcholine-peroxyl radicals by high-performance liquid chromatography with chemiluminescent detection. Lipids 35, 1405–1410.

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15 Capillary Electrochromatographic Analysis of Tocopherols Salvatore Fanali Istituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche, Area della Ricerca di Roma I, Via Salaria Km 29, 300, 00016 Monterotondo Scalo (Rome), Italy

Abbreviations: BGE, background electrolyte; BHT, butylated hydroxytoluene; CE, capillary electrophoresis; CEC, capillary electrochromatography; EM, electromigration method; EOF, electro-osmotic flow; HPLC, high-performace liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MeCN, acetonitrile; MeOH, methanol; o-CEC, open capillary electrochromatography; ODS, octadecylsilica; OS, octylsilica; p-CEC, packed capillary electrochromatography; PFPS, pentafluorophenylsilica; SFC, supercritical fluid chromatography; TCS, triacontylsilica.

Abstract This mini-review summarizes the analysis of tocopherols by using capillary electrochromatography. The general principles of this recently introduced electromigration technique are briefly discussed pointing out the separation principles and the instrumentation, including the preparation of the capillary used for the analytical separations. The analyses of tocopherols in human serum and in oil extracts are also reported.

Introduction Electromigration methods (EMs) are relatively old separation tools utilized for both analytical and preparative purposes in the field of separation science. With these methods, a relatively high electric field is applied for the movement of mobile phase (or background electrolyte, BGE) and analytes. In the last two decades, a great deal of attention was paid by a number of researchers to the development of nanoscale techniques allowing the application of relatively high electric fields for the separation of both charged and uncharged compounds. This was possible because of the use of: (i) capillaries of low internal diameter (i.d.; -tocopherol). Retention time, lnk, and resolution factor of tocopherols were studied by changing the MeOH/MeCN ratio. The optimum experimental conditions allowing the complete separation of the three tocopherol in the shortest analysis time were found to be MeCN–MeOH (70:30, v/v) with 0.01% ammonium acetate. The same standard mixture was injected in the short capillary end and analysed. Baseline separation of BHT and the three tocopherols was achieved in 10 M (Fig. 82.6). A similar effect is observed for -tocopherol and -tocopherol, and may relate to the increased number of foci observed at higher concentrations. It is interesting to note that -, - and -tocopherols are seldom found at levels exceeding 10 M in humans, whereas -tocopherol levels normally are found to be approximately 30 M, with considerably higher levels often observed in individuals taking tocopherol supplements.

Proliferation and apoptosis Inappropriate proliferation of cells is an important mechanistic agent in the pathogenesis of a tumour cell as well as in characterizing the essence of what a tumour cell is. The effects of DNA damage leading to the genetic changes necessary for neoplasia are enhanced when DNA synthesis associated with cell division occurs simultaneously (Ames and Shigenaga, 1992). Mutations occurring in the pathways regulating cell proliferation in turn lead to cells that proliferate without the normal growth controls that govern cell and tissue function. Tocopherols affect many of these pathways and processes associated with the regulation of cell growth and death suggesting potential mechanistic explanations for the preventive and treatment properties of the tocopherols with respect to cancer. Evidence for differential effects of tocopherols on cellular proliferation and apoptosis have been found for a number of cell lines (Chatelain et al., 1993; Azzi et al., 2000). Of most interest has been the observed effects on normal and tumorigenic prostate cell lines in which -tocopherol is observed to inhibit tumour cell proliferation (Galli et al., 2004), possibly through downregulation of cyclins (Gysin et al., 2002), and induce apoptosis in human prostate cancer cells (Jiang et al., 2004). In the latter study, -tocopherol was shown synergistically to enhance the effect of -tocopherol, whereas -tocopherol had little or no effect. Similarly, Galli et al. (2004) demonstrated approximately 70% inhibition of prostate tumour cell proliferation by -tocopherol at 10 M, while -tocopherol was significantly less effective, a situation mirroring the observed inhibition of transformation in vitro. Shah et al. (2003) provide further evidence that the apoptotic effects of vitamin E are mediated through caspase-8

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Y. Tanaka and R.V. Cooney

Fig. 82.4. Inhibition of IFN/LPS-induced DNA double-strand breaks and isoprostane formation by tocopherols. (A) Confluent dishes of C3H 10T1/2 fibroblasts were treated with the indicated tocopherol (10 M) at the time of a weekly medium change. After 7 days, cells were given another medium change, tocopherol re-treatment and were then treated with IFN/LPS (IFN at 10 ng/ml and LPS at 10 g/ml dissolved in phosphate-buffered saline (PBS)). Cells were then harvested 2 days later and DNA double-strand breaks determined (A). Data are expressed as the mean fraction of DNA 2 IU/day was required to reduce transgene loss while a lesser dose (1–2 IU/day) was able to reduce the mutation frequency (Haqqani et al., 2002b; Soo et al., 2004). This may be due to a difference in the mechanisms by which these two forms of genetic instability develop. For example, both disruption of the neutrophil–tumour cell interactions and scavenging of neutrophil-derived RNOS by -tocopherol may be necessary to prevent transgene loss, whereas only the latter property may be necessary to inhibit mutations. Based on our experimental findings (Haqqani et al., 2000, 2001), we postulate that IL-8 transgene loss is a result of a complex interaction between IL-8-producing tumour cells and tumour-infiltrating neutrophils. In response to the high level of IL-8 produced by Mutatect TM-28 cells early after subcutaneous injection into the animals, a large number of neutrophils is attracted to the injection site. These neutrophils release a large quantity of genotoxic and cytotoxic RNOS. Exposure of the tumour to these species causes large-scale deletion mutations in (i.e. loss of) the IL-8 transgene. In addition, neutrophils may be selectively cytotoxic towards IL-8-producing tumour cells (Haqqani et al., 2000). The combined effects may explain why TM-28 cells lacking the transgene eventually dominate the tumour. If IL-8 transgene loss occurs early in tumour development during the initial high influx of neutrophils and Hprt mutagenesis occurs later, it may require a higher dose of dietary -tocopherol to inhibit the former. Alternatively, the type of RNOS involved in the two genetic instabilities may be different, i.e. a higher dose of -tocopherol may be required to scavenge the type of RNOS involved in IL-8 transgene loss than the type involved in Hprt mutations. Thus, the differences between the individual mechanisms of in vivo-arising IL-8 transgene loss and Hprt mutations are

911

likely to be responsible for the differential protective effect of dietary -tocopherol seen in the two instabilities. Effects of ␥-tocopherol on in vivo-arising genetic instabilities and neutrophil distribution in Mutatect tumours -Tocopherol is a form of vitamin E that is most prevalent in North American diets, whereas -tocopherol is the form that is most readily found in dietary supplements. While most studies have focused on the effects of -tocopherol, very little attention has been paid to -tocopherol probably because it is taken up poorly compared with the  form. Physiologically, -tocopherol has been considered to be the most active form and it is the predominant form present in human tissues. However, -tocopherol has been shown to be a better scavenger of some forms of RNOS and is more protective against malignant transformation of cells than -tocopherol (Cooney et al., 1993). Once -tocopherol was shown to be protective against in vivo-arising genetic instability in Mutatect tumours, we performed similar experiments to determine the relative effectiveness of tocopherol. The effectiveness of dietary -tocopherol was compared with that of -tocopherol in reducing Hprt mutations and IL-8 transgene loss and on neutrophil distribution. In our first experiment, mice were given different dietary doses of a mixed tocopherol enriched in -tocopherol, (17% -tocopherol, 10% -tocopherol, 56% -tocopherol and 17% -tocopherol). Doses were given such that mice received 0, 0.5, 1 or 2 IU of -tocopherol per day. None of the doses administered showed any protection against either in vivo-arising Hprt mutation frequency or loss of the IL-8 transgene, nor any effect on neutrophil levels in either single cell or stromal fractions. Since the presence of other tocopherol forms (mainly -tocopherol) can affect the absorption of -tocopherol by the liver, we carried out a second experiment using a vitamin E-free diet supplemented with only pure -tocopherol. Again, tumours of mice receiving 2 IU of -tocopherol/day failed to exhibit any significant change in Hprt mutation frequency, IL-8 transgene loss or neutrophil distribution. Thus, in contrast to the dramatic protections by dietary -tocopherol, dietary -tocopherol does not appear to be protective. At least part of the difference in biological effectiveness of - and -tocopherol may be related to differences in their bioavailability. While an -tocopherol level of 4–20 nmol/ml in plasma and 5–40 nmol/g wet weight in tumours was achieved in mice fed 2 IU of pure -tocopherol/day, a -tocopherol level of only 1–2 nmol/ml in plasma and 4–12 nmol/g wet weight in tumours was achieved in mice fed 2 IU of pure -tocopherol/day (Soo et al., 2004), i.e. -tocopherol appears to be four- to tenfold more readily absorbed than -tocopherol. Our findings on the lack of effect of dietary -tocopherol in our animal model may simply reflect our inability to achieve a sufficiently high level of -tocopherol in either plasma or tumours. Thus, our results are not inconsistent with suggestions that -tocopherol might have important health benefits (Jiang et al., 2001).

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Conclusions and Significance of Findings from the Effects of Vitamin E on Mutatect Tumours Results from the Mutatect mouse model have provided a glimpse into the role of the tumour microenvironment on genetic instability and have revealed new details about the benefits of dietary vitamin E. Some of the key results have been summarized in Table 85.3. Dietary -tocopherol is able to protect against in vivo-arising genetic instabilities in a dose-dependent manner, an effect that is much more marked in tumours with high levels of inflammatory cells and RNOS. We therefore postulate that some of the reported discrepancies in effectiveness of vitamin E on human cancers may depend upon the degree to which inflammation and RNOS are important in the initiation and progression of the specific cancer. Protection by dietary -tocopherol against in vivo-arising genetic instability seems to involve not just its ability to scavenge RNOS but also its ability to affect neutrophil distribution (Fig. 85.4). Thus, the non-antioxidant effect of vitamin E should also be taken into account when examining the protective effect of vitamin E. Since vitamin E is currently being used in a large human prevention trial (SELECT) for prostate cancer (Klein et al., 2001; Lieberman, 2001), our findings about the dose-dependent protective effects of -tocopherol may have implications for the design and interpretation of such studies. Mutatect is an experimental animal tumour system. As with every animal model, extrapolation to the human

condition has its limitations. However, Mutatect tumours show some interesting similarities to some types of human cancers. Mutatect tumours exhibit a high level of genetic instability, a hallmark of progression of human tumours. Mutatect tumours are infiltrated with inflammatory cells that are a source of RNOS, as is found in many human tumours. Furthermore, Mutatect tumours show evidence of RNOSmediated damage, also observed in many human tumours. The non-antioxidant effects of -tocopherol on Mutatect tumours (i.e. neutrophil redistribution) are consistent with reports of the evidence in humans and human cells that vitamin E can affect cellular functions in ways that are independent of its antioxidant/radical-scavenging ability (Azzi and Stocker, 2000), including effects on neutrophil– or monocyte–endothelial cell adhesion interactions (Yoshikawa et al., 1998; Jialal et al., 2001). Thus, the findings from the Mutatect model about the mechanisms of in vivo-arising genetic instability and the mechanisms of protection by vitamin E should be considered in the context of human studies.

Acknowledgements This work was supported by grants from the Canadian Institutes for Health Research and the Cancer Research Society.

Table 85.3. Summary of the effects of dietary vitamin E on various parameters in Mutatect tumoursa. Vitamin E tested

Parameters

Effect

Conclusions

-Tocopherol

Genetic instability: mutations at the Hprt locus Genetic instability: IL-8 transgene loss Tumour-infiltrating neutrophils

Protective at low and high doses; more protective in inflammatory tumours Protective at only highest dose tested

Dietary -tocopherol is anti mutagenic

Redistribution (protective)

Genetic instability: mutations at the Hprt locus Genetic instability: IL-8 transgene loss Tumour-infiltrating neutrophils

None

Results suggests an additional mechanism of protection by vitamin E Either -tocopherol is not anti mutagenic or it was not able to reach the target

-Tocopherol -Tocopherol -Tocopherol -Tocopherol -Tocopherol

None None

effects of dietary supplementation of - or -tocopherol were tested on various parameters in Mutatect tumours. Animals were maintained on a supplemented diet from 1 week prior to tumour formation until when the tumours were excised. Hprt, hypoxanthine phosphoribosyl transferase; IL-8, interleukin-8. a The

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86 Tocopherols and Lung Cancer Victor Cohen1, Shaheenah Dawood1 and Fadlo R. Khuri2 1Sir

Mortimer B Davis–Jewish General Hospital, McGill University School of Medicine, Department of Oncology, 3755 Cote Ste. Catherine Road, Suite E-177, Montreal, Quebec, Canada H3T-1E2; 2Winship Cancer Institute, Emory University, 1365 Clifton Road NE, Bldg C 3094, Atlanta, GA 30322, USA

Abbreviations: ATBC, Alpha-Tocopherol, Beta-Carotene Cancer Prevention; CI, confidence interval; NSCLC, non-small cell lung cancer; RR, relative risk; SCLC, small cell lung cancer.

Abstract Lung cancer is the most common cause of cancer mortality worldwide and continues to be a major health problem. In addition to being the biggest cancer killer, lung cancer is one of the few cancers with a well defined aetiology, namely the inhalation of tobacco smoke. Diet has also been implicated in the development of lung cancer, although the specific nutrients remain to be elucidated. Vitamins with antioxidant activity including -carotene (the most efficient provitamin A), and vitamins E and C have received much attention. Vitamin E is a collective term used to refer to a group of eight different naturally occurring compounds known as tocopherols and tocotrienols as well as synthetic vitamin E. Prospective epidemiological studies have shown both positive and negative correlations between plasma concentrations of -tocopherol and lung cancer risk. The findings from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention study (ATBC) study do not support the hypothesis that supplementation with this molecule would reduce the incidence of lung cancer. Thus, supplementary vitamin E in any form cannot be recommended for prevention of lung cancer outside of a clinical study. The most effective method of reducing the risk of lung cancer development remains avoidance of cigarette smoking.

Introduction Lung cancer is among the most commonly occurring malignancies in the world and is one of the few that continues to show an increasing incidence. Approximately 1.2 million cases were diagnosed in 2002 (12.3% of all new cancer cases) and >1.5 million are anticipated by 2005 (Stewart and Kleinhues, 2003). In the USA, the disease has been the leading cause of cancer death in men for years and, since 1988, it has also become the number one cause of cancer death in women. It is estimated that lung cancer was responsible for approximately 164,440 deaths during 2004, in comparison with 127,210 deaths from the combined mortality of breast, prostate and colorectal cancer over the same period (Jemal et al., 2004). Primary carcinoma of the lung appears to develop from a pluripotent stem cell involved in the generation of the

bronchial epithelium and capable of differentiation along several pathways (Sekido et al., 2001). The biology of this process is based on two themes: field cancerization and multi-step carcinogenesis. Field cancerization denotes diffuse epithelial injury resulting from carcinogenic (e.g. tobacco smoke) exposure in an entire epithelial field or region, setting off a chronic pattern of tissue damage and wound healing where changes can be detected at the gross, microscopic and molecular levels. The clinical importance of this phenomenon is best illustrated in aerodigestive cancers for which both synchronous and metachronous second primary tumours are common. Chronic carcinogenic insult sets off a multi-step process characterized by the occurrence of initiation, promotion and progression events occurring over latent periods of a decade or more (Fig. 86.1). These events produce an accumulation of genetic and epigenetic alterations of at least three groups

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Fig. 86.1. Multi-step lung carcinogenesis. Illustration of the accumulation of multiple genetic alterations that can potentially progress to carcinoma.

of genes: proto-oncogenes, tumour suppressor genes and mutator genes resulting in imbalances between cellular proliferation, apoptosis and shedding (Sekido et al., 2001). An imbalance in cellular population kinetics promotes a build-up of cells that, if sufficiently abnormal, have malignant capability. Numerous systems including repair, replacement or recruitment, replication and redundancy mechanisms become operational to help restore structural and functional integrity. In some instances, however, these mechanisms fail or are overwhelmed, and unrepaired injury not only occurs but also is propagated, resulting in the triggering of a transformation from normal to pre-malignant cells and eventually to frank invasive carcinoma. Although multiple cell types are often found within a single lung tumour, one type usually predominates. Two major subdivisions are recognized: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). This is due to the major clinical differences in presentation, metastatic spread and response to therapy. SCLC accounts for 15–25% of all lung cancers and although the disease is sensitive to both chemotherapy and radiotherapy, the duration of response is usually short lived. The majority of SCLC patients die from progressive disease (Chute et al., 1999; Janne et al., 2002). NSCLC accounts for approximately 75–85% of all cases and comprises a heterogeneous aggregate of at least three histological subtypes including adenocarcinoma, squamous cell carcinoma and large cell carcinoma (Sekido et al., 2001; Page et al., 2002). About 30% of patients with NSCLC present with stage I and II disease. Surgery is currently the treatment of choice for these patients and represents the best chance of a cure. A further 25–30% of patients have locally advanced or stage III disease at presentation. While multimodality therapy is routinely recommended for this patient group, its exact nature and sequence remain controversial. In the past, radiation therapy was considered the standard of

care; however, long-term survival with this approach was poor, in the range of 5–10% with poor local control and early development of distant metastatic disease. Recent studies indicate that the addition of chemotherapy improves survival in these patients; however, the magnitude of improvement is small (Furuse et al., 1995, 1999; Dillman et al., 1996; Curran et al., 2000; Sause et al., 2000). The outcome for patients with stage IV disease is particularly bleak. Systemic chemotherapy has been used in an attempt to prolong symptom-free survival. Treatment with modern cisplatin-containing regimens improves median survival by a modest 6–12 weeks, and 1-year survival from 5–15% with best supportive care alone to 30–40% in treated patients (Non-Small Cell Lung Cancer Collaborative Group, 1995). Therefore, despite improvements in diagnostic imaging, surgery, radiotherapy and chemotherapy, the overall survival for NSCLC remains poor, with only about 14% of patients surviving 5 years from diagnosis. Furthermore, it appears unlikely that additional marked improvements with these practices alone will occur in the near future. This grim overview argues powerfully for alternative approaches such as prevention strategies for controlling lung cancer.

Risk Factors In addition to being the biggest cancer killer, lung cancer is one of the few cancers with a well defined aetiology, namely the inhalation of tobacco smoke. Cigarette smoking is estimated to be responsible for approximately 87% of lung cancer cases, and evidence for this link is indisputable (Wingo et al., 1999). Estimates of the relative risk of disease in the long-term smoker vary from ten- to 30-fold. The cumulative lung cancer risk among heavy smokers may be as high as 30% compared with a lifetime risk of 1% or less in non-smokers (Samet et al., 1988; Samet, 1991). The risk of

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Tocopherols and Lung Cancer

carcinoma increases with the number of cigarettes smoked, years of smoking, earlier age of onset, degree of inhalation, tar and nicotine content, and use of unfiltered cigarettes. Other risk factors for lung cancer include exposure to asbestos, haloethers, polycyclic aromatic hydrocarbons, nickel, arsenic, genetic factors and the presence of underlying benign forms of parenchymal lung disease, especially pulmonary fibrosis (Sekido et al., 2001). Recent interest has focused on the potential roles of exposure to environmental tobacco smoke (passive exposure to second-hand smoke) and to radon.

Dietary Risk Factors Diet has also been implicated in the development of lung cancer. Numerous studies have shown that the incidence of lung cancer can be inversely related to the intake of many food groups or to low serum concentrations of many micronutrients. In this chapter, available evidence concerning the relationship between vitamin E and lung cancer is reviewed and evaluated. To this end, relevant studies regarding vitamin E intake (including that from dietary and supplemental sources) and vegetable and fruit consumption are taken into account and summarized.

Vitamin E The term ‘vitamin E’ encompasses four common tocopherols (-, -, - and -tocopherols), four common tocotrienols (-, -, - and -tocotrienols) and synthetic vitamin E (a chemical mixture composed of a naturally occurring RRR--tocopherol and stereoisomers).

Vitamin E and Lung Cancer: What is the Evidence? Observational epidemiological studies of lung cancer and vegetables, fruit and vitamin E An extensive body of literature suggests that an increased consumption of fruits and vegetables is associated with a lower risk of lung cancer. Several case–control studies have shown at least a 25% reduction in lung cancer risk for comparisons of the highest versus lowest intake category for fruits or vegetables (Maclennan et al., 1977; Ziegler et al., 1986; Fontham et al., 1988; Koo, 1988; Le Marchand et al., 1989; Jain et al., 1990; Kalandidi et al., 1990; Candelora et al., 1992; Forman et al., 1992; Gao et al., 1993; Mayne et al., 1994; Sankaranarayanan et al., 1994; Axelsson et al., 1996; Lei et al., 1996; Agudo et al., 1997; Ko et al., 1997; Pawlega et al., 1997; Pillow et al., 1997; Nyberg et al., 1998; De Stefani et al., 1999; Brennan et al., 2000; Marchand et al., 2002). Other case–control studies have shown weaker or insignificant associations (Ziegler et al., 1986; Le Marchand et al., 1989; Pierce et al., 1989; Jain et al., 1990; Kalandidi et al., 1990; Forman et al., 1992; Swanson et al., 1992, 1997; Alavanja et al., 1993; Mayne et al., 1994; Agudo et al., 1997;

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Hu et al., 1997; Ko et al., 1997; Pillow et al., 1997; Nyberg et al., 1998; Mohr et al., 1999; Brennan et al., 2000; Kubik et al., 2001; Marchand et al., 2002; Wright et al., 2003). Although risk estimates from these reports could be exaggerated by biased recall of diet among the case subjects and by selective participation of more health-conscious control subjects, results from retrospective and prospective studies have been quite similar. In a report on two American cohorts (the Nurses Health Study of women and the Health Professionals’ Follow-up Study of men), an effect of fruit and vegetable intake was found in women but not in men; in European multi-country cohorts, fruit intake was inversely related to mortality in male smokers and lung cancer risk; and in a study in The Netherlands, inverse associations were found for both fruits and vegetables (Feskanich et al., 2000; Voorrips et al., 2000; Jansen et al., 2001). Inverse associations with lung cancer risk were also found for fruits in cohort studies in China and Japan (Xu et al., 1996; Ozasa et al., 2001). It has been hypothesized that this association is because of the presence of naturally occurring macronutrients and micronutrients or trace compounds, which act as inhibitors of carcinogenesis. If the food ingredients responsible for this ‘prevention’ activity can be recognized, modification of the diet to include foods rich in these substances or supplementation with the specific agent or agents may be an effective approach of lung cancer prevention. The challenge has been to identify the relevant dietary constituents. One approach used to identify candidate prevention nutrients is the analysis and comparison of serum obtained from persons who later developed lung cancer with those from matched healthy controls. Using this methodology, inverse correlations have been described between the serum concentrations of many dietary micronutrients and lung cancer incidence. A number of studies have examined the association between dietary intake or blood levels of vitamin E and lung cancer. Vitamin E occurs in nature in at least eight structurally related forms; four tocopherols (, ,  and ) and four tocotrienols (, ,  and ), all of which are potent membrane-soluble antioxidants. -Tocopherol is the predominant and most active form of vitamin E in humans and it is thought to inhibit carcinogenesis through its antioxidant activity (Halliwell, 1994). There is a growing body of evidence suggesting that this molecule may also inhibit carcinogenesis through various alternative mechanisms including inhibition of cell proliferation, induction of apoptosis, inhibition of angiogenesis and enhancement of immune function (Meydani and Beharka, 1996; Shklar and Schwartz, 1996; Sigounas et al., 1997). These multiple functions may allow it to inhibit tumorigenesis at various stages, from initiation and promotion to progression and tumour growth. In general, null or weak inverse associations of serum vitamin E and subsequent risk of lung cancer have been observed. Case–control studies have generally been supportive of reduced lung cancer risk among persons having higher blood levels of -tocopherol, and pre-diagnostic serum levels have been shown to be inversely associated with lung cancer in some but not all cohort investigations.

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Six prospective studies have been reported. Menkes et al. (1986) studied the relationship of serum vitamin A (retinol), -carotene, vitamin E and selenium to the risk of lung cancer using serum that had been collected during a large blood collection study performed in Washington County, Maryland in 1974. Levels of the nutrients in serum samples from 99 persons who were subsequently found to have lung cancer were compared with levels in controls who were matched for age, sex, race, month of blood donation and smoking history. Mean levels of vitamin E were lower among cases than controls when all histological types of cancers were considered. In addition, a linear trend in risk was found so that persons with serum levels of the vitamin in the lowest quintile had a 2.5 times higher risk of lung cancer than persons with levels in the highest quintile. Inverse associations between low levels of vitamin E and the risk of lung cancer were found in two other studies; in the 12-year follow-up of the Basel study, low levels of lipidadjusted vitamin E in 2974 healthy male volunteers were associated with a significantly increased risk for lung cancer and subsequent overall and disease-related mortality, and in the other study a modest non-significant or trivial difference was noted (Stahelin et al., 1991; Comstock et al., 1997). Conversely, Knekt et al. (1988), Wald et al. (1987) and Nomura et al. (1985) showed no statistically significant inverse associations. In 1999, Woodson et al. reported the results of a study examining the relationship between prospectively collected serum -tocopherol and lung cancer in the AlphaTocopherol, Beta-Carotene Cancer Prevention (ATBC) Study cohort. This investigation is unique in that it evaluated prospectively within the same cohort the potential impact of both chronic vitamin E status (as assessed by serum -tocopherol, dietary -tocopherol and vitamin E) and a randomized, placebo-controlled test of daily -tocopherol supplementation. The study’s size and number of events provided sufficient power to control tightly for confounding and to evaluate effect modification by several relevant study factors. A 19% reduction in lung cancer incidence was observed in the highest versus lowest quintile of serum -tocopherol (relative risk (RR) = 0.81; 95% confidence interval (CI) = 0.67–0.97). There was a stronger inverse association among younger men, among men with less cumulative tobacco exposure and among men receiving -tocopherol supplementation. In interpreting the findings of many of these studies, it must be recognized that smoking status may be an important potential confounder (because of the powerful influence of smoking on lung cancer incidence and the correlations between many smoking characteristics and diet) and, therefore, must be assessed rigorously. In analyses with adequate control for smoking characteristics, data regarding modification of this effect have been inconsistent. Comstock et al. (1997) observed no differences in lung cancer risk associated with serum -tocopherol across smoking categories (non-smokers, former smokers or current smokers) whereas two other studies found that a higher serum -tocopherol was more protective among male nonsmokers and younger subjects (Kneckt et al., 1988; Kneckt, 1993).

Randomized intervention trials of vitamin E and lung cancer Randomized intervention trials provide highly pertinent, specific and convincing evidence regarding supplemental nutrients or dietary habits and cancer risk, and have an important role in the development of related nutrition recommendations. They test specific nutrients, nutrient combinations or dietary interventions through randomized experimental designs that avoid most of the biases inherent in observational studies. Only one controlled trial has reported the results on the effect of supplementary vitamin E on the risk of lung cancer (Table 86.1). The ATBC study used a 2  2 factorial design to test -tocopherol and -carotene in 29,133 Finnish chronic male smokers aged 50–69 years (Alpha-Tocopherol, BetaCarotene Cancer Prevention Study Group, 1994; Albanes et al., 1996). Subjects were randomized to one of four groups: -tocopherol 50 mg/day alone, -carotene 20 mg/day alone, both -tocopherol and -carotene at the above doses, or placebo, and were followed for 5–8 years. Supplementation with -tocopherol produced no overall effect on lung cancer events (RR 0.99, 95% CI 0.87–1.13, P = 0.86). Secondary analyses suggested that participants having longer exposure to -tocopherol supplementation may have accrued some marginal benefit (i.e. a 10–15% reduction in incidence). This latter finding, however, must be viewed as questionable at best given the problematic nature of post hoc secondary analyses and the high likelihood of type I and II error rates. Table 86.1. Summary of major design elements and results from the AlphaTocopherol Beta-Carotene (ATBC) study: RRs for supplemented versus placebo group comparisons. Population Design Intervention Duration of intervention Primary cancer end-point

Secondary end-point

29,133 male smokers Aged 50–69 years 2  2 factorial randomized double-blind placebo-controlled -Tocopherol and/or -carotene 6.1 years Lung cancer incidence, RR = 0.99 (AT) RR = 1.16 (BC)* Total death, RR = 1.02 (AT) RR = 1.08 (BC) Prostate cancer incidence, RR = 0.68 (AT)* RR = 1.23 (BC) Colorectal cancer incidence, RR = 0.78 (AT) RR = 1.05 (BC) Urothelial cancer incidence, RR = 1.1 (AT) RR = 1.0 (BC) Kidney cancer incidence, RR = 1.1 (AT) RR = 0.8 (BC) Stomach cancer incidence, RR = 1.21 (AT) RR = 1.26 (BC)

AT, -tocopherol; BC, -carotene; RR, relative risk. *P- value is statistically significant (P < 0.05).

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Tocopherols and Lung Cancer

Although -tocopherol showed no effect on lung cancer risk overall, two very important and unexpected findings emerged from this controlled trial that warrant some discussion. The first is the observation of a 32% decrease in prostate cancer incidence and 41% decrease in prostate cancer mortality among men receiving -tocopherol compared with placebo. This result for prostate cancer as a secondary end-point has provided new leads for further studies which are now underway. An example is the Selenium and Vitamin E Cancer Prevention Trial, a large, prospective trial of selenium and -tocopherol in >30,000 men. The second was the finding of a harmful effect (excess lung cancer incidence and overall mortality) for -carotene. By the end of the ATBC study, and as reported in the final report for lung cancer, lung cancer was diagnosed in 482 men in the -carotene-supplemented group and 412 in the group not receiving -carotene (RR 1.16, 95% CI 1.02–1.33, P = 0.02). The adverse effect of -carotene appeared to be stronger in those who were heavy smokers of at least 20 cigarettes per day (RR 1.25, 95% CI 1.07–1.46) than in those who smoked 19 cigarettes or less per day (RR 0.97, 95% CI 0.76–1.23). The significance of this finding was heightened by the fact that the -carotene group also experienced increased overall mortality (by 8%) including an apparent increase in ischaemic heart disease mortality. A similar result for -carotene was subsequently reported in the BetaCarotene and Retinol Efficacy Trial or CARET, which halted its intervention of -carotene and retinyl palmitate after an observed increase in lung cancer incidence and total mortality in the supplemented group (Omenn et al., 1996). The results of these two trials were in sharp contrast to the epidemiologic data (as with vitamin E, -carotene was found to be inversely related to the risk of lung cancer in prospective epidemiological studies, especially in studies measuring serum concentrations of -carotene), emphasizing the importance of carefully controlled intervention trials to confirm epidemiological studies and in determining the role of dietary supplements or any intervention agent.

γ-Tocopherol Whereas -tocopherol is the predominant form of vitamin E in tissues and the primary form in supplements, -tocopherol

is the most abundant form of vitamin E in the North American diet. This molecule for a variety of reasons has received little attention since the discovery of vitamin E by Evans and Bishop in 1922 and is not included in the current dietary intake recommendations. Recently, however, researchers have discovered that -tocopherol may be important to human health and that it possesses properties that are not shared by its confreres (these unique qualities are probably a result of its distinct chemical reactivity, metabolism and biological activity). Some human and animal studies indicate that plasma concentrations are inversely associated with the incidence of cardiovascular disease and certain types of cancers including prostate cancer (Jiang et al., 2001). With regards to lung cancer, however, the relationship between -tocopherol and disease risk remains to be clarified. Epidemiological studies conducted so far have been unrevealing.

Summary and Conclusions Observational studies of diet and lung cancer strongly suggest that increased vegetable and fruit intake is associated with reduced risk of lung cancer. Prospective studies have shown both positive and negative correlations between plasma concentrations of -tocopherol and disease risk. The findings from the ATBC study do not support the hypothesis that supplementation with this molecule would reduce the incidence of lung cancer. Thus, supplementary vitamin E in any form cannot be recommended for prevention of lung cancer outside of a clinical study. The most effective method of reducing the risk of lung cancer development remains avoidance of cigarette smoking. Quitting smoking causes approximately a tenfold drop in this risk, whereas the decrease in the risk of lung cancer associated with increased vegetable and fruit intake is, at most, twofold. However, a diet rich in vegetables and fruits containing many potentially beneficial nutrients and phytochemicals is to be recommended in order to reduce the risk of lung and other cancers and also other chronic diseases, and certainly such dietary modification is unlikely to be harmful.

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Nyberg, F., Agrenius, V., Svartengren, K. et al. (1998) Dietary factors and risk of lung cancer in never-smokers. International Journal of Cancer 78, 430–436. Omenn, G.S., Goodman, G.E., Thornquist, M.D. et al. (1996) Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. New England Journal of Medicine 334, 1150–1155. Ozasa, K., Watanabe, Y., Ito, Y. et al. (2001) Dietary habits and risk of lung cancer death in a large-scale cohort study (JACC Study) in Japan by sex and smoking habit. Japanese Journal of Cancer Research 92, 1259–1269. Page, N.C., Read, W.L., Tiemey, R.M. et al. (2002) The epidemiology of small cell lung carcinoma. Proceedings of the American Society for Clinical Oncology 2, 305a. Pawlega, J., Rachtan, J. and Dyba, T. (1997) Evaluation of certain risk factors for lung cancer in Cracow (Poland). Acta Oncologica 36, 471–476. Pierce, R.J., Kune, G.A., Kune, S. et al. (1989) Dietary and alcohol intake, smoking pattern, occupational risk, and family history in lung cancer patients: results of a case–control study in males. Nutrition and Cancer 12, 237–48. Pillow, P.C., Hursting, S.D., Duphorne, C.M. et al. (1997) Case–control assessment of diet and lung cancer risk in African Americans and Mexican Americans. Nutrition and Cancer 29, 169–173. Samet, J.M. (1991) Health benefits of smoking cessation. Clinics in Chest Medicine 12, 669–679. Samet, J.M., Wiggins, C.L., Humble, C.G. et al. (1988) Cigarette smoking and lung cancer in New Mexico. American Review of Respiratory Diseases 137, 1110–1113. Sankaranarayanan, R., Varghese, C., Duffy, S.W. et al. (1994) A case–control study of diet and lung cancer in Kerala, South India. International Journal of Cancer 58, 644–649. Sause, W., Kolesar, P., Taylor, S. IV et al. (2000) Final results of phase III trial in regionally advanced unresectable non-small cell lung cancer. Radiation Therapy Oncology Group, Eastern Cooperative Oncology Group, and Southwest Oncology Group. Chest 117, 358–364. Sekido, Y., Fong, K. and Minna, J. (2001) Cancer of the lung. In: DeVita, V.T. Jr, Hellman, S. and Rosenberg S.A. (eds) Cancer: Principles and Practice of Oncology, 5th edn. Lippincott-Raven, Philadelphia, pp. 917–1018. Shklar, G. and Schwartz, J.L. (1996) Vitamin E inhibits experimental carcinogenesis and tumor angiogenesis. European Journal of Cancer B Oral Oncology 32B, 114–119. Sigounas, G., Anagnostou, A. and Steiner, M. (1997) DL-Alpha-tocopherol induces apoptosis in erythroleukemia, prostate, and breast cancer cells. Nutrition and Cancer 28, 30–35. Stahelin, H.B., Gey, K.F., Eichholzer, M. et al. (1991) Plasma antioxidant vitamins and subsequent cancer mortality in the 12-year follow-up of the prospective Basel study. American Journal of Epidemiology 133, 766–775. Stewart, B.W. and Kleinhues, P. (2003) World Cancer Report 2003. WHO and International Agency for Research on Cancer. IARC Press, Lyon, pp. 182–187. Swanson, C.A., Mao, B.L., Li, J.Y. et al. (1992) Dietary determinants of lung cancer risk: results from a case–control study in Yunnan Province, China. International Journal of Cancer 50, 876–880. Swanson, C.A., Brown, C.C., Sinha, R. et al. (1997) Dietary fats and lung cancer risk among women: the Missouri Women’s Health Study (United States). Cancer Causes and Control 8, 883–893. Voorrips, L.E., Goldbohm, R.A., Verhoeven, D.T. et al. (2000) Vegetable and fruit consumption and lung cancer risk in the Netherlands Cohort Study on diet and cancer. Cancer Causes and Control 11, 101–115. Wald, N.J., Thompson, S.G., Densem, J.W. et al. (1987) Serum vitamin E and subsequent risk of cancer. British Journal of Cancer 56, 69–72. Wingo, P.A., Reis, L.A., Giovino, G.A. et al. (1999) Annual report to the nation on the status of cancer, 1973–1996, with a special attention on lung cancer and tobacco smoking. Journal of the National Cancer Institute 91, 675–690. Woodson, K., Tangrea, J.A., Barrett, M.J. et al. (1999) Serum -tocopherol and subsequent risk of lung cancer among male smokers. Journal of the National Cancer Institute 91, 1738–1743. Wright, M.E., Mayne, S.T., Swanson, C.A. et al. (2003) Dietary carotenoids, vegetables, and lung cancer risk in women: the Missouri Women’s Health Study (United States). Cancer Causes and Control 14, 85–96. Xu, Z., Brown, L.M., Pan, G.W. et al. (1996) Cancer risks among iron and steel workers in Anshan, China. II. Case–control studies of lung and stomach cancer. American Journal of Internal Medicine 30, 7–15. Ziegler, R.G., Mason, T.J., Stemhagen, A. et al. (1986) Caroteoid intake, vegetables, and the risk of lung cancer among white men in New Jersey. American Journal of Epidemiology 123, 1080–1093.

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87 Gastrointestinal Cancers and Vitamin E Farin Kamangar Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892–8314, USA

Abbreviation: FFQ, food frequency questionnaire.

Abstract The role of vitamin E in preventing gastrointestinal cancers has been examined in observational and experimental studies in both animals and humans. The organs studied most extensively are the oesophagus, stomach and colon. The evidence reviewed includes animal studies, human observational studies and human experimental studies. There is consistent evidence that vitamin E reduces the risk of oesophageal squamous cell carcinoma, but not oesophageal adenocarcinoma. The effect of vitamin E on oesophageal squamous cell carcinoma appears to be stronger in younger than in older individuals. Current evidence suggests a lack of association between vitamin E and gastric cancer. Although some studies have shown an inverse association between dietary intake or serum levels of vitamin E with colon cancer, others have failed to confirm these associations. Therefore, an association between vitamin E and colon cancer is uncertain. There have been no or very few studies on the associations between vitamin E and other gastrointestinal cancers, such as hepatocellular carcinoma or pancreatic cancers.

Introduction The role of vitamin E in chemoprevention of gastrointestinal cancers has been examined in several studies. The concept of chemoprevention, first proposed in 1976, refers to any intervention with natural or synthetic pharmacological agents to prevent the development of invasive cancer (Sporn et al., 1976). In proposing this concept, Sporn and colleagues based the rationale in the multi-stage model of carcinogenesis and the possibility of modifying either the earliest stages (initiation) or the later stages (promotion and progression) of carcinogenesis, and thereby preventing or postponing the occurrence of clinical cancer. For gastrointestinal cancers, a further rationale for chemoprevention can be found in the concept of field cancerization. The field cancerization concept, introduced in 1953, suggests that the process of carcinogenesis occurs over a large field of epithelia exposed to carcinogens and leads to multi-focal primary carcinomas (Slaughter et al., 1953). Reviewing 783 tumours of the upper aerodigestive tract, Slaughter and colleagues observed that: (i) in all cases, 922

the benign epithelium beyond the limits of the malignant tumour was abnormal; (ii) the majority of tumours were spread laterally rather than invading the deeper parts of the tissue; and (iii) second primary tumours had developed in 88 (11.2%) of the cases. The researchers concluded that ‘…surgically we are seldom beyond the limits of abnormal epithelium … radiation notoriously is less effective on benign epithelium and leukoplakia …eventually the prevention of the disease would appear to be a more practical method of control’. Field cancerization implies a multi-focal origin of cancers and the need for interventions that will have an effect across at-risk epithelial surfaces. The effect of vitamin E in preventing gastrointestinal cancers has been examined in observational and experimental studies in both animals and humans. The organs studied most extensively are the oesophagus, stomach and colon. The evidence reviewed includes animal studies, human observational studies and human experimental studies. All of the animal studies reviewed in this chapter are experimental studies conducted in mice and rats.

© CAB International 2007. The Encyclopedia of Vitamin E (eds V.R. Preedy and R.R. Watson)

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Gastrointestinal Cancers and Vitamin E

Gastrointestinal tracts of animals may have substantial differences from those of humans. For example, the oesophagi of rats and mice have substantially fewer epithelial cell layers than human oesophagus and, unlike human oesophagus, oesophagi of rat and mice are keratinized. Therefore, the results of animal studies should be generalized to humans with caution. Under human observational studies, we have discussed ecological studies, retrospective case–control studies, and prospective cohort and nested case–control studies. In ecological studies, high- and low-risk geographic areas for cancer are compared for dietary intake or serum levels of vitamin E. Although ecological studies help in generating hypotheses, these studies provide only weak evidence for an association. In retrospective case–control studies, typically cancer cases and controls are compared for their dietary intake of vitamin E using food frequency questionnaires (FFQs); cases are usually asked to report their usual diet prior to their disease. Evidence from case–control studies is considered stronger than that from ecological studies. However, case–control studies suffer from several methodological problems, such as recall bias; cancer cases may selectively recall intake of certain foods that they consider associated with their disease. Other methodological problems, including inaccuracies in response to FFQs and biased results due to confounding factors, are also possible. For example, subjects who report lower dietary intake of vitamin E may also have lower intakes of other vitamins and nutrients and their cancer may be related to these associated factors. Data from prospective cohort studies are considered strongest among observational studies. Healthy individuals are asked about their dietary intake using FFQs or 24 h recalls. Individuals who subsequently develop gastrointestinal cancers will be compared with those who do not. It is also possible to obtain baseline serum and compare baseline serum - or -tocopherol between those who do and those who do not subsequently develop cancer. Since in this study design, exposure data are collected before disease occurrence, cohort studies are free from recall bias. However, other problems such as confounding factors are still possible. Experimental studies in humans (clinical trials) are the strongest epidemiological studies to investigate the association between vitamin E and cancers. Several studies have randomized individuals to -tocopherol pills or placebo and have compared the incidence of gastrointestinal cancers between the study arms. Clinical trials do not suffer from recall bias or confounding. However, their results may be restricted to populations that are similar to the study population. In this chapter, we will review the evidence for the association between vitamin E and gastrointestinal cancers. Data will presented separately for oesophageal, gastric and colon cancers.

Oesophageal Cancer Oesophageal cancers are histologically classified into two categories: oesophageal squamous cell carcinomas and

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oesophageal adenocarcinomas. Oesophageal squamous cell carcinoma and oesophageal adenocarcinoma have different aetiological factors and are considered as two different types of cancers. Therefore, we will discuss them separately. Until 30 years ago, oesophageal squamous cell carcinoma was the dominant histological type of oesophageal cancer in all countries of the world, with >90% of all oesophageal cancer being of this type. In countries where oesophageal cancer is very common and most oesophageal cancer studies are conducted, such as China and Iran, oesophageal squamous cell carcinoma is still the predominant histological type of oesophageal cancer (Blot et al., 1993; Islami et al., 2004). Therefore, more studies have been conducted on the association between vitamin E and oesophageal squamous cell carcinoma and fewer on vitamin E and oesophageal adenocarcinoma. During the past 30 years, however, the incidence of oesophageal adenocarcinoma has dramatically increased in the USA and other Western countries (Brown and Devesa, 2002). In the USA, for example, oesophageal adenocarcinoma now counts for slightly over 50% of all oesophageal cancer cases (Devesa et al., 1998). Hence, the association between vitamin E and oesophageal adenocarcinoma is becoming more important.

Oesophageal squamous cell carcinoma There is consistent evidence that vitamin E reduces the risk of oesophageal squamous cell carcinoma. Animal studies have all shown that intervention with vitamin E reduces the risk of oesophageal squamous cell carcinoma. -Tocopherol reduced the risk of oesophageal squamous cell carcinoma in rats treated with Nnitrososarcosine by almost 50% (Bespalov et al., 1989). Dietary intake of vitamin E significantly reduced the number and size of N-nitrosomethylbenzylamide-induced oesophageal tumours in mice (Odeleye et al., 1992a, b). Also diet supplemented with high levels of vitamin E suppressed ethanol-induced promotion of tumours in mice possibly via inhibiting free radical formation (Watson et al., 1992; Eskelson et al., 1993). Ecological studies have suggested that nutritional deficiencies, including vitamin E deficiency, may be major aetiological factors in the aetiology of oesophageal squamous cell carcinomas, especially in high-risk areas of the world for this disease. High-risk areas of the world for oesophageal squamous cell carcinoma include Golestan province, Iran, and Linxian county, China. A low intake of vitamins was reported in high incidence areas of Iran (Hormozdiari et al., 1975). Similarly, laboratory analyses and dietary surveys confirmed poor nutrition and deficiencies in vitamins and micronutrients in Linxian (Yang et al., 1982); half the population were reported to be low or deficient for -tocopherol (Yang et al., 1984). Observational studies in humans have confirmed the association between dietary intake or serum levels of vitamin E with oesophageal squamous cell carcinoma. Several retrospective case–control studies used questionnaires to compare dietary intake of vitamin E between oesophageal cancer cases and control subjects (Table 87.1). These studies

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Table 87.1. Vitamin E and oesophageal squamous cell carcinoma. Study design

Number of cases/controls

Location

Exposure

Result

Tuyns et al. (1987)

Case–control

743/1975

France

Dietary intake (FFQ)

Barone et al. (1992)

Case–control

133/264

USA

Launoy et al. (1998)

Case–control

108/339

France

Vitamin supplement use Dietary intake (FFQ)

Terry et al. (2000)

Case–control

165/815

Sweden

Dietary intake (FFQ)

Franceschi et al. (2000)

Case–control

304/743

Italy

Dietary intake (FFQ)

Bollschweiler et al. (2002) Case–control

52/100

Germany

Dietary intake (FFQ)

Taylor et al. (2003)

Cohort

590/1053

China

Taylor et al. (2004)

Clinical trial

1515/~30,000

China

Serum -tocopherol and serum -tocopherol Intervention with a cocktail of vitamin E, selenium and -carotene

Dose-dependent risk reduction; 73% reduction comparing heavy versus low intake 60% risk reduction before, and 30% reduction after adjustment for other vitamins Dose-dependent risk reduction; 77% risk reduction comparing the highest versus lowest intake (>16 versus 90% risk reduction comparing the highest versus lowest intake (>17 versus 17 versus