New Research on Food Habits [1 ed.] 9781608764396, 9781604568646

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

NEW RESEARCH ON FOOD HABITS

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

NEW RESEARCH ON FOOD HABITS

KAITO HASEGAWA AND

HARUTO TAKAHASHI

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

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LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA New research on food habits / Kaito Hasegawa and Haruto Takahashi (editor). p. cm. ISBN 978-1-60876-439-6 (E-Book) 1. Food habits--Research. I. Hasegawa, Kaito. II. Takahashi, Haruto. GT2850.N49 2008 394.1'2072--dc22 2008023331

Published by Nova Science Publishers, Inc. Ô New York

CONTENTS

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Preface

vii

Chapter 1

Potential Use of Egg White Protein in Bitterness Suppression Kenji Maehashi

Chapter 2

What Should We Eat: Contradictory Researches and the Confused Consumer! Poonam C. Mittal

1

17

Chapter 3

The Effect of Diet on Human Bodily Odors Jan Havlicek and Tamsin Saxton

35

Chapter 4

The Natural History of Food Allergy in Infancy Kostas N. Priftis, Dimitrios Hatzis, Michael B. Anthracopoulos and Eva Mantzouranis

45

Chapter 5

Parental and Childhood Obesity: The Role of Food Habits Rena Kosti and Demosthenes Panagiotakos

65

Chapter 6

Scientificated Eating and Commodified Healthiness Mari Niva and Johanna Mäkelä

85

Chapter 7

“Energy Drinks” and Work Bartosz Bilski

Index

101 111

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

PREFACE Food habits are constantly being impacted by the perpetually increasing amount of information available to every person. Print and electronic media is full of advise regarding what should be eaten and why. Recommendations are often conflicting. Many foods are declared detrimental to health on one day and beneficial the next. The average consumer ends up being confused or even disillusioned by the scientific method. A major reason for the confusion is that any food item is a heterogeneous mixture of hundreds of compounds, and the effect of one isolated compound may be the opposite of another compound in the same food. Taking the case of chocolate, if studied for the effect of sugar, it is declared harmful, but when studied for its flavonoids, it is declared desirable because it contributes to reduction in oxidative stress. This book presents new research on the field of food habits and regarding the impact of some common foods on health. Chapter 1 - Bitter substances are generally avoided because they may be toxic. Nevertheless, excessively bitter foods must be avoided because they are less likely to be consumed even if they have a high nutritional value. Therefore, the food and pharmaceutical industry shares a common aim of developing debittering techniques. Several such techniques have been developed, e.g., use of bitterness inhibitors, treatment with enzymes that degrade bitter compounds, and encapsulation and entrapment of bitter substances. Recently, riboflavin-binding protein (RBP), which is a monomeric phosphorylated glycoprotein with a molecular weight of 35 kDa and a well-known source of riboflavin in developing chick embryos, was found to have unique taste-modifying characteristics. RBP has a selective sweet inhibition toward protein sweeteners and broadly tuned bitterness inhibition. The selective sweetness inhibition of RBP may be a useful tool for understanding the mechanism of sweet taste perception. On the other hand, bitterness inhibition of RBP is not only useful for basic research but also has practical industrial applications. Several bitter inhibitors are currently used in the industry; however, a natural protein-based bitterness inhibitor might prove more useful for several food applications where excessive bitterness is an issue. This paper first reviews debittering techniques and then describes the characteristics and potential applications of a novel bitterness inhibitor, RBP, in bitterness suppression. Chapter 2 - Food habits are constantly being impacted by the perpetually increasing amount of information available to every person. Print and electronic media is full of advise regarding what should be eaten and why. Recommendations are often conflicting. Many foods are declared detrimental to health on one day and beneficial the next. The average consumer ends up being confused or even disillusioned by the scientific method.

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

viii

Kaito Hasegawa and HarutoTakahashi

A major reason for the confusion is that any food item is a heterogeneous mixture of hundreds of compounds, and the effect of one isolated compound may be the opposite of another compound in the same food. Taking the case of chocolate, if studied for the effect of sugar, it is declared harmful, but when studied for its flavonoids, it is declared desirable because it contributes to reduction in oxidative stress. Confusion also arises due to the fact that findings regarding effect of various food components on health are based on a variety of experimental and empirical models. These include epidemiological and experimental studies on humans, animal models, and in vitro studies of several types, involving tissue culture, cell isolates, and organelle isolates. But what may work in a simple in vitro model may not be validated by a more complex living system with more elaborate feedback controls. Another reason may be that experimental studies generally employ much higher concentrations of an isolated compound than is found in the natural food. Adding to the confusion are questions regarding the absorbability of the active compound and the role and feedback responses of the recipient system, which is ultimately the human body as a whole. Further, differences may exist between short term and long-term feedback responses from the recipient system, thus affecting the practical application of a laboratory finding. The present review will seek to identify controversies regarding the impact of some common foods on health, study conflicting reports from existing research, and seek reasons to explain them in the light of the foregoing. It may finally help us to conclude that dogmas with regard to food habits are harmful, but may get reinforced if the average, educated consumer gets a feeling that research is always conflicting and therefore not to be relied upon. Chapter 3 - One of the most underestimated and poorly understood aspects of diet is its influence on human body odor. Humans have a distinct odor signature which arises from a combination of genetic and environmental factors. The link between genes and body odor is well appreciated, and an understanding of the influences of environmental factors such as emotional state, reproductive phase, and health status, is underway. Research into the impact of diet upon bodily odors, however, has been somewhat neglected. A recent study found that consumption of red meat for a period of two weeks decreased the pleasantness and increased the intensity of male axillary odor when judged by opposite-sex raters. Changes in bodily odors have also been linked to the consumption of food such as soya beans, plants from the Alliaceae or Brassicaceae families, strongly-flavored spices, fish, and milk-based products. This chapter critically reviews the literature on various aspects of food influencing human body odor and suggests several avenues for future research. Chapter 4 - Food allergy is primarily a problem of infants and young children with a decreasing prevalence in the years that follow. The natural history of specific food allergies varies substantially. Children who have become sensitized to cow’s milk, hen’s egg, wheat, and soybean through the gastrointestinal tract will usually lose this sensitivity as they grow older. On the other hand, certain food allergies, such as peanut, tree nut, and seafood usually continue throughout life. In this chapter the findings of a case control study of infants allergic to egg and/or fish followed to school age and the risk of wheezing illness and bronchial hyperreactivity (BHR) are presented and discussed. Sixty nine schoolchildren allergic to egg (N=60) and/or fish (N=29) in early life were recruited. They were followed for one year and were evaluated by parental questionnaire, skin prick testing, spirometry, and metacholine bronchial challenge. Another 154 (70 sensitized to inhaled allergens) were recruited selectively from a general

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Preface

ix

population sample with no history of food allergy during their first three years and served as controls. Twenty three children (38.3%) maintained their sensitization to egg and 19 (65.5%) to fish; the prevalence of sensitization to ≥1 inhaled allergen(s) increased from 59.4% to 71% during childhood. Current asthma symptoms were reported more frequently in the study group than in either control groups. Index cases showed more often BHR compared to the control group as a whole; the difference was statistically indicative when index cases were separately compared to the sensitized controls. Multivariate logistic regression analysis showed that BHR as well as reported current asthma symptoms were associated with early wheezing and early sensitization to inhaled allergens but not with atopic dermatitis in infancy or persistence of egg or fish allergy. Chapter 5 - The correlation that emerged between parental and adolescent’s BMI values shows an influence of both parents’ BMI on their offspring, probably due both to genetic conditions and unhealthy dietary and lifestyle habits [smoking, physical inactivity], indicating that familial disposition has to be taken into serious consideration in order to identify risk groups for preventive measures. Thus, a renewed focus on the family environment may provide information about behavioural factors that contribute to familial aggregation of obesity. Chapter 6 - Research on the links between nutrition and health has, in the past few years, yielded increasingly detailed knowledge about the ways in which health and wellbeing can be promoted by means of diet. While health has become a fundamental principle structuring life and one of the pillars of modern consumerism, healthiness has established itself as a conspicuous frame of reference in the debate surrounding food. The principles of healthy eating are part of the everyday knowledge regulating eating practices and notions of food. New research findings and advanced food technologies have led to the development of novel, healthier foods. In Finland, the case of our study, products known as functional foods first became known in the 1990s, when lactic acid bacteria drinks promoting the wellbeing of the stomach and cholesterol-reducing spreads appeared on the market. Functional foods are generally taken to mean products that promote health and reduce the risk of contracting a particular disease, though few countries have developed a precise definition of a ‘functional food’ in their national food regulations. Functional foods are a multidimensional phenomenon. They carry great expectations, but their targeted healthfulness differs in many ways from ordinary, healthy food. They are technological products developed specifically to be functional and to some extent they conflict with the ideal of ‘natural’ healthiness. Our chapter is based on the premise that functional foods do not become part of everyday eating in the way that ordinary foods do. Their controversial nature demands a different appropriation process in which functional foods acquire their meanings. By appropriation we mean both the conceptualisations, viewpoints and ideas relating to functional foods and the consumption of functional foods as part of the daily diet. Our article examines the conditions and prerequisites for appropriation. We begin by describing the commodification of healthy food and the grounds for and criticism of the development of functional foods. Drawing on Finnish empirical data we analyse how consumers appropriate functional foods and the significance of trust in this process. We then examine how targeted healthfulness, technology and science are domesticated into functional products. Finally we address the everyday knowledge about healthy eating and its relationship to targeted health-promoting products and the visions of bioscience.

x

Kaito Hasegawa and HarutoTakahashi

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Chapter 7 - Coffee and other “energy drinks” are consumed very commonly by workers for well-being and improvement of mental and manual efficiency at work. The author discusses the biochemical activity of the most popularingredients, the influence on mental and manual efficiency, and hazards connected with using these substances.

In: New Research on Food Habits Editors: K. Hasegawa and H. Takahashi

ISBN: 978-1-60456-864-6 © 2009 Nova Science Publishers, Inc.

Chapter 1

POTENTIAL USE OF EGG WHITE PROTEIN IN BITTERNESS SUPPRESSION Kenji Maehashi Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

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ABSTRACT Bitter substances are generally avoided because they may be toxic. Nevertheless, excessively bitter foods must be avoided because they are less likely to be consumed even if they have a high nutritional value. Therefore, the food and pharmaceutical industry shares a common aim of developing debittering techniques. Several such techniques have been developed, e.g., use of bitterness inhibitors, treatment with enzymes that degrade bitter compounds, and encapsulation and entrapment of bitter substances. Recently, riboflavin-binding protein (RBP), which is a monomeric phosphorylated glycoprotein with a molecular weight of 35 kDa and a well-known source of riboflavin in developing chick embryos, was found to have unique taste-modifying characteristics. RBP has a selective sweet inhibition toward protein sweeteners and broadly tuned bitterness inhibition. The selective sweetness inhibition of RBP may be a useful tool for understanding the mechanism of sweet taste perception. On the other hand, bitterness inhibition of RBP is not only useful for basic research but also has practical industrial applications. Several bitter inhibitors are currently used in the industry; however, a natural protein-based bitterness inhibitor might prove more useful for several food applications where excessive bitterness is an issue. This paper first reviews debittering techniques and then describes the characteristics and potential applications of a novel bitterness inhibitor, RBP, in bitterness suppression.

2

Kenji Maehashi

DEBITTERING TECHNIQUES Masking of Bitter Substances Because many drugs elicit a bitter taste, development of techniques for reducing bitterness is desirable, especially in the pharmaceutical industry. Currently existing effective methods for reducing bitterness of drugs include physical masking of the drug by coating with sugar or polymers [1], or chemical modification of the drugs into insoluble derivatives [2]. The merit of these methods is that the structure and biological activity of the bitter substance are not altered. A bitterness-masking method that uses spray-congealing with a spray dryer was developed by Yajima et al. [3]. This is an effective method of taste-masking as it is cost effective and requires no solvent. Additionally, it can produce a denser film than films made by other methods without drying or rearranging materials. They investigated a particle design for good release and satisfactory taste-masking, with manufacturing feasibility.

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Degradation of Bitter Substances Enzymatic degradation of bitter substances is a suitable method when the bitter principles and the specific enzymes that target them are known. Naringin is the main bitter component of citrus juices [4]. Degradation of naringin in citrus juices has obvious industrial implications. Naringinase, a debittering enzyme with α-rhamnosidase activity, splits naringin into rhamnose and prunin, which in turn is hydrolyzed by β-glucosidase into naringenin and glucose, resulting in a reduction in bitterness [5]. Many techniques for reducing naringin content in grapefruit juice with naringinase have been investigated [6-10]. Puri and Banerjee [11] reviewed naringinase and its essential role in the commercial processing of citrus fruit juice. Limonin is a bitter member of a group of limonoids that are chemically related to triterpene derivatives found in Rutaceae and Meliaceae [12]. The bitterness in juices due to limonin gradually develops after extraction from oranges and is referred to as delayed bitterness. Hasegawa et al. [13] and Brewster et al. [14] isolated an enzyme from Arthrobacter globiformis and Pseudomonas sp., respectively, which oxidizes the limonate Aring lactone to a nonbitter product, 17-dehydrolimonate A-ring lactone. The 17dehydrolimonate A-ring lactone exists for short time in juices before it is converted to limonin and does not attack the closed ring bitter limonin. Hasegawa et al. [13] showed that A. globiformis cells immobilized in acrylamide gel were capable of reducing the bitter limonin content in navel orange juice. Vaks and Lifshitz [15] immobilized Acinetobacter sp. bacteria, which are capable of growth using limonin as the sole carbon source, in a dialysis sac for removal of limonin from orange juice. They described that a total of 120 mg (dry weight) of bacteria is needed for the conversion of 1 L of bitter orange juice (at pH 4.5 containing 18 ppm of limonin) to drinkable juice. The fact that protein hydrolysates are accompanied by the formation of an intense bitter taste limits their use in the food processing industry. On the other hand, protein hydrolysates have many advantages in physiological functions, such as antihypertensive activity [16,17]

Potential Use of Egg White Protein in Bitterness Suppression

3

and antioxidative activity [18]. Murray and Baker [19] found that the hydrolysis of casein with various proteases caused an increase in the quantity of bitter peptides. Several attempts have been made to reduce the bitterness of protein hydrolysates, such as employing hydrophobic amino acid specific peptidases, because bitterness is correlated to the hydrophobicity of these peptides [20-22]. To remove bitterness from protein hydrolysates, hydrolysis with exopeptidases and the plastein reaction have been carried out. Minagawa et al. [23] found a highly thermostable aminopeptidase (aminopeptidase T), which had a broad substrate specificity but preferentially hydrolyzed hydrophobic amino acid residues at the N-terminal of peptides, from Thermus aquaticus YT-1. They investigated a technique for removal of the bitterness of subtilisin, papain, and trypsin hydrolysates of casein by further hydrolyzing the substrate with aminopeptidase T. Izawa et al. [24] identified an aminopeptidase from Aeromonas caviae T64 and discovered that this enzyme hydrolyses the peptides containing hydrophobic amino acid residues at the N-terminal or at an adjacent position with high efficiency. Tan et al. [25] investigated the degradation of a mixture of peptides, obtained from βcasein by trypsin hydrolysis, using general aminopeptidase (aminopeptidase N) from Lactococcus lactis subsp. cremoris WG2. They found that the degradation of the bitter tryptic digest by aminopeptidase N resulted in a decrease in hydrophobic peptides and a drastic decrease of bitterness of the same reaction mixture. Kodera et al. [26] analyzed the storage protein degradation process during soybean germination and obtained a novel cysteine protease D3 from germinating soybean cotyledons. The enzyme preferred hydrophobic amino acids with substrate specificity similar to cathepsin L and cathepsin K. Because of its substrate specificity, most hydrophobic amino acid residues in the D3 hydrolysates are presumed not to be located at the peptide terminals. The D3 hydrolysates were found to be significantly less bitter than the other enzymatic hydrolysates. Matsuoka et al. [27] obtained aminopeptidase II from Penicillium caseicolum, which has been used as a mold starter for manufacturing Camembert and Brie cheese. They showed that aminopeptidase II was able to decrease the bitterness of the peptide fractions from whole casein with peptic digestion. Umetsu et al. [28] isolated carboxypeptidase from wheat bran. The enzyme can eliminate bitter taste in enzymatic protein hydrolysates by releasing hydrophobic amino acids from bitter peptides.

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Removal of Bitter Substances from the Bitter Product Protein hydrolysates have been important not only in food but also in pharmaceuticals designed to supplement the nutritional requirements of patients who cannot consume or are allergic to certain dietary proteins. However, the bitter flavor of extensively hydrolyzed proteins is a major hindrance for its use in food as well as in pharmaceuticals. It is known that the bitter taste of peptides is attributed to their hydrophobic amino acid content [29]. To remove the bitter peptides from protein hydrolysates, a variety of hydrophobic materials have been investigated; e.g., talc [30], hexylsepharose [31], phenolic formaldehyde resin [32], active carbons, glass materials, hexylepoxy cellulose [33], octadecyl siloxane (C18) column [34], etc.

4

Kenji Maehashi

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Specific Bitterness Inhibitors Several bitter inhibitors have been reported; e.g., sodium salts [35], zinc sulfate [36], lipoprotein [37], flavanones [38], and benzylamides [39]. Breslin and Beauchamp [35] investigated taste interactions between salts and bitter compounds. They found that perceived bitterness was suppressed by salts in most cases, and the salt suppressed the bitterness of urea effectively. They proposed that a key component in the effect was sodium or lithium ion. Keast et al. [40] examined the influence of a variety of cations and anions on the bitterness of selected oral pharmaceuticals and bitter taste stimuli. They concluded that sodium was the most successful cation, and glutamate and adenosine monophosphate were the most successful anions at inhibiting bitterness. Konno et al. [41] reported on the bitterness reduction of naringin and limonin with βcyclodextrin. They showed that the bitterness reduction was due to the formation of an inclusion complex between β-cyclodextrin and naringin or limonin. Ley et al. [38] reported that the flavanones—homoeriodictyol, its sodium salt, and eriodictyol isolated from Herba Sanata—showed remarkable bitter masking effects without exhibiting any additional strong taste or flavor. Furthermore, they found that hydroxybenzoic acid vanillylamides were able to reduce the bitterness of caffeine with 2,4-dihydroxybenzoic acid vanillylamide as an inhibitor of caffeine bitterness, demonstrating a clear dose-dependent activity [39]. Katsuragi et al. [42] found that a lipoprotein consisting of phosphatidic acid (PA) and βlactoglobulin (LG) completely suppressed the taste responses to the bitter substances without affecting the responses to other taste stimuli such as salts, acids, and sugars in experiments using both frogs and humans. They examined the inhibitory activities of lipoproteins composed of various lipids such as phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylcholin (PC), diacylglycerol (DG), triacylglycerol (TG), and various proteins such as α-lactalbumin (LG), bovine serum albumin (BSA), ovalbumin (OVA), and casein, and found that the lipoproteins containing PA were most effective in inhibiting bitterness and those containing PS were moderately effective [43]. They prepared lipoproteins composed of LG and various lipids and examined their effects on the bitterness of 0.5 mM quinine. Results showed that the bitterness decreased with increasing PA-LG concentration, whereas lipoproteins of LG with other lipids only partly suppressed the bitterness. However, LG itself had only a small suppressive effect on the bitterness of quinine and no effect on that of caffeine. PA alone is not soluble in water; however, PA-LG is easily dispersed in water and produces a transparent solution with only slight turbidity. A 3.0% solution of PA-LG greatly suppressed the bitterness of propranolol, promehtazine, quinine, denatonium, glycyl-Lleucine, caffeine, L-phenylalanine, naringin, and theophylline. To test the possibility that the suppression was partly caused by the direct interaction of the bitter substances with PA-LG in the medium when tasted in its presence, the binding of the bitter substances to PA-LG was measured. Bitter substances were dissolved in 3.0% PA-LG solution, and the resultant solutions were filtered with 5000 MW cut-off filters by centrifugation. The percentage of the quantity of a bitter substance in the filtrate with respect to the total quantity of the substance indicated that propanolol, promethazine, quinine, brucine, and strychnine were mostly bound to PA-LG in the medium.

Potential Use of Egg White Protein in Bitterness Suppression

5

EGG WHITE PROTEIN FOR TASTE MODIFICATION

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Survey of a Taste-Modifying Protein in Egg White Proteins have important functions not only as nutritious components of food but also of altering many physicochemical properties of foods [44], such as their viscosity, gelation, cohesion, adhesion, elasticity, emulsification, and foaming abilities. Because almost all proteins are apparently tasteless themselves, it is believed that proteins contribute to the palatability of food only through their textural effects. However, several researchers have found that lysozyme, a bacteriolytic enzyme [45] in egg white, has a sweet taste [46]. Most other sweet proteins such as monellin [47], thaumatin [48], and brazzein [49] have been discovered from tropical fruits. This would suggest that proteins having taste functions may be more common than previously thought, although we have never found that egg white itself is sweet in spite of 0.3% lysozyme content [50], which is enough to elicit sweetness. This might be attributed to the presence of a sweet-inhibiting substance in egg white. There are several taste-modifying proteins found in tropical fruits, such as miraculin [51] and curculin [52]. Miraculin transforms sour taste into sweet taste, and curculin induces a sweet taste with water. However, it is important to discover a taste-modifying protein in common and widely consumed foods because that information could help us understand the interaction or the competition between food and its components and how they affect the taste of food. Maehashi et al. [53] surveyed sweet-suppressing proteins from egg whites because egg whites are not sweet even though they contain lysozyme, a sweet protein. Egg white contains mainly ovalbumin and small amounts of lysozyme and trypsin inhibitor. Using ion-exchange chromatography with CM-Sepharose and DEAE-Sepharose gel columns, they separated the egg white protein preparation into 5 fractions: flow through, CM-1, CM-2, DEAE-1, and DEAE-2. The fractions were desalted and lyophilized for sensory evaluation. CM-2 elicited a strong sweetness because of the presence of lysozyme, which was confirmed by an assay for its bacteriolytic activity. The sweetness of egg white is undetectable unless egg white is fractionated and the individual components are concentrated. The other four fractions were substantially tasteless. The 4 fractions—flow-through, CM-1, DEAE-1, and DEAE-2—were examined for their sweet-masking effect on the sweetness of lysozyme. Although the DEAE-1 and CM-1 fractions had no effect on the taste of lysozyme, the DEAE-2 fraction suppressed the sweetness of 0.2% lysozyme completely. Therefore, the DEAE-2 fraction probably contained a protein having sweet-suppressing activity. We further purified the DEAE-2 fraction, by hydrophobic chromatography on a Phenyl Sepharose CL-4B column, to identify it. The peak fraction containing the sweet-suppressing activity was collected and isolated further by gel filtration chromatography. After the active fraction was dialyzed, a yellow colored protein was obtained, which appeared as a single band on SDS-PAGE with an estimated molecular weight of 35 kDa. Given its yellow color and molecular weight, it was hypothesized to be a riboflavin-binding protein (RBP). To confirm this hypothesis, a partial amino acid sequence was determined by sequencing the peptide fragments from digests of V8 protease and trypsin. Because the sequences of the fragments

6

Kenji Maehashi

were identical to that of RBP, the sweet-suppressing protein purified from chicken egg white was identified as RBP. RBP percentage in chicken egg white is 0.09% [54], which is relatively abundant amongst egg white proteins. However, this concentration of RBP is not enough to suppress the sweetness of lysozyme, which is present at 0.3% [50] in egg white. This is based on the result of Maehashi et al. [53], who showed 0.35–3.5% of RBP was required to suppress the sweetness of 0.14% lysozyme. Egg white contains a number of proteins as indicated by Rhodes et al. [55], who fractionated egg white into 14 peaks on CM-cellulose column. These proteins include ovomucoid, flavoprotein, ovalbumin, globulin, conalbumin, avidin, and lysozyme. Because interactions between lysozyme and other proteins such as ovomucin, a high molecular weight glycoprotein in egg white, are known to occur [56], it is likely that other proteins may play a role in masking the sweetness of lysozyme in egg white besides RBP. If Maehashi et al. [53] had not separated the egg white, the existence of RBP, a protein suppressing the sweetness of lysozyme, would not have been identified.

Selective Sweet Suppression

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Several non-protein sweet inhibitors such as lactisole [57], gymnemic acid [58], ziziphin [59], hodulcin [60], and zinc sulfate [61] have been identified and studied. Most of the sweet inhibitors are effective against any sweetener including different sugars and their derivatives, terpenoids, peptides, and proteins. However, Schiffman et al. [62] reported that lactisole suppressed the sweetness of sucrose, aspartame, and stevioside, whereas it did not suppress the sweetness of thaumatin, a protein sweetener. Moreover, Keast et al. [61] found that zinc sulfate inhibited the sweetness of most compounds used in their experiments, whereas it did not inhibit the sweetness of Na-cyclamate. These observations suggest that the sweet inhibitors exhibit selectivity. A difference in the mechanism of inhibition among the sweeteners could explain the selectivity. Interestingly, RBP was found to be effective in inhibiting the sweetness of proteins such as lysozyme, monellin, and thaumatin but not the sweetness of low-molecular sweeteners such as sucrose, aspartame, cyclamate, saccharin, stevioside, D-phenylalanine, and glycine [53]. Therefore, RBP appears to be a sweet inhibitor for protein sweeteners only.

Structure and Physicochemical Property of RBP RBP in chicken egg white is the first riboflavin specific carrier to be identified [63], and thus, the most extensively studied. It is a monomeric phosphorylated glycoprotein [64] consisting of 219 amino acid residues with 9 disulfide bonds [65]. It binds one molecule of riboflavin [66] and is involved in the transport of riboflavin from the serum compartment into the chicken oocyte and from the oviduct into the egg white [67]. It is known that the family of chicken RBP includes 3 proteins from different organs: egg white, egg yolk, and plasma. These proteins are coded for by the same genes, but they undergo different post-translational modifications. Egg white RBP is synthesized in oviduct, plasma RBP is synthesized in liver, and egg yolk RBP results from proteolytic cleavage of the C-terminal 11 or 13 amino acids

Potential Use of Egg White Protein in Bitterness Suppression

7

from plasma RBP. The amino acid sequence of egg yolk RBP is identical to that of egg white RBP, except that the C-termini of these proteins are different [68]. It is also known that egg yolk RBP has the same characteristics as white RBP, such as carbohydrate chains attached to both Asn36 and Asn147 residues and phosphate groups bound to serine residues in the sequence from Ser85 to Ser197 as a cluster [64]; however, carbohydrate composition in yolk RBP differs from that in white RBP [68]. Maehashi et al. [53] showed that yolk RBP exhibited sweet-suppressing activity with the same selectivity toward protein sweeteners; however, the activity was lower than that of egg white RBP. The differences between the 2 RBPs are the C-terminal amino acid sequence and the structure of oligosaccharide chains. These structures might have a relationship to the sweet-suppressing activity; however, that relationship is not likely to be essential because the structural differences result in only a reduction of activity, not a complete loss.

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Broadly Tuned Bitter Inhibition Maehashi et al. [53] accidentally found that RBP exhibited not only selective inhibition toward protein sweeteners but also inhibited the bitterness of quinine. RBP inhibited bitterness of 0.125 mM quinine; however, it did not inhibit the sweetness of 0.15 M sucrose, the saltiness of 0.15 M sodium chloride, the umami quality of 25 mM monosodium glutamate, or the sourness of 10 mM citric acid. Thus, RBP acts as a selective inhibitor of quinine bitterness [69]. Besides inhibiting the bitterness of quinine, RBP inhibits the bitterness of various other bitter compounds. Six bitter compounds were chosen as representatives of the bitter principles found in food, i.e., naringin for grapefruits, caffeine for coffee, glycyl-L-phenylalanine for protein hydrolysates (a model for fermented food), and theobromine for cacao. Denatoniuim benzoate and quinine hydrochloride were chosen as representatives of bitter compounds found in pharmaceuticals. To examine the effect of RBP on compounds with the same level of bitterness, concentration of each bitter compound equivalent to the moderate bitterness of 0.125 mM quinine was determined [53]. The bitterness of all bitter compounds including 1 mM naringin, 0.1 μM denatonium, 14 mM caffeine, 60 mM glycyl-L-phenylalanine, 6.5 mM theobromine, and 0.125 mM quinine was inhibited by RBP in a concentration-dependent manner. Even at higher concentration of quinine such as 0.25 mM and 0.5 mM, 1 mM RBP completely suppressed the bitterness. Selectivity was not observed in the inhibition of bitterness unlike the inhibition of sweetness of protein sweeteners. RBP thus showed a broad selectivity of bitterness inhibition towards structurally diverse compounds.

Relationship between Bitterness Suppression and Structure of RBP To examine the specificity of RBP as the bitterness suppressor, three commercially available proteins—ovalbumin, α-lactalbumin, and bovine serum albumin—all tasteless themselves, were compared with RBP for their effect on the bitterness of 0.125 mM quinine or 1 mM naringin [69]. Ovalbumin, α-lactalbumin, and bovine serum albumin did not substantially affect the bitterness. These results indicate that bitterness inhibition is not a common attribute of all proteins; instead, it is a relatively unique property of RBP. Katsuragi

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et al. [43] showed that the bitterness of 0.5 mM quinine was greatly suppressed by a lipoprotein composed of phosphatidic acid and α-lactalbumin but not by α-lactalbumin alone. They suggested that for α-lactalbumin to suppress the bitterness of quinine, complex formation with phosphatidic acid is required. The binding of riboflavin occurs between Tyr75 and Trp156 of RBP [70]. Because no difference was detected in the activity of the apo- and holo- forms of RBP, it is thought that riboflavin itself does not elicit bitter inhibition, and the riboflavin-binding site within RBP molecule does not participate in this activity. RBP is contained not only in egg white but also in egg yolk, as mentioned previously. There are some minor differences between the RBPs found in egg white and yolk. The egg white and yolk RBPs were compared for their effect on the bitterness of 0.125 mM quinine [69], and it was found that yolk RBP also inhibits the bitterness but the concentration for complete inhibition is 5 times higher than that required for egg white RBP. This difference in bitterness inhibition may be due to the difference in the C-terminal structure or the difference in the oligosaccharide chain between them. Because both RBPs inhibit bitterness, the Cterminal of RBP does not play a role in bitterness inhibition. RBP is phosphorylated at Ser185 to Ser197 as a cluster [64]. These phosphate groups have some physiological functions such as transportation of RBP to the oocyte [71]. The role of the phosphate groups in bitterness inhibition was examined [69]. Dephosphorylation of RBP was conducted using acid phosphatase, and resultant dephosphorylated RBP was isolated by anion exchange chromatography. Although dephosphorylated RBP showed different electric charge on native polyacrylamide gel electrophoresis, its bitterness inhibition was almost the same as native RBP. The negative charges of phosphate groups of RBP do not participate in bitterness inhibition.

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Possible Mechanism of Inhibition of Sweetness and Bitterness The detection of sweetness of most or all sweet tasting compounds is mediated by a heteromeric receptor complex composed of T1R2 and T1R3, a family of G protein-coupled receptors selectively expressed in subsets of taste receptor cells [72]. Lactisole suppresses the sweet taste perception by humans but not by rats [73]. Recent experiments with heterologous expression of the human/mouse chimeras of T1R3 suggest that the molecular basis for species-specific sensitivity to protein sweeteners and lactisole depend on a site within the human T1R3 molecule [74,75]. The sweet taste receptor is suggested to have a lactisolesensitive and a lactisole-insensitive site. RBP is an acidic protein with isoelectric pH 3.9–4.1 [54] attributed to its phosphate groups. RBP exhibits selective inhibition toward basic protein sweeteners, with isoelectric pH 9.3–11.7 [76]. Therefore, there was a possibility that the sweet inhibition was the result of the direct interaction of the sweet compounds with RBP. To examine the molecular interactions involved, a mixture of RBP and lysozyme was run on the Sephadex G-75 gel permeation chromatography column and its elution profile was compared with each of those eluted singly [53]. A mixture of 1 mM RBP and 0.1 mM lysozyme showed neither a shift nor a complex of peaks suggesting that there is no chemical interaction between lysozyme and RBP. This result indicates that RBP interacts with the sweet receptor site on the taste cell and interferes with the access of the sweet protein. The property of RBP that inhibits sweetness of protein

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Potential Use of Egg White Protein in Bitterness Suppression

9

sweeteners but not of sugars is opposite to that of lactisole; however, it is consistent with the existence of multiple interaction sites within a single sweet receptor. Another general property of proteins is hydrophobicity. Because most of the bitter compounds are hydrophobic [43], there is a possibility that the bitter inhibition is the result of hydrophobic interactions of bitter compounds. Thus, the possible binding of the bitter compounds with RBP was examined [69]. The bitter compound solution and the mixture solution with RBP were filtered with 10,000 MW cut-off filters by centrifugation, followed by comparison of the concentrations of bitter substances in both filtrates. If the bitter compound binds RBP, the complex should not pass through the membrane. The results showed that 14 mM caffeine, 1 mM naringin, 6.5 mM theobromine, and 60 mM glycyl-Lphenylalanine, all of which were inhibited by 2 mM RBP, did not substantially bind to RBP, whereas quinine was bound to 0.2 mM and 0.5 mM RBP, respectively. Quinine was the only compound bound to RBP among bitter compounds. Interestingly, ovalbumin, which did not exhibit bitter inhibition, bound to quinine as well as RBP did. These results indicate that the binding of quinine with proteins does not correlate well with bitter inhibition. Instead, they suggest that masking on the tongue surface, probably at the level of the bitter receptor cells, is more likely to be responsible for the bitterness inhibition by RBP. Bitter taste is detected by members of approximately 30 divergent G protein-coupled receptors, termed T2Rs [77,78]. Ligands of some of the receptors have been reported, e.g., human (h) T2R4 responds to denatonium [79], hT2R16 responds for salicin [80], etc. [81-83]. Each bitter compound may have the highest affinity for a specific T2R receptor. However, the binding sites of bitter receptors have not been identified. It is likely that there are several specific binding sites for each bitter compound in the T2R receptors. Because RBP inhibited only the bitter taste modality and several other proteins did not alter bitterness, the interaction of RBP with receptor cells appears to be rather specific. For hydrophobic quinine [43], there would be an interaction between RBP and quinine that prevents any interaction with the bitter receptor cell to inhibit bitterness. Katsuragi et al. [43] suggested that lipoprotein PA-LG, which is composed of phosphatidic acid (PA) and βlactoglobulin (LG), was effective in suppressing the bitterness of quinine, promethazine, and brucine. This is mostly due to the binding of these compounds to PA-LG by hydrophobic interactions. However, they proposed that the bitter suppressive effects of RBP on caffeine, glycyl-L-leucine, and naringin are probably caused by masking of the target sites on the tongue for bitterness with PA-LG. RBP is likely to have properties similar to PA-LG. The bitterness of quinine was only inhibited by RBP, although quinine bound to both RBP and ovalbumin; this might be because of the higher affinity of RBP to the bitter receptor sites than that of ovalbumin. Additionally, RBP binds to bitter receptor sites with a higher affinity than quinine does. The mechanism of bitter inhibition of RBP is therefore a competition for binding to bitter receptor sites with bitter compounds. In the case of quinine, it includes the direct binding and sequestering of quinine. RBP exhibits a broad range of bitter inhibition toward structurally diverse bitter substances but does not inhibit other taste modalities. Therefore, it is likely that RBP interacts with bitter receptor proteins or bitter receptor cells with a high affinity to inhibit access of any other structurally diverse bitter substances to them.

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Perspective for Practical Use of RBP in Bitterness Inhibition When a novel substance is considered for use as a food additive, it is important to assess its safety first and then its economic advantage. RBP is present in egg white naturally and has been consumed safely since prehistoric times. However, the evaluation of the influence of high dose of RBP is still necessary because much higher amount than that present in egg would be required for complete inhibition of bitterness. RBP is obtained from egg white using a simple procedure, which was established by Miller et al. [84] and partially modified by Maehashi et al. [53]. This procedure includes sonication, batch adsorption to DEAE-cellulose (or DEAE-sepharose), salt precipitation, and DEAE-cellulose (or DEAE-sepharose) chromatography. These steps result in a pure preparation of yellow holoprotein of RBP. On the other hand, the preparation using salt precipitation adjusted at pH 3.14 and adsorption on a CM-cellulose (or CM-sepharose) column, followed by elution at pH 5.8, produces colorless apoprotein of RBP [84]. Holo-RBP has an advantage in practical use in food, in that it supplies riboflavin as vitamin B2, because there is no difference between holo- and apo-RBP in bitterness inhibition. One of the major problems for use of proteins in food processing is their stability, especially thermal stability. RBP has 8 disulphide bonds, which confer heat stable properties to it [84]. Maehashi et al. [69] do not make any mention of the thermal stability of RBP while describing its bitterness inhibition.

CONCLUSION

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Several techniques are used for the inhibition of bitterness in food and pharmaceuticals. One of the effective tools is the use of bitterness inhibitors. Among the various bitterness inhibitors that have been developed to date, RBP is the only one that is a simple protein naturally found in ordinary food. It has many advantages over previously developed inhibitors, such as its broad specificity to bitter compounds, ease of availability, and safety. The discovery of RBP as a bitterness inhibitor implicates a novel function of food proteins in modifying the sensory properties of food. This will stimulate further studies on the sensory functions of food proteins, which had previously not been considered. It will also provide us valuable information for understanding how taste stimuli work within the complex matrix that is food.

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In: New Research on Food Habits Editors: K. Hasegawa and H. Takahashi

ISBN: 978-1-60456-864-6 © 2009 Nova Science Publishers, Inc.

Chapter 2

WHAT SHOULD WE EAT: CONTRADICTORY RESEARCHES AND THE CONFUSED CONSUMER! Poonam C. Mittal* Biochemistry Department, University of Allahabad, Allahabad – 211002, India

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ABSTRACT Food habits are constantly being impacted by the perpetually increasing amount of information available to every person. Print and electronic media is full of advise regarding what should be eaten and why. Recommendations are often conflicting. Many foods are declared detrimental to health on one day and beneficial the next. The average consumer ends up being confused or even disillusioned by the scientific method. A major reason for the confusion is that any food item is a heterogeneous mixture of hundreds of compounds, and the effect of one isolated compound may be the opposite of another compound in the same food. Taking the case of chocolate, if studied for the effect of sugar, it is declared harmful, but when studied for its flavonoids, it is declared desirable because it contributes to reduction in oxidative stress. Confusion also arises due to the fact that findings regarding effect of various food components on health are based on a variety of experimental and empirical models. These include epidemiological and experimental studies on humans, animal models, and in vitro studies of several types, involving tissue culture, cell isolates, and organelle isolates. But what may work in a simple in vitro model may not be validated by a more complex living system with more elaborate feedback controls. Another reason may be that experimental studies generally employ much higher concentrations of an isolated compound than is found in the natural food. Adding to the confusion are questions regarding the absorbability of the active compound and the role and feedback responses of the recipient system, which is ultimately the human body as a whole. Further, differences may exist between short term and long-term feedback responses from the recipient system, thus affecting the practical application of a laboratory finding. The present review will seek to identify controversies regarding the impact of some common foods on health, study conflicting reports from existing research, and seek reasons to explain them in the light of the foregoing. It may finally help us to conclude *

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Poonam C. Mittal that dogmas with regard to food habits are harmful, but may get reinforced if the average, educated consumer gets a feeling that research is always conflicting and therefore not to be relied upon.

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INTRODUCTION In the early stages of evolution, eating was a biological act, without any capacity for thinking about what should be eaten and what should be avoided. Selection pressures must have favored those who got to be programmed with better ability to eat desirable foods and avoid undesirable ones. At some stage, the capacity must have developed for observing the effects of eating different things, memorizing, and passing the information so gained to others, particularly to offspring. Natural selection would now have favored those who developed this capacity better. It is no wonder that eating habits form an important component of all cultures. Probably in all cultures, food habits, though different, center around the consumption of cereals and legumes together, making full use of the complementary nature of their amino acid composition. The ancient seers must have deduced the importance of various foods and their combinations by keen observation. Records of some of these have been found, the Vedas being among the earliest records which originated in the last years of the second millennium and the early years of the first millennium BC. They contain much food related information as part of the indigenous Indian system called ayurveda. Nonetheless, both the biological and the cultural determinants of food habits are inadequate in today’s world. There are several reasons for this. Biologically, we are tuned to living in a food scarce world. By and large, cultural habits have also evolved for such a world. However, for large numbers of people in today’s world, any amount of food is available just for the asking, without any struggle, without any realistic fear of having to go hungry the next day. Biological adjustment cannot be expected to evolve in a short time. Cultural habits have also barely changed to accommodate this new reality. As a result, there are more people suffering from an excess of food and consequent obesity than from the problem of scarcity and hunger. Another recent phenomenon is that the modern world has a huge food processing industry which introduces artificial foods and molecules at a very rapid rate, compared to the slow assimilation of food habits as an evolutionary process. Then there is the pronounced intermingling of cultures due to movement of people and food products. More and more people are enjoying foods of other cultures. It is not clear what effects food from other cultures has, when taken by a body that has evolved in a different niche. Clearly, in the modern world, neither biology (my taste buds must have evolved to know what is best for me) nor conventional wisdom about eating practices (our society has learnt the right eating practices over centuries best suited to our environment) can provide adequate guidance. Today science is an important component of human culture. The scientific method is expected to provide objective, bias free and reliable guidance to modify our food habits in order to live a healthier life.

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Lavoisier who lived in the 18th century is credited with having laid the foundations of modern nutrition. Through the late 19th century till the early part of the 20th century, major developments took place in the area of nutrition, linking observations of deficiency symptoms to possible causes, leading to discovery of many vitamins. This was followed by more quantitative concepts to answer questions related to how much of each nutrient is required, and nutrition became a more organized science. This involved dependence on other sciences such as physics and technology for hitherto unknown methodologies, and interaction with newly developing subjects such as biochemistry for understanding the mechanisms by which food functioned in the body. A large part of the 20th century had seen war, famine and strife. Naturally, nutritional studies focused on food shortage and nutrient deficiencies. It was only towards the seventh decade that the links between affluence, obesity and consequent harmful effects on health became important issues requiring attention. The advent of the internet and its easy availability for the common man in the last two decades, has led to unprecedented access to information. Advice regarding what should be eaten and why, occupies substantial space in print and electronic media. Food habits are constantly being influenced by the perpetually increasing amount of information. Despite this, or because of it, there are more dogmas and misconceptions related to food and diet related issues than to most other areas of scientific study. Recommendations are often conflicting, so that many foods are declared detrimental to health on one day and beneficial the next. To inculcate rationality with regard to food habits, and to ensure that the average consumer is not disillusioned by the scientific method, it is desirable to examine some of the reasons for this trend. Scientific analysis of food related questions can be divided into two approaches. The first approach deals with analysis of different food items as chemical compounds and understanding the mechanisms and processes that take place when these compounds enter the body. The second approach is empirical. It tries to study the effects of a particular food on a large experimental group, as compared to a control group using statistical analysis. Of course, the two approaches interact with each other, knowledge gained from one aiding the design and interpretation of the other.

EVERY FOOD IS A MIXTURE OF ‘HEALTHY’ AND ‘UNHEALTHY’ COMPOUNDS: Conventional wisdom was mostly based on observation, propagated by folklore and handed down generations. Much of folklore may not withstand the test of modern scientific methods, for example, the classification of foods into hot and cold categories. This concept is found in many cultures such as Indian, Chinese and Latin American, but agreement with regard to details is lacking. For example, rice is considered cold in the Indian ayurvedic system but neutral in the Chinese system [1]. On the other hand, the utility of some traditional preparations like Chyawanprash, has been vindicated by present methods of science. Chyawanprash, an ayurvedic preparation, said to have been assembled by ancient sages two millenia ago, has the Indian gooseberry as its main ingredient, now known to be one of the richest sources of ascorbic acid, and also contains scores of herbs which contribute to the

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micronutrients required by the body. It has been found to prevent steroid-induced cataracts [2] induce a greater beneficial effect on glucose tolerance and lipoprotein profile as compared to vitamin C alone [3]and show genoprotective efficacy on smokers [4. Developments in chromatographic, colorimetric, spectroscopic, microbiological and many other techniques have led to an understanding of biological phenomenon and biochemical mechanisms. These have found widespread applications to study foods and their effects on the body. They have also led to better methods of analysis and characterization of large numbers of compounds in a food. One of the major reasons for confusion with regard to benefits or harmful effects of any food is that almost every food item is a heterogeneous mixture of hundreds of compounds, and the effect of one isolated compound may be the opposite of another compound in the same food. Therefore, the reported desirability of a particular food would depend on which of its components has been investigated. Another investigation based on a different component could lead to a contrary recommendation. Also, if a food is a mixture of several compounds, and the experiment is designed to ensure that intake of just one of these compounds is different, the effect attributed to that compound may actually be because the compound under study interacts with some ingredient in the food to produce the effect but may not function if taken alone as a supplement. Such fluctuating reports can leave the general public confused. Chocolate is just one example that can help explain this statement. Chocolates were long condemned as junk foods, bad for the teeth, major causative for dental caries, an unhealthy source of empty calories, full of sugar and saturated fat. Then it came to be known that chocolates are a rich source of polyphenols such as (-)-epicatechin and (+)-catechin, and their oligomers called procyanidins, all of which belong to the class of plant compounds known as flavanols. So they started being investigated for their possible healthy properties. This was because the health promoting properties of vegetables and fruit were attributed to the various flavonoids in them [5] Flavonoids belong to a class of compounds known as polyphenolics. There are thousands of polyphenolics in the plant kingdom, of which flavonoids are the most abundant [6] Soon there was a surge of studies to investigate the relationship between flavonoids in chocolate and health. The early years of this decade reported their role in protection against cardiovascular diseases [6], hypertension [7] nd diabetes [8]. Chocolates have also been recognized as ‘feel good’ food because of their phenylethylamine content, which however, has also been implicated in the migraine triggering effect of chocolate. According to a 1998 report [9] nalysts have detected more than 300 chemicals in chocolate. It is not clear whether this includes the polyphenols. Chocolates also contain alkaloids caffeine and the chemically similar theobromine [10] affeine has been implicated as the reason of popularity of several drinks such as tea, coffee and colas because it raises heart rate, blood pressure, and stimulates the brain by raising dopamine. Theobromine has been implicated in the cough controlling function of chocolate as well as in its anti-hypertensive function [11,12] Chocilate also contains saturated fat and cholesterol, long considered harmful. However, it is now reported that cocoa butter which is the saturated fat in chocolate, does not raise bad cholesterol and is actually beneficial because it prevents the chocolate from sticking to teeth and causing dental caries. Moreover, it has a melting point just below body temperature at 350 C, which gives the melt-in-the-mouth quality to the final product. Apart from the constituents of the cocoa bean, chocolate also contains milk solids and sugar, so any attributes of chocolates have to be examined in the total product. Chocolates, as

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marketed, are also said to promote pimples and acne because of the milk fat that they contain, but since cocoa contains an abundance of antioxidant properties, the overall effect on the skin remains an open question [13]. The high sugar content of chocolates has also been a cause for concern due to a large number of detrimental effects attributed to sugar [14, 15, 16]. However, the conclusions of a workshop [17] in 2003 on ‘Sugars and Health’ failed to establish health concerns for which a direct association with sugar could be established. The only confirmatory evidence available at reasonable intakes of sugar was its link to an increased risk and incidence of dental caries. At high levels of intake of more than 125 g (the equivalent of 25 teaspoons) on a 2000 kcal diet, sugar was found to result in lowering of micronutrient intake (especially calcium). The final verdict was that “negative energy balance (for example, sedentary lifestyles) are more important in weight management, insulin sensitivity, and blood lipid concentrations than is the inclusion or exclusion of any particular dietary component, including sugars.” In this context, chocolate is also a calorie-rich food with a high fat content, so daily intake of chocolate requires reducing caloric intake of other foods. It is pertinent to note that the beneficial effects of chocolate on insulin sensitivity and blood pressure, described by Grassi and associates [8], were on intakes of 100g of dark chocolate, providing 480 kcal. The controversies continue, even as a recent study has reported that older women who consumed chocolate daily had lower bone density and strength [18]. These are just a few of the studies, based on some of the compounds in one food product which demonstrate how research findings can confuse a consumer. The Indian food table [19] lists about six hundred raw foods items, which can be combined in thousands of ways. Each food, be it wheat, rice or one of hundreds of vegetables, contains thousands of compounds, which can be studied for their individual properties, and if the findings of each of these studies makes media headlines, the ensuing confusion can well be imagined. In this context, it is noteworthy that there is an explosion of research studies that are easily accessible to scientists as well as others, due to the internet. More than 15 million citations are available in MEDLINE, and 10 to 20 thousand are added every week [20]. These studies have varying methodology, are conducted on different populations, use differing test conditions and interventions [21]. Even highly accepted studies are refuted by subsequent work by the same and/or other investigators [22], so that experts are also confused by different answers to the same question. Other reasons for complications with regard to research findings that have been extensively discussed elsewhere include the biases introduced due to financial and/or career interests of the researchers, the funding agencies and the food industry [23, 24, 25, 26].

VARIED MODELS OF RESEARCH MAKE SIMILAR EYE-CATCHING HEADLINES Confusion also arises because findings regarding effect of various food components on health are based on a variety of diverse experimental models and empirical studies. Experimental models may involve in vitro techniques, where the experiment is performed in a controlled environment, for example in cell isolates or organelle isolates, or where the cell is made to grow outside the living organism in tissue culture. Since test conditions may not

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correspond to those inside a living organism, results may not always replicate in vivo conditions. Studies may also be conducted in situ, which is intermediate between in vivo and in vitro conditions, for example in a cell within the intact organ, but after the animal is sacrificed, or ex vivo, where experimentation is done in or on living tissue in an artificial environment outside the organism. Experiments are also conducted on non-human animals such as rats, mice, dogs or guinea pigs, and results applied to humans, because there is large physiological similarity between them. Such studies enable researchers to perform experiments that are not ethical or feasible in humans. However, in all these models, there is the element of extrapolation. Studies involving humans are typically empirical, where research is based on evidence and not just theory. These include limited experimental studies involving intervention and clinical assessment, but are largely based on epidemiological models such as cohort, casecontrol and observational studies. The variables in such studies are enormous, so findings attributed to a particular event or phenomenon may actually be caused by another reason. Varied applications of statistics to such studies can also often yield diverse results for the same data. We will seek to illustrate this conjecture, based on recent researches on flavonoids, because in recent years, flavonoids are unparalleled in the media attention that they have attracted as wonder molecules, and have been at the centre stage of investigation for their role as anti-oxidants. The advice available to a consumer with regard to this group of compounds has perhaps occupied more newsprint than any other, with people being advised to consume large amounts of tea, chocolates, beer, wine, soybean, various berries. The list of flavonoidrich healthy foods can go on. Over 5000 naturally occurring flavonoids, responsible for color and flavor, have been characterized from various plants, and the USDA database [27] lists the content of several selected flavonoids in 225 commonly consumed foods. As mentioned earlier, the beneficial effects of fruits, vegetables and beverages such as tea, coffee, beer, wine and fruit drinks have been attributed more to flavonoids than to traditional antioxidant vitamins. This is partly because foods contain quantitatively more flavonoids than the antioxidant vitamins, ascorbic acid, the tocopherols, the carotenoids [28, 29, 30]. Consequently, the dietary intakes of ascorbic acid, the tocopherols, the carotenoids have been found to be one order of magnitude less (70, 7-10 and 2-3 mg respectively) than that of total flavonoids. Early studies reported flavonoid intakes to be 1 and 1.1 g/day [31]. Later studies, using better analytic methodologies, corroborate that the recommended nine daily servings of fruits and vegetables and moderate amounts of tea, coffee, wine, beer, or chocolate can provide well over 1000 mg of total phenols per day [32]. A large number of studies have demonstrated that ingestion of flavonoid-rich foods raises the antioxidant levels of blood and tissues, which in turn is responsible for their antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor functions, and also for prevention of cancer and cardiovascular diseases [33, 34]. These conclusions have been derived from studies on animals [35, 36, 37], cell isolates [37, 38, 39], in vitro models of human or animal tissues[40, 41]. Thus the strong antioxidant capacity and free-radical scavenging activities of flavonoids in vitro seems indubitable and interest in the possible health benefits of flavonoids has increased. Studies on humans have mostly indicated a protective effect of flavonoids [42, 43, 44] even though there are a few reports to the contrary [45]. Yet, epidemiologic studies exploring

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the role of flavonoids in human health have been inconclusive. Some studies support a protective effect of flavonoid consumption in cardiovascular disease and cancer, other studies demonstrate no effect, and a few studies suggest potential harm. Because there are many biological activities attributed to the flavonoids, some of which could be beneficial or detrimental depending on specific circumstances, it has been suggested that further studies in both the laboratory and with populations are required [46]. Questions have also been raised with regard to their absorption, and the emerging view is that their absorption is much lower and their half-life much shorter than that of other dietary antioxidants such as ascorbic acid and tocopherols, suggestive of the viewpoint that their capability to act as antioxidants in vivo is limited [47]. However, in vivo studies have consistently shown an increase in total antioxidant capacity of plasma on ingestion of flavonoids rich foods, leading to more questions regarding these conflicting findings. It has now been postulated that the increase in total plasma antioxidant capacity on ingestion of a wide variety of flavonoid rich fruits and vegetables is a secondary phenomenon resulting from their extensive metabolism to urate in vivo, and that the macro- and micronutrients present in fruits and vegetables may directly or indirectly affect the total antioxidant capacity of plasma [32]. So the question regarding the in vivo efficacy of flavonoid-rich foods for their health promoting functions remains wide open, even as, in addition to the natural sources, the market is replete with nutritional supplements of specific flavonoids, such as quercetin, isoflavones, catechins and various other bioflavonoids. In this context, the question arises whether an isolated flavonoid will provide the same health benefit as it would, if it were present in the whole food, and even whether the isolated compound may actually be harmful [48]. Notwithstanding whether flavonoids have the beneficial effects attributed to them in recent times or not, it is likely that if each of the studies on flavonoids made newspaper headlines, as they have been found to do, dietary advise to the public to drink ‘n’ number of cups of tea / coffee / wine / juice of any of several berries, and include ‘x’ bars of chocolate and ‘y’ servings of tofu, yoghurt, salads, honey, fish, green chillies, olive oil etc. etc. per day would add up to several kilograms of food. It is more likely than not that this would translate into an excessive intake of energy, consequent obesity, and damage to health!

QUANTITY MATTERS! PHYSIOLOGICAL VS. PHARMACOLOGICAL LEVELS OF INTAKE Experimental studies generally employ much higher concentrations of an isolated compound than is found in the natural food. When findings of such researches reach the common man, he looks for ways to achieve them, and is led to non-dietary sources of the nutrient. Thus nutritional supplements, called nutraceuticals, have become very popular. The amounts of various nutrients, especially vitamins and minerals, recommended by national and international agencies such as the Food and Nutrition Board (US) [49, 50] the FAO [51] and the ICMR [52] are generally much lower than those supplied as supplements. They are also not in the proportions as recommended, or as found in natural foods. The question arises whether our metabolic machinery is designed to handle amounts that are

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larger than can be obtained from diet. It is well known that fat-soluble vitamins are absorbed readily, stored for prolonged periods in the liver but cannot be readily excreted in urine. What is not clear is whether the large stores impact the metabolism in the body. A recent metaanalysis of studies dealing with vitamin E supplementation spanning from 1966 to 2004 revealed that high dosage >400 IU/d of vitamin E supplements may increase all-cause mortality and should be avoided, even as lower doses (< 150 IU/d )may have beneficial effects [53]. The amounts typically available from dietary sources are approximately 14 IU/d) [54]. Similarly, high doses of β-carotene supplementation have been reported to increase mortality rates [55]. Supplements of minerals are also common, but it is well accepted that minerals can have negative interactions with other minerals, which can impact their intestinal absorption, transport, utilization and storage. Calcium and iron are two minerals, the absorption of which are known to depend on need of the body, because they are heavy metals, not easily excreted in the urine due to low solubility of their salts. Iron deficiency is the most common nutritional deficiency in the world, and has been implicated in a wide range of problems, such as developmental delays, behavioral disturbances and cognitive deficits in children and increased risk for a preterm delivery and low-birth weight babies in women. Supplementation of iron has been prescribed, almost universally, for pregnant women, adolescent girls, and newborns. However, concerns have been raised with regard to its possible harmful effects [56], and maternal complications consequent to oxidative stress have been reported when iron is given as a prophylactic to pregnant women who do not have iron-deficiency anemia [57]. The necessity of routine iron supplementation during pregnancy has been debated in industrialized countries, but is still advocated in developing countries, because traditional diets provide inadequate iron and where malaria and other infections causing increased losses are endemic [58]. Ascorbic acid is routinely administered with inorganic iron supplements to keep the ferrous salt in the reduced state. However, this has been reported to have adverse effects, such as oxidative DNA damage and consequently cancer in well nourished adults [59] and increased oxidative stress in the gastrointestinal tract [60]. High tissue iron concentrations have been associated with the development and progression of several pathological conditions, including certain cancers, liver and heart disease, diabetes, hormonal abnormalities, and immune system dysfunctions, due to free radical-mediated tissue damage, even as ingestion of antioxidant rich foods may prevent or delay primary and secondary effects associated with iron overload-related diseases. [61]. Absorption of zinc is known to get reduced by non-heme iron supplementation, although postabsorptive interactions between these nutrients are less clear [62]. Excessive intake of zinc can reduce copper absorption, and excessive copper intake can result in reduced absorption of manganese, zinc, and iron [63]. Among the macrominerals, calcium has attracted maximum attention as a desirable supplement, especially for elderly women who are at risk for osteoporosis. Dietary calcium deficiency has not been established as the major etiological factor for osteoporosis, yet supplements are being taken by most postmenopausal women and even elderly males as a prophylactic measure against osteoporosis. Apart from its benefits on bone health, calcium supplementation has been linked to increase in high density lipoprotein cholesterol and

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decrease in low density lipoprotein cholesterol [64]. However, calcium supplementation has been reported to adversely affect vascular health [65] by accelerating vascular calcification [66, 67, 68, 69]. More recently, calcium supplementation in healthy postmenopausal women has been found to be associated with upward trends in cardiovascular event rates [70]. It has also been implicated in development of brain lesions [71]. Such examples, of positive and negative consequences of supplements providing doses of known nutrients, which are nutritionally unattainable, abound in literature. Distinction is required between physiological and pharmacological doses. It is therefore important that pharmacological doses should not be taken without sufficient reason, that the consumer should be informed of possible harmful effects, that they should be administered under supervision, and that their potentially detrimental effect should be balanced against the likely benefits. Amounts obtained from a balanced diet cannot be excessive, and therefore are unlikely to cause harm, so they should be encouraged. Pharmacological doses of nutritional supplements should also be regulated like other medicines, to be taken only under medical advice.

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SHORT-TERM RESPONSES ARE DIFFERENT FROM LONG-TERM RESPONSES It is generally assumed that more is better, and this stands true for nutrients, growth rates and parameters such as blood hemoglobin, even as it has been argued that functional criteria are preferable [72]. However, calorie restriction is beneficial in the long run, and promotes longevity [73, 74, 75], even though in the short run, growth is compromised. It is possible that short-term responses to any deficiency are different from the long term responses. So in case of energy deficits, for example, the immediate response is slowing down of growth, but in the long run, as an adaptive measure, the organism is known to reduce metabolic rates and achieve catch up. Life has evolved through shortages, and the physiology of all organisms is designed to hold on to nutrients. Our digestive system is designed to absorb several times the amounts of dietary fuels, carbohydrate, protein and fat, normally present in the diet. However, it is unable to adapt to the relatively recent phenomenon of availability of excessive food by reducing digestion and absorption. Our adipose tissue is designed to hold on to the absorbed excess. Anyone who has lost weight knows how difficult it is to keep it off. There is intricate metabolic regulation to conserve body fat, and regain it even if availability of energy remains low, as in continued dieting. It has been suggested that the human body is likely to have a genetically determined set-point weight that is controlled by metabolic hormones and fat cell enzymes [76]. On the other hand, in countries such as India, poor children continue to grow on dietary energy intakes barely above those required for basal metabolism, and the gestation and lactation performance of poor women compares favorably in many respects with that of upper class women [77]. In animal studies it has been shown that the nutritional inadequacy of the diet is, to a large extent, compensated for, by better utilization of dietary energy, protein and improved efficiency of nitrogen utilization [78, 79, 80, 81]. Also, rats switched from a low protein diet in early life to a moderate protein diet, and back to a low protein diet were found to display a more efficient and rapid adaptation to the switches, as judged by growth as well

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as rate of serum protein turnover, than those switched from a high protein to a low protein diet after an intervening period on a moderate protein diet [82]. This suggests that adaptive mechanisms that come into play during periods of protein deprivation appear to persist later when the stress is reduced, and may in fact be beneficial to the organism. Similarly, it is interesting to find that rats fed carotene as the only source of vitamin A in early life were more efficient in converting it to vitamin A later in life, than those fed preformed vitamin A throughout [83] emphasizing the long term responses of the organism to nutrient utilization. The difference between short term and long-term feedback responses from the recipient system are also likely to affect the practical application of laboratory findings, because of elaborate feedback controls found in the complex living system.

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THE ROLE OF FEEDBACK RESPONSES OF THE RECIPIENT SYSTEM: THE COMPLEX HUMAN BODY The recipient system of any nutrient can consist of a fairly homogenous system such as found in a cell isolate or tissue culture, or it can be as heterogeneous and complex as the human body. The human body is a conglomerate of about a hundred trillion cells (which incidentally is four orders of magnitude more than all the Homo sapiens on earth!) and they work in marvelous unison to maintain homeostasis, (or is it homeodynamics?). To do this, there are intricate feedback mechanisms, so the effect of a particular compound on the homogenous system may not get extrapolated to the complex system. In biochemistry, it is generally found that if one set of chemicals drives the system in a given direction, another set of chemicals will drive it in an opposing direction. If one or both of these opposing influences are non-linear, an equilibrium point(s) results to which the system gravitates. Any perturbation, generally in the form of a chemical stimulus resulting from the influx of a nutrient, nutrient supplement, drug, or even thought, can trigger a negative feedback system to reestablish the equilibrium. Since ultimately the system moves to a predetermined equilibrium, there may be results of an immediate response to an intervention, which are often not sustained in the long run, as feedback systems come into play. As a consequence of negative feedback mechanisms, many qualitatively different stimuli oppose each other, and finally arrive at a point close to the equilibrium point. By positive feedback, the system responds in the same direction as the perturbation, so the signal is amplified. In biological systems, the controls are typically based on negative feedback, through varied mechanisms. Many multienzyme pathways in metabolism are regulated; the final product inhibits an early reaction in the pathway, after it is formed in sufficient quantities, thus preventing excessive amounts of its own formation. Hormonal regulation of blood sugar, blood pressure, body temperature and erythropoiesis are but a few examples of well documented negative feedback controls. Disruption of negative feedback can lead to undesirable results. Once the principle that equilibrium is to be maintained in the long run is acknowledged, it is logical to accept that all perturbations will eventually get attenuated, explaining why many drugs, supplements and nutrients produce the expected result in short term studies, but in the long run, the efficacy is lost.

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Most traditional methodologies used in biological sciences have adopted the reductionist approach to knowledge. However, as discussed in relation to feedback approaches, the system is likely to behave differently when impacted by diverse stimuli, than when it is in a controlled environment. One of the great current debates in biology concerns whether the observed behavior of a system can be accounted for in terms of the behaviors of its subcomponents, and it has been suggested that holistic approaches may be more predictive and make for better understanding of the functioning of the body. Systems biology develops these concepts and attempts to understand the integrated function of complex, multicomponent biological systems ranging from interacting proteins that carry out specific tasks to whole organisms [84] Scientists dealing with varied aspects of food, nutrition and nutritional biochemistry have more recently been involved in developing new approaches to accommodate the complexities of the organism, leading to the development of new disciplines such as nutrigenomics, which has the potential to prescribe tailored dietary regimens specific to the individuals’ requirements. Nutrigenomics links genomics, transcriptomics, proteomics and metabolomics to human nutrition. Genomics allows the study of the genetic abilities of an individual to metabolize nutrients based on its entire genome. It includes the study of genome-nutrient interactions, including the role of nutrients and dietary components in regulation of genome structure, expression and stability and the role of genetic variation on individual nutrient requirements. Transcriptomics allows the global study of gene expression at the RNA level, which will ultimately determine the extent of transcription of a gene. Knowing the proteomics will help in determining whether the enzymes and other required proteins have the desired conformation to perform optimal catalytic function as required. Finally, it includes metabolomics, the development of which began in 1970, to investigate the ideas of Linus Pauling with a view to studying relationships between biological variability and wide ranges of nutritional requirements. Metabolomics [85], specifically nutritional metabolomics, is concerned with metabolic pathways and networks and includes regulation of metabolic pathways and networks by nutrients and other food components. It summates all the metabolites in body fluids, which are impacted by endogenous factors such as age, sex, body composition, genetics, underlying pathologies, circadian rhythms and resting metabolic rate and exogenous factors such as diet, including all known and hitherto unknown nutrients as well as non-nutrients such as dietary fiber, additives, pollutants, drugs etc., and the large number of signals from the intestinal microflora. A very large number of compounds make the metabolome, which can be likened to a metabolic fingerprint which reflects the balance of an individual’s metabolism. These compounds need to be identified, quantified and their relative proportions analyzed and interpreted. This has led to the development of metabonomics which is concerned with the quantitative measurement of the metabolome [85]. Metabolomics requires the establishment of analytical methods that can profile human serum and urinary metabolites to assess nutritional imbalances and disease risk. Such analysis will require the application of sensitive techniques such as nuclear magnetic resonance, functional magnetic resonance imaging and high performance liquid chromatography, and handling of a large amount of mathematical data. It should be clear from the foregoing, that the utilization of compounds from food in the body is an extremely complex issue, with large individual variations due to interactions

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between genetics and overall food habits. It is generally not true that every laboratory finding is applicable to every one in the population. Therefore, categorical statements about the benefits, or otherwise, of a food do not apply universally. Such statements are inherently misleading and should be avoided.

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LIMITATIONS OF THE METHODOLOGY OF SCIENCE: THE ROLE AND RESPONSIBILITY OF THE MEDIA Statistics forms the basis for evaluating the results of modern scientific studies, whether based on observation or on varied experimental designs. But even the basic assumptions, on which statistical tests are based, are not devoid of controversy. Usually, inferences are drawn, in a mechanical manner, using statistical techniques, without appreciation of the assumptions on which these techniques are based. Very often the techniques are based on assumptions of normal distribution, even for such data as are not drawn from normal distribution. If different samples are taken from a normal population, they will usually yield different sample means. One of the most frequently used statistical techniques, the null hypothesis testing, provides the probability that two samples have been drawn from the same normal distribution. If this probability is less than an arbitrarily chosen number p (usually 0.05, 0.01, or 0.001), the two samples (experimental group and the control group) are regarded as drawn from different populations. It is usual to interpret this by the statement that the treatment given to the experimental group has been effective at the significance level p. The null hypothesis testing is used in a very wide variety of experimental designs because it allows a crisp decision – to accept or reject a hypothesis. It is used to compare an experimental group with a control group, to determine whether they belong to the same normal population, with regard to the parameter under investigation. It is assumed that the populations under study are matched with regard to all other variables except the one being investigated. Results are presented as dichotomous –the null hypothesis is accepted or not, thereby concluding that the effect is / is not observed. In the above procedure, the conventional choice of p = 0.05, 0.01 or 0.001 seems to be arbitrary. The choice of p should depend on the nature of the question asked and an assessment of the benefits and risks involved. The practice of using the p value for testing the null hypothesis has been criticized for arbitrarily dividing results into significant and not significant [86, 87]. Extremely small differences can become statistically significant if the sample size is sufficiently large, and find their way into scientific writings of repute. Null hypothesis significance testing has been called a ‘research quality assurance test’ and is a requirement for experimental research to be published. However, it is not always appreciated that this widely used statistical methodology is not without its problems when extended to practical advice; small statistically significant differences may not be of practical significance. It is important that reports of scientific research are conveyed to the consumers in a reasonably accurate manner. As discussed earlier, there is an extremely large, and often conflicting, amount of scientific data being generated, and the consumer ends up being confused.

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It is important that the consumer is guided to be able to make informed choices with regard to healthy food and desirable food habits. Media should desist from giving the misleading impression that everyone who eats a particular food, or follows a particular practice, will benefit from it. It is therefore suggested that media should report scientific findings to the consumer only when reasonable consensus has been reached on a particular issue. In the absence of such a consensus, the reports should include full details of the study design and findings in a language that is comprehensible to the typical educated consumer yet does not give misleading impressions. For example, they should give the sample size and the extent of the effect observed, how many among the experimental group/s exhibited a response in a direction opposite to the group average and how many among the control group showed the response even without the intervention. The consumer should also be made aware of the limitations of the scientific methodology. The tendency to reduce a study to a simple statement expressing that ‘x’ food / habit causes ‘y’ effect needs to be restricted, both by scientists and media. The common man should be encouraged to take his own decisions regarding whether to try out a particular food for a particular condition. The media should facilitate such decision making, by improving its information content, and seek to educate the consumer to take informed decisions, rather than make categorical headlines about foods as if they apply to all individuals in a society. For example instead of saying ‘Eat chocolates for lowering blood pressure’, media should be encouraged to say that of ‘n’ persons who ate ‘m’ grams of chocolate everyday, ‘x’ persons showed a ‘y-z’ percent reduction in blood pressure. Headlines based on laboratory findings based on in vitro or animal studies can be even more damaging. For example if a purified compound from a plant shows a health benefit on rats, but makes media headlines that everyone who consumes a food containing that compound will benefit from it, the result can sometimes even be harmful. We need to have guidelines for media to refrain from making potentially irresponsible headlines.

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CONCLUSION In the final analysis, we can conclude that there is a never-before quantum of information available at the click of a button, but uncertainties and methodological limitations are inherent in the scientific method. This is certainly not to denigrate research per se, which, despite conflicting observations, or because of them, certainly promotes understanding of foodphysiological interactions and leads to wide applications in disease amelioration and promotion of good health. Nevertheless, it is important to emphasize that advice about food is a complex issue. In complicated medical conditions, food choices can even make the difference between life and death. Therefore, it is necessary to caution the media from creating euphoria over viewpoints that may not extend to in vivo functionality based on superficial reports of research findings. Education, in the modern world, must endeavor to make people understand the strengths and limitations of the scientific method in general and nutritional research in particular.

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[20] National Library of Medicine: Fact Sheet Medline. http://www.nlm.nih.gov/pubs/factsheets/medline.html. Accessed 2007. November 15. [21] Garg , AX; Hackam, D; Tonelli, M. Systematic Review and Meta-analysis: When One Study Is Just not Enough. Clin. J. Am. Soc. Nephrol. 2008. 3: 253–260. [22] Ioannidis, JP: Contradicted and initially stronger effects in highly cited clinical research. J. Am. Med. Assoc. 2005. 294: 218–228. [23] Nestle, M. Eating made simple. Sci. Am. 2007, Sept. 60-69. [24] Nestle, M. Food Politics: How the Food Industry Influences Nutrition and Health. Revised edition. University of California Press, 2007. [25] Hilden, J; Jorgensen, AW; Gotzsche, PC. Cochrane reviews compared with industry supported meta-analyses and other meta-analyses of the same drugs: Systematic review. Br. Med. J. 2006. 333: 782. [26] Lesser, LI; Ebbeling, CB; Goozner, M; Wypij, D; Ludwig, DS. Relationship between Funding Source and Conclusion among Nutrition-Related Scientific Articles. PLoS Medicine. 2007. 4, 1: e5, 41–46. [27] Nutrient Data Laboratory; Beltsville Human Nutrition Research Center, USDA. Nutrient database for standard reference, release. USDA Database; 2003. Available at http://www.nal.usda.gov/fnic/foodcomp). [28] Vinson, JA; Su, X; Zubik, L; Bose, P. Phenol antioxidant quantity and quality in foods: fruits. J. Agric. Food Chem. 2001. 49:5315–5321. [29] Lotito, SB; Frei, B. Relevance of apple polyphenols as antioxidants in human plasma: contrasting in vitro and in vivo effects. Free Radic. Biol. Med. 36:201–211; 2004. [30] Aaby, K; Skrede, G; Wrolstad, RE. Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa). J. Agric. Food Chem. 2005. 53:4032–4040. [31] Kuhnau J. The flavonoids: a class of semi-essential food components: their role in human nutrition. World Rev. Nutr. Diet. 1976. 24:117–91. [32] Frei, B and Lotito, BS. Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: Cause, consequence, or epiphenomenon? Free Radic. Biol. Med. 2006. 41: 1727–1746. [33] Aviram, M; Fuhrman, B. Wine flavonoids protect against LDL oxidation and atherosclerosis. Ann.. N. Y. Acad. Sci. 2002. 957:146–161. [34] Rietveld, A; Wiseman, S. Antioxidant effects of tea: evidence from human clinical trials. J. Nutr. 2003. 133:3285S–3292S. [35] Goyarzu, P; Malin, DH; Lau, FC; et al. Blueberry supplemented diet: effects on object recognition memory and nuclear factor-kappa B levels in aged rats. Nutr. Neurosci. 2004. 7(2):75-83. [36] Frei, B; Higdon, JV. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J. Nutr. 2003. 133(10):3275S-3284S. [37] Kavanagh, KT; Hafer, LJ; Kim, DW; et al. Green tea extracts decrease carcinogeninduced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture. J. Cell Biochem. 2001. 82(3):387-398. [38] Spencer, JP; Schroeter, H; Crossthwaithe, AJ; Kuhnle, G; Williams, RJ; Rice-Evans, C. Contrasting influences of glucuronidation and O-methylation of epicatechin on hydrogen peroxide-induced cell death in neurons and fibroblasts. Free Radic. Biol. Med. 2001. 31(9):1139-1146.

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[39] Spencer, JP; Chen, JJ; Ye, ZQ; Koo, MW. Growth inhibition and cell cycle arrest effects of epigallocatechin gallate in the NBT-II bladder tumour cell line. BJU Int. 2004. 93(7):1082-1086. [40] Cheng, IF; Breen, K. On the ability of four flavonoids, baicilein, luteolin, naringenin, and quercetin, to suppress the Fenton reaction of the iron-ATP complex. Biometals. 2000. 13(1):77-83. [41] Polagruto, JA; Schramm, DD; Wang-Polagruto, JF; Lee, L; Keen, CL. Effects of flavonoid-rich beverages on prostacyclin synthesis in humans and human aortic endothelial cells: association with ex vivo platelet function. J. Med. Food. 2003. 6(4):301-308. [42] Geleijnse, JM; Launer, LJ; Van der Kuip, DA; Hofman, A; Witteman, JC. Inverse association of tea and flavonoid intakes with incident myocardial infarction: the Rotterdam Study. Am. J. Clin. Nutr. 2002. 75(5):880-886. [43] Yochum, L; Kushi, LH; Meyer, K; Folsom, AR. Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women. Am. J. Epidemiol. 1999. 149(10):943-949. [44] Liu, RH. Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am. J. Clin. Nutr. 2003. 78(3 Suppl):517S-520S. [45] Freese, R; Vaarala, O; Turpeinen, AM; Mutanen, M. No difference in platelet activation or inflammation markers after diets rich or poor in vegetables, berries and apple in healthy subjects. Eur. J. Nutr. 2004. 43(3):175-182. [46] Ross, JA; Kasum, CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu. Rev. Nutr. 2002. 22:19-34. [47] Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C. Bioavailability and bioefficacy of polyphenols in humans: I. Review of 97 bioavailability studies. Am. J. Clin. Nutr. 2005. 81:230S–242S. [48] Lotito, SB; Frei, B. Relevance of apple polyphenols as antioxidants in human plasma: contrasting in vitro and in vivo effects. Free Radic. Biol. Med. 2004. 36:201–211. [49] Food and Nutrition Board. Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D and fluoride. Washington DC: National Academy Press, 1997. [50] Food and Nutrition Board. Dietary reference intakes: Thiamine, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington DC: National Academy Press, 1998. [51] WHO/ FAO/ IAEA. Trace elements in human nutrition and health. Geneva: World Health Organization. 1996. [52] Indian Council of Medical Research. Nutrient requirements and recommended dietary allowances for Indians, National Institute of Nutrition, Hyderabad, 1995. [53] Miller, III ER; Pastor-Barriuso, R; Dalal, D; Riemersma, RA; Appel, LJ; Guallar, E. Meta-Analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann. Intern. Med. 2005; 142:37-46. [54] Institute of Medicine. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. A report of the Panel on Dietary Antioxidants and Related Compounds, Subcommittees on Upper Reference Levels of Nutrients and Interpretation and Uses of Dietary Reference Intakes, and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Food Nutrition Board. Washington, DC: National Academies Pr; 2000.

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[55] Vivekananthan, DP; Penn, MS; Sapp, SK; Hsu, A; Topol, EJ. Use of antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomized trials. Lancet. 2003; 361:2017-23. [56] Scholl, TO; Reilly, T. Anemia, iron and pregnancy outcome. J. Nutr. 2000.130(2S Suppl):443S-447S. [57] Scholl,TO. Iron status during pregnancy: setting the stage for mother and infant Am. J. Clin. Nutr. 2005. 81(5):1218S-1222S. [58] Mungen, E. Iron supplementation in pregnancy. J. Perinat. Med. 2003. 31(5):420-6. [59] Rehman, A; Collis, CS; Yang, M; Kelly, M; Diplock, AT; Halliwell, B; Rice-Evans, C. The Effects of Iron and Vitamin C Co-supplementation on Oxidative Damage to DNA in Healthy Volunteers. Biochem. Biophys. Res. Comm. 1998. 246, 1: 293-298. [60] Fisher, AEO; Naughton, DP. Iron supplements: the quick fix with long-term consequences. Nutr. Journal. 2004. 3:2. [61] Fraga, CG and Oteiza, PI. Iron toxicity and antioxidant nutrients. Toxicology. 2002. 180: 1, 23-32. [62] Donangelo, CM; Woodhouse, LR; King, SM; Viteri, FE; King, JC. Supplemental Zinc Lowers Measures of Iron Status in Young Women with Low Iron Reserves. J. Nutr. 2002.132:1860-1864. [63] Anderson, JJB. Minerals. In Krause’s Food, Nutrition and Diet therapy. Mahan LK, Escott-Stump S (Eds) WB Saunders Co. Philadelphia, 2000. p 111. [64] Reid, IR; Mason, B; Horne, A; Ames, R; Clearwater, J; Bava, U; et al. Effects of calcium supplementation on serum lipid concentrations in normal older women: a randomized controlled trial. Am. J. Med. 2002.112:343-7. [65] Reid, IR; Schooler, BA; Hannon, S; Ibbertson, HK. The acute biochemical effects of four proprietary calcium supplements. Aust. N Z J. Med. 1986.16: 193-7. [66] Pletcher, MJ; Tice, JA; Pignone, M; Browner, WS. Using the coronary artery calcium score to predict coronary heart disease events: a systematic review and meta-analysis. Arch. Intern. Med. 2004. 164: 1285-92. [67] Chertow, GM; Burke, SK; Raggi, P. Treat to Goal Working Group. Sevelamer attenuates the progression of coronary and aortic calcification in hemodialysis patients. Kidney Int. 2002. 62: 245-52. [68] Block, GA; Spiegel, DM; Ehrlich, J; Mehta, R; Lindbergh, J; Dreisbach, A; et al. Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis. Kidney Int. 2005. 68:1815-24. [69] Asmus, HG; Braun, J; Krause, R; Brunkhorst, R; Holzer, H; Schulz,W; et al. Two year comparison of sevelamer and calciumcarbonate effects on cardiovascular calcification and bone density. Nephrol Dialysis Transplant. 2005. 20:1653-61. [70] Bolland, MJ; Barber, PA; Doughty, RN; Mason, B; Horne, A; Ames, R; Gamble, GD; Grey A and Reid, IR. Vascular events in healthy older women receiving calcium supplementation: randomised controlled trial. Br. Med. J. published online 15 Jan 2008. [71] Payne, ME; Anderson, JJB; Steffens DC. Calcium and vitamin D intakes are positively associated with brain lesions in depressed and nondepressed elders. FASEB J. 2007. 21:837. [72] Beaton, GH. Iron needs during pregnancy: Do we need to rethink out targets? Am.. J. Clin. Nutr. 2000. 72 (suppl):265S-71S. [73] Weidruch, R. Calorie restriction and aging. Sc Am. January 1996, p32-38.

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[74] Fontana, L. Excessive adiposity, calorie restriction and aging in humans. J. Am. Med Assoc. 2006. 293:13. [75] Heilbron, LK;, de Jonge L; Frisard, MI; DeLany, JP; Enette, D; Meyer, L; Rood, J; Nguyen, T; Martin, CK; Volaufova, J; Most, MM; Greenway, FL; Smith, SR; Williamson, DA; Deutsch, WA; Ravussin, E. Effect of 6-month calorie restriction on biomarkers of aging, metabolic adaptation and oxidative stress in overweight subjects. J. Am. Med. Assoc. 2006. 293:13. [76] Pi-Sunyer FX. Obesity. In Modern Nutrition and health and disease. Eds. Shils ME, Olson JA, Shike M, Ross AC. Ninth edition. Williams and Wilkins, Baltimore. 1999. 1395-1418. [77] Mittal PC. Pregnancy Outcome In Relation To Anemia, Nutrient Intakes and Socioeconomic Status. Abstract no. M16, p51. Micronutrient Forum Meeting, Istanbul, 16-18 April 2007. [78] Mittal PC. Lactation performance, maternal behavior and activity of rats subjected to neonatal and/or post weaning food restriction. Nutr. Rep. Int. 1984. 30, 305-310. [79] Mittal, PC. Nitrogen balance during reproduction of rats fed diets simulating those consumed by different population groups. Nutr. Rep. Int.1984. 30, 1355-65. [80] Mittal, PC. Response of rats to lysine deficiency at different ages. Nutr. Rep. Int. 1985. 32, 453-461. [81] Mittal, PC. Nitrogen balance during reproduction of second-generation rats fed diets typical of different socio-economic strata. Nutr. Rep. Int. 1986. 33, 25-32. [82] Mittal, PC. Response of rats to variations in dietary protein content. Nutr. Rep. Int. 1985. 31, 521-533. [83] Mittal, PC. β - Carotene utilization in rats fed either Vitamin A or carotene in early life. Nutr. Rep. Inter. 1983. 28, 181-188. [84] Strange, K. The end of "naive reductionism": rise of systems biology or renaissance of physiology? Am. J. Physiol. Cell Physiol. 2005. 288: 968-974. [85] Gibney, M J; Walsh, M; Brennan, L; Roche, HM; German, B; van Ommen, B. Metabolomics in human nutrition: opportunities and challenges. Am. J. Clin. Nutr. 2005. 82: 497-503. [86] Sterne, JAC; Smith, GD. Sifting the evidence—what's wrong with significance tests? Br. Med. J. 2001. 322 – 27. [87] Ioannidis, JPA. Why most published research findings are false. PLoS Med. 2005. 2(8): e124.

In: New Research on Food Habits Editors: K. Hasegawa and H. Takahashi

ISBN: 978-1-60456-864-6 © 2009 Nova Science Publishers, Inc.

Chapter 3

THE EFFECT OF DIET ON HUMAN BODILY ODORS Jan Havlicek1,* and Tamsin Saxton2 1

2

Department of Anthropology, Charles University, Prague, Czech Republic School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom

ABSTRACT

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One of the most underestimated and poorly understood aspects of diet is its influence on human body odor. Humans have a distinct odor signature which arises from a combination of genetic and environmental factors. The link between genes and body odor is well appreciated, and an understanding of the influences of environmental factors such as emotional state, reproductive phase, and health status, is underway. Research into the impact of diet upon bodily odors, however, has been somewhat neglected. A recent study found that consumption of red meat for a period of two weeks decreased the pleasantness and increased the intensity of male axillary odor when judged by opposite-sex raters. Changes in bodily odors have also been linked to the consumption of food such as soya beans, plants from the Alliaceae or Brassicaceae families, strongly-flavored spices, fish, and milk-based products. This chapter critically reviews the literature on various aspects of food influencing human body odor and suggests several avenues for future research.

INTRODUCTION It is a widely acknowledged fact that human eating habits affect physical appearance and attractiveness. As appearance has a bearing on human relations, diet may be relevant to social interactions, albeit indirectly. However, eating habits may modify not only human visual appearance, but also, and more directly, human olfactory qualities. Although odor is probably the least understood of the senses, human bodily odor may well be influential within human social interaction. Individual body odor profiles are thought to be fairly stable across time [1], and these odor profiles may impact upon human partner choice [2-5] and familial recognition [1, 6, 7]; specific odors of bodily origin may also have even more direct influences on human *

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behavior [8-10]. In spite of this, researchers have paid only limited attention to the effects of eating habits on bodily odors. The aim of this chapter is to review our current knowledge on how various components of food influence human bodily odors and to show directions for future research. We will start with a short introduction of the sources and physiology of nondietetic body odor, then continue with a more detailed analysis of dietetic factors.

ODOR SOURCES IN THE HUMAN BODY General human body odor originates from body parts such as the axilla, scalp, mouth and lungs, genital and anal regions, feet etc. Skin is colonized by a number of bacteria and eukaryotic Malassezia; their metabolic activity on either exfoliating skin cells or chemicals produced in the skin glands results in external body odor [11]. Skin glands are categorized as three different types: sebaceous glands, eccrine glands and apocrine glands [12]. Sebaceous glands produce oily chemicals whose main function is to protect the skin from environmental stress. The main function of the eccrine glands is perspiration. They produce mostly water, salts and some amino acids, although the actual amount and composition varies according to physical activity, environmental temperature, hydration levels, etc. Under normal conditions, neither sebaceous nor eccrine glands have a large impact upon body odor; rather, this is left to the apocrine glands, which are mainly concentrated in the face, genital region and, in particular, the armpits. These glands produce short chain fatty acids and androstene steroids together with other compounds. Fresh apocrine gland secretions are milky in color and odorless; the smell itself results from bacterial activity. Armpit bacteria include coryneforms, propionibacteria and micrococci [13].

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GENETIC FACTORS Human body odor is specific to each individual and thus has been characterized as an “odor signature” [6, 14], shaped in part by genetic factors. Genetic influences have been demonstrated in twin experiments, where both humans and dogs can detect the similarity in the body odor of twins, even when they live apart [15, 16]. Other studies show that the body odor of parents and offspring or individual siblings can be matched at better-than-chance levels by individuals not acquainted with the body odor donors [17]. Further, research on body odor attractiveness has found that it is influenced by genes of the Major Histocompatibility Complex or MHC [2, 18]. This is a highly variable gene complex whose products play a crucial role in the immune response. Across species, there is evidence that individuals tend to prefer mates whose MHC genes differ from their own; MHC-based mate choice may derive from odor cues [19, 20].

ENVIRONMENTAL FACTORS Environmental influences on bodily odor include 1) emotional state, 2) reproductive status, 3) health and 4) diet; for a review, see [21]. The results of several studies suggest that

The Effect of Diet on Human Bodily Odors

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emotional states such as fear or happiness can be perceived by others through the medium of smell. Most of these studies are based on emotions induced by comedies or horror movies while the target individuals wear cotton pads in their armpits to collect odor samples [22, 23]. Individuals not acquainted with the targets are able to distinguish happiness and fear on the basis of the odor at levels greater than chance. Although ovulation in humans is not apparent in the same way as in some other primates [24], recent evidence shows that female body odor fluctuates across the menstrual cycle, becoming most attractive around the time of ovulation [25, 26]. These changes are not restricted to axillary odor but have also been observed in vaginal smells [27]. Axillary odor changes were not found in women taking oral contraceptives, suggesting that the changes are under the control of steroid hormones [25]. Various diseases are accompanied by a distinctive smell, a fact acknowledged by ancient physicians for diagnostic purposes. Some genetic disorders cause changes in metabolic pathways due to the absence or scarcity of a particular enzyme. This may result in a distinctive smell caused by a specific by-product or its metabolites. An example of such a disorder would be trimethylaminuria, the inability to transform trimethylamine to trimethylamine N-oxide [28]. The ingestion in particular of choline-containing food by affected individuals can result in the excess, unmetabolised trimethylamine being excreted in breath, urine and sweat, giving rise to an unpleasant smell which, in extreme cases, is similar to that of decomposing fish. Various infectious diseases can also cause changes in the bodily odor of the patient. This smell is mostly caused by the metabolic activity of the infectious agent and/or by the immune response. For instance, skin diseases such as wounds or ulcers are accompanied by a distinctive foul smell [29].

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EFFECT OF DIET Healthy human body odor is also affected by diet. The first observations of the effects of food on odor were carried out in rodents. Beauchamp [30] found that female guinea pigs preferred the odor of male guinea pigs fed on a diet designed for guinea pigs over that of guinea pigs fed on a diet for laboratory rats. More recently, it was shown in meadow voles that scents from the anogenital marks, feces and urine of males fed with lower protein portions were smelled by females for a shorter time (i.e. demonstrating lower mating interest) than were the scents of males fed on a high protein diet [31]. A further study by the same team investigated the effect of food deprivation on body odor attractiveness in meadow voles and found that the odors of females deprived for 24h were found less attractive. 48h after they were re-fed they became as attractive as at baseline [32]. The effect of diet-related odors is not restricted to mammalian species, but has also been found to influence odor preferences in fish. More specifically, juvenile Arctic charr (Salvelinus alpinus) prefer siblings on the same diet over those on different diets [33]. Although the rodent studies neatly demonstrate the principle of dietary influence on odor, their direct relevance to humans is questionable, as the main source of rodent chemical communication is urine, where metabolites from digested food can be directly detected. In contrast, in humans, the most significant source of body odor is probably the armpit region; urine is of minor relevance.

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A demonstration of the general effect of diet on human bodily odor was made in the 1970s by Wallace who found that human smellers could distinguish monozygotic twins from their hand odors only if the twins consumed different diets [34]. Similarly, dogs cannot discriminate infants on the same (breast- or bottle-fed) diet [15]. Researchers in human body odor studies tend to ask their subjects to avoid specific foods thought to affect body odor, including garlic, onion, chilies, pepper, vinegar, blue cheese, cabbage, radish, fermented milk products and marinated fish [35-37], yet this list is based more on everyday experience than on experimental data. In fact, our knowledge of the effect of specific foods on human bodily odors is highly limited. Below, we are going to review both the direct and indirect evidence on this issue. One source of bodily odors derives from the digestive processes themselves. Gas is produced within the digestive system by the action of bacteria on endogenous sources such as intestinal mucins and on dietary material which has not been digested in the small intestine [38]. This consists mainly of dietary fiber and complex polysaccharides, as are found in relatively large quantities in legumes such as soya and other beans [39]. These gases eventually emerge as flatus. A large portion of flatus consists of non-odorous gases such as oxygen, nitrogen, carbon dioxide, hydrogen and methane [40]. Flatus malodor is associated with sulphurous gases; one study that provoked flatulence by the consumption of pinto beans and lactulose found that concentrations of hydrogen sulphide, whose odor is reminiscent of rotten eggs, was most strongly correlated with flatus malodor, and that methanethiol, smelling of decomposing vegetables, probably also contributed [40]. Foods containing particularly high levels of sulphur include some breads, dried fruits, brassicas such as broccoli and cabbage, and soya flour [41]. There are also aromatic foods of plant origin that impact upon bodily odors. The distinctive post-ingestive malodor of plants from the Alliaceae family, including garlic and onion, is associated with various sulphurous gases such as allyl methyl sulfide [42]. The consumption of garlic, particularly raw garlic, affects breath odor [43], and gives rise to reports of changes in body odor [44]. Breath odor is affected both by gases from the oral cavity, probably due to trapped particles of garlic, and also from allyl methyl sulfide in the gut, an odor source which may linger for several hours after garlic consumption [43]. Analytic studies suggest indirectly that plants of the Brassicaceae family (e.g. broccoli, cauliflower, kohlrabi), which are a significant component of various European cuisines, may influence human breath and axillary odor, since they emit high amounts of dimethyl trisulphide and other sulphur compounds when boiled [45]. In humans, dietary odors have also been shown to enter into the domain of mother-infant interactions. Studies in mice and humans have shown that maternal food choices during gestation can affect offspring food preference postnatally [46, 47]. Mice whose mothers were fed garlic during gestation preferred garlic over onion, unlike control mice whose mothers were fed a garlic-free diet [47]. In humans, the infants of mothers who consumed anise during pregnancy showed a post-natal preference for the odor of anise [46]. Such preferences may be learnt prenatally either by stimulation of the olfactory receptors via the bloodstream, or from direct exposure to the amniotic fluid [47]. Certainly, adult raters can detect recent maternal garlic consumption from the odor of amniotic fluid [48], and the odor of maternal amniotic fluid is known to exert a calming influence on human infants [49]. Postnatally, the mother’s diet can also affect milk taste and odor. Carrot, vanilla, garlic and alcohol flavors have all been shown to cross from the mother’s diet to her milk, affecting suckling behavior [50-54].

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For instance, infants whose mothers ingested garlic capsules breastfed longer and sucked more compared to controls. Also, two hours after garlic consumption by the mother, adult panelists rated the odor of the milk as smelling stronger and more like garlic [53]. There are also a few clinical reports of peculiar body odors in newborn babies that appear to result from the mothers’ diet. Cumin, fenugreek or curry have been detectable in separate instances in the odor of newborn infants, following consumption of spicy food by the mother [55]. In a similar case in Turkey, a newborn baby smelled of maple syrup, which is a diagnostic sign of maple syrup urine disease [56]. Laboratory tests did not confirm the diagnosis and further discussion with the mother revealed instead that before the delivery she ate food containing fenugreek. Leaves of this plant (family Fabaceae) are widely used as a spice in traditional cuisine in Mediterranean countries such as Turkey and contain 3-hydroxy4,5-dimethyl-2(5H)-furanone which is responsible for its distinctive odour [57]. Early food preferences may persist through childhood [54], and it is therefore conceivable that maternal odors induced by diet should have long-term or even life-long effects on the diet of the offspring [58].

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SUBJECTIVE PERCEPTIONS OF BODILY ODORS INDUCED BY DIET As far as we know there is only one study that focused directly on how ratings of human body odor are affected by diet, or more specifically, by the consumption of red meat [59]. The authors asked half of their male odor donors to keep to a meat-free diet and half to eat red meat every day for a period of two weeks. For the last four days of the diet, all of the food, differing only in meat content, was provided to the participants. On the final day, participants wore cotton pads in their armpits for 24h. The odor of the pads was then rated by a group of female students for its intensity, pleasantness and attractiveness. A month later the whole procedure was repeated; this time, the donors previously on a meat diet consumed a meat-free diet, and vice versa. Data analysis showed that the odor of the donors when on the red meat diet was judged as less pleasant and more intense compared to their odor when on a non-meat diet (figure 1). At this point it is not clear what amount of meat must be eaten to be discernable in body odor. Neither do we know for how long such an effect lasts. It would also be interesting to test whether eating poultry or fish produces a similar effect. Some anthropologists have remarked that individuals in communities where fish are eaten on a daily basis smell of fish [12], possibly due to trimethylamine, an odorous substance abundant in fish. Likewise, anecdotally, consumers of products based on cows’ milk smell unpleasant to people in cultures where cows’ milk is not consumed. This second finding is particularly interesting because of the link between genetic and cultural variation. The ability to digest milk is mediated by the lactase gene, a gene which is only maintained in adults amongst populations with a history of animal domestication and adult milk consumption [60]. Alcohol, too, is associated with population-level differences in ethanol metabolic ability [61], and, anecdotally, affects body odors. The co-occurrence of genetic differences and dietetic practices allows for an interesting gene-culture evolution. Could body odor be one of the proximate mechanisms by which cultural and genetic dietetic differences are maintained? Direct explorations of these phenomena remain to be made.

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Figure 1.Mean ratings (±S.E.) of axillary odor pleasantness, attractiveness and intensity when body odor donors were on a “meat” diet (white bars) and when on a “non-meat” diet (grey bars). Differences are significant at p=0.01 (repeated measures ANOVA). Reprinted from Havlicek and Lenochova (2006) with permission from Oxford University Press.

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CONCLUSION Eating habits affect social interactions both directly (e.g. via culturally-based food practices) and indirectly (e.g. due to the effects of weight on attractiveness). However, since food choices affect bodily odors, eating habits may also influence social interactions in a more subtle way. For instance, body odor is thought, albeit amidst some controversy, to influence human partnership formation by providing information on MHC compatibility [1820]. Experimental work in rodents has demonstrated that diet may be more salient than MHC type in affecting bodily odors [62, 63], and spiny-mouse pups have been shown to prefer the odor of heterospecific females fed on a familiar diet compared to the odor of conspecific females fed on an unfamiliar diet [64]. These results suggest that diet may interact with, or mask, individually-specific odors denoting gender, species etc. Unfortunately at this point, we can only speculate about the effects of specific dietetic compounds on subjective perceptions of human bodily odors, and the interaction between diet and individual genetically-based body odors. Different dietetic habits between cultures and communities may also contribute to differences in body odors between populations, and contribute to xenophobic beliefs that individuals of other cultures smell bad, although much of the aversive effect of some odors is likely to be a simple effect of unfamiliarity [65]. For instance, Chinese people living in the United States report conscious efforts to reduce odors associated with their traditional foods

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in the domestic and personal domains [66]. However, if genetic and cultural practices remain linked, the study of food choices and genetic diversity may be an inspiring model for gene and cultural interactions.

ACKNOWLEDGMENTS We wish to thank Jindra Havlickova for her valuable comments on the manuscript. JH was supported by grant GACR 406/06/P377 and Czech Ministry of Education grant 0021620828.

REFERENCES

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Lenochova P; Havlicek, J. Human body odour individuality. In: Hurst JL, Beynon, RJ, Roberts, SC and Wyatt TD, ed. Chemical Signals in Vertebrates IX. New York: Springer; 2008; 189-98. [2] Wedekind C; Seebeck T; Bettens F, Paepke AJ. MHC-dependent mate preferences in humans. Proceedings of the Royal Society of London Series B-Biological Sciences.. 1995 260, 245-9. [3] Herz RS; Inzlicht M. Sex differences in response to physical and social factors involved in human mate selection - The importance of smell for women. Evolution And Human Behavior. 2002 23, 359-64. [4] Herz RS; Cahill ED. Differential use of sensory information in sexual behavior as a function of gender. Human Nature. 1997 8, 275-86. [5] Havlicek J; Saxton TK; Roberts SC; Jozifkova E; Lhota S; Valentova J; Flegr J. He sees, she smells? Male and female reports on sensory reliance in mate choice and nonmate choice contexts. Personality and Individual Differences. 2008 45, 564-69. [6] Porter RH; Cernoch JM; Balogh RD. Odor signatures and kin recognition. Physiology and Behavior. 1985 34, 445-8. [7] Porter RH. Olfaction and human kin recognition. Genetica. 1998 104, 259-63. [8] Hays WST. Human pheromones: have they been demonstrated? Behavioral Ecology and Sociobiology. 2003 54, 89-97. [9] Thorne F; Neave N; Scholey A; Moss M; Fink B. Effects of putative male pheromones on female ratings of male attractiveness: Influence of oral contraceptives and the menstrual cycle. Neuroendocrinology Letters. 2002 23, 291-7. [10] Saxton TK; Little AC; Roberts SC. Ecological validity in the study of human pheromones. In: Hurst JL, Beynon, RJ, Roberts, SC and Wyatt TD, ed. Chemical Signals in Vertebrates. New York: Springer; 2008; 111-20. [11] Bojar RA; Holland KT. Review: the human cutaneous microflora and factors controlling colonisation. World Journal of Microbiology and Biotechnology. 2002 18, 889-903. [12] Stoddart DM. The scented ape - The biology and culture of human odour. Cambridge: Cambridge University Press 1990.

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[13] Wilson M. Microbial inhabitants of humans. Cambridge: Cambridge University Press 2005. [14] Penn DJ; Oberzaucher E; Grammer K; Fischer G; Soini HA; Wiesler D, et al. Individual and gender fingerprints in human body odour. Journal of the Royal Society Interface. 2007 4, 331-40. [15] Hepper PG. The discrimination of human odor by the dog. Perception. 1988 17, 54954. [16] Roberts SC; Gosling LM; Spector TD; Miller P; Penn DJ; Petrie M. Body odor similarity in noncohabiting twins. Chemical Senses. 2005 30, 1-6. [17] Porter RH; Moore JD. Human kin recognition by olfactory cues. Physiology and Behavior. 1981 27, 493-5. [18] Havlicek J; Roberts, SC. MHC correlated mate choice in humans: A review. Psychoneuroendocrinology. In press. [19] Penn DJ; Potts WK. The evolution of mating preferences and major histocompatibility genes. American Naturalist. 1999 153, 145-64. [20] Penn DJ. The scent of genetic compatibility: Sexual selection and the major histocompatibility complex. Ethology. 2002 108, 1-21. [21] Havlicek J; Lenochova P. Environmental effects on human body odour. In: Hurst JL, Beynon, RJ, Roberts, SC and Wyatt TD, ed. Chemical Signals in Vertebrates XI. New York: Springer; 2008; 199-212. [22] Chen D; Haviland-Jones J. Human olfactory communication of emotion. Perceptual and Motor Skills. 2000 91, 771-81. [23] Ackerl K; Atzmueller M; Grammer K. The scent of fear. Neuroendocrinology Letters. 2002 23, 79-84. [24] Burley N. The evolution of concealed ovulation. The American Naturalist. 1979 114, 835-58. [25] Kuukasjarvi S; Eriksson CJP; Koskela E; Mappes T; Nissinen K; Rantala MJ. Attractiveness of women's body odors over the menstrual cycle: the role of oral contraceptives and receiver sex. Behavioral Ecology. 2004 15, 579-84. [26] Havlicek J; Dvorakova R; Bartos L; Flegr J. Non-advertized does not mean concealed: Body odour changes across the human menstrual cycle. Ethology. 2006 112, 81-90. [27] Doty RL; Ford M; Preti G; Huggins GR. Changes in the intensity and pleasantness of human vaginal odors during the menstrual cycle. Science. 1975 190, 1316-7. [28] Chalmers RA; Bain MD; Michelakakis H; Zschocke J; Iles RA. Diagnosis and management of trimethylaminuria (FMO3 deficiency) in children. Journal of Inherited Metabolic Disease. 2006 29, 162-72. [29] Finlay IG; Bowszyc J; Ramlau C; Gwiezdzinski Z. The effect of topical 0.75% metronidazole gel on malodorous cutaneous ulcers. Journal of Pain and Symptom Management. 1996 11, 158-62. [30] Beauchamp GK. Diet influences attractiveness of urine in guinea-pigs. Nature. 1976 263, 587-8. [31] Ferkin MH; Sorokin ES; Johnston RE; Lee CJ. Attractiveness of scents varies with protein content of the diet in meadow voles. Animal Behaviour. 1997 53, 133-41. [32] Pierce AA; Ferkin MH. Re-feeding and the restoration of odor attractivity, odor preference, and sexual receptivity in food-deprived female meadow voles. Physiology and Behavior. 2005 84, 553-61.

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[33] Olsen KH; Grahn M; Lohm J. The influence of dominance and diet on individual odours in MHC identical juvenile Arctic charr siblings. Journal of Fish Biology. 2003 63, 855-62. [34] Wallace P. Individual discrimination of human by odor. Physiology and Behavior. 1977 19, 577-9. [35] Gangestad SW; Thornhill R. Menstrual cycle variation in women's preferences for the scent of symmetrical men. Proceedings of the Royal Society of London Series BBiological Sciences. 1998 265, 927-33. [36] Havlicek J; Roberts SC; Flegr J. Women's preference for dominant male odour: effects of menstrual cycle and relationship status. Biology Letters. 2005 1, 256-9. [37] Rikowski A; Grammer K. Human body odour, symmetry and attractiveness. Proceedings of the Royal Society of London Series B-Biological Sciences. 1999 266, 869-74. [38] Suarez F; Furne J; Springfield J; Levitt M. Insights into human colonic physiology obtained from the study of flatus composition. American Journal of PhysiologyGastrointestinal and Liver Physiology. 1997 35, G1028-G33. [39] Suarez FL; Springfield J; Furne JK; Lohrmann TT; Kerr PS; Levitt MD. Gas production in humans ingesting a soybean flour derived from beans naturally low in oligosaccharides. American Journal of Clinical Nutrition. 1999 69, 135-9. [40] Suarez FL; Springfield J; Levitt MD. Identification of gases responsible for the odour of human flatus and evaluation of a device purported to reduce this odour. Gut. 1998 43, 100-4. [41] Florin THJ; Neale G; Goretski S; Cummings JH. The sulfate content of foods and beverages. Journal of Food Composition and Analysis. 1993 6, 140-51. [42] Suarez F; Springfield J; Furne J; Levitt M. Differentiation of mouth versus gut as site of origin of odoriferous breath gases after garlic ingestion. American Journal of Physiology - Gastrointestinal and Liver Physiology. 1999 276, G425-30. [43] Tamaki T; Sonoki S. Volatile sulfur compounds in human expiration after eating raw or heat-treated garlic. Journal of Nutritional Science and Vitaminology. 1999 45, 213-22. [44] Stevinson C; Pittler MH; Ernst E. Garlic for treating hypercholesterolemia: A metaanalysis of randomized clinical trials. Annals of Internal Medicine. 2000 133, 420-9. [45] Buttery RG; Guadagni DG; Ling LC; Seifert RM; Lipton W. Additional volatile components of cabbage, broccoli, and cauliflower Journal of Agriculture and Food Chemistry. 1976 24, 829-32. [46] Schaal B; Marlier L; Soussignan R. Human foetuses learn odours from their pregnant mother's diet. Chemical Senses. 2000 25, 729-37. [47] Hepper PG. Adaptive fetal learning: prenatal exposure to garlic affects postnatal preferences. Animal Behaviour. 1988 36, 935-6. [48] Mennella JA; Johnson A; Beauchamp GK. Garlic ingestion by pregnant women alters the odor of amniotic fluid. Chemical Senses. 1995 20, 207-9. [49] Winberg J; Porter RH. Olfaction and human neonatal behaviour: Clinical implications. Acta Paediatrica. 1998 87, 6-10. [50] Mennella JA; Beauchamp GK. Maternal diet alters the sensory qualities of human milk and the nursling's behavior. Pediatrics. 1991 88, 737-44. [51] Mennella JA; Beauchamp GK. The transfer of alcohol to human milk: Effects on flavor and the infant’s behavior. New England Journal of Medicine. 1991 325, 981-5.

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[52] Mennella JA; Beauchamp GK. The human infants' response to vanilla flavors in mother's milk and formula. Infant Behavior and Development. 1996 19, 13-9. [53] Mennella JA. Mother's milk: A medium for early flavor experiences. Journal of Human Lactation. 1995 11, 39-45. [54] Mennella JA; Jagnow CP; Beauchamp GK. Prenatal and postnatal flavor learning by human infants. Pediatrics. 2001 107, e88. [55] Hauser GJ; Chitayat D; Berns L; Braver D; Muhlbauer B. Peculiar odours in newborns and maternal prenatal ingestion of spicy food. European Journal of Pediatrics. 1985 144, 403. [56] Yalcin SS; Tekinalp G; Ozalp I. Peculiar odor of traditional food and maple syrup urine disease. Pediatrics International. 1999 41, 108-9. [57] Podebrad F; Heil M; Reichert S; Mosandl A; Sewell AC; Bohles H. 4,5-dimethyl-3hydroxy-2[5H]-furanone (sotolone) - The odour of maple syrup urine disease. Journal of Inherited Metabolic Disease. 1999 22, 107-14. [58] Skinner JD; Carruth BR; Bounds W, Ziegler PJ. Children's food preferences: A longitudinal analysis. Journal of the American Dietetic Association 2002 102, 1638-47. [59] Havlicek J; Lenochova P. The effect of meat consumption on body odour attractiveness. Chemical Senses. 2006 31, 747-52. [60] Tishkoff SA; Reed FA; Ranciaro A; Voight BF; Babbitt CC; Silverman JS, et al. Convergent adaptation of human lactase persistence in Africa and Europe. Nature Genetics. 2007 39, 31-40. [61] Chan AWK. Racial differences in alchohol sensitivity. Alcohol and Alcoholism. 1986 21, 93-104. [62] Brown RE; Schellinck HM; West AM. The influence of dietary and genetic cues on the ability of rats to discriminate between the urinary odors of MHC-congenic mice. Physiology and Behavior. 1996 60, 365-72. [63] Schellinck HM; Slotnick BM; Brown RE. Odors of individuality originating from the major histocompatibility complex are masked by diet cues in the urine of rats. Animal Learning and Behavior. 1997 25, 193-9. [64] Porter RH; Doane HM. Dietary-dependent cross-species similarities in maternal chemical cues. Physiology and Behavior. 1977 19 129-31. [65] Zajonc RB. Attitudinal effects of mere exposure. Journal of Personality and Social Psychology. 1968 9, 1-27. [66] Manalansan IV MF. Immigrant lives and the politics of olfaction in the global city. In: Drobnick J, ed. The Smell Culture reader. Oxford: Berg; 2006; 41-52.

In: New Research on Food Habits Editors: K. Hasegawa and H. Takahashi

ISBN: 978-1-60456-864-6 © 2009 Nova Science Publishers, Inc.

Chapter 4

THE NATURAL HISTORY OF FOOD ALLERGY IN INFANCY Kostas N. Priftis*,1 Dimitrios Hatzis,2 Michael B. Anthracopoulos3 and Eva Mantzouranis4 1 2

Department of Allergy-Pneumonology, Penteli Children’s Hospital, P. Penteli, Greece First Department of Pediatrics, “Aghia Sophia” Children’s Hospital, Athens University Medical School, Athens, Greece 3 Respiratory Unit, Department of Paediatrics, University of Patras, Patras, Greece 4 Department of Pediatrics, University of Crete, Heraklion, Greece

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ABSTRACT Food allergy is primarily a problem of infants and young children with a decreasing prevalence in the years that follow. The natural history of specific food allergies varies substantially. Children who have become sensitized to cow’s milk, hen’s egg, wheat, and soybean through the gastrointestinal tract will usually lose this sensitivity as they grow older. On the other hand, certain food allergies, such as peanut, tree nut, and seafood usually continue throughout life. In this chapter the findings of a case control study of infants allergic to egg and/or fish followed to school age and the risk of wheezing illness and bronchial hyperreactivity (BHR) are presented and discussed. Sixty nine schoolchildren allergic to egg (N=60) and/or fish (N=29) in early life were recruited. They were followed for one year and were evaluated by parental questionnaire, skin prick testing, spirometry, and metacholine bronchial challenge. Another 154 (70 sensitized to inhaled allergens) were recruited selectively from a general population sample with no history of food allergy during their first three years and served as controls. Twenty three children (38.3%) maintained their sensitization to egg and 19 (65.5%) to fish; the prevalence of sensitization to ≥1 inhaled allergen(s) increased from 59.4% to 71% during childhood. Current asthma symptoms were reported more frequently in the study group than in either control groups. Index cases showed more often BHR compared to the control group as a whole; the difference was statistically indicative when index cases were separately compared to the sensitized controls. Multivariate logistic regression analysis showed that BHR as well as reported *

152 36 P. Penteli, Greece,E-mail:[email protected] Tel. +30-210-8036484, Fax +30-210-6131173

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Kostas N. Priftis, Dimitrios Hatzis, Michael B. Anthracopoulos et al. current asthma symptoms were associated with early wheezing and early sensitization to inhaled allergens but not with atopic dermatitis in infancy or persistence of egg or fish allergy.

INTRODUCTION

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Immune responses to a particular allergen vary, depending on the type of exposure and the processing of the food [1, 2]. Sensitization to food allergens develops in the order of exposure. Therefore, the prevalence of the reaction to different foods depends in part on the type and order of introduction of foods to infants and young children in a given population [3, 4]. In early infancy the most commonly encountered food allergy is cow’s milk protein allergy followed by allergy to wheat, egg, soybean and fish [1, 4, 5]. Risk factors associated with the development of food allergy include personal or family history of atopy or food allergy in particular, maternal consumption of major food allergens during pregnancy and the breastfeeding period, presence of atopic dermatitis, and transdermal food exposure [6-8]. The reactions that occur in infants after ingestion, inhalation, or skin contact with foods or food additives may vary from mild, gradually developing symptoms limited to the gastrointestinal tract or skin to severe, rapidly progressing, life threatening anaphylactic reactions [1, 7-12]. The natural history of specific food allergies varies substantially. Children who have become sensitized to cow’s milk, hen’s egg, wheat, and soybean through the gastrointestinal tract will usually lose this sensitivity as they grow older. On the other hand, certain food allergies, such as peanut, tree nut, and seafood usually continue throughout life [13, 14]. Diagnosis of childhood asthma and airway hyperreactivity is associated with early sensitization to inhaled allergens [15, 16]; conversely, respiratory manifestations in some food allergies such as cow’s milk allergy, seems to be unrelated to the persistence of food allergy into school age [17]. In the present chapter we attempt to offer further insight into natural history of food allergy developed in the first three years of life based on the findings of our own research. Our experience is derived from a case control study of infants allergic to egg and/or fish followed to school age evaluated for the risk of wheezing illness and bronchial hyperreactivity at school age [18].

NOMENCLATURE For the purpose of this review firstly we provide specific definitions of the most commonly used terms to describe a range of reproducible adverse reactions to food according to the Nomenclature Review Committee of the World Allergy Organization updating the European Academy of Allergology and Clinical Immunology Revised Nomenclature for Allergy Position Statement relevant [19, 20]. The term hypersensitivity should be used to describe objectively reproducible symptoms or signs initiated by exposure to a defined stimulus at a dose tolerated by normal persons. By this definition are included all kinds of controversial adverse reactions to food and food additives.

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Allergy is a hypersensitivity reaction initiated by specific immunologic mechanisms, antibody-mediated or cell-mediated. In most patients, the antibody typically responsible for an allergic reaction belongs to the IgE isotype and these patients may be said to suffer from IgE-mediated allergy. Antigens stimulating hypersensitivity mediated by an immunologic mechanism are referred to as allergens. Most allergens reacting with IgE and IgG antibodies are proteins, often with carbohydrate side chains. An allergic epitope denotes a specific peptide domain within a protein associated with allergenic potential. Atopy is a personal and/or familial tendency, usually in childhood or adolescence, to become sensitized and produce IgE antibodies in response to ordinary exposures to allergens. As a consequence, these persons can develop typical symptoms of asthma, rhinoconjunctivitis, or eczema. The term atopy should be reserved to describe the genetic predisposition to become IgE-sensitized to allergens commonly occurring in the environment and to which everyone is exposed but to which the majority do not produce a prolonged IgEantibody response. Thus, atopy is a clinical definition of an IgE-antibody high-responder. The term atopy can not be used until an IgE sensitization has been documented by IgE antibodies in serum or by a positive skin prick test. An adverse reaction to food is called food hypersensitivity. When immunologic mechanisms have been demonstrated, the appropriate term is food allergy, and, if the role of IgE is highlighted, the term is IgE-mediated food allergy. All other reactions are referred to as nonallergic food hypersensitivity. Anaphylaxis is a severe, life-threatening, generalized or systemic hypersensitivity reaction. The term allergic anaphylaxis is used when an immunologic mechanism can be shown to be important, e.g., IgE, IgG, and immune complex complement-related. An anaphylactic reaction mediated by IgE antibodies, such as peanut-induced food anaphylaxis, may be referred to as IgE-mediated allergic anaphylaxis. Tolerance toward food and inhaled allergens, known as oral and respiratory tolerance, respectively, is the suppression or down-regulation of immune effector cell responses (T and B) to an antigen by prior administration of the antigen by mucosal (e.g., oral) route.

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FOOD ALLERGENS Allergenic food proteins are typically water-soluble glycoproteins resistant to proteases, acids, and heat. Their molecular weight ranges from 10 kDa to 70 kDa. Two classes of food allergens exist: class 1 allergens are capable of sensitizing as well as triggering allergic reactions via the oral route. In contrast, class 2 food allergens do not sensitize orally because they are easily digested and thereby lose their sensitization potential. For the same reason, they trigger IgE mediated reactions only at the primary contact sites [1, 21]. In young children, milk, eggs, soy, peanut, and wheat account for more than 90% of food reactions, whereas in older children and adults peanuts, fish, shellfish, and tree nuts account for the vast majority of reactions. However, during the last decades eating habits have changed in modern societies, and rare vegetables and fresh fruits from all over the world are now available in every modern society. This has led to an increase in allergic reactions to fruits and vegetables such as papaya, kiwi, and seeds in the Western world [1, 12].

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Major allergenic epitopes in many foods have been isolated and characterized, and genes for some of the major allergens have been cloned and sequenced. The technology of food processing has been blamed for the enhancement of the allergenicity of some food proteins, while it reduced and the antigenicity of others. As an example, strong heat (≥121oC) treatment, through conformational changes, eliminates the allergenicity of the milk whey proteins, but only tempers that of other caseins. In general, heat treatment only reduces, but does not eliminate the allergenicity of milk proteins. Similarly, neither pasteurization nor homogenization alters the allergenicity of cow’s milk protein [3, 12]. Previously, an increased allergenicity of partially digested food proteins was suggested, but recent investigations do not support this hypothesis. The apparently high allergenicity of some food proteins may be due to timing of exposure, frequency of exposure, or possibly due to cross-reactivity with other proteins rather than to increased allergenicity per se [6].

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CLINICAL PRESENTATION OF FOOD ALLERGY IN INFANCY In early infancy the most common food allergy is cow’s milk protein allergy. Most infants develop symptoms at the very first months of age, often within one week after introduction of cow’s milk based formula. The majority of infants have cutaneous, gastrointestinal, and some present with respiratory symptoms [1, 3]. Furthermore, exclusively breastfed infants may react to food protein in their mother’s milk and in these infants the predominant clinical presentation is atopic eczema or proctocolitis (visible specks or streaks of blood mixed with mucus in the stool) [22, 23]. Symptoms occurring within a few minutes to less than 2 hours after food exposure, i.e. "immediate reactions" are mostly IgE-mediated, whereas symptoms occurring more than 2 hours after food intake are classified as "delayed reactions" [1]. Late reactions may occur after many hours and even up to a few days later. Most often delayed reactions are non-IgE-mediated [24]. Anaphylaxis has been reported with varying frequencies reflecting differences in patient selection. Immediate IgE-mediated reactions to foods often involve two or more target organs, e.g. the gastrointestinal tract, the skin, and the lungs, and may result in a variety of symptoms that potentially lead, eventually, to life-threatening reactions, including asthma exacerbation, laryngeal oedema, and anaphylaxis with cardiovascular collapse [2, 25]. An exception is the Oral Allergy Syndrome (OAS), for lack of a better term, which can be thought as the mucosal equivalent of urticaria [26]. After ingestion of specific foods (fresh fruits and vegetables) itching and swelling in the mouth and oropharynx occurs, in some patients. Owing to these symptoms, cessation of intake of the offending food may often ensue, as a self-established preventing measure by these patients. OAS is, commonly, associated with seasonal allergic rhinitis and/or rhinoconjunctivitis due to allergy to pollen/s, especially birch, grass, ragweed, and mugwort [27-29]. Cross-reactivity occurs when two or more allergens share epitopes and therefore bind to the same IgE antibodies [30]. As a result, patients sensitized to one allergen may also react to other, with same structure, without previous exposure and sensitization.

The Natural History of Food Allergy in Infancy

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NATURAL HISTORY Sensitization to hen’s egg in early life has been proposed as a predictor of subsequent sensitization to inhalant allergens that in itself constitutes a strong determinant of asthma [31, 32]. Early onset of IgE mediated food allergy, especially if associated with atopic dermatitis, indicates a Th2 dominant lymphocytic response pattern and a cytokine profile that facilitates an IgE response to environmental inhaled antigens; respiratory allergy is the consequence of the interaction between these inhaled allergens and high risk genetic background [33]. Allergy to common food allergens, such as cow’s milk, egg and fish, begins predominantly before the second year of life demonstrating a clear temporal relationship with the introduction of these foods into the children’s diet. Over time, most food allergy is lost, although the possibility of such loss depends on the individual child and the specific food allergen. In contrast to cow’s milk and egg, allergies to fish are usually not outgrown [34-36]. It is not clear whether infants with food allergies of different natural history are at a different risk of developing asthma at school age. The mechanisms that predispose children with food allergy to develop asthma at a later age remain poorly understood. It is speculated that the immunologic basis of specific organ syndromes such as allergic rhinitis and asthma actually results from a systemic dysregulation of immunity [37, 38]; eczema and, at a later age, hyperreactive airways could be manifestations of different target organs within the frame of the same systemic disease. Most studies that address this issue are observational and focus on the so called “allergic march”, i.e. the clinical expression of atopy that begins with eczema and sensitization to foods and evolves into asthma and allergic rhinitis in association with sensitization to airborne allergens [39]. Few studies have focused on bronchial hyperresponsiveness (BHR) that does not always parallel the clinical expression of atopy and may remain latent [40, 41].

Our Research

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

In our research, we hypothesized that children allergic to common food allergens in infancy are at increased risk of wheezing illness and BHR during school age; i.e., sensitization to food allergens with different natural histories may carry a different risk of developing asthma or hyperreactive airways at school age.

Study Population The present case-control study evaluated three groups of children (table 1). The study group consisted of 69 school age children who presented to our outpatient Allergy clinic during the first three years of life for a variety of symptoms from January 1995 until the end of 1998. The diagnosis of food allergy was based on a positive skin prick test (SPT) result or a positive serum-specific immunoglobulin E test to hen’s egg or/and fish (>0.35 IU/mL), a well-documented history of reaction to relevant food(s) and, in thirteen cases, on immediate symptoms following open challenge with suspected food. Sixty children were allergic to hen’s egg (egg white and yolk) and 29 to fish (20 were sensitized to both). Open food challenges were also performed when appropriate to investigate development of tolerance [42, 43].

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Kostas N. Priftis, Dimitrios Hatzis, Michael B. Anthracopoulos et al.

Table 1. General characteristics of the study population (index cases and two control groups).

Male (%) Median age, years (range) Atopic family history (%) Passive smoking (%) Central house heating (%) Lifetime asthma symptoms (%) Current asthma symptoms (%) Lifetime chronic rhinitis (%) Current chronic rhinitis (%) Lifetime atopic dermatitis (%) Current atopic dermatitis (%)

Study group N=69 52 (75.4) 9.9 (7.2-13.3) 41 (59.4)* 45 (65.2) 59 (85.5) 38 (55.1)* # 30 (43.5)* # 27 (39.1) § 20 (29.0) § 38 (55.1)* # 7 (10.1) #

Sensitised controls N=70 44 (62.8) 10.3 (8.0-13.5) 35 (50.0) ¶ 46 (65.7) 56 (80.0) 20 (28.5) ¶ 17 (24.3)¶ 23 (32.8) 21 (30.0) § 20 (28.5) 19 (27.1)¶

Non-sesnsitised controls N=84 50 (59.5) 10.0 (7.5-13.1) 21 (25.0) 56 (66.7) 67 (79.8) 9 (10.7) 5 (5.9) 19 (22.6) 13 (15.5) 13 (15.5) 8 (9.5)

* In comparison to non-sensitised controls: p