Advances in Food and Nutrition Research Volume 33

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 33

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ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 33

Edited by JOHN E. Institute of Cornell Ithaca,

KINSELLA Food Science University New York

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

San Diego New York Berkeley Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

COPYRIGHT 0 1989 BY ACADEMlC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

ACADEMIC PRESS, INC. San Diego, California 92101 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NW 1 7DX

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ISSN

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ISBN 0-12-016433-7

(alk. paper)

PRINTED IN THE UNITED STATES OFAMERICA 89 90 91 92

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8 7 6 5 4 3 2

I

CONTENTS

CONTRIBUTORS TO VOLUME 33 ......................................................... PREFACE..................................................................................... EMILM . MRAK ...........................................................................

vii ix

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Chemical and Nutritional Aspects of Folate Research: Analytical Procedures. Methods of Folate Synthesis. Stability. and Bioavailability of Dietary Folates Jesse F . Gregory I11 I . Introduction .................................................................................. I1 . Chemical Properties of Folates ......................................................... Ill . Determination of Folate in Foods and Other Biological Materials ........... IV . Stability and Chemical Behavior of Folates in Foods ........................... V Bioavailability of Dietary Folate ....................................................... V1 . Conclusion ................................................................................... References ....................................................................................

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2 4 13 40 52 80 80

Calcium in the Diet: Food Sources. Recommended Intakes. and Nutritional Bioavailability Dennis D . Miller 1. Introduction .................................................................................. ....................................... I1 . Calcium Intakes and Food Sources .... 111. Recommended Dietary Allowances ................................................... ...................................... IV . Calcium Homeostasis ........ V . Intestinal Absorption of Calcium .............................................. VI . Calcium Bioavailability ................................................................... ............................. v11. Summary and Conclusions ............. VIII . Research Needs .................................................................... References ....................................................................................

103 105 108 112 114 119 147 148 149

Dietary and Biochemical Aspects of Vitamin E Robert S . Parker I . Introduction .................................................................................. I1 . Dietary Sources. Stability. and Intake of Tocopherols .......................... 111. Intestinal Absorption of Tocopherols .................................................

V

158 159 168

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CONTENTS

IV . V. VI . V11. VI11.

1X .

Transport of Tocopherols in Blood ................................................... Tocopherols in Tissues ................................................................... Tocopherol Binding and Transfer Factors .......................................... Tocopherol Metabolism .................................................................. Functional Aspects of Tocopherols in Biomembranes ........................... Future Research Directions ............................................................. References ....................................................................................

174 178 191 199 207 220 222

Oxldatlon of Polyunsaturated Fatty Acids: Mechanisms. Products. and inhibition with Emphasis on Fish

R . J . Hsieh and J . E . Kinsella 1. 11. 111. IV . V. V1. VII .

Introduction .................................................................................. Mechanisms of Oxidation of Polyunsaturated Fatty Acids ..................... Products of Lipid Oxidation ............................................................. Other Effects of Lipid Oxidation ...................................................... Lipid Oxidation in Fish ................................................................... Control of Lipid Oxidation .............................................................. Conclusions .................................................................................. References ....................................................................................

234 234 270 281 286 302 320 320

Proteins in Whey: Chemical. Physical. and Functional Properties

J . E . Kinsella and D . M . Whitehead I . Introduction .................................................................................. I1 . Whey Products .............................................................................. 111. Structure of Whey Proteins ............................................................. 1v. Whey Protein Preparation and Isolation ............................................. V . Functional Properties of Whey Proteins ............................................. v1. Hydration and Solubility ................................................................. VI1. Thermal Properties of Whey Proteins ................................................ VIII . Gelation ....................................................................................... IX . Surface Activity of Whey Proteins .................................................... X . Foams ......................................................................................... XI . Emulsions .................................................................................... XI1. Ligand Binding by Whey Proteins ..................................................... XI11. Modification of Whey Proteins ......................................................... XIV . Nutritional Aspects of Whey Proteins ................................................ xv . Summary and Conclusions .............................................................. Research Needs ............................................................................. References ....................................................................................

344 346 349 360 364 373 376 381 391 397 405 408 416 422 423 424 425

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INDEX

CONTRIBUTORS TO VOLUME 33 Numbers in parentheses indicate the pages on which the authors' contributions begin.

Jesse F. Gregory 111, Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611 (1) R. J. Hsieh, Campbell Institute for Research and Technology, Campbell Soup Company, Camden, New Jersey 08103 (233); formerly at the Institute of Food Science, Cornell University, Ithaca, New York, 14853 J. E. Kinsella, Institute of Food Science, Cornell University, Ithaca, New York 14853 (233, 343) Dennis D. Miller, Institute of Food Science, Cornell University, Ithaca, New York 14853 (103) Robert S . Parker, Division of Nutritional Sciences and Department of Food Science, Cornell University, Ithaca, New York 14853 (157) D. M. Whitehead, institute of Food Science, Cornell University, Ithaca, New York 14853 (343)

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PREFACE

This is the first volume of Advances in Food and Nutrition Research, formerly called Advances in Food Research, the leading publication for comprehensive reviews on important topics in food science. The expanded title recognizes the integral relationships between food science and nutrition and reflects the intention to present reviews of topics in nutrition as well as in food science. This change also encourages nutritionists and food scientists to become more familiar with relevant advances in these respective and interrelated areas. Future volumes will endeavor to emphasize relationships between production, processing, formulation, and fabrication of foods and the nutrient content, nutritional value, bioavailability, safety, and public health. Accelerating developments in analytical methods are providing new data concerning the nutrient and antinutrient content, bioavailability,and the effects of processing and storage on nutrient and non-nutrient interactions. Such methods are timely, as new sources and varieties of foods are being developed via the emerging “new” biotechnologies. In addition, new processing technologies should facilitate the design and fabrication of foods with a more balanced nutrient profile. This will underscore the need for nutritional and toxicological expertise in the food processing sector and the concomitant need for the timely interchange of information in the relevant discipline areas. Advances in Food and Nutrition Research will endeavor to meet this need. The increasing emphasis on science and nutrition, combined with the breadth of knowledge required to be a food scientist, makes food science a very challenging field, with specialization in particular areas becoming a prerequisite for maintaining competence. With this continuing challenge, comprehensive reviews that summarize scientific developments in relevant topic areas and integrate this information into contemporary food science and nutrition knowledge are extremely important. This is a primary objective of Advances in Food and Nutrition Research, and the editorial board welcomes contributions from scientists active in the various disciplines encompassed by food science and nutrition. Suggestions of suitable topics, possible authors, and themes for future issues are solicited, and scholarly contributions are welcome. JOHN E. KINSELLA

ix

EMILM. MRAK ( 1901-1 987)

EMlL M. MRAK

Emil Marcel Mrak, Chancellor Emeritus of the Davis campus of the University of California, died April 9, 1987, concluding a vigorous academic and public career spanning nearly 60 years. A respected teacher and researcher, an energetic and imaginative administrator, and a skilled advisor to government and private agencies, Emil led the Davis campus through the decade of its greatest growth. His stewardship brought him lasting respect from faculty, staff, students, administrators, alumni, parents, and educational and political leaders. Emil’s leadership was an important contributing factor to the University of California, Davis, in gaining national and international recognition. Emil Mrak was born in San Francisco on October 27, 1901, and was reared in the Santa Clara Valley. On graduation from Campbell High School, his lifelong career in the University of California system began. In 1926, he received his B.S. degree in food technology from the Berkeley campus and received his M.S. degree in 1928. By the time he completed his Ph.D. in botany and mycology, also at Berkeley, in 1936, he had authored or co-authored nearly 40 articles. His creative application of science to the solution of practical problems in food processing-the hallmark of his professional career-was clearly established. Appointed as an instructor in food technology in 1937, and rising to the rank of professor and department chairman in 1948, Emil transferred with most of his staff to a new building on the Davis campus in 1951 to reorganize and expand the Department of Food Science and Technology. Under his leadership, the department grew in size and diversity of research areas and eventually became one of the top institutions of food research in the world. In spite of his administrative responsibilities, Emil continued his active participation in basic research, which dealt with ecological problems and classification of yeasts. Two yeast species have been named for him; Williopsis mrakki and Zygosaccharomyces mrakki. Emil’s vigor and intellectual curiosity made him one of the truly challenging and highly respected teachers on both’ the Berkeley and Davis campuses. These

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same traits fostered a rich record of editorial contributions including Advances in Food Research, for which he served as editor and occasional author. In 1959, Emil was appointed chancellor of the University of California, Davis. His personal commitment to fostering quality scholarship in all disciplines, his respect for education in the professions, and his perception of what constituted an appropriate intellectual, social, and physical environment for faculty and students guided the deveropment of the Davis campus through its most critical formative period. His values were clearly and frequently communicated to the campus community. Informality combined with a high degree of warmth and sensitivity toward faculty, student, and staff needs were carefully balanced with his inherent drive and impatience with those who were slower to recognize and seize opportunities to strengthen the campus. His style is perhaps best captured in his inaugural address: “The only way to keep this world a good world, and make it better, is to assert creative and constructive individualism, which is, to me, another way of saying ‘leadership’.” Emil’s enormous energy, practical understanding, and his ability to communicate with widely diverse groups made him a favorite with the public and particularly with public officials and his administrative colleagues. Emil Mrak contributed extensively, nationally and internationally, in his professional field throughout his career. He was president and fellow of the Institute of Food Technologists, president of the International Academy of Environmental Safety, president of the International Academy of Sciences, fellow of the American Association for the Advancement of Science, fellow of the American Public Health Association, fellow of the American Academy of Arts and Sciences, and co-founder and president of the International Congress of Food Science and Technology. He chaired numerous national oganizations and commissions both public and private. Retiring from the university in 1969, Emil devoted his energies to increased public service, serving on a number of scientific advisory cornmitees to agencies, such as the U.S. Public Health Service, the National Institutes of Health, the Environmental Protection Agency, and the National Science Foundation, and on special assignments for the White House related to food quality and safety, environmental quality, and relations with Latin America. He was awarded honorary degrees from the University of California, Davis, Michigan State University, the Swiss Federal Institute of Technology, and Rutgers University. His major professional and industrial awards exceed 20, and include the Nicholas Appert Medal, and the Babcock-Hart, Spencer, Atwater, Tanner, and C. R. Fellers awards, the most prestigious in the field of food

EMIL M. MRAK

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science. The administration building on the Davis campus was named Emil M. Mrak Hall in 1969. Notwithstanding his accomplishments and stature, Emil maintained an open-door policy throughout his career, which significantly extended his personal influence on the lives of students and his associates. He was particularly known for his compassion for people and his intense personal and institutional loyalties. Emil’s colleagues found his friendship and scientific involvements very valuable to them and to the department at the University of California, Davis. He was one of the unique leaders in the field of food science and technology in our time. He had a special feeling also for Advances in Food Research, which he helped initiate and for which he served as senior editor. He never let up his intensity, even in the recent years before his death at age 86. In spite of the pace of his life, Emil was deeply devoted to his family and significantly influenced their educational and career development. Emil Mrak was indeed a scientist, a leader, and a humanist.

C. 0. MCCORKLE, JR. M. W. MILLER R. C. PEARL H. J. PHAFF B. SCHWEIGERT

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ADVANCES IN FOOD A N D NUTRITION RESEARCH. VOL.

33

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE RESEARCH: ANALYTICAL PROCEDURES, METHODS OF FOLATE SYNTHESIS, STABILITY, AND BlOAVAlLABlLlTY OF DIETARY FOLATES JESSE F. GREGORY 111

Food Science und Humun Nirtrition Depurtment Instiiirie of Food und Agricrrltural Sciences University of Florida Guinesville, Floridu 3261 I

I.

Introduction Chemical Properties of Folates A. Structural, Ionic, and Inherent Chemical Properties B. Synthesis of Experimentally Significant Folates 111. Determination of Folate in Foods and Other Biological Materials A. Introduction B. Preparative Phases of Folate Determination C. Methods of Folate Determination D. Comparison of Analytical Methods E. Discussion and Conclusion IV. Stability and Chemical Behavior of Folates in Foods A. Introduction B. Intrinsic Stability of Folates C. Stability of Folates in Foods and Related Materials V. Bioavailability of Dietary Folate A. Introduction B. Physiological and Biochemical Aspects of Folate Absorption C. In Vivo Kinetics of Folate Metabolism D. Methods for the Assessment of Folate Bioavailability E. Bioavailability of Folate VI. Conclusion References 11.

I Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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1.

INTRODUCTION

It has long been established that a nutritional requirement exists for the group of compounds of which folic acid is the base structure. The generic term for this group of organic micronutrients is folate or folacin (the former currently preferred). These compounds, which cannot by synthesized de novo by mammalian cells, function coenzymatically as carriers of one-carbon units in a variety of reactions involved primarily in the metabolism of certain nucleic acids and amino acids. The mechanism of action of folate cofactors has largely been elucidated (Benkovic, 1978, 1980). The chemical structures of the folates are summarized in Fig. 1. All folates are similar in structure to folic acid, which comprises a fully aromatic pteridine coupled via a carbon-nitrogen (C-9-N-10) bond to p-aminobenzoic acid, which in turn is coupled via an amide bond through its carboxyl moiety to the amino group of L-glutamic acid. The pteridine moiety of folates can exist in three oxidation states: fully oxidized (aromatic), or as the reduced 7,g-dihydro (H,folate) or 5,6,7,8-tetrahydrofolate(H,folate) forms. Tetrahydrofolates are the coenzymatically active forms of the vitamin. One-carbon substituent groups can be present at either the N-5 or N-10 positions or linking these positions. The one-carbon groups also differ in their oxidation state, with folates existing as derivatives of formate (5-formyl-H,folate, 10-formyl-H,folate, 5,10-methenyl-H4folate,and 5-formimino-H4folate),methanol (5-methyl-H4folate),or formaldehyde (5,lO-methylene-H,folate).In addition to these structural variables, most naturally occurring folates exist as y-linked polyglutamate conjugates, with chain lengths typically in the range of five to seven glutamate residues. Over 100 distinct forms of the vitamin may exist naturally as a result of this chemical diversity, although less than 50 principal folates exist in most animal and plant tissues. Analytical and experimental methods have been a limitation in the study of the chemical, biochemical, and nutritional properties and functions of this vitamin. In addition, the content of the vitamin in foods, its stability and chemical behavior, and the factors affecting its bioavailability are not fully understood. It is the intent of this chapter to review and critically appraise current knowledge concerning (1) the inherent chemical properties of folates and methods of synthesis of experimentally important forms of the vitamin, (2) analytical methods for measurement of folates in foods and other biological materials, (3) the stability and chemical behavior of folates in foods, (4) the folate requirement of humans and the nutritional adequacy of human diets, ( 5 ) methods of assessment of the bioavailability of the vitamin, and (6)the mechanism of folate absorption

CHEMICAL A N D NUTRITIONAL ASPECTS OF FOLATE

3

A

C

D

FIG. 1. Structures of folates. Each of the compounds shown also exists in poly-y-glutamate forms. (A) Folic acid, X=H; 10-formyl-folate, X=CHO. (B) 5,6-H, folate, R = H ; 5-methyl-5,6-H2folate, R=CH,. (C) 5,6,7,8-H, fol-ate, R = H, X = H ; 5 methyl-H, folate, R = CH,, X = H; 5-formyl-H, folate, R = CHO, X = H; 5-formimino-H, folate, R = CHNH, X = H ; 10-formyl-H,folate, R = H , X=CHO. (D) Pterioc acid. 5,10-Methylidene(methenyl)H, folate and 5,10-methylene-H, folate have -CH= and 4 H 2 - bridges between N-5 and N-10 positions, respectively.

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JESSE F. GREGORY Ill

and factors affecting the bioavailability of dietary folate. These topics have been the subject, in part, of earlier reviews (Rodriquez, 1978; Anderson and Talbot, 1981). II. CHEMICAL PROPERTIES OF FOLATES

A. STRUCTURAL, IONIC, AND INHERENT CHEMICAL PROPERTIES The structure of the various folates has been firmly established on the basis of ultraviolet, infrared, and proton magnetic resonance spectral studies, selective degradation reactions, oxidation/reductionstudies, isotopic hydrogen exchange experiments, and specific methods of synthesis. The reader is referred to reviews by BIakley (1969) and Huennekens (1968) for further information in this regard. Descriptive terminology for folates has been recently formulated by the IUPAC-IUB Commission on Biochemical Nomenclature (Blakley, 1987). An important structural aspect of the 5,6,7,8-H4folatesis the stereochemical orientation at the C-6 asymmetric carbon of the pyrazine ring. In vivo vitamin activity of H,folates and in vitro coenzymatic activity is only observed with the (6S)-H,folate isomers (formerly called 6 1 isomers). As discussed later, the different biochemical properties of 6R and 6 s isomers often complicate analytical and nutritional studies. Methods of chemical synthesis of H,folates, whether by catalytic hydrogenation or chemical reduction, yield a racemic product. Stereochemical nomenclature for the reduced folates is described by Blakley (1987). The site of unsaturation on the pyrazine ring of 7,8-H2folateshas been determined by several experimental approaches (Huennekens, 1968). H,folate derived from the chemical reduction of folk acid with dithionite or borohydride, which is identical to naturally occurring H,folate, can act as substrate for dihydrofolate reductases, with the formation of H,folate which exhibits full biological activity (i.e., in the 6 s isomeric form). This observation provided strong evidence against the 5 ,6-H2folate isomer, which would have yielded a racemic (6-ambo)-H,folate product in the dihydrofolate reductase reaction. The identification of the 7,8- double bond was also supported by labeling studies (Zakrzewski, 1966; Scrimgeour and Vitols, 1966) and NMR spectra (Poe, 1973). Folates are ionogenic and amphoteric molecules. The pK, values of the various ionizable groups have been reported (Kallen and Jencks, 1966; Kallen, 1971; Poe, 1977). Ionogenic groups of particular significance in

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

5

the range of pH values relevant to foods and biological systems are the N-5 position of H,folate (pK, = 4.8) and the glutamate carboxyl groups (y pK, = 4.8; a pK, = 3.5). Folates generally reach minimum solubility in the mildly acidic pH range (e.g., pH 2-4), with monocationic and neutral species predominating, while solubility generally increases in proportion to the pH above this range (mainly anionic species). It should be noted that the solubility of folates is high at very low pH (e.g., 6 M HCI, pH -0.781, in which the highly cationic species exist. Marked effects of pH are observed on the UV spectra of all folates (Blakley, 1969), reflecting charge and tautomerization effects on the pteridine moiety. Pteroic acid (folic acid minus the glutamic acid) is markedly less soluble than folic acid over most of the pH scale. The greater solubility of folic acid presumably reflects the influence of its additional carboxyl group, which provides additional polar and hydrophilic character. Polyglutamyl folates, because of the free a-carboxyl group associated with each glutamate residue, exhibit greater anionic character than the monoglutamyl forms at intermediate and higher pH values. By contrast, long-chain polyglutamyl folates exhibit greater hydrophobic character than their shorter-chain analogs when the glutamate carboxyl groups are predominantly protonated (e.g., pH 2) (Bush et al., 1979). The stability of the various folates is markedly affected by the pH of the medium, as will be discussed later. The influence of pH on the behavior of folates is perhaps most pronounced in the case of the formyl H,folates. 5,l0-Methenyl-H4folate is formed spontaneously from either 5-formyl-H4folateor 10-formyl-H,folate at low pH, with the rate proportional to the acidity of the medium (Robinson, 1971). This reaction goes to completion at pH <2.0. Following neutralization of a solution of 5,10-methenyl-H4folate,hydrolysis occurs to yield 10-formyl-H,folate, with minor amounts of 5-formyl-H4folate. The rate of hydrolysis of 5,l0-methenyl-H4folateis strongly enhanced by the presence of inorganic or organic buffer ions at typical concentrations of 0.05 to 0.1 M ,while an inhibitory effect is observed at higher buffer concentrations (Robinson, 1971). Ascorbate ions retard the hydrolysis of 5,10-methenyl-H4folateat levels commonly used to protect folates from oxidation (>5 rnM) (Brown et al., 1973a). The nonenzymatic reaction of H,folate with formaldehyde to form 5,lOmethylene-H,folate is another strongly pH-dependent phenomenon. This reaction involving the H,folate species with the N-5 position unprotonated (pK, = 4.8) is approximately 40-fold more favorable than the same reaction involving an N-5-protonated H,folate (Kallen, 1971). Although evidence of the reaction of formaldehyde with the N-5position to form 5-hydroxymethyl-H4folate has been reported, the equilibria favor the for-

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JESSE F. GREGORY 111

mation of the 5,IO-methylenederivative by 1000-fold (Kallen, 1971). The reaction of formaldehyde at the N-10 position appears equally unfavorable. Folates have been shown to undergo a concentration-dependent selfassociation phenomenon. In proton magnetic resonance studies by Pastore (1971), Poe (1973), and Khaled and Krumdieck (1983, the occurrence of concentration-dependent up-field shifts in resonances was evidence of monomer-dimer ,equilibria at high folate concentrations (1-20 mM). These associations can be envisioned as a hydrophobic stacking of folate molecules in solution. Folic acid, being more aromatic, exhibited a greater tendency to dimerize than H2folate,which in turn was more prone to self-association than H,folate (Khaled and Krumdieck, 1985). Although these associations may complicate certain kinetic studies of folate enzymes, the reported association constants (Khaled and Krumdieck, 1985) suggest that there is little in vivo significance. B. SYNTHESIS OF EXPERIMENTALLY SIGNIFICANT FOLATES Many forms of folate are not commercially available; thus, the ability to synthesize needed forms of the vitamin becomes an essential element of folate research. The folates available from commercial biochemical suppliers include folk acid, H,folate, H,folate, 5-methyl-H4folate,and 5formyl-H,folate. Because the commercial H,folate derivatives are chemically reduced, they are racemic at the C-6 asymmetric carbon (i.e., 6ambo forms).

I. Synthesis of Polyglutamyl Folates Methods for the synthesis of polyglutamyl folates have been devised using either solid-phase or liquid-phase methods for the peptide synthesis. These methods have been thoroughly reviewed by Krumdieck et al. (1983b). Research concerning the nutritional properties, chemistry, and metabolism of folates will be greatly facilitated when polyglutamyl folates are available on a commercial basis. The solid-phase synthesis, which was developed by Krumdieck and Baugh (1969, 1980a), is a modification of the standard procedure of Marshall and Merrifield for peptide synthesis (1965). By this procedure, Ntert-butyloxycarbonyl-glutamicacid-a-benzyl ester is esterified to the beads of a chloromethylated resin. The y-glutamyl peptide chain is elongated in stepwise fashion by reaction of activated N-tert-butyloxycarbonyl-glutamic acid-a-benzyl ester (as the isobutylchloroformate mixed an-

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

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hydride) with the deprotected amino group of the resin-bound glutamic acid. Because of the solid-phase nature of the reaction, all steps can be conducted conveniently in a single reaction vessel. This facilitates the addition of reagents, mixing, and washing steps. When the desired polyglutamyl chain length is achieved, pteroic acid (as the N-10-trifluoroacetyl, isobutylchloroformate mixed anhydride) is coupled. The removal of protective groups and cleavage of the polyglutamyl folate from the resin is best accomplished by saponification in an alkaline acetone solution. The product is then recovered by precipitation and is purified by ion-exchange chromatography. In spite of the fact that an integral step in the peptide synthesis is an acetylation step to block unreacted amino groups prior to introduction of the next glutamate residue, the solid-phase procedure inevitably yields a product that is contaminated with the shorter chain (n - I ) polyglutamate (typically several mole percent; e.g., Matthews, 1986). Several minor modifications were recently described by Rowe and Lewis (1986) which improved the overall yield and reproducibility of the synthesis. The major limitations of the solid-phase method concern the following ( I ) the method is highly sensitive to water, thus requiring anhydrous conditions and distillation of reagents; (2) yields are frequently low, although this can be compensated for by adjustment of the quantity of starting resin and reagents; (3) as with all methods of synthesis, the preparation and purification of sufficient pteroic acid is often rate limiting. Liquid-phase methods (e.g., Godwin et al., 1972; Rabinowitz, 1983)for the synthesis of polyglutamyl folates offer the potential advantage of yielding a more homogeneous product. This is achieved by purification of the newly synthesized polyglutamyl peptide derivative after each elongation step, which increases the time and effort required and may decrease the yield of the procedures. At this time, the liquid-phase methods offer little advantage over the more widely used solid-phase synthesis. Regardless of the method of peptide synthesis employed, the preparation of pteroic acid is a difficult but essential step in the synthesis of polyglutamyl folates. Because of the expense of commercially available pteroic acid, this compound is best prepared in large scale by bacterial fermentation using a strain of Pseudomonas (ATCC 25301) (Houlihan et al., 1972; Scott, 1980b). This organism is capable of growing in a liquid medium containing folic acid as its sole source of carbon and nitrogen and can yield over 90% conversion of folk acid to pteroic acid. A procedure for the preparation of pteroic acid by chemical cleavage of folic acid has been developed by Temple et al. (1981), while the total synthesis of pteroic acid also has been reported (Nair et al., 1981). These procedures offer no advantage over the microbiological method and are substantially more laborious. In addition, lengthy purification of pteroic acid by ad-

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JESSE F. GREGORY I11

sorption chromatography on nonionic cellulose (Houlihan et al., 1972; Scott, 1980b)is required following either microbiological or chemical synthesis. Smaller-scalepreparations can be performed by enzymatically catalyzed cleavage of folic acid using carboxypeptidase G 1 (Bacher and Rappold, 1980), which is now commercially available.

2. Preparation of 5,6,7,8-Tetrahydrofolateand 7,8-Dihydrofolate A variety of interconversion reactions is available for the preparation of monoglutamyl and polyglutamyl folates that are not commercially available. Considerations in the selection of methods mainly involve the quantity of product and the stereochemical form required. A major problem in biological and chemical studies of reduced folates is the instability of H,folate and impurity of commercial sources (typically 70%, although highly variable), Various methods have been reported for the preparation of H,folate by reduction of folic acid by catalytic hydrogen,ation (as used commercially) or chemical reduction. Hydrogenation of folic acid in glacial acetic acid with a platinum oxide catalyst yields racemic (6-ambo)-H4folateif rapidly stabilized with 2-mercaptoethanol (Huennekens et al., 1963b).The major limitations of this procedure are the tendency for oxidative degradation during filtration and uncertainty concerning the end point of the hydrogenation reaction. A more convenient method for the preparation of H,€olate is the chemical reduction of 7,8-H2folate, prepared by dithionite reduction of folic acid, with sodium borohydride (Scrimgeour, 1980). Using this reduction method, (6-ambo)-H,folate can be prepared with approximately 95% yield at ambient temperature and neutral pH. Following decomposition of residual borohydride, the H,folate is protected by the addition of 2-mercaptoethanol to a concentration of 0.2 M. The reaction of folic acid and sodium dithionite has been exploited widely in folate research. Dithionite rapidly reduces folic acid to 7,8-H2folate at ambient temperature as originally described by Futterman (1957, 1963)and modified by Blakley (1960), with nearly 100% yield when ascorbate is present as a protective agent. When folic acid and dithionite are reacted at 75°C for 90 min in the presence of ascorbate, or at ambient temperature in glacial acetic acid, (6-ambo)-H4folateis the primary product (Zakrzewski and Sansone, 1971; Davis, 1968). These methods offer no advantage over the preparation of H,folate using the dithionite and borohydride under milder conditions (Scrimgeour, 1980). Alternatively, folic acid dissolved in glacial acetic acid can be reduced to H,folate using sodium cyanoborohydride (Tatum et al.,1980). This method gives H,folate in 60-75% yield and is well suited for large-scale preparations.

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9

Electrochemical reduction is an attractive method for the preparation of (6-ambo)-5,6,7,8-H4folates from folic acid or pteroylpolyglutamates (Rowe and Lewis, 1986).In this procedure, the folate (dissolved in a volatile triethylammonium bicarbonate buffer, pH 9.25) is reduced under nitrogen gas with a gradually decreasing potential (final potential -0.95 V). It was reported that pteroylpentaglutamate, after reduction, stabilization with 2-mercaptoethanol (0.2 M ) and lyophilization, gave H,folate pentaglutamate with a final yield of 96%. Enzymatically catalyzed reduction using dihydrofolate reductases is necessary to provide H,folate that is stereochemically pure as the biologically active 6 s isomer. The original procedure of Matthews and Huennekens (1960), which yielded effective (83%) reduction of 7,8-H2folate,was based on the use of glucose-6-phosphate dehydrogenase to provide continual regeneration of the NADPH coenzyme. A similar method was reported for the enzymatic preparation of polyglutamyl (6S)-5,6,7,8-H4folates (Kisliuk et al., 1981) with overall yields of 40% (including reduction and purification). Matthews (1986) described a procedure for the formation of H,folate polyglutamates from fully oxidized polyglutamyl folates, with 50-70% net yield after chromatographic purification. The use of immobilized dihyrofolate reductase provides an alternative that may be used in batchwise or continuous formation of H,folates (Ahmed and Dunlap, 1984). It should be noted that the source of dihydrofolate reductase can be an important consideration if the intended procedure involves a direct enzymatic reduction of fully oxidized folic acid or pteroylpolyglutamates rather than H,folates. The reductase from Lactobacillus casei is substantially more effective in reducing oxidized folates than the enzyme from other commercially available sources. 3. Formation of One-Carbon-Substituted Folates

Unlabeled, monoglutamyl forms of (6-ambo)-5-methyl-H4folateand (6ambo)-5-formyl-H,folate are commercially available in suitable purity and, thus, their synthesis is generally not required for most aspects of folate research. This commercial form of (6-ambo)-5-formyl-H4folate serves as a convenient precursor for racemic forms of 5,IO-methenylH,folate and 10-formyl-H,folate (methods described previously; Robinson, 1971). However, the formation of other one-carbon substituted H,folates is frequently required in studies involving (1) polyglutamyl folates which are chemically synthesized as pteroylpolyglutamates (i.e., folic acid polyglutamates) and (2) tritiated or deuterium-labeled forms of folic acid. Several approaches are available for the formation of each of the substituted folates, as reviewed by Krumdieck et al. (1983b).

10

JESSE F. GREGORY I11

The formation of formyl-H,folates can be accomplished by formylation before or after pteridine reduction. In view of the interconvertibility of these compounds (Rabinowitz, 1963; Robinson, 197I ) , the synthesis of either 5- or 10-formyl-H,folate or 5,lO-methenyl-H,folate permits the preparation of the other forms. For formylation prior to reduction, folic acid is incubated in 98% formic acid at ambient temperature for 2 days (Blakley, 1959) or at 50-60°C for 3 hr (Huennekens et al., 1963a). The product (10-formyl-folkacid) was obtained in good purity, while only minor amounts of unreacted folic acid and other contaminants are observed. Catalytic hydrogenation of 10-formyl-folic acid yields (6-ambo)-10formyl-H,folate (Blakley, 1957; Huennekens et al., 1963a). Alternatively, H4folate can be formylated by incubation in 98% formic acid (in 2% v/v 2-mercaptoethanol),which forms 5,lO-methenyl-H4folate(Rowe, 1971). Several additional procedures for the preparation of (6-ambo)-5-formylH,folate have been reported. H,Folate can be formylated by a carbodiimide-mediated reaction with formic acid to yield the 5-formyl isomer (Moran and Colman, 1982; Moran et al., 1986). Tatum et al. (1980) developed a procedure in which H,folate (produced by folate reduction with sodium cyanoborohydride) is formylated by incubation in a mixture of formic acid, acetic acid, and 2-mercaptoethanol, which yields 5,lO-methenyl-H,folate. Following ion-exchange purification under acidic conditions, the product can be crystallized. The advantages of this procedure are its simplicity and the stability of 5,10-methenyl-H4folatefor use as a convenient precursor of the more labile 10-formyl-H,folate. Forsch and Rosowsky (1985) devised an innovative method in which folic acid is incubated with a combined dimethylamine-borane reagent in formic acid. The bifunctional nature of this reagent is responsible for the simultaneous formylation pteridine reduction which occurs without formation of the 5,lOmethenyl derivative. The chemical synthesis of 5-methyl-H4folate is most readily accomplished by incubation of H,folate with formaldehyde, followed by reduction of the 5,10-methylene-H4folate intermediate with borohydride (Donaldson and Keresztesy, 1962; Gupta and Huennekens, 1967). This procedure is convenient because it can yield the product in 6 s or 6-ambo form, depending on the form of H,folate employed. Matthews (1986) has reported procedures suitable for the formation of the 5-methyl- and 5,lO-methylene-H4folate polyglutamates based on these original methods. These methods also have been adapted for the synthesis of tritiated reduced folates from high-specific-activity tritiated folic acid (Horne et al., 1977, 1980; Nixon and Bertino, 1971a, 1980).

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

II

4. Preparation of Radioiso topically Labeled Folates

Folates are commercially available in radiolabeled form as [3',5',7,9'Hlfolic acid (20-50 Ci/mmol) and [2-'4C]folic acid (50-60 mCi/mmol). The low specific activity of the carbon-I4 labeled material precludes its use in many studies of folate binding, transport, and metabolism in which low quantities or concentrations of the vitamin must be used. The high specific activity of the commercially available [3H]folicacid make it well suited for direct use in biologicalhiochemical studies and as a precursor in the synthesis of other folates (Horne et al., 1977, 1980; Nixon and Bertino, 1971a, 1980; Moran and Colman, 1982). This compound is prepared by catalytic dehalogenation of a 3',5'-dihalo (bromo or iodo) derivative of folic acid using tritium gas. Zakrzewski et al. (1970) and Evans et al. (1979) examined the labeling pattern of the reaction and reported evidence that the labeling reaction was not specific. ['HIFolic acid prepared by this method typically exhibits 35-50% of the tritium at the 3 ' 3 ' positions, with the remainder at the C-7 and C-9 positions. This distribution of tritium must be noted in any studies with labeled folates that require analytical methods involving cleavage of the C-9-N-10 bond. (6S)-H,Folates labeled specifically at C-6 with tritium or deuterium can be prepared by reduction of unlabeled folate with dihydrofolate reductase using ['HINADPH or ['HINADPH as the coenzyme (Pastore, 1980a). Subsequent conversion of this product to 5-formyl-H4folatewas accomplished enzymatically using N-formylglutamate and 5-formyl-H4folate: glutamate transformylase. Similar methods have been reported for enzymatic reduction and tritium labeling of polyglutamyl folates (Paquin et al., 1985). Biological synthesis is a very useful tool for the preparation of longchain reduced (6s) polyglutamyl folates. Curthoys et al. (1972) reported the use of Clostridium acidiurici as a source of radiolabeled polyglutamyl folates. The major limitation of this organism was that the principal intracellular forms of the vitamin were triglutamates, with negligible amounts of longer chain species. Buehring et al. (1974) employed Lactobacillus casei and Streptococcus faecium grown in folate-deficient medium supplemented with 1-10 ng/ml [3H]folicacid for a biological synthesis of longer-chain folates (mainly octa and nonoglutamates). No dilution of the label occurs because these species lack the ability to synthesize folates de novo. Abad and Gregory (1988) employed this method for the preparation of long-chain [3H]folatesfor use in nutritional experiments. Following thermal lysis of the cells (I5 min at 100°C) and purification by gel filtration on Sephadex G-25, high-performance liquid chromatographic (HPLC) analysis revealed that 8040% were 5-formyl-H4folates,with the remain-

12

JESSE F. GREGORY 111

der 5-methyl-H,folates. The high proportion of 5-formyl derivatives obtained in this procedure may reflect interconversions that may have occurred during thermal lysis. A similar preparation of labeled bacterial folates has been reported by Darcy-Vrillon et al. (1986).

5 . Preparation of Stable-Isotopically Labeled Folates The labeling of folates with deuterium or other stable isotopes (e.g., I3C)has been useful in studies of enzyme mechanisms and, recently, for in vivo studies of folate bioavailability. Pastore (1980b) reported that [7'H]-7,8-HZfolate could be prepared by reduction of folic acid by dithionite in deuterium oxide (D20). This deuterated H,folate could then be converted to folic acid, if needed, by iodine-mediated oxidation. An additional cycle of dithionite reduction in D 2 0 yielded [7-ZH,]-7,8-H2folate with approximately 1.6 deuterium atoms per molecule. Plante et al. (1980) reported methods for the synthesis of folic acid labeled with I3C at the benzoyl carbonyl group. This lengthy synthesis involved the preparation of [~arboxyl-'~C]pteroic acid by coupling derivatized forms of pteridineacid, followed by mixed an6-aldehyde and [carboxyl-'3C]-p-aminobenzoic hydride coupling of trifluoroacetyl [13C]pteroicacid to diethylglutamate. Procedures for the synthesis, in vivo application, and mass spectral analysis of deuterated folates in human urine have been recently developed (Gregory and Toth, 1988a,b; Toth and Gregory, 1988). Because the mass spectral procedures involve analysis of the derivatized p-aminobenzoylglutamate fragment after C - S N - 1 0 bond cleavage, the deuterium atoms in folates synthesized for in vivo use must be present in that portion of the folate molecule. In addition, mass spectral analysis is facilitated if the mass of the labeled folate is two or more units greater than that of the parent (unlabeled)compound. Within these constraints, two approaches to the synthesis of deuterium-labeled folates have been employed. Trifluoroacetylpteroic acid was coupled to dimethyl-[2,2,3,3,-2H,]glutamic acid to form glutamate-labeled[2H,]folicacid with over 90% yield following saponification in 0.1 M sodium deuteroxide. The product contained approximately 3.52 deuterium atoms per molecule (Gregory and Toth, 1988b). Catalytic dehalogenation of 3' 3'-dibromofolic acid was also employed for the deuterium labeling (Gregory and Toth, 1988c) in a procedure analogous to that employed for tritium labeling (Zakrzewski et al., 1970). When the reaction was performed in 0.1 M NaOH, the folic acid produced exhibited 7675% labeling of the 3'3' positions (approximately 1.5 deuterium atoms per molecule). In contrast to the distribution of the isotope in [ 'Hlfolic acid, nuclear magnetic resonance (NMR)analysis indicated that this product is labeled only at the 3'3' positions (Gregory and Toth, 1988b). The extent of labeling can be increased to over 95% by performing

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

13

the catalytic dehalogenation in 0.1 M sodium deuteroxide in D,O. The yield of pure product in the 3’3‘-deuteration is typically 15-20% (Gregory and Toth, 1988c), as observed in preparation of [3H]folateby catalytic dehalogenation (Zakrzewski et al., 1970). As an alternate route, over 90% labeling of the 3’3’ positions can be obtained by acid-catalyzed exchange (Hachey et al., 1978). Because of the low yield of the catalytic dehalogenation reaction, the direct deuteration of polyglutamyl folates would be prohibitively inefficient and expensive. However, 3’,5’-’H2-labelingof a polyglutamyl folate has been accomplished through the labeling of pteroic acid via the catalytic dehalogenation procedure described above (Gregory and Toth, 1980a)and the use of this compound in the solid-phase synthesis (Krumdieck and Baugh, 1980b). Over 90% labeling of the 3’3’ sites of pteroic acid was achieved. Following purification and derivatization, this material was coupled to the resin-bound hexaglutamyl peptide to yield [3’,5’2H,]pteroylhexaglutamate. The potential for racemization of the glutamate a-carbon of the folate product is a concern in all syntheses involving alkaline reaction conditions. In the methods for synthesis of deuterated monoglutamyl folates described above, the products were found to exhibit proton NMR spectra with a proton signals identical to those of authentic pteroyl-L-glutamic acid reference compounds (Gregory and Toth, 1988a,b). These results suggest little or no isomerization occurred during the synthesis of glutamate-labeled [*H,]folic acid and [3’,5’-2H,]folicacid. In addition, the alkaline conditions employed in the deprotection phase of the solid-phase poiyglutamate synthesis apparently do not cause significant racemization in view of the ability of synthetic polyglutamyl folates to serve as substrates in enzymatic reactions specific for (poly)-L-glutamylfolates (C. L. Krumdieck, personal communication),and their ability to support the growth of folate-dependent bacteria and animals in bioassays. These observations concerning the chiral stability of mono- and polyglutamyl folates are consistent with the report that the L-glutamic acid is comparatively resistant to racemization in alkaline-treated food proteins and synthetic polymers (Liardon and Friedman, 1987). Ill. DETERMINATION OF FOLATE IN FOODS AND OTHER BlOLOGICAL MATERIALS A.

INTRODUCTION

The determination of folate in foods is of primary concern in view of the need for accurate data in establishing the folate requirement, assessing the bioavailability of the vitamin, and evaluating diet-health corre-

14

JESSE F. GREGORY I11

lates. Considerable information has been published regarding the content of folate in foods (for example, Hoppner et al., 1972; Perloff and Butrum, 1977;Paul and Southgate, 1978;U.S.D.A. Agricultural Handbook No. 8-3, 1978; Thenen, 1982). However, more complete data bases are needed, in addition to the need for reassessment of existing data as improved methodologies become available. Improved methods are also needed to facilitate studies of folate metabolism, nutritional requirements, and the metabolic function of the vitamin. The complexity, diversity, and instability of folates are substantial obstacles encountered in the development and selection of analytical methods. Difficulties in sample preparation, including extraction, deconjugation, and extract purification, further complicate analytical methods for folate. There are several analytical approaches that may be employed for the measurement of total folate and/or the distribution of individual folates in foods and other biological materials. The objective of this section is to provide a discussion of (I) the strengths and limitations of various methods of analysis, (2) further research needs concerning folate analysis, (3) potential problems affecting the validity of analytical results, and (4) considerations in the selection and adaptation of existing analytical methods. Additional reviews of analytical methods have been published elsewhere (Krumdieck et al., 1983b; Keagy, 1985a; Gregory, 1984, 1985b). B. PREPARATIVE PHASES OF FOLATE DETERMINATION 1 . Extraction

The preparative phase of the determination of folate in foods and other biological materials has received the least attention of all aspects of the analysis. Complete extraction of folates from the sample matrix is essential, while deconjugation of polyglutamyl folates is required to obtain a quantitative response in many analytical procedures (discussed later). In methods based on the analysis of intact polyglutamyl folates or their paminobenzoylglutamatederivatives, an additional requirement is that extraction conditions must prevent enzymatic or chemical deconjugation. The conditions presently used for the extraction of folate from biological samples have not been determined systematically. The major method of extraction, which is the basis of many determinations of folate in foods and animal tissues, is heating a buffered sample homogenate, generally either at pH 4.5-4.9 or at 7.0-7.85. These pH values were selected largely because they were near the pH optima of enzymes used for subsequent deconjugation. This mode of extraction is intended to cause thermal dena-

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLArE

15

turation of folate-binding proteins and enzymes that may catalyze folate degradation or interconversion. Because of the effect of the pH and ionic strength of the extractant on the net charge of folates and the extent of interaction with the insoluble components of the sample, pronounced effects of those variables on extraction efficiency are predicted. Detailed evaluations have not yet been performed, however. A requirement for rapid heat treatment to at least 70°C has been reported to preserve the integrity of folates in biological materials (Wittenberg ef al., 1962). Studies of extraction variables in the analysis of food folates have been recently conducted in the author's laboratory (Engelhardt, 1988). Radiolabeled forms of folk acid, mixed bacterial monoglutamyl H,folates, and mixed bacterial polyglutamyl H,folates were added to selected plant- and animal-derived foods prior to homogenization and heating (60 min at I00"C) at pH 4.5 or 7.0. Extractions were performed twice (i.e., the residue from the first extraction was resuspended and centrifuged), and the recovery of the isotope determined at each stage. In these studies no clearly optimal conditions were found. The recovery depended on the form of folate added, the pH, and the type of food sample. A double extraction markedly enhanced the recovery of the labeled folates for most samples. These data suggest that extraction of endogenous food folates by standard single extraction methods may often be incomplete, and that double extraction should be adopted as a routine procedure. A preservative is required in all folate determinations to inhibit oxidative losses of the labile reduced folates. Ascorbic acid and, to a lesser extent, 2-mercaptoethanol have been used widely for this purpose. Each can be effective at ambient temperature if samples are deoxygenated with nitrogen or other inert gas. Wilson and Horne (1983)reported that formaldehyde and/or other products of the thermal degradation of ascorbate at neutral pH can cause interconversion of reduced folates, especially the conversion of H,folate to 5,10-methylene-H4folateand 5-methyl-H4folate. It was later reported that an extractant containing both ascorbate and 2mercaptoethanol could alleviate the problem (Wilson and Horne, 1984, 1986). It should be noted that the stability of ascorbate is much greater at pH 4-5 than at pH 7. The effect of pH on the stability of folates during extraction and analysis is not fully clear and is dependent on the inherent pHdependent stability of reduced folates (O'Broin ef al., 1975),as well as on the pronounced effect of pH on the stability of ascorbic acid. Acceptable stability of reduced folates can be obtained during thermal extraction at pH 4.9 with 1.0% ascorbate (lOO"C, 60 min; Gregory et al., 1984a), with over 90% retention of the highly labile H,folate under these conditions. Recent studies have substantiated these findings with respect to extrac-

16

JESSE F. GREGORY Ill

tion of radiolabeled folates in livers from rats previously injected with [3H]folicacid. Greater stability was obtained during extraction at pH 4.9 (versus pH 7.0) using 1.0% ascorbate as an antioxidant. The lower pH also minimized apparent enzymatic interconversion reactions of folates that occurred immediately prior to heating liver homogenates (Engelhardt, 1988). On the basis of these studies, mildly acidic buffers containing ascorbate appear to be fully suitable for extraction in the determination of folates in foods and other biological materials. As an alternate extraction medium, Wilson and Horne (1984, 1986) reported that the use of a pH 7.85 Ches/HEPES buffer containing 2% ascorbate and 0.2 M 2-mercaptoethanol permits the use of thermal extraction in more neutral medium without ascorbate-mediated interconversions (Wilson and Horne, 1984, 1986). It was hypothesized that the presence of 2-mercaptoethanol was beneficial by complexing formaldehyde formed during ascorbate oxidation as a hemithioacetal, which rendered it unreactive. However, the effectiveness of this buffer with respect to its extraction efficiency has not yet been examined. Regardless of the buffer employed, double extraction would appear to be advisable, unless otherwise determined in preliminary studies. It should be noted that heat inevitably causes the conversion of 10formyl-H,folate and its polyglutamates to the 5-formyl-H4folateisomers (Robinson, 1971). As a result, distributions of formyl folates are ambiguous in many thermally extracted samples, and it would be difficult to determine whether 5-formyl-H4folate detected was naturally occurring or the result of thermal isomerization. In addition, chromatographicanalysis would be complicated in situations where partial interconversion occurred. Gregory et al. (1984a) approached this problem by extracting animal tissue samples under conditions that yielded complete conversion of 10-formyl-H,folate to the 5-formyl isomer. A noteworthy property of the pH 7.85 extractant employed by Wilson and Horne (1984)is that no interconversion of 10-formyl-and 5-formyl-H4folateoccurs during thermal extraction. Nonthermal extraction procedures have employed trichloroacetic acid (Reed and Scott, 1980; Selhub et al., 1980; Shane, 1982, 1986) and 0.1 N HCI (Eto and Krumdieck, 1981, 1982). In view of the lability of H,folate at low pH, such methods do not appear to be viable for the routine analysis of intact folates in foods and other biological materials. As discussed later, extraction with trichloroacetic acid or HCI is fully compatible with procedures in which folates are intentionally subjected to C-9-N- 10 bond cleavage, such as the methods of Shane (1982, 1986) and Eto and Krumdieck (1981, 1982). Folates in fibroblast cultures, following thermal

CHEMICAL A N D NUTRITIONAL ASPECTS OF FOLATE

17

lysis of cells and conjugase treatment, have been extracted by deproteination with methanol (Kashani and Cooper, 1985). This would not be a viable approach to folate extraction from solid samples requiring subsequent enzymatic deconjugation.

2 . Enzymatic Deconjugation of Folates Folates in sample extracts must be enzymatically deconjugated to permit a complete response in microbiological assays and to provide unambiguous separations in many chromatographic methods. In addition, competitive-binding assays of folates in foods and certain other biological materials would require deconjugation because of the dependence of the binding affinity on the polyglutamyl chain length of the analyte (Shane et al., 1980). The folate content of foods has been traditionally reported as “free” and total folate values, which represent the L. casei activity measured without and with enzymatic deconjugation, respectively. The use of “free” folate values should be discontinued because of the variable and indistinct nature of this term. An indicated previously, L. casei responds most readily to mono-, di-, and triglutamyl folates, such that “free” folate represents the response to any of these species. Leichter et al. (1979) showed clearly that the action of endogenous conjugases of vegetable samples can cause an increase in the apparent “free” folate during extraction of the sample. Thus, “free” (i.e., short chain) folate is only a valid concept if care is taken to inactivate conjugases before homogenization. The pteroylpolyglutamate hydrolases (folate conjugases) used in folate deconjugation differ in their pH optima, mode of action, and type of folate product. Conjugase from chicken pancreas exhibits a neutral pH optimum and yields a folate diglutamate product (Leichter et al., 1977). Hog kidney conjugase exhibits an acidic pH optimum (pH 4.5-4.9) and sequentially attacks folate polyglutamate substrates to yield ultimately a monoglutamate product (Bird et al., 1946; Engelhardt, 1988). Folate conjugase of blood plasma (human or rat) also exhibits a pH optimum of 4.5 and yields a monoglutamyl folate product (Lakshmaiah and Ramasastri, 1975; Horne et al., 1981). As an alternative to the use of an exogenous conjugase, incubation of tissue homogenates under conditions providing autolytic deconjugation with endogenous conjugase(s) has been employed. Conjugase activity suitable for complete or partial deconjugation is present in many plant (Chan et al., 1973; Leichter et al., 1979) and animal (Slink et al., 1975)

18

JESSE F. GREGORY I11

tissues. Autolysis has been used effectively for the deconjugation of folates prior to microbiological assay; however, autolysis procedures should be approached with caution because of the potential for undesirable interconversion and/or enzymatic degradation of folates by other enzymes. The potential for extensive interconversion of folates always exists in the use of autolysis for deconjugation in chromatographic analysis of either unlabeled or radiolabeled folates in tissues, although Davidson et al. (1975) experimentally ruled out interconversion of folates during autolysis in their study. Variability between samples in the activity of endogenous conjugase(s) also represents a problem in the use of autolysis. The selection of an enzyme for the deconjugation of polyglutamyl folates depends mainly on the requirements of the analytical method used. The microbiological assay using L. casei requires only that sample folates be triglutamate or shorter in chain length (Tamura et al., 1972; Shane et al., 1980). Thus, relatively little control of the deconjugation step is needed, provided the reaction proceeds at least to the folate triglutamate level of hydrolysis. In contrast, chromatographic methods that separate and measure the individual folates in food extracts require that all folates be fully deconjugated to the monoglutamate level. The use of the chicken pancreas conjugase (which yields a diglutamyl folate product) or incomplete deconjugation when using other conjugases would yield ambiguous separations (as appear in the early literature) and erroneous quantitation. Relatively little information is available concerning the efficacy of enzymatic deconjugation in folate analysis. It has been reported that hog kidney conjugase is more susceptible to inhibition by food components than the enzyme from chicken pancreas (Eigen and Shockman, 1963). This, coupled with the commercial availability of a dehydrated chicken pancreas preparation (Difco Laboratories), has been the basis of the generally preferred use of the pancreatic enzyme in most food analysis. Kirsch and Chen (1984) examined the effectiveness of conjugases from chicken pancreas and hog kidney for the deconjugation of polyglutamyl folates in spinach extracts. Substantial variation in the rate and final extent of deconjugation was observed with each enzyme, depending on the pH and type of buffer. Significantly, citrate ions exhibited greater inhibitory effects on the chicken pancreas conjugase than on the enzyme from hog kidney. Optimal deconjugation of spinach folates (reflected in maximal folate values) could be obtained with either enzyme under appropriate conditions (buffer, pH, amount of enzyme, reaction time). In contrast, Phillips and Wright ( 1983) observed slightly greater apparent folate values in a variety of foods when assayed following deconjugation with the hog kidney enzyme compared to the same extracts treated with

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

19

chicken pancreas. Recently, Pedersen (1988) examined the deconjugation of folates in food extracts and concluded that, under optimal reaction conditions, the type of conjugase (hog kidney vs chicken pancreas) did not affect the measured concentration of folate. The susceptibility of hog kidney conjugase to inhibition by components of various plant- and animal-derived foods was recently examined in our laboratory (Engelhardt, 1988). In these studies, synthetic pteroyltriglutamate (PG-3) was added to food extracts, followed by HPLC evaluation of the progress of deconjugation during incubation with hog kidney conjugase (Fig. 2). Each sample examined (including various citrate solutions, and extracts of orange juice, milk, boiled cabbage, chicken liver, kidney beans, and whole wheat flour) yielded significant inhibition relative to the control (PG-3 in buffer alone). Essentially complete deconjugation could be obtained, however, with sufficiently increased amount of enzyme added, extended reaction time, or both. McMartin et al. (1981) reported very low recovery of liver folate during deconjugation with crude hog kidney conjugase at pH 4.7. Complete deconjugation could be obtained at pH 6.0, with improved recovery, although these conditions were far from the pH optimum of the enzyme. These results were interpreted as indicating binding of folates to protein in the enzyme preparation at the lower pH. This effect has not been observed in our laboratory when using a slightly more purified form of hog kidney conjugase. The use of human plasma as a source of conjugase activity has been reported but not extensively examined in food analysis. Lakshmaiah and Ramasastri (1975) performed a limited comparison of food folate values using chicken pancreas, rat liver, and human plasma conjugase. After determining conditions for quantitative deconjugation, equivalent values for total folate in several foods were obtained with these enzymes. The susceptibility of the plasma enzyme to inhibition was not determined. Rat plasma also has been used effectively as a source of conjugase activity in HPLC analysis of folates in biological materials (Wilson and Horne, 1984, 1986; Kashani and Cooper, 1985). Overall, these studies indicate that conjugases from a variety of sources would be suitable for microbiological assay of folate in many foods and other biological materials. Because of the potential influence of the components of any sample on the rate of enzymatic deconjugation, preliminary studies are essential to determine appropriate conditions. In the absence of such verification to ensure adequate deconjugation, incomplete hydrolysis with resulting underestimation of folate content could easily occur. The accuracy of many published data is unclear in that regard.

20

JESSE F. GREGORY I11

A n

n

W

z 0

i W

0

z 0

0

s 4 W

K

0

20

: 40

60

: 80

:

:

100

:

: 120

:

:

140

REACTION TIME (min) B

s W

z 0

s5 W

0

z 0

0

E4 W

K

REACTION TIME (min) FIG. 2. Relative distribution of pteroyltriglutamate (PG-3). diglutamate (PG-2). and folic acid (PG-I) versus incubation time with hog kidney conjugase in (A) control in 0.05 M sodium acetate buffer, pH 4.9) or (B) extract of boiled cabbage in the same buffer fortified with 5 p M PG-3. (0) PG-3. ( 0 )PG-2, and (A) PG-I. [From the data of Engelhardt (1988).]

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

21

C . METHODS OF FOLATE DETERMINATION I . Microbiological Methods To date, the majority of the determination of folate in foods, clinical specimens, and other biological materials has been performed using turbidimetric bacterial growth assays employing L. casei (reviewed by Herbert and Bertino, 1%7; Keagy, 1985a). This organism has been employed because of its similar response to common naturally occurring reduced folates and folk acid. The organism responds nearly equally to monothrough triglutamyl folates, although growth in response to longer-chain folates (n > 3) decreases markedly in proportion to chain length (Tamura et al., 1972). Thus, for accuracy in the L. casei assay of total folate in biological materials, the following are essential: (1) the molar response of the standard and all folates present in the food extract must be equivalent, (2) enzymatic deconjugation must be adequate to yield folate products of sufficiently short chain that full growth response is obtained, and (3) stimulation or inhibition of bacterial growth by nonfolate components of the extract must not occur. Typical data concerning the microbiological determination of total folate in selected foods are shown in Table I. Although L. casei is generally reported to exhibit equivalent growth response to various monoglutamyl folates, Phillips and Wright (1982, TABLE I RESULTS OF MICROBIOLOGICAL (I!,. casei) DETERMINING OF TOTAL FOLATE IN SELECTED FOODSO

Total folate (nmol/g or ml) Perloff and Butrum (1977)

Gregory

Engelhardt

Food

er al. (1984a)b

( 1988)b

Orange juice Whole milk Cabbage (raw) Whole wheat flour Calf liver Chicken liver Red kidney beans

1.90 0.027 0.29 1.59 36.3

2.40 0.14 0.5 I I .57

I .2 0.1 I 1.5 I .2 5.0

5.99 5.15

8.2 3.0

These results illustrate the variability of folate content of foods as determined by standard analytical procedures. 'Samples were extracted at pH 4.9 and deconjugated using hog kidney conjugase. Microbiological assays were performed as described by Phillips and Wright (1983).

22

JESSE F. GREGORY I11

1985) have shown that 5-methyl-H4folateyielded a concentration-dependent reduced response, relative to folic acid, under conventional assay conditions (i,e,, initial pH 6.8). Adjustment of the initial pH of the assay medium to 6.2 yields approximately equivalent response. Consequently, assays performed with media initially at pH 6.8 may underestimate the folate content of foods that contain predominantly 5-methyl-H4folate (Phillips and Wright, 1983, 1985). Wilson and Horne (1984) reported evidence that the microbiological assay provided a uniform molar response to various reduced folates, while Newman and Tsai (1986) reported slight differences in the molar response of L . casei to folk acid, 5-methyl-H4folate, and 5-formyl-H4folate. In view of these contradictory findings, preliminary investigation of the response of different folates in L . casei assays is warranted prior to the use of this assay. Several modifications have been reported that will facilitate further application of microbiological assays. The use of cryoprotected preparations of L. casei eliminates the need for a new inoculum in each assay (Grossowicz et al., 1981; Wilson and Horne, 1982). Several continuousflow systems have been reported for semiautomated assays (Davis et al., 1970; Tennant, 1977), although the procedures are complex and their application has been limited. Assay systems also have been reported, using multitube or plate-type semiautomated equipment, that appear to be well suited for routine use with heavy sample loads (Keagy, 1986; Newman and Tsai, 1986). As an alternative to turbidimetric methods, a radiometric microbiological procedure has been reported for the L. casei assay that involves the measurement of I4CO, evolved from the bacterial metabolism of [l-’4C]gluconate(Chen et al., 1978, 1983). Little research has been conducted to examine the specificity of L. casei as employed in the analysis of foods and other biological materials. Stimulation of the growth of the organism by nonfolate factors of food extracts would yield erroneously high response, while antagonistic factors would inhibit the response of the assay. The determination of the recovery of folate in routine analysis would be partially effective in evaluating such effects. The observation of “drift,” or nonlinear assay response to graded levels of the sample, is also evidence of such stimulatoryhnhibitory effects. The availability of chromatographic methods will permit comparative studies to evaluate more fully the specificity of the L. casei assay procedure.

2. Ligand Binding Methods Competitive binding isotope dilution methods for the measurement of folate in biological materials (mainly blood serum) were developed

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

23

shortly after the discovery of folate-binding proteins (Herbert and Colman, 1985). These procedures are based on competition of radiolabeled folate and unlabeled folate(s) from standards or samples for a folate-binding protein. The distribution of protein-bound and free species of the labeled folate is reflective of the concentration of unlabeled folate present. Many variations on this basic procedure have been reported, as reviewed previously (Gregory, 1985a). The major differences among competitive binding procedures include the source of folate-binding protein (milk, plasma, or hog kidney), pH of the assay medium, the isotope used for the labeled folate (3H, '"I, or '%e), the form of folate used as the standard (folic acid or 5-methyl-H4folate), and the method of separation of free and protein-bound forms of labeled folate. Generally, the folate-binding protein is used in its native soluble form. Recently, Hansen e l d . (1987) reported an assay in which purified folate-binding protein from cow's milk is immobilized to plastic beads which could be readily separated from the medium by centrifugation or filtration. The use of purified folate-binding protein in immobilized form provided a technically simple assay procedure which was effective in assays of erythrocyte folate. Hansen and Holm (1988)later reported an enzyme-linked ligand sorbent assay in which a biotinylated folate-binding protein is employed along with an avidinphosphatase conjugate. This permits colorimetric quantification on a microtiter plate reader and eliminates the need for radioisotopes. (It should be noted that the soluble folate-binding protein of milk whey is distinctly different from p-lactoglobulin, although the folate-binding protein is present as a contaminant in commercial p-lactoglubulin preparations. Preparative aspects of immobilized folate-binding proteins will be discussed later in this chapter.) Although any high-affinity folate-binding protein would be applicable to competitive binding assays, the selectivity of the assay varies depending on the characteristics of the particular protein employed (e.g., relative affinity for folic acid and reduced folates, effect of polyglutamyl chain length on binding affinity, pH effects on binding affinity, and affinity for the biologically inactive 6R isomer if racemic 5-methyl-H4folate is used as a standard). Shane et af. (1980) examined these factors with respect to the response of four commercially available assay kits. The substantial variation among the kits in these studies would be reflected in similar variation in their accuracy in any applications. Although folk acid is more stable than 5-methyl-H4folate, folic acid appears to be inappropriate as a standard in the range of pH 7.2-8.0 because of its higher affinity for the widely used folate-binding protein from milk (Givas and Gutcho, 1975). Folk acid and 5-methyl-H4folate exhibit equivalent affinity for the milk folate-binding protein at pH 9.3. However, Waxman and Schreiber

24

JESSE F. GREGORY 111

pointed out that assays conducted at pH 9.3 are susceptible to error because of the marked variation in binding affinity that occurs with small changes in pH near 9.3. In selection of a radioassay procedure to be run “from scratch” or using commercial reagents, one must carefully consider the properties of the binding protein, the type of folates present in the samples to be analyzed, and whether the molar response of the standard employed is consistent with that of the folates present in the samples. Competitive binding assay procedures are widely used for the rapid measurement of plasma and red cell folate in clinical settings. If appropriately standardized and periodically validated against L. casei microbiological procedures, these procedures can be a convenient alternative to microbiological methods. In a recent comparative study involving L. casei assays in comparison with five commercial radioassay kits (pH after incubation with binding protein of 9.1-9.3), generally acceptable correlations were observed for serum and erythrocyte folate values (Gilois and Dunbar, 1987). In spite of the good correlations between assays, considerable variation was found with respect to the ability of various assays to detect folate deficiency based on serum folate values. These results illustrate the suitability of competitive binding procedures and also point out the need to determine the “normal range” for any assay procedure employed. The interassay agreement for erythrocyte analysis was found to be more consistent than that for serum assays (Gilois and Dunbar, 1987). Application of competitive binding assays to other aspects of biological and food analysis has received little attention to date, largely because of the potential problems associated with variation of binding affinity among the various folates for the folate-binding protein (Shane et al., 1980). Reports concerning the application of competitive binding methods to foods and animal tissues are inconsistent. Tigner and Roe (1979) and Graham et al. (1980) reported good correlation between the results of a competitive binding procedure and L. casei assays applied to rat liver and various convenience foods. Mandella and DePaola (1984) also reported good correlation in a comparative study of rat liver folates. It should be noted, however, that Mandella and DePaola (1984) incorrectly assumed that the milk folate-binding protein used in their assay only bound the 6s isomer of the 5-methyl-H4folatestandard, contrary to the findings of Shane et al. (1980). The correctly calculated competitive binding data of Mandella and DePaola would actually indicate values for liver folate approximately twice those of microbiological assays. Klein and Kuo (1981) found significantly higher values for spinach folate when determined by a competitive binding method. When applied to a fortified food (oat breakfast cereal), a competitive binding assay yielded folate values similar to those obtained

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

25

with L. casei and fluorometric HPLC assays (Gregory et al., 1982). In the same study, folate values for cabbage obtained by the competitive binding method were over 2-fold higher than those obtained by microbiological assay. Competitive binding assay values were markedly lower than corresponding microbiological and HPLC data in an infant formula, possible due to active folate-binding protein in the milk-based formula. At the present time, competitive binding methods do not appear to fulfill the need for a rapid assay procedure for general use in analysis of foods and other biological materials. On the basis of the known specificity of the bovine milk folate-binding protein, it is difficult to rationalize the studies reporting good correlation between the results of competitive binding and microbiological assays. Another limitation of the analysis of animal tissues using competitive binding methods is that the folate-binding protein of milk does not exhibit significant affinity for 5-formyl-H4folate(Fig. 3). Although 5-formyl-H4folate is a minor form of the vitamin in most tissues, thermal extraction procedures can cause extensive conversion to this compound from 10formyl-H,folate. Thus, the folate content of samples containing 5-formylH,folate (naturally occurring or formed during thermal processing or extraction) would tend to be underestimated by competitive binding assay methods. An alternate approach to folate analysis by traditional competitive

0 0.200

1 .ooo

10.000

ANALYTE CONCENTRATION (nM) FIG. 3. Typical binding curves in folate radioassay at pH 9.2 using cow’s milk folatebinding protein. Note the relative lack of affinity of 5-formyl-tetrahydrofolate for the folatePterin-6-COOH. (0)pABG, (A) 5-formyl-H4folate, and (A)folic acid. binding protein. (0) [Redrawn from the data of Gregory et ul. (1982).]

26

JESSE F. GREGORY 111

binding analysis involves the formation of a ternary covalent complex of 5,lO-methylene-H,folate with [3H]fluorodeoxyuridylate and thymidylate synthase (Priest et al., 1982). The reactive 5,lO-methylene-H,folate is quantified directly by liquid scintillation counting following separation of the complex from unreacted [3H]fluorodeoxyuridylateon Sephadex G-25. Other forms of folate, including H,folate, H,folate, folic acid, 5-methylH4folate, and formyl folates, can be analyzed by this method by incorporating prior interconversion to the 5,lO-methylene derivative (Doig et al., 1985). While these methods have been successful for measurement of folates in animal tissues and cell cultures, they have not been attempted in other aspects of food and biological analysis. Application of these methods to the determination of the chain length of polyglutamyl folates is described in Section III,C,4. Radioimmunoassay methods also have been devised for the rapid measurement of certain folates. In these procedures, antibodies are elicited against covalent complexes of protein and a folate molecule. Various procedures have been described for the radioimmunoassay of folic acid (DaCosta and Rothenberg, 1971;Hendel, 1981),5-methyl-H,folate (Langone, 1980),and methotrexate (Hendel et al., 1976).The high degree of specificity of these assays may also be a limitation if there is a need for measurement of all folates in a biological specimen.

3. Separation and Analysis of Folates in Monoglutamyl Form While methods have been devised for the separation of folates by paper and thin-layer chromatography and paper electrophoresis (Eigen and Shockman, 1963; Godwin et al., 1972; Brown et al., 1973b; Scott, 1980a), problems of low resolution and instability of certain folates preclude the use of these procedures in most aspects of folate analysis. Column electrophoretic separations also have been reported (Usdin and Porath, 1957), although the technique is not well suited for the analysis of multiple samples. Shortly after the development of ion-exchange column chromatography, its usefulness for the separation of monoglutamyl folates was demonstrated. Usdin and Porath (1957) reported anion-exchange separations of several monoglutamyl folates using Dowex 2 and triethylaminoethyl (TEAE)-cellulose. They also showed that mono-, di-, and triglutamyl folates could be separated using TEAE-cellulose. Ion-exchange chromatography on TEAE-cellulose was employed in initial studies of the folate content of blood (Usdin, 1959). The further extension of ion-exchange techniques to the separation of polyglutamyl folates will be discussed in Section III,C,4. Applications of TEAE-cellulose have been surpassed by the use of

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

27

commercially available diethylaminoethyl (DEAE)-cellulose, DEAESephadex and DEAE derivatives of other beaded carbohydrates, and quaternary aminoethyl (QAE)-Sephadex (Silverman et al., 1961; Nixon and Bertino, 1971b; Parker et al., 1971). The first apparent use of DEAEcellulose for the separation of folates in biological materials was reported by Silverman et al. (1961). Since that time, ion-exchange chromatography has been used extensively as a routine technique for the preparative and analytical separation of monoglutamyl and polyglutamyl folates. Most separations involve the use of a gradient of sodium chloride in a neutral buffer or a gradient of an anionic buffer ion (e.g., phosphate). Elution using a volatile buffer system (triethylammonium bicarbonate) also has been employed for the preparative separation of folate compounds (Parker et d . , 1971; Chan et d . , 1973). [It should be noted that triethylammonium bicarbonate buffers should be made from freshly redistilled triethylamine if folates are being purified for in vivo use. Distillation removes nonvolatile impurities which are toxic to rats (Gregory et al., I984b).] Although HPLC methods have largely replaced conventional column chromatography for analytical separations of monoglutamyl folates, DEAE-cellulose and related sorbents are still extremely valuable for preparative purposes in view of their substantially higher capacity than common silica-based HPLC columns. In addition, carbohydratebased chromatographic sorbents are more compatible with operation at mildly alkaline pH values that promote the solubility of certain folates during certain preparative separations. Advances have been made in the HPLC analysis of folates as either the monoglutamyl forms (following deconjugation) or according to the chain length of polyglutamyl folates. In the context of most food analysis, the separation and quantitation of folates in their monoglutamyl form (i.e., following enzymatic deconjugation) is the approach used almost exclusively. Approaches to the HPLC determination of folate have been reviewed (Gregory, 1985a). For the determination of total folate in foods and other biological materials, the most direct procedure involves the HPLC analysis of extracted folates in their monoglutamyl forms. Monoglutamyl folates can be separated readily by reverse phase, with or without ion-pairing agent, or by ion-exchange modes of HPLC. An HPLC method using a column of bovine serum albumin immobilized to silica is capable of resolving the 6R and 6 s stereoisomers of reduced folates (Choi and Schilsky, 1988), which will be useful in the analysis and small-scale preparative separation of synthetic folates. The major problems in the development HPLC methods for application to biological materials lie mainly in the preparative phases (extraction, deconjugation, and extract purification) of the analysis. As

28

JESSE F. GREGORY I11

indicated previously, complete deconjugation of polyglutamyl folates to the monoglutamyl forms is required for accurate analysis. Purification of extracts can be performed by ion-exchange using small columns of DEAE-Sephadex A-25 (Gregory et al., 1984a) or other weak anion exchangers (Rebello, 1987). A cation-exchange method has been reported for extract purification (Gregory et al., 1982; Duch et al., 1983), although low recovery of 5-formyl-H4folateprecludes many applications. Alternatively, sample purification using affinity chromatography columns prepared with immobilized folate-binding protein from milk (Selhub et al., 1980, 1988) can be employed. This approach has been successfully adapted to sample purification in the analysis of urinary folates (Gregory and Toth, 1988a), although only limited quantitative data have been reported concerning its use in the analysis of other biological materials (Selhub and Malec, 1986; Selhub et al., 1988). It should be noted that the immobilized folate-binding protein does not bind 5-formyl-H4folate,thus requiring preliminary conversion to 10-formyl-H,folate in samples containing this form of the vitamin (Gregory and Toth, 1988a). Methods for the detection of eluted folates include direct ultraviolet absorbance (280 nm), fluorescence, and electrochemical techniques, and microbiological assay of collected fractions. UV absorbance is a uniform detection method which responds to all folates, although its sensitivity may be insufficient for samples having low folate concentration. HPLC with UV absorbance has been shown to permit the measurement of folates in certain biological materials following ion-exchange purification (Duch et al., 1983; Rebello, 1987)or affinity chromatographicpurification of sample extracts or human urine (Gregory and Toth, 1988a; Selhub et al., 1988). The effectiveness of affinity chromatography is illustrated in the chromatogram of Fig. 4,in which the only peaks observed are ascorbate and folates present in the urine sample (Gregory and Toth, 1988a). The comparatively low sensitivity of UV absorbance is a problem when excessive dilution is encountered during extract purification. For example, Duch et al. (1983) had to lyophilize the purified extracts to concentrate the folates prior to HPLC, thus increasing the analysis time and the potential for oxidative losses of labile folates. Hoppner and Lampi (1983) reported a reversed-phase HPLC analysis of 5-methyl-H4folatein plasma and whole blood using absorbance at 280 nm. Sample extracts were purified using sequential columns of Bio-Beads SM-2 (a hydrophobic adsorbent) and DEAE-cellulose. Although values derived by HPLC correlated well with the results of L. casei assays, the lengthy nature of the sample purification would prevent the use of this method for routine analysis. The fluorescence of reduced folates in acidic media (Uyeda and Rabinowitz, 1963) provides a sensitive and specific means of HPLC detection,

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

0

4

8

12

16

29

20

Retention Time ( min 1 FIG. 4. Determination of urinary folates by reversed-phase HPLC with ultraviolet absorption (280 nm) detection following affinity chromatographic purification. [Reprinted with permission from Gregory and Toth (1988a); copyright (1988) Academic Press, Inc.]

although folic acid does not fluoresce (Gregory ef al., 1984a). An example of the fluorometric HPLC determination of reduced folates in rat liver is shown in Fig. 5 . Samples containing folic acid (e.g., extracts of fortified foods) can be analyzed fluorometrically by the introduction of a postcolumn derivatization system involving hypochlorite to cleave folic acid (and H,folate and H,folate) oxidatively to fluorescent pterins. When this postcolumn oxidation system is connected in series downstream from a fluorometer monitoring the native fluorescence of the reduced folates, all folates can be monitored fluorometrically. Fluorometnc detection can provide substantially greater specificity and sensitivity than UV absorbance, which is important in many applications. HPLC detection by postcolumn fluorogenic derivatization also has been adapted to the determination of polyglutamamyl folates for HPLC assays of folate conjugase activity (Gregory ef al., 1987). In this procedure, the substrate, pteroyltriglutamate, and the products pteroyldiglutamate and folk acid are detected fluorometrically (Fig. 6), which provides a sensitive procedure that is an economical alternative to the radiometric assay (Krumdieck and Baugh, 1980b). Microbiological assay of collected HPLC fractions permits the analysis of folates in crude biological extracts (McMartin ef al., 1981; Wilson and Horne, 1983, 1984, 1986) (Fig. 7). Although the method has not been em-

30

JESSE F. GREGORY 111

0

5

10

15

20'' 0

5

1

I

I

10

15

20

Retention Time (min) FIG. 5. Determination of reduced folates following ion-exchange purification by reversedphase HPLC with direct fluorornetricdetection. Liver folates were converted to monoglutamy1 form using hog kidney conjugase. (A) Standards and (B) rat liver. [Reprinted with permission from Gregory ef al. (1984a); copyright (1984) American Institute of Nutrition.]

ployed in food analysis, it appears to be directly applicable. Potential limitations include the additional time and effort required for the microbiological assay and possible instability of H4folate over the extended time period of this analysis. Wilson and Horne (1984) reported that over 90% of the microbiologically detectable folate activity in a liver extract was accounted for by the folate peaks in analysis by the HPLC/microbiological method. Electrochemical detection is a potentially powerful method for the sensitive and specific monitoring of HPLC separations. Lankelma et d . (1980) first reported the use of electrochemical detection in folate analysis. These investigators employed a two-column system for anion-exchange on-column concentration and purification followed by reversedphase separation and electrochemical quantitation of 5-methyl-H,folate in plasma and spinal fluid. The response to 5-formyl-H4folate,folic acid, and methotrexate was negligible (< 0.1%) under under the conditions of detection (+ 0.3 V). The oxidation potentials for folates under conditions of electrochemical detection in a reversed-phase HPLC system have been reported (Kohashi et al., 1986) (Table 11). Electrochemical detection may be suitable as a nonspecific method for the detection of all folates. However, relatively high potentials would have to be applied (ca. + l V) for the uniform electrochemical detection of all folates. At this high oxidation

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

I

I

I

31

1

i, 5 10 is Retention Time ( min) FIG. 6. Determination of pteroyltriglutamate (PG-3). diglutamate (PG-2), and folic acid (PG-I) in conjugase assay mixture by reversed phase HPLC. Fluorometric detection was performed by postcolumn fluorogenic oxidation with a sodium hypochlorite reagent. [Reprinted with permission from Gregory ef a / . (1987); copyright (1987) Pergamon Journals Ltd.]

potential, interferences from sample components and preservatives (e.g., ascorbate or 2-mercaptoethanol)could be a serious problem. Kohashi et al. (1986) published chromatograms that showed substantial response from nonfolate components using an applied potential of + 0.35 V to provide sensitive detection of 5-methy1-H4folate.Similarly, Lankelma et al. (1980) observed a higher background response at an applied potential of +0.5 V compared to +0.3 V, even following anion-exchange purification of plasma samples. The suitability of electrochemical methods for the determination of multiple folates in foods and other biological materials has not been evaluated. Because of the potential for interference from oxidizable nonfolate compounds, substantial requirements for purification of sample extracts are anticipated. The data of Kohashi et al. (1986) indicate that the limits of detection of pure folate compounds are significantly lower for electrochemical methods than other means of detection. Although somewhat less sensitive, the greater specificity of fluorometric

J

B

FRACTION NUMBER FIG. 7. Determination of folates by reversed-phase ion-pair HPLC with detection of eluted folates by microbiological assay. Peak identity: 1, lO-formyl-H,folate; 2, H, folate; 3, 5formyl-H,folate; 4, H2folate; 5, 5,10-methylene-H4folate; 6, 5-methyl-H,folate; and 7, folic acid. [Reprinted with permission from Wilson and Home (1983); copyright (1983) Wilson and Home.]

TABLE I1 OXIDATION POTENTIAL OF FOLATES AS DETERMINED BY HPLC WITH ELECTROCHEMICAL DETECTION USING VARIED APPLIED DETECTOR

Folate

POTENTIAL^

Oxidation potential (volts)* (potential yielding half-maximal response)

~~

THF 5-Methyl-THF 10-Formyl-THF 5-Formyl-THF Folk acid

0.10

0.25 0.62 0.65 0.85

“(Adapted from Kohashi ef a / . (1986). *Approximatevalues estimated from graph.

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

33

detection appears to be a distinct advantage over electrochemical techniques for the determination of folates in the presence of other biological compounds. 4. Analysis of Polyglutamyl Folates

The determination of naturally occurring folates in their intact polyglutamyl form is a difficult analytical problem because of the complex array of compounds differing in chain length, pteridine ring oxidation state, and one-carbon substituents present. A comprehensive review of analytical methods for polyglutamyl folates has been published by Krumdieck et al. ( 1983b). Within any single class of folate compound, polyglutamyl forms can be separated as a function of chain length by gel filtration chromatography on Sephadex G-15 or (3-25 (Shin et al., 1972a). The mode of separation was not, however, simple size exclusion. These dextran-based chromatographic media exhibited selective adsorption as a function of the oxidation state and one-carbon substituents, in addition to apparent electrostatic repulsion of the anionic polyglutamyl side chain (Shin et al., 1972a). Consequently, separations using gel filtration are valuable in the characterization or purification of homologous families of synthetic folates but are ambiguous for use in characterizing the chain length of mixed naturally occurring polyglutamyl folates. It should be noted that the interaction of monoglutamyl folates with Sephadex G-10 has been used as an alternative to ion exchange for isocratic separations of certain monoglutamy1 folates (Kas and Cerna, 1976). The most effective characterizations of polyglutamyl folates in biological materials on the basis of conventional chromatography have been those employing ion-exchange chromatography, either alone or parallel to gel filtration, with analysis by differential microbiological assay for quantification and identification. By reanalysis of samples or collected peaks following complete deconjugation, more conclusive identification of the folate coenzymes is obtained. This type of analysis has been employed in the identification of naturally occurring folates in a wide range of materials, including certain bacteriophages (Kozloff et al., 1970), yeasts (Rao and Noronha, 1978) and bacteria (Buehring et al., 1974), rat and sheep liver (Shin et al., 1972b; Osborne-White and Smith, 1973), rat kidney and erythrocytes (Shin et al., 1974), rat brain (Brody et al., 1976), milk and soybeans (Shin et al., 1975), orange juice (Tamura et al., 1976), and cabbage (Chan et al., 1973). Similar techniques have been employed in many studies of the metabolism of radiolabeled folates in animal tissues and microbial and mammalian cell cultures.

34

JESSE F. GREGORY 111

Quantitative studies of ion-exchange separations of polyglutamyl folates were conducted by Osborne-White and Smith (1973) and Rao and Noronha (1978). In each study, the retention behavior of naturally occurring polyglutamyl folates was examined as a function of the polyglutamyl chain length, oxidation state, and one-carbon substituent. Within a homologous series of polyglutamyl folates (H,folates, 5-methyl-H4folates,5formyl-H,folates, and 10-formyl-H4folates)the logarithm of the phosphate concentration in the eluting buffer was linearly related to the number of glutamyl residues. Through the combined use of differential microbiological assays and quantitative evaluation of retention behavior, chain length and identity could be ascertained with reasonable certainty. The extremely laborious nature of the many microbiological assays required does, however, limit the use of these procedures. The procedures of Priest and associates involving the formation of a covalent ternary complex between thymidylate synthetase, [3H]fluorodeoxyuridylate, and 5 , 10-methylene-H,folate have been effectively used for the electrophoretic evaluation of the polyglutamyl chain length of folates in biological materials. As described previously, the chemical and/ or enzymatic conversion of the various classes of folates to the 5,lO-methenyl derivative permits extension of the method to other forms of the vitamin. Quantification is based on densitometry of slab gels following electrophoretic separation and autoradiography (Priest et al., 1980, 1981, 1982; Doig et al., 1985). These methods permit the determination of the distribution of polyglutamyl chain length with high sensitivity. Direct electrophoretic separation of synthetic folic acid polyglutamates in 40% (w/v) polyacrylamide gels also has been reported (Zorzopulos et al., 1983). This procedure would be inadequate for the analysis of complex mixtures of polyglutamyl folates in biological materials because of uncertain resolution and the lack of a specific detection method. An important advance in analytical methodology was the development of HPLC methods for the determination of the chain length of folates in biological materials (Eto and Krumdieck, 1982; Shane, 1982). These procedures have been applied to animal tissues and mammalian and bacterial cell culture systems, but have not been used in food analysis. The basis of these methods is cleavage of the C-9-N-10 bond of extracted folates using sequential oxidative and reductive procedures, followed by formation of the naphthylethylenediamine azo dye derivative of the resulting p-aminobenzoyl(po1y)glutamates.After formation of the azo dye derivatives, purification is performed by column chromatography on BioGel P-2. This purification is based on the adsorptive properties of the Bio-Gel material in acidic solution, rather than its traditional use in gel filtration. Although Brody et al. (1979) reported a method in which Bio-

35

CHEMICAL A N D NUTRITIONAL ASPECTS O F FOLATE

Gel P-4 is used for analytical separation of the purified, derivatized p aminobenzoyl(poly)glutamates, HPLC methods have been devised which yield faster and more efficient separation and quantification. Eto and Krumdieck (1982) performed direct reversed-phase HPLC analysis following this purification (Fig. 81, while Shane (1982) performed reductive ZdHCI cleavage to remove the azo dye moiety from the p-aminobenzoyl(po1y)glutamatesprior to ion-exchange HPLC (Fig. 9). While these methods are sufficiently sensitive for the determination of the unlabeled folates in animal tissues and bacteria, the HPLC detection by UV (Shane, 1982) or visible (Eto and Krumdieck, 1982) absorption lacks the sensitivity required for measurement of folates in many biological materials. Krumdieck et al. (1983a) reported that the sensitivity of the method can be enhanced by the use of tritium-labeled naphthylethylenediamine for the derivatization, which permits HPLC quantification of naturally occurring folates by liquid scintillation counting of collected fractions. Alternatively, Loewen (1986)and associates developed a method in which folates of bacterial and fungal cells were determined fluorometrically as the fluorescamine derivatives of the p-aminobenzoyl(po1y)glutamate cleavage products. This method has been employed following separation of derivatized species using ion-exchange chromatography on DEAE-Sephadex A-25, but has not yet been applied to HPLC. The advantage of this fluorometric method is that it provides substantially greater sensitivity than corresponding procedures using azo dye derivatives. A disadvantage of analytical methods involving C-+N-IO bond cleavage is that the nature of the pteridine moiety cannot be readily deterC6

I 0

I

1

1

10

20

30

I

1

I

40

50

60

tlmo (mln)

FIG. 8. Analysis of polyglutamyl folates as the azo dye derivative of p-aminobenzoyl(polylglutarnates (G 1 4 7 . monoglutamate-heptaglutamate) by reversed-phase HPLC. [Reprinted with permission from Eto and Krumdieck (1982); copyright (1982) Academic Press. Inc.] Bar, 8 x 10.’ a.u. at 560 nm.

36

JESSE F. GREGORY I11

Time (minutes) FIG. 9. Analysis of polyglutamyl folates as the p-aminobenzoyl(po1y)glufamatederivatives by ion-exchange HPLC. Peak identity: 9 min, pABGlu-I; 22 min, pABGlu-2; 29 min, pABGlu-3; 35 min, pABGlu-4; 39.5 min, pABGlu-5; 45 min, pABGlu-6; 49 min, pABGlu-7. [Reprinted with permission from Shane (1982); copyright (1982) American Society for Clinical Nutrition.

mined. However, the method of Eto and Krumdieck (1981, 1982) does permit the selective cleavage of each of three pools of folates (Pool I = H,folates, H,folates, and 5,lO-methylene-H,folates;Pool 2 = 5-methylH,folates; Pool 3 = 5 - and 10-formyl-H,folates and 5,10-methenyl-H,folates), which provides information in this regard. Many other HPLC methods have been developed for the quantitative analysis of polyglutamyl folates in comparatively simpler applications (e.g., in vitro enzyme reactions and studies of folate chemistry). Cashmore et al. (1980)reported ion-exchange and reversed-phase methods for the separation of polyglutamyl folates as well as paminobenzoyl(po1y)glutamates of varying chain length. Significantly, these authors showed that the degree of retention and order of elution could be manipulated by varying the pH of the eluent in the pH 2-6 range in reversed-phase HPLC (Bush et al., 1979) using isocratic elution or acetonitrile-gradientelution. These findings were consistent with decreased retention as a result of the ionization of glutamyl carboxyl groups (Fig. 10). An example of this reversed-phase method is its application to the assay of folate conjugase

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

37

FIG. 10. Relationship between capacity factor (k’ = relative retention time) versus mobile phase pH for reversed-phase HPLC of pteroylpolyglutamates(Pt Glu). (m) Pt Glu-7, ( 0 )Pt Glu-5, (A) Pt Glu-3, and (0) Pt Glu-1 folk acid A. [Reprinted with permission from Bush et al. (1979); copyright (1979) Elsevier Science Publishers.]

activity (Fig. 6; Day and Gregory, 1985; Gregory et al., 1987). Anionexchange HPLC also yielded convenient of separations, with retention proportional to polyglutamyl chain length (Stout et al., 1976; Cashmore et al., 1980). An ion-pair HPLC procedure has been reported that yields separations similar to those of the anion-exchange technique (Matthews,

38

JESSE F. GREGORY 111

1986). In this procedure, HPLC is performed using tetrabutylammonium phosphate as the ion-pairing agent, and elution is effected with an acetonitrile gradient. Several procedures were initially employed for the determination of the polyglutamyl chain length of folates in biological materials following oxidative cleavage with alkaline permanganate or reductive cleavage with ZnlHC1. A large body of literature regarding the distribution of polyglutamy1 folates was generated on the basis of these methods. Quantitative studies by Maruyama et al. (1978)and Lewis and Rowe (1979) concerning these reactions clearly demonstrated that the widely used cleavage procedures were unable to cleave 5-methyl-H,folate, and that oxidative procedures yielded 10-formyl-folk acid from 5-formyl-, 10-formyl-, and 5,lOmethenyl-H4folates.Consequently, data obtained using conventional oxidative or reductive cleavage methods would be in error due to incomplete cleavage and, potentially, incomplete separation of the remaining intact polyglutamyl folates from p-aminobenzoyl(poly)glutarnates.Baugh et al. (1979) reported a method for the oxidative cleavage of 5-methyl-H4folate using a peracetic acid reagent in an effort to improve methods for this approach to folate analysis. Later, Foo et al. (1980) and Eto and Krumdieck (1980) evaluated oxidative and reductive methods and determined conditions that were suitable for the conversion of all common folates to their respective p-aminobenzoyl(po1y)glutamates. These studies served as the fundamental basis for the effective methods for the analysis of polyglutamyl folates discussed previously. Recently, Selhub and associates (Selhub et al., 1988; Selhub and Fell, 1988) have reported an innovative approach to the analysis of folates in foods and other biological materials in which affinity chromatography is used to purify extracted folates, followed by reversed-phase HPLC separation of the various folates in their intact polyglutamyl forms. Because of the complex effects of polyglutamyl chain length, pteridine oxidation state, and one-carbon substituents on retention, the separation is complex and many peaks are not fully resolved. Spectral analysis with a diode array HPLC detector aided the identification and quantification of the eluting folates. Quantitative data have not yet been reported, and extraction procedures have not been validated. D. COMPARISON OF ANALYTICAL METHODS Few studies have provided comparative data over a wide range of food samples. As stated previously, several comparisons have been made between competitive binding methods and microbiological assays (Tigner and Roe, 1980; Graham et al., 1980; Klein and Kuo, 1981) and between

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

39

microbiological assays employing different modes of enzymatic deconjugation (Phillips and Wright, 1983; Kirsch and Chen, 1984; Pedersen, 1988). The comparative data accumulated in the author’s laboratory indicate substantial variability between microbiological, competitive binding, and HPLC methods for many foods, even though the precision within a method is ordinarily adequate (Gregory e f al., 1982, 1984a; Engelhardt, 1988). In the analysis of naturally occurring folates in selected foods, the folate content determined by microbiological assay was frequently greater than that determined by HPLC analysis ofthe same extract (Gregory et al., 1984a; Engelhardt, 1988). The potential influence of nonfolate components of the sample extract on the response of the microbiological assay may have been involved, as suggested by studies of urinary folate analysis (Gregory and Toth, 1988a). Variation between published values concerning the folate content of selected foods and the results of microbiological assays is illustrated by the data in Table I. E. DISCUSSION AND CONCLUSION The objective of this review of analytical methods has been to illustrate the current status of folate analysis and to point out the many uncertainties. Because of the questions concerning sample preparation and assay response in the microbiological methods used for the generation of current data bases regarding the folate content of foods, one must conclude that these data are, at best, highly questionable. Substantial improvement in methods for the measurement of folate in foods is needed, followed by analysis of a broader range of foods. In addition, the evidence of incomplete extraction of folates from foods and rat liver (Engelhardt, 1988) suggests that many studies of folate metabolism may have underestimated the content of labeled or unlabeled folates in animal tissues. A further limitation of existing data bases concerning folate in foods is the inherent variability of folate content of foods. For example, variation in folate content approaching +/- 100% for raw vegetables is not uncommon (Table 111; Mullin et al., 1982). In addition, many foods undergo losses of folate during food processing and preparation (e.g., losses by oxidation and/or extraction into cooking water, as discussed in Section IV,C,l). In view of the inherent variability and the potential for variable losses dependent on conditions of food preparation, prediction of the folate content of foods as consumed becomes a formidable task. Improved analytical methodology will only partially correct the problems of accuracy and precision of food composition data bases.

40

JESSE F. GREGORY Ill

TABLE I11 TOTALFOLATECONTENTOFSELECTED SPINACH AND SWISS CHARD SAMPLES”

Sample

Total folate ((Ld100g)

Store-bought spinach Greenhouse spinach A Greenhouse spinach B Field-grown spinach C Field-grown spinach D Field-grown spinach E Field-grown spinach F Field-grown Swiss chard A Field-grown Swiss chard B Field-grown Swiss chard C

23 I 304 183 155 204 194 180 191 140 168

T h e s e data illustrate the problem of inherent variability in folate content among naturally occurring sources of the vitamin (Mullin et a/., 1982).

IV. STABILITY AND CHEMICAL BEHAVIOR OF FOLATES IN FOODS

A.

INTRODUCTION

As previously indicated, there is a need for more complete data concerning the folate content of foods. However, considerable information has been gathered concerning the general forms of the vitamin present in many foods. As illustrated by the representative data presented in Table IV, the majority of naturally occurring folates in foods are methyl, formyl, and unsubstituted H,folates of various polyglutamyl chain lengths. Fully oxidized folates (i.e., folic acid and its polyglutamates) exist in only trace quantities in plant- and animal-derived foods, probably as oxidation products of H,folates. In considering the chemical behavior and net retention of the vitamin in foods, one must emphasize the properties of the various reduced folates. This discussion will first cover the intrinsic modes of degradation of the principal folates, followed by an examination of the reactivity of folates in foods.

B. INTRINSIC STABILITY OF FOLATES Folates are subject to chemical modifications which destroy their vitamin activity largely through cleavage of the C-!I-N-lO bond. The susceptibility of folk acid to cleavage was first demonstrated in studies by Stok-

TABLE IV DISTRIBUTION OF FOLATES IN SELECTED FOODS

Polyglutamyl chain length (74)

Form of folate (after deconjugation)(%)” Food

THF

5-Methyl-THF

Formyl-THF

Folic acid

I

2

3

4

5

6

7 or longer

Reference”

Cow’s milk Cow’s milk Soybean Cabbage Cabbage Broccoli Calf liver, frozen Chicken liver Orange juice from concentrate Orange juice from concentrate Orange juice fresh Whole wheat flour Red kidney beans

ND ND ND ND 38 65 89 12 ND

92 100 15 91 62 35 6 50 100

8 ND 85 3 ND ND 4 23 ND

ND ND ND ND ND ND I 15 ND

60

6

8

4

8

6

8

53

16

3.5

3.5

19

5 32

62

I 2 I 3 2 2 2 4 5

ND

100

ND

ND

2

ND ND ND

100 ND 100

ND ND ND

ND 100 ND

2 4 4

30-40

(15-30% di- to tetra-glu)

40-50

“ND, Not determined; THF, tetrahydrofolate. bReference: I , Shin er a / . (1975);2, Gregory et a / . (1984a); 3, Chan er a / . (1973); 4, Engelhardt (1988);5 . Tamura cr

N/.

(1975).

42

JESSE F. GREGORY 111

A

H

Tetrohydrofolote

Quinonoid - dihydrofolote

0

+ p-Aminobenzoyl

HxY:>

gIuto mate + HCHO

H2N

I

H

7,8- Dihydropterin

f? = ~ C O N H C H ( CI H 2 ) 2 C O O H COOH

FIG. I I . Proposed mechanisms of the oxidative degradation of tetrahydrofolic acid. The reactions causing the formation of (A) 7,8-dihydropterin and (B)6-formyltetrahydropterin predominate in acidic and alkaline media, respectively. [Reprinted with permission from Reed and Archer (1980);copyright (1980) American Chemical Society.]

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

43

B

H

Te t r o h y d rof o Io t e

li

Nlo- nitrenium ion I-H+

C,-

ti Nio- Schiff base hydrolysis

0 H '

I

H

6 - Formyttetrohydropterin

+

p - Arninobenzoyl ~ l u t o m a t e

44

JESSE F. GREGORY I11

stad et al. (1948) and Hutchings er al. (1948), who identified pterins and diazotizable amines as products following oxidative or reductive treatments. It was also shown that folic acid was susceptible to photochemically catalyzed cleavage (Stokstad et al., 1947) in addition to reductive cleavage mediated by sulfurous acid or zinc dust in acid (Hutchings et al., 1948). The reduced unsubstituted folates (H,folate and H,folate) are markedly less stable than folic acid (O’Broin et al., 197% although their mode of degradation via oxidative cleavage is similar to that of folic acid (Chippel and Scrimgeour, 1970; Blair and Pearson, 1974; Pearson, 1974; Reed and Archer, 1980). In detailed studies of the oxidative degradation of H,folate by air, Reed and Archer ( 1980)observed that p-aminobenzoylglutamate was the major oxidation product at pH 4, 7, and 10. The partial conversion of H,folate to 7,8-H2folatecan also occur at high pH in the presence of air or other oxidant (Chippel and Scrimgeour, 1970; Reed and Archer, 1980). The identity of the pterin fragment produced during oxidative cleavage varies depending on the pH, with pterin predominating at pH 4, and 6-formylpterin as the major pterin at pH 7 and 10 (Reed and Archer, 1980). Mechanisms were proposed for these cleavage reactions that involved a labile quinoid-dihydrofolate intermediate at pH 4 and a labile C-9-N- 10 Schiff base at pH 7 and 10 (Fig. 11) (Reed and Archer, 1980). A quinoid-dihydrofolate intermediate presumably occurs during the oxidation of H,folate and tetrahydropteridines by air and oxidants such as ferricyanide and Fe3+ (Vonderschmitt and Scrimgeour, 1967; Archer and Scrimgeour, 1970; Chippel and Scrimgeour, 1970; Reed and Archer, 1980). Blair and Pearson (1974) reported that the degradation of H,folate in the presence of oxygen was approximately first-order with respect to oxygen. Increased lability of H,folate at high pH was observed, which was attributed to increased oxidation rate in proportion to the extent of ionization of the 3,4-amide group (pK, of amide = 10.5; Kallen and Jencks, 1966). The reactions of nitrite ions with various folates have been studied to determine further the behavior of this reactive food component (Reed and Archer, 1979). H,folate in the presence of nitrite ions is cleaved to p-aminobenzoylglutamate and pterins. Folic acid and 5-formyl-H4folatewere converted to their 10-nitroso derivatives. 10-Nitrosofolic acid has been reported to be carcinogenic in the rat (Wogan et al., 1975). The nitrosation of folk acid was reported to be second-order with respect to nitrite, as is the case with other nitrosation reactions of secondary amines. Ascorbic acid tended to inhibit the nitrite-mediated degradation of H,folate. The significance of these reactions with respect to folate stability and food safety is unclear. Most foods that are subjected to nitrite-containing curing treatments are low in folate concentration (e.g., bacon).

45

CHEMICAL AND NUTRITIONAL ASPECTS O F FOLATE

The degradation of folates via a free radical mechanism has been proposed on the basis of kinetic data (Blair and Pearson, 1974; Pearson, 1974), although no hydroperoxy folate intermediates were detected (Pearson, 1974). Taher and Lakshmaiah (1987) have recently reported that folic acid is degraded, via C-9-N-10 cleavage, by hydrogen peroxide or organic hydroperoxides in the presence of cytochrome c. This finding suggests that this heme protein, which generates free radicals as a result of its peroxidase activity, can mediate folate degradation. The role of such a reaction in the degradation of folates in vivo and in foods is unclear at this time. Great differences are observed between the various folates with respect to their resistance to oxidative degradation (Table V). The resistance to C-9-N-10 bond cleavage of folates with one-carbon substituents at the 5 or 10 positions is substantially greater than that of unsubstituted H,folate. This stabilizing effect of the substituent groups is presumably due to the effect of the one-carbon substituent in interfering with the formation of resonance forms involved in oxidative degradation. In addition, Pearson (1974) hypothesized that the substituent group would sterically hinder the approach of the oxidant. The mechanism of oxidation of 5-methyl-H4folatehas been the subject of considerable uncertainty and ambiguous nomenclature. The primary TABLE V COMPARATIVE STABILITY OF VARIOUS MONOGLUTAMYL FOLATES IN AQUEOUS SOLUTION AT ROOM TEMPERATUREU

Half-time for loss of folate activity (hr)bat pH: Folate THF 5-Methyl-THF 10-Formyl-TH F 5-Formyl-THF 5.10-Methenyl-THF Folic acid

2 1.1

28

-

384 80

4

6

8

0.5 28 132 552

0.6 35 33 696 68

4.2 250 216 192

-

56

-

136

“From O’Broin et a/. (1975). bHalf-time values represent storage time required for 50% loss of folate activity as determined by L . cosei assay (0.3% ascorbate in assay medium). No attempt was made to determine the extent of interconversion or oxidation of folates during storage. Significantly lower stability was observed in several cases in phosphate buffers, compared to other ions tested, at near neutral pH. The data shown represent the following buffers: pH 2, 0.1 M HCI/KCI; pH 4, 0.05 M citratelphosphate; pH 6, 0.05 M phosphate; pH 8, 0.05 M phosphate.

46

JESSE F. GREGORY 111

oxidation product of 5-methyl-H4folateunder mild conditions (e.g., oxygen or dilute hydrogen peroxide) was reported to be an H,folate derivative identified as 5-methyI-5,6,-H2folate(Larrabee et al., 1961, 1963; Donaldson and Keresztesy, 1962). This compound, which is inactive in microbiological assays, cannot be reduced in vitro by dihydrofolate reductase, although facile nonenzymatic reduction to 5-methyl-H4folate occurs in the presence of ascorbate or 2-mercaptoethanol or by reduction with borohydride or catalytic hydrogenation (Larrabee et al., 1961;Donaldson and Keresztesy, 1962; Bertino et al., 1965). Gregory e? al. (1984b) reported that 5-methyl-5,6-H,folate exhibits nearly complete folate activity when given orally to the rat and chick, presumbly via nonenzymatic reduction in vivo. In a similar experiment, the results of Kennelly et al. (1982) suggested that some degradation of 5-methyI-5,6-H2folate may however, occur in the gut. The action of ascorbate and 2-mercaptoethanol in reducing this compound is analytically significant because, if 5-methyl5,6-H2folatewere present in foods as an oxidation product of 5-methylH,folate, rapid reduction to the parent compound would occur during extraction of the sample. It should be noted that conversion of 5-methylH,folate to 5-methyl-5,6-H2folateis useful in analytical methods requiring C-9-N-10 bond cleavage in view of the spontaneous cleavage upon acidification (Foo et al., 1980; Eto and Krumdieck, 1980). Oxidation of 5-methyl-H4folate under more severe conditions (e.g., higher concentration of hydrogen peroxide) yields, apparently via a 5methyl-5,6-H2folateintermediate, a compound originally identified as 4ahydroxy-5-methyl-H4folate (Gapski et al., 1971; Blair et al., 1975). The “4a-hydroxy-” compound could not be converted to biologically active by chemical reduction or catalytic hydrogenation. Upon reexamination of the identity of this compound and further structural investigation, Jongejan et al. (1979) determined that the actual structure of “4a-hydroxy-5-methyl-H2folate” was a pyrazino-s-triazine structure derived from rearrangement of 5-methyl-5,6-dihydrofolate(Fig. 12). This compound has been detected as a folate excretory product (identified as “4ahydroxy-”) in rat urine (Barford et al., 1978). The triazine derivative has been shown to be inactive in folate metabolism and rapidly excreted (Kennelly et al., 1982). 10-Formyl-H,folate, the main naturally occurring formyl folate compound, is readily oxidized to 10-formyl-folic acid, a stable form of the vitamin which exhibits folate activity in microbiological and animal assays (Gregory et al., 1984b). As discussed previously, 10-formyl-H,folate is also subject to conversion to 5-formyl-H4folate,a form of the vitamin which exhibits great stability. Thus, although 10-formyl-H,folate is inherently unstable, there is frequently little loss of folate activity because

47

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

unidentified pterin U

t

!-.

/ p-aminobenzoylglutamate

5 -methyl - dihydro - pyrazino- s - trazine compound

FIG. 12. Proposed mechanism for the oxidative degradation of 5-methyl-tetrahydrofolate. [Reprinted with permission from Gregory ( 1985a); copyright (1985) Van Nostrand Reinhold.]

of the comparative stability of 10-formyl-folicacid and 5-formyl-H,folate (O’Broin el al., 1975). While the oxidative degradation of many folates (e.g., H,folate) causes loss of vitamin activity, limited oxidation of 10formyl-H,folate and 5-methyl-H4folateappears to have little detrimental effect (Gregory et al., 1984b). C. STABILITY OF FOLATES IN FOODS AND RELATED MATERIALS

I. Losses of Folate during Cooking and Thermal Processing. The stability of folate in foods has been difficult to characterize fully because of the multiple forms of the vitamin present and variation in their susceptibility to degradation and the influence of various environmental factors (pH, oxygen concentration, metals ions, etc.). In some cases, extensive losses of folate have been reported even after short exposure to heat, while in similar cases relatively little loss of folate activity is observed (Table VI). Much of this variability is probably due to differences in oxygen exposure during cooking, inherent differences between foods in ascorbic acid content (which would exert a protective effect), and the amount of water present. Folates, like many water-soluble vitamins and minerals, are subject to leaching from foods into surrounding cooking water in addition to chemical modes of degradation. Extensive losses of folate have been reported in boiled foods (Hurdle et al.. 1968). Leichter et al. (1978) examined the leaching of folate during cooking ( 10-min boiling) of several vegetables

48

JESSE F. GREGORY 111

TABLE VI RETENTION OF FOLATE IN SELECTED FOODS

Material

Treatment

Retention (%)

Reference"

Corn meal, fortified Corn grits fortified Rice, fortified premix Bread, made with fortified flour Bread, made with fortified flour Bread, made with fortified flour

Storage 6 months, room temperature (6.5% water) Storage 6 months, room temperature (1 1.4% water) Storage 6 months, room temperature

100

1

100

I

LOO

1

80-100

2

100

2

61

3

86-92

3

90-100

3

Bread, made with fortified flour Flour, fortified with folic acid Breakfast cereal, fortified Potato Broccoli Porridge oats Asparagus Broccoli Brussels sprouts Cabbage Cauliflower Spinach Garbanzo beans Garbanzo beans Garbanzo beans Milk, cow's Milk, cow's Milk, cow's Milk, human

Baking Baking

+ 5 days at room temperature

Baking (refers to stability of all naturally occurring folate in flour, milk, and synthesized by yeast) Baking (refers to stability of added folic acid) Storage 12 months, 38°C Storage 3 months, 40°C Storage 6 months, 22°C Boiled Fried (not adjusted for water loss in frying) Boiled Boiled Boiled 10 rnin Boiled 10 min Boiled 10 rnin Boiled 10 rnin Boiled 10 min Boiled 10 min Steam blanch 20 min Water blanch 20 min Retorted 118°C. 30-53 min in brine (retention relative to blanched beans) Retorted 118"C, 30-53 min + 0.2% ascorbate (retention relative to blanched beans) HTST pasteurization (71.7"C, I5 sec) UHT pasteurization (138.145"C, 2 4 sec) Boiled 1 min Storage -20", 3 months

80

4

100

67 275 41 20 84 38 73 54 75 22 76 54 89-93

5 5 6 6 6 6 6 6 7 7 7

89-98

7

90-100

8 9

40-97 35 45

5

10

II

"Reference: I , Rubin er a / . (1977); 2, Cort et a / . (1976); 3, Keagy er a / . (1975); 4, Anderson e t a / . (1976); 5 , Hurdle er a / . (1968); 6, Leichter er a/. (1978); 7, Lin et a / . (1975);8, Burton er a / . (1967); 9, Ford er a / . (1969); 10, Ek and Magnus (1980); I I , Bank et al. (1985).

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

49

and observed from 22 to 84% of the initial folate in the cooking water. In addition, the sum of folate retained in the cooked vegetable plus that of the cooking water was nearly equivalent to the total folate of the raw vegetables in most cases. These results suggest that aqueous extraction, not oxidation or thermal degradation, is frequently responsible for losses of folate during cooking. Similarly, Lin et al. (1975) reported that the folate content of canned retorted garbanzo ( 1 18"C, 30-53 min) was approximately 70% whether or not 0.2% ascorbic acid was added to the canning brine. Because ascorbic acid would retard oxidative degradation of folates, these results provide strong evidence for a lack of folate oxidation in this instance. Regardless of the mechanism of loss of folate from foods, substantial losses can occur, particularly during home cooking. It should be noted that Cooper et al. (1978) reported that 5-methyl-H4folatein solution was degraded more rapidly during microwave heating than during conduction heating. However, the significance of this observation with respect to food preparation is questionable. Variable and sometimes conflicting conclusions have been reached concerning the stability of folates in foods and model food systems. The thermal degradation of 5-methyl-H4folatein citrate buffers and fruit juices was examined and was found to exhibit pseudo-first-order kinetics under conditions of excess or partially limiting oxygen (Mnkeni and Beveridge, 1982, 1983). Similar results also have been reported for 5-methyl-H4folate as well as other folates (Paine-Wilson and Chen, 1979; Garrett, 1956; Chen and Cooper, 1979). In view of the involvement of oxygen in the oxidative degradation of folates, one would expect deviation from pseudo-first-order behavior under conditions of limiting oxygen concentration. This was observed by Ruddick et al. (1980) in studies of the effect of oxygen concentration on the stability of 5-methyl-H4folate. Day and Gregory (1983) also reported evidence of second-order kinetics in studies of the thermal degradation of folk acid and 5-methyl-H4folatein liquid model systems at low oxygen concentration (1-2 ppm). These levels of oxygen are comparable to the low levels encountered during the retorting of canned foods. In addition to the effect of dissolved oxygen, several other factors relevant to effects of food composition on folate degradation have been reported. Day and Gregory (1983) examined the stability of folic acid and 5-methyl-H4folatein liquid model systems containing potassium caseinate and lactose at pH 7.0, which were designed to simulate commercially available canned infant formulas. When examining the effects of ascorbate and/or iron (ferrous sulfate) fortification at nutritionally relevant levels, these components tended to improve the retention of the folates. It

50

JESSE F. GREGORY I11

was hypothesized that these effects of ascorbate and Fe3+ were due in part to a reduction oxygen concentration of the model systems. The energy of activation has been reported for the oxidative degradation of various folates, as summarized in Table VII. With a few exceptions, the energy of activation values are high, which reflects a strong TABLE VII ENERGY OF ACTIVATION FOR DEGRADATION OF FOLATES IN AQUEOUS SOLUTION

Folate compound Tetrahydrofolate (THF) 5-Methyl-THF 5-Methyl-THF

5-Methyl-THF

5-Methyl-THF Folic acid

Folk acid Folic acid

ExgeCimental conditions 0.1 M phosphate, pH 7.3

(unlimited oxygen) 0.1 M phosphate, pH 7.3 (unlimited oxygen) Citrate, ionic strength 0.1 (unlimited oxygen) PH 3 PH 4 PH 5 PH 6 Citrate, ionic strength 0.1 (limited oxygen) PH 3 PH 4 PH 5 PH 6 Juices with limited oxygen Apple juice, pH 3.4 Tomato juice, pH 4.3 Citrate, ionic strength 0.1 (limited oxygen) PH 3 PH 4 PH 5 PH 6 Fortified juices Apple, pH 3.4 Tomato, pH 4.3 Accelerated storage test of a multivitamin syrup, pH 3.2 (pseudo-first order)

Energy of activation (kcalhol)

References"

13.9

1

7. I

2

3 19.0 17.0 19.7 19.8 3 13.6 11.8 13.2 13.3 9.5 10.6

22.5 19.8 18.0 17.5 4 20.0 19.7

16.8

5

"Reference: I , Blair and Pearson (1974);2. Ruddick et a / . (1980); 3, Mnkeni and Beveridge (1983); 4. Mnkeni and Beveridge (1982); 5, Garrett (1956).

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

51

temperature dependence on the rate of folate degradation. The low energies of activation reported for 5-methyl-H4folatein apple juice and tomato juice (Mnkeni and Beveridge, 1983) are suggestive of either a different mechanism of oxidation in these products or, more likely, a reaction governed by the rate of oxygen diffusion. The y-peptide bonds of polyglutamyl folates appear to be stable during food preparation and processing, although they are readily cleaved by endogenous conjugases present in many foods in the raw state. Reed et al. (1976) reported little or no change in polyglutamyl chain lengths of chicken liver folates during cooking. 2.

Stability of Folate in Milk Products

The stability of folate in dairy products has been studied extensively. Over 90% of the folate in cow's milk is 5-methyl-H4folate(Karlin, 1969; Shin et al., 1975; Dong and Oace, 1975; Gregory et al., 1984a), with 60% of the total in monoglutamate form (Shin et al., 1975). Ghitis (1966) conducted the first systematic study of folate stability in milk. He observed three fractions of folate in milk that could be differentiated according to their stability in the presence or absence of added copper (used to oxidize ascorbic acid). Burton et al. (1967), in an extension of these studies, found that the degradation of folate in milk was potentiated by prior heat treatment, which caused oxidation of ascorbate. In addition, Ford (1967) reported that exclusion of oxygen markedly reduced the losses of folate in milk during conventional sterilization. The pasteurization of milk by conventional and ultrahigh-temperature (UHT) methods has been shown to cause little (<20%) loss of total folate activity (Karlin, 1969; Burton et al., 1967, 1970; Ford and Scott, 1968; Ford et al., 1969). The concentration of residual oxygen was shown to be a critical factor affecting the stability of folate in milk following UHT pasteurization (Ford et al., 1969; Burton et al., 1970). Ek and Magnus (1980) reported evidence of folate deficiency in infants that was secondary to losses of folate during the preparation of formula made from boiled cow's milk. The stability of folate in human milk during refrigerated or frozen storage also has been investigated (Bank et al., 1985). No change in folate content was observed during 24 hr storage at 4°C. By contrast, a progressive decline in folate concentration occurred during storage at - 20"C, with approximately 55% loss of total folate over 3 months. These findings indicated that frozen storage of human milk for 1 month would yield losses of sufficient magnitude that the Recommended Dietary Allowance of folate would not be provided to the infant.

52

JESSE F. GREGORY 111

3. Folic Acid in Food Fortification

Folic acid is the form of the vitamin used in food fortification because of its excellent stability characteristics. Anderson er al. (1976) and Cort et al. (1976) examined the retention of folic acid in fortified breakfast cereals and vitamin-mineral premixes and found little or no loss during storage periods of up to 6 months at ambient temperature. Similar stability of folic acid has been found in applications in baking. High stability has been observed during storage of fortified flour and fortified grains and during the baking of bread products (Colman et al., 1975a; Rubin et al., 1977; Cort et al., 1976; Keagy et al., 1975). Folic acid also exhibits a high degree of stability in foods of high moisture content (Colman, 1982; Day and Gregory, 1983). The efficacy of added folic acid as a source of available folate has been demonstrated by Colman and associates (Colman et al., 1975a,b; Colman, 1982). V. BlOAVAlLABlLlTY OF DIETARY FOLATE

A. INTRODUCTION

The Recommended Dietary Allowance (RDA) of folate for adults is 400 Fg per day folic acid equivalents, increased to 800 Fg for allowance for pregnancy and 500 pg for lactation (Food and Nutrition Board, 1980).The RDA of 400 pg was based on the assumption that 100-200 pg folic acid equivalents was required daily to meet the minimum requirement and that the bioavailability of dietary folate was only 25-50%. As apparent from the ensuing discussion, a great deal of uncertainty exists in our knowledge of folate bioavailability, and the accuracy of the estimated 25-50% bioavailability is unclear. Recent reviews by Herbert (1987) and Reisenauer and Halsted (1987) have summarized information concerning the folate requirement. The conclusion of each review was that the best estimate of the daily folate requirement in normal humans is 3 pg folate equivalents (6.8 nmol) per kilogram of body weight. Herbert (1987) proposed recommended dietary intakes (RDI) for folate that were calculated on this basis. The nutritional status of an individual is a function of the metabolic requirement for the vitamin, the amount of folate ingested, and its bioavailability. With respect to folate, the bioavailability of dietary folate is the least understood of these factors. The term bioavailability, in the context of the utilization of dietary folates, refers to the extent of intestinal absorption, the metabolic function of folate coenzymes, and the kinetics

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

53

of their clearance via catabolic or excretory processes. Thus, the bioavailability of dietary folates is affected by factors potentially influencing the extent of intestinal absorption, including dietary and physiological variables, as well as intrinsic factors affecting metabolism and excretion of the vitamin. B. PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS OF FOLATE ABSORPTION The intestinal absorption of folates at physiological concentrations is a complex process involvingtransport of monoglutamyl forms of the vitamin. Folate transport across the brush border membrane occurs mainly in the jejunum by a saturable, pH-dependent process (Rose et al., 1978; Rosenberg, 1976, 1981; Cooper, 1977; Rosenberg et al., 1979; Halsted, 1980; Selhub et al., 1983). In addition to the saturable absorption mechanism, there is also evidence of nonsaturable absorption which involves passive diffusion (Strum, 1981; Selhub et al., 1983). Intestinal transport of folates, whether in intact intestine or isolated brush border membrane vesicles, generally exhibits K, values in the low micromolar range for the various monoglutamyl folates. The minor differences in K, values for transport of the various folates (Selhub et al., 1983) do not appear to be of sufficient magnitude to yield significant differences in the rate of uptake among various forms of the vitamin. Competitive inhibition between folic acid, 5methyl-H,folate, 5-formyl-H4folate, and methotrexate is evidence of a single transport protein for pteroylmonoglutamates (Chungi et al., 1979; Selhub and Rosenberg, 1981; Selhub et al., 1983, 1984; Said et a1.,1985, 1987; Strum, 1981; Schron et al., 1988). Folate transport is saturated at luminal concentrations of 10-20 pM, although absorption by the nonsaturable mechanism continues linearly through much higher concentrations (Selhub et al., 1984b). Folate absorption is markedly pH dependent. The pH optimum of folate transport in brush border membrane vesicles is 4.9-5.5, while that of intact intestine preparations is 6.0-6.3 (Selhub et al., 1984b). This difference between isolated membrane vesicles and whole intestine probably reflects a role of the unstirred water layer or mucosal coat in controlling the pH at the intestinal absorptive surface (Smithson et al., 1981). The implications of the acidic pH optimum and strong pH dependence have been the subject of much debate. Blair and associates (Blair and Matty, 1974; Blair et al., 1981) proposed that the mildly acidic pH range is necessary to minimize the charge on the folate molecule, and thus, promote passage across the brush border membrane. The subsequent discovery of the transport process provided evidence for a pH effect on the transport

54

JESSE F. GREGORY 111

protein. In addition, recent evidence indicates that the pH gradient present between the acidic absorptive surface (e.g., pH 5.5) and the neutral cytosol (pH 7.7) drives folate transport through a folate/hydroxyl exchange process (Schron et al., 1985, 1988). It therefore appears that the pH at the absorptive surface influences the rate of folate transport in several ways. A folate-binding protein is present in the jejunal brush border membrane (Leslie and Rowe, 1972; Selhub et al., 1979; Reisenauer et al., 1986). Although a role of this protein in folate transport has been proposed, such a function has not been confirmed. Reisenauer (1988) recently demonstrated that covalent affinity labeling of the intestinal folatebinding protein caused a concomittent loss of folate-binding capacity and folate transport. These results suggest that the folate-binding protein is either the transport protein or, at least, is intimately associated with the transport process. Prior to entry into the portal blood, folic acid is largely reduced to H,folate and either methylated or formylated in the mucosal cells (Perry and Chanarin, 1973; Strum, 1979; Selhub et al., 1973, 1983). The transport and distribution of absorbed folates to the liver and other tissues has been described by Steinberg (1984). Approximately 10-20% of the absorbed folates in reduced substituted form is taken up by the liver on the first pass, while the remainder distributes to other tissues. Any folic acid entering the portal blood is taken up almost exclusively by the liver (Steinberg, 1984). The nature and function of various folate-binding proteins involved in the circulation and tissue absorption of folates has been reviewed by Wagner (1982, 1985). The liver has been shown to secrete folate as monoglutamyl 5-methylH4folatein the bile (Steinberg et al., 1979; Steinberg, 1984). Although this provides the potential for continual enterohepatic circulation of folate, the magnitude of biliary secretion and the extent of subsequent reabsorption of secreted folate has not been well established in humans. Studies by Krumdieck et al. (1978) concerning the routes of excretion of I4C following administration of ['4C]folicacid to a human adult provide relevant information. These researchers observed that fecal excretion accounted for as much or more of the total isotopic clearance than did urinary excretion. It thus appears that a significant portion of the biliary folate is not reabsorbed, although additional studies are needed for verification. Tamura and Halsted (1983) reported data indicating that the fecal route comprises approximately 10% of the isotopic excretion in normal monkeys. The deconjugation of dietary polyglutamyl folate is a requisite step in the absorption process. Studies of in situ folate absorption in intestinal segments of anesthetized dogs suggested that the deconjugation of poly-

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

55

glutamyl folates may occur at the surface of the absorptive cells (Baugh et al., 1971, 1975). The observation of intact polyglutamyl folates in the mucosal cells suggested that absorption involved at least partial transport of folates prior to intracellular deconjugation in the dog. Further perfusion studies with humans established that the human intestinal mucosal surface is able to deconjugate polyglutamyl folate (Halsted et al., 1975; Halsted, 1979, 1980). In these studies, perfusion of radiolabeled polyglutamyl folate yielded an array of shorter-chain polyglutamates in the luminal contents, although little or no soluble conjugase activity was detected in the aspirated intestinal fluid assayed at pH 6.8. The accumulation of polyglutamyl folates in the intestinal lumen also provided evidence that the rate of deconjugation is, in the absence of inhibitory factors, greater than the rate of the absorption mechanism. Substantial variability occurs among various animal species with respect to the location, activity, and properties of conjugases associated with the digestive system. Intestinal conjugase activity was first identified and found to be associated with the lysosomal fraction of the enterocytes (Hoffbrand and Peters, 1969), a finding that was later confirmed by Wang et al. (1986). A soluble conjugase has been identified in chicken intestine. which is an endopeptidase that forms a pteroyldiglutamate product (Saini and Rosenberg, 1974; Rosenberg and Neumann, 1974). Studies of the intestinal mucosa from the rat, other rodents, and monkeys have shown little or no conjugase activity associated with the brush border membrane (Day and Gregory, 1984; Wang et al., 1985), whereas most of the activity is in the soluble or lysosomal fraction with a pH optimum of 4-5 (Hoffbrand and Peters, 1969; Elsenhans et al., 1984; Wang et af., 1985). In addition, in vivo studies of folate absorption in dogs suggest that little or no brush border conjugase activity is present in this species (Baugh et al., 1971, 1975). Although similar findings were initially reported for human mucosal tissue (Halsted et al., 1977), a brush border conjugase activity that was distinctly different from the intracellular enzyme was later detected by Reisenauer et al. (1977). The brush border enzyme differed from the intracellular conjugase by virtue of its neutral pH optimum (pH -6.5), lack of inhibition by p-hydroxyrnercuribenzoate (a strong inhibitor of the intracellular enzyme), and chromatographic behavior. These findings have been extended in studies showing a high degree of similarity between the brush border conjugases of both swine and humans (Day and Gregory, 1984; Wang et al., 1985; Chandler et al., 1986a,b; Gregory et al., 1987), with clear differences between the properties of these enzymes and an intracellular conjugase purified from rat intestinal mucosa (Elsenhans et af., 1984). These differences in the properties of intestinal conjugases of various species are summarized in Table VIII. On the basis of

56

JESSE F. GREGORY Ill

TABLE VIII COMPARATIVE PROPERTIES OF INTESTINAL PTEROYLPOLYGLUTAMATE HYDROLASES (CONJUGASES) OF THE RAT, MONKEY, PIG. AND HUMANY

Parameter Brush border membrane pH optimum Specific activity (pmol/hr/mg protein) Effect of I mM Zn'+ Effect of pHMB Molecular weight (11OOO) Mode of action K, for PG-3 (pW High-speed supernatant (Cytosol) pH optimum Specific activity (pmol/hr/mg protein) Effect of 1 mM Zn" Effect of pHMB Molecular weight (/IOOO) Mode of action

Rat

Monkey

Pig

Human

Referenceh

-

-

0

0

6.5-7.0 I78

6.5-7.0 34 1

1-5 2

Stim None 240-400 Exo 1.0-1.7

Stim None 700 Exo 0.6-1.8

I. 2 1, 2 3-5 3, 4 3. 4

4.5 84

4.5-5.0 I02

4.5-5 .O 89

4.5-5.0 214

lnhib lnhib 80 Mixed

Inhib lnhib ND Mixed

Inhib lnhib ND Mixed

lnhib lnhib 75 Mixed

"Stim, Stimulated; Exo, exohydrolytic; Inhib, inhibited; ND, not determined. "Reference: I , Day and Gregory (1984); 2, Wang et at. (1985); 3, Gregory et al. (1987); 4, Chandler el a / . (1986a); 5, Chandler er a1. (1986b);6, Elsenhans et al. (1984); 7, Wang et a / . (1986).

these findings, it appears that the pig would be the ideal animal model in terms of similarity to the human. Surprisingly, the monkey did not exhibit significant intestinal brush border conjugase activity (Wang et af., 1985). The nutritional significance of these differences among species has not been determined. Reisenauer and Halsted (1987) have recently calculated that the activity of human jejunal brush border conjugase is in sufficient excess that the hydrolysis of dietary polyglutamyl folates is not rate limiting in the absorption process. Because of the necessity for deconjugation prior to folate absorption, inhibition of the enzyme by food components or pharmaceuticals could, however, reduce folate absorption (see later discussion). The source of conjugase activity functioning in the hydrolysis of dietary polyglutamyl folates is unclear in animals lacking an intestinal brush border conjugase, and the action of nonmucosal conjugases in the human and pig has not been determined. Conjugase activity has been reported in the bile (Bernstein and Gutstein, 1969; Home et al., 1981) and pancreatic fluid (Jagerstad et al., 1972; Jagerstad and Westesson, 1974; Kesavan and

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

57

Noronha, 1983) of rats and humans. The biliary and pancreatic conjugases were not fully characterized and their activity has not yet been adequately quantified. The results of Horne et al. (1981) and Kesavan and Noronha (1983) suggest that biliary and pancreatic conjugases may be responsible for the deconjugation of dietary polyglutamyl folates in the rat in view of the absence of a brush border conjugase. S. D. Bhandari and J. F. Gregory (unpublished data) have examined conjugase activity of porcine bile and pancreatic fluid and have found insignificant activity in bile relative to that of pancreatic fluid. The pancreatic conjugase of pigs was present in sufficient quantity that, in spite of its pH optimum at pH 4.5, this enzyme could be involved in the initiation of folate deconjugation in concerted action with the brush border conjugase. As with other digestive enzymes in pancreatic fluid, the secretion of pancreatic conjugase in pigs was stimulated by food consumption. Pancreatic conjugase from pigs was found to exhibit endohandom specificity in the deconjugation of polyglutamyl folates, in contrast to the exohydrolytic mode of the brush border enzyme (Chandler et al., 1986a; Gregory et al., 1987). This finding is consistent with a function of the pancreatic enzyme in initiating the deconjugation of dietary folates in the pig and potentially in humans prior to complete hydrolysis by the brush border conjugase. Conflicting reports exist concerning the conjugase activity in human intestinal fluid. Klipstein (1967) detected conjugase activity in semiquantitative assays of aspirated intestinal fluid, while assays by Halsted et al., (1975) at pH 6.8 showed little activity. The negative results of Halsted et al. (1975) do not exclude the presence of pancreatic conjugase activity in view of its acidic pH optimum (Jagerstad et al., 1976; Bhandari and Gregory, unpublished data ). Further study is needed to clarify the physiological roles of the various conjugases in folate digestion and absorption.

C . In Vivo KINETICS OF FOLATE METABOLISM The design of studies investigating the bioavailability of folate must be based on a consideration of the in vivo turnover kinetics. When physiologically small doses of radiolabeled folates are administered to humans (Krumdieck et al., 1978; Russell et al., 1983) or animals (Wilkinson and Shane, 1982; Tamura and Halsted, 19831, biphasic excretion curves have been consistently reported. Although variable half-time (t1,2)values have been reported for isotopic elimination, two folate pools are consistently observed. On the basis of urinary excretion of I4C derived from [I4C]folate, Krumdieck et al. (1978) calculated a I , , ~of 100 days for the kinetically slow body pool and 3 1.5 hr for the elimination of newly absorbed folate. As indicated in Section V, B, fecal excretion of [14C] was nearly

58

JESSE F. GREGORY 111

*

equivalent to urinary excretion (t,,2 = 63 20 days as calculated from their data concerning the fecal excretion). Tamura and Halsted (1983) reported similar kinetic data for biphasic folate elimination (calculated from combined urinary and fecal excretion) in monkeys (t,,, = 1.1 days and 119 days). In contrast to the many studies published concerning excretion kinetics, only one study apparently has been reported concerning plasma folate kinetics. Loew et al. (1987) determined plasma folate concentration following an intramuscular injection of unlabeled folic acid. They reported a f,,, value of 1.4 hr for the clearance of folate from the plasma following attainment of peak concentration 0.7 hr after injection. The rapid plasma clearance is largely due to distribution of the vitamin to the tissues, rather than extensive excretion.

D. METHODS FOR THE ASSESSMENT OF FOLATE BIOAVAILABILITY Methods for the assessment of vitamin bioavailability, as recently reviewed (Gregory, 1988), include bioassays with animals (rat and chick), bioassays with humans, intestinal perfusion studies in animals or humans, and in vivo isotopic methods in animals or humans. Intestinal perfusion and cell culture studies have been extremely useful in elucidating the behavior of nutrients as presented to the absorptive surface; however, such procedures are of limited utility in the study of the bioavailability of vitamins in foods. 1 . Bioassays with Animals and Humans

The question of an appropriate animal model for the study of folate bioavailability has not been fully resolved. To date, the rat and chick have been employed as primary bioassay animals (Herbert and Bertino, 1967; Keagy, 1985a). As discussed previously, the pig may be better suited in view of its physiological similarity to the human, although its size and expense may preclude the use of the pig in many studies. The rat has been used frequently as a bioassay animal, although many applications are complicated by the ability of the intestinal microflora to synthesize folate which may be available to the animal by either coprophagy or direct absorption from the lower intestine (Teply et al., 1947; Abad and Gregory, 1987). Whereas oral antibiotics have been employed to suppress the microbial synthesis of the vitamin (Grossowicz et al., 1964; Babu and Lakshmaiah, 1987), the use of antibiotics introduces further questions as to the appropriateness of the animal model in reflecting fo-

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

59

04 -1

0

1

2

3

DIETARY FOLIC ACID (nrnol/g)

FIG. 13. Dose-response curves of plasma folate versus dietary folate concentration in a rat bioassay performed (0) with and (0)without the use of tailcups to prevent coprophagy. [From the data of Abad and Gregory (1987).]

late bioavailability in normal humans. Tail-cup devices that collect feces and prevent coprophagy have been shown to reduce the contribution of microbial synthesis to the folate nutriture of the rat (Fig. 13), although bioassays using tail-cups are not without ambiguity (Abad and Gregory, 1987). Depletion-repletion bioassay procedures for available folate using the rat have been reported by Keagy and Oace (1982) and Hoppner and Lampi (1985). These procedures, which do not employ oral antibiotics, were reported to yield a dose-response relationship between dietary folate and liver folate. Abad and Gregory (1987) employed a slight modification of these procedures in studies of the bioassay response. In these studies, liver folate was found to be an inconsistent indicator of available dietary folate, while fasting plasma folate was a highly sensitive criterion. Abad and Gregory (1987) also observed that the rat bioassay was sensitive to overestimation of available folate when conducted with or without the prevention of coprophagy. However, successful applications of rat bioassays have been reported (Keagy and Oace, 1984; Hoppner and Lampi, 1986a,b, 1988; Babu and Lakshmaiah, 1987). The factors responsible for inconsistency of liver folate as a response criterion have not been determined. The chick has been used for folate bioassays because of its sensitivity to and rapid onset of folate deficiency, ease of handling, and high rate of growth. Chick bioassays have been based mainly on the diets and protocols developed by Scott ef al. (1969). Although growth is often not a specific indicator of available dietary folate, plasma and liver folate are

60

JESSE F. GREGORY 111

highly sensitive criteria (Ristow et al., 1982a). Variations of this procedure have been employed in the study of bioavailability of folate in foods and in the assessment of the folate activity of various forms of the vitamin (Graham et al.. 1980; Ristow et al., 1982a,b; Gregory et al., 1984b). Potential limitations in the use of the chick as an animal model are mainly concerned with the differences in intestinal mechanism of enzymatic deconjugation of polyglutamyl folates (Rosenberg and Neumann, 1974). The first use of bioassays involving human subjects was reported by Melnick et al. (1945) for the study of the bioavailability of ascorbic acid, niacin, riboflavin, and thiamin. These procedures were based on quantitation by measurement of urinary excretion over a 24-hr period following ingestion of a reference dose or test substance. This approach was employed in bioassays by Tamura and Stokstad (1973) to study folate bioavailability. These researchers observed a weak and inconsistent relationship between ingested folate and urinary excretion in unsupplemented subjects. In contrast, linear relationships between ingested and excreted folate were found when the subjects were maintained in a state of folate saturation by the administration of 2 mg per day of folk acid orally. Folate saturation has been used in additional bioassays involving the use of urinary excretion as the primary criterion of response (Tamura et a f . , 1976). Alternatively, the short-term (1-2 hr) change in plasma folate concentration following an oral dose of the vitamin has been used in many studies as a short-term indicator of available folate. Primary examples of studies of this type were reported by Perry and Chanarin (1970, 1972), Brown et al., (1973a), and Colman et al. (1975a). Although these methods may be useful under controlled conditions, caution must be exercised. The implied assumption in the use of such methods is that the rate of absorption is equivalent between reference and test doses, such that the plasma concentration at any time is reflective of the quantity of folate absorbed (Gregory, 1988). The potential flaw in such methods is that the bioavailability of the vitamin may still be high (i.e., complete absorption) even when there is a significantly reduced rate of absorption. Thus, methods based on a limited number of plasma folate measurements would be prone to underestimation of the bioavailability of folate in materials from which absorption is slower than that of the reference dose. Keagy et al. (1988) recently employed such a procedure in a study of folate bioavailability and provided an appropriate discussion of the procedural limitations. Whenever possible, multiple blood samples should be taken for up to 8 hr or more to permit an evaluation of the area under the plasma concentration versus time curve (Bailey et al., 1988). Such an approach provides greater pharmacokinetic validity in the experimental design and interpre-

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

61

tation. It should be noted that comparatively large doses are required to obtain a quantifiable rise in plasma folate (Bailey et al., 1988).

2. Isotopic Methods Experimental methods in which the vitamin of interest is labeled with radioisotopes or stable isotopes provide several important advantages over conventional bioassay methods (Gregory, 1988).The presence of the isotope permits specific detection of the administered dose or its metabolites in blood, excreta, and/or tissues. As described in Section 11, B, methods are available for the synthesis of a.wide variety of folates labeled with tritium or deuterium. While the use of radiolabeled folates provides a powerful tool for the study of folate bioavailability in experimental animals and for diagnostic procedures in humans, stable-isotopically labeled folates are better suited for use in normal human subjects because of the safety of these compounds. Isotopic methods for the study of the bioavailability of dietary folates are in their early stages, although radioisotopic methods have been used widely for the investigation of folate metabolism and in vivo kinetics. Further applications of both radioisotopes (in animals) and stable isotopes (in humans) will provide important new information concerning the bioavailability, in vivo kinetics, and requirements of folate. Abad and Gregory (1988), in a single-dose protocol with rats, used various [3H]folatesto examine intrinsic differences in bioavailability between various forms of the vitamin and the effect of various foods on the utilization of the labeled folates. A limitation of studies such as this using radiolabeled folates is the lack of labeled folates suitable for dual-isotope studies. Although [2-'4C]folicacid is commercially available (-55 mCi/mmol), its specific radioactivity is too low to be useful in certain in vivo studies of folate bioavailability at nutritionally relevant doses. Consequently, many radioisotopic studies are limited to those in which a single tritiated form of the vitamin is administered. Quantification of stable-isotopic enrichment requires mass spectrometry. Proton nuclear magnetic resonance, an alternate approach, lacks the sensitivity required for analysis at concentrations of folates routinely encountered in biological samples. Folates in intact form are poorly suited for many forms of mass spectrometry because of their nonvolatility and thermal instability. These problems have been overcome through cleavage of the C-9-N-I0 bond, followed by isolation of the p-aminobenzoylglutamate fragment (Toth and Gregory, 1988; Gregory and Toth, 1988a). Derivatization of p-aminobenzoylglutamate using trifluoroacetic anhydride-trifluoroethanol forms an internal lactam that is well suited for gas

62

JESSE F. GREGORY I11

chromatography-mass spectrometry (GCMS) in the negative-ion chemical-ionization electron-capture mode (Fig. 14) (Toth and Gregory, 1988). A critical aspect of the analysis is the initial sample preparation. Affinity chromatography using immobilized folate-binding protein (Selhub et al., 1980) provides suitable purification and concentration of urinary folates (Gregory and Toth, 1988a). This GCMS method is fully suitable for use in studies employing [3’,5’-*H,J- and [gl~tamate-~H,]-labeledfolates (Gregory and Toth, 1988a,b). The advent of alternative modes of mass spectral ionization (e.g., thermospray mass spectrometry and fast atom bombardment) and improved HPLC interfaces may simplify the analysis in the future. Eicke et al. (1983a,b) reported that underivatized folates could be analyzed by secondary-ion mass spectrometry. Clifford et al.

Chemlcal ClMVOgO d4-pABG

+ PWnS

TFA Anhydride t Trit luoroethanol

fl

CF, C-

Dorivotired d4-pABG Loctom

FIG. 14. Conversion of [gl~tamate-~H,]folic acid (d,-folic acid) to d,-pABG lactam derivative in preparationfor mass spectrometric analysis. Unlabeled (do)and d,-folate would yield do- and d,-pABG lactam derivatives, respectively. [Reprinted with permission from Gregory and Toth (1988a); copyright (1988) Academic Press, Inc.]

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

63

(1986) indicated the feasibility of mass spectral quantification of either underivatized or methylated folate standards by fast atom bombardment tandem mass spectrometry. The applicability of these techniques in biological studies has not been established. Protocols for stable isotopic studies in humans have not yet been fully optimized. With present mass spectral methodology (Gregory and Toth, 1988a; Toth and Gregory, 1988), experiments are largely limited to the quantification of the urinary excretion of deuterated folates or catabolic products. This is because the low concentrations of the vitamin in plasma generally preclude measurement of the isotope in this readily accessible in vivo pool. As discussed previously, a predose saturation regimen appears necessary to accentuate the excretion of the experimental dose in the form of intact folate. Such a saturation regimen may still yield incomplete urinary excretion of the isotope, however. Using a 2-mg per day saturation dose for the preceding 7 days, the recovery of intact deuterium-labeled folates has been found to be 5-20% over 48 hr postdose following the administration of [*H,]- or [‘H,]folate (Gregory and Toth, 1988a; Gregory et al., 1989). The metabolic equivalence of these two labeled forms of the vitamin still permits direct comparisons in dual-label studies of folate bioavailability even with this comparatively low isotopic recovery (Gregory et al., 1989). Alternative saturation protocols, employed in studies with unlabeled folates, have involved the administration of 10-15 mg of folk acid per day for several days pretrial (Perry and Chanarin, 1970, 1972; Colman et al., 1975a), although their efficacy in isotopic studies has not been determined. Studies with radioactive folate have provided information that is potentially useful in establishing stable-isotopic protocols. Russell et al. (1979, 1986) employed a saturation dose of 15 mg of folic acid injected intramuscularly 24 hr prior to absorption test with [’H]folic acid. The effectiveness of this procedure was shown by the recovery of typically 55-60% of the ingested tritium in 24-hr urine collected from normal subjects. The use of a flushing dose was originated by Klipstein (1963), who employed a 15mg parenteral injection of unlabeled folic acid 3 hr after administration of the oral dose of [’Hlfolate. Other investigators have used a “flushing” dose with 10-15 mg of folic acid injected intramuscularly within 6 hr postdose, to enhance the urinary excretion of the administered isotope(s) (Godwin and Rosenberg, 1971; Lane et al., 1976; Halsted et al., 1977; Bailey et a / . , 1984). Questions to be addressed in optimizing experimental design in stableisotopic studies of folate bioavailability include the following. (1) What is the appropriate saturation protocol? (2) What, if any, is the appropriate flushing dose? (3) To what extent are single-dose protocols, rather than

64

JESSE F. GREGORY 111

those involving chronic administration, useful in studies of nutrient bioavailability? (4) In single-dose protocols, what proportion of the dose enters the kinetically slow body pool (tic! 100 days) versus the kinetically rapid pool (tllz31.5 hr) (Krumdieck et al., 1978)? (5) Are there secondary isotope effects (Connor et al., 1980), i.e., differences in the rate of absorption, metabolism, and/or excretion, that occur in the use of deuteriumlabeled folates? With respect to (9, it has been shown recently that [3’,5‘ H2]and [glutamate - H,]-forms of folk acid exhibit equivalent bio-availability in normal humans (Gregory et al., 1989). This finding suggests that secondary isotope effects, if any, are insignificant with these forms of the vitamin. Similarly, the data of Connor et al. (1980) indicate that differences in the rate of absorption between [3H]and [14C]-labeled folates would be small.

-’

’

E. BIOAVAILABILITY OF FOLATE Assessment of the bioavailability of dietary folate is complicated by potential differences in inherent bioavailability among the various folates, the effects of various dietary components and pharmaceuticals on the intestinal deconjugation and absorption processes, and the effects of various digestive abnormalities that may affect the extent of absorption. Additional research concerning each of these factors is required to provide a more accurate description of the bioavailability of folate. Even when more complete data are available, the many interactive factors involved would tend to preclude attempts to make highly precise estimates of folate bioavailability in typical diets. The following is a discussion of present knowledge concerning this topic. The bioavailability of folate has been reviewed recently by Bailey (1988) and Sauberlich (1985).

I . Inherent Bioavailability of Various Folates Superimposed upon any influence of diet composition are potential differences in the apparent bioavailability of the various folates. Although this topic has been examined, the reported data are often contradictory. This may be due to differences in the experimental protocols used, as well as the purity and stability of the compounds employed. The effective absorption of folic acid under fasting conditions has been well established, and the relative bioavailability of purified monoglutamyl and polyglutamyl folates has been extensively investigated. Experiments with radiolabeled folates administered orally or by intestinal perfusion have been used to compare the bioavailability of radiolabeled monoglutamy1 and polyglutamyl forms of folic acid. The extent of absorption of

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

65

polyglutamyl folate in normal individuals, as evaluated by urinary excretion of the isotope following a flushing doSe, is typically 6680% of that observed for monoglutamyl folic acid (Godwin and Rosenberg, 1975; Corcino er al., 1976; Halsted er al., 1975, 1977, 1978; Russell et al., 1979, 1986; Bailey er af., 1984). In contrast, Rosenberg and Godwin (1971a) reported equivalent utilization of [3H]folicacid and polyglutamyl [3H]folate under the conditions of their study. Bioassays quantified by urinary folate in folate-saturated humans have indicated mean bioavailability values of 8540% for PG-3 and PG-7 relative to folk acid (Tamura and Stokstad, 1973). Keagy et al. (1988) recently reported that the bioavailability of PG-7 chronically administered in formula diets to humans was 63% relative to folk acid, on the basis of urinary excretion. Contradicting these studies showing relatively effective utilization of polyglutamyl folates, Perry and Chanarin (1972) reported approximately 25% apparent bioavailability, relative to folk acid, of polyglutamyl folates isolated from yeast. Whether this low response was due to inhibitors in the yeast folate or the large dose employed (45 nmol/kg body weight) is unclear. Although the rat lacks a brush border conjugase, effective utilization of polyglutamyl folates occurs. Rat bioassays have indicated little or no difference in the bioavailability of folk acid and polyglutamyl folates when fed chronically in dehydrated diets (Keagy, 1985b; Abad and Gregory, 1987). Using single-dose administration in hydrated gelled diets, the bioavailability of radiolabeled polyglutamyl reduced folates was approximately 80% that of monoglutamyl reduced folates (Abad and Gregory, 1988). The comparative bioavailability of the various monoglutamyl folates has not been fully studied. Experiments with tritiated folates administered orally to the rat have indicated that the bioavailability of (6S)-5formyl-H,folate and 10-formyl-folic acid is greater than that of folic acid (Gregory er al., 1984b; Abad and Gregory, 1988). The factors responsible for the more effective utilization of formyl folates may be related to differences in the extent of polyglutamylation and subsequent retention in the liver following absorption (Steinberg, 1984). The results of chick bioassays indicated that the bioavailability of 5-methyl-H,folate was equivalent to that of folic acid (Ristow er af., 1982b). The various monoglutamyl folates have been reported to exhibit slight differences in K,,, or K, values for the intestinal folate transport system in whole intestine or brush border membrane preparations (Selhub and Rosenberg, 1981; Selhub er af., 1984; Said et af., 1987). Studies directly comparing the transport properties of all of the monoglutamyl folates have not, however, been performed. Much of the published data is conflicting because of differing experimental conditions and, in some cases,

66

JESSE F. GREGORY I11

the use of racemic (6-ambo)-H,folates rather than the naturally occurring diastereomers. Similarly, the inherent bioavailability of the various monoglutamyl folates in humans or animals has not been adequately determined. The conflicting nature of the data (summarized in Table IX) may be due to the imprecise nature of certain bioassay methods, and as stated above, uncertain purity of the tested compounds, and the use of racemic forms of H,folates. The physiological factors responsible for these differences have not been determined, although this topic is being investigated in the author's laboratory. Any differences in the net bioavailability of the various forms of the vitamin must be determined before the influence of other variables can be fully understood. TABLE IX APPARENT BlOAVAlLABlLlTY OF VARIOUS MONOGLUTAMYL FOLATES: SUMMARY OF

PUBLISHED DATA

Species Human Human

Human Human Rat Rat

Folate"

Response criterion

Urine folate Urine folate Urine folate Serum folate Serum folate Serum folate Serum folate (6R,S)-5,IO-Methenyl-THFSerum folate Serum folate DHF (6R,S)-THF Urine folate (6R,S)-5-Formyl-THF Urine folate Urine folate (6R,S)-SMethyLTHF (6R.S)-THF Serum folate (6R,S)-S-Formyl-THF Serum folate (6R,S)-S-MethyI-THF Serum folate ['HI 10-Formyl-FA Liver ['Hlfolate ['H](6S)-5-Methyl-DHF Liver ['Hlfolate Liver ['Hlfolate ['H](6S)-5-Formyl-THF

(6S)-THF (6S)-S-Methyl-THF (6S)J-Formyl-THF (6R,S)-S-Formyl-THF (6R,S)-5-Methyl-THF (6R,S)-THF (6R,S)-10-Fo1myl-THF

Apparent bioavailability'

Reference' I

I05 121 70 101 135

2

40

159 I50 156 I20 179 218 26 59 78 175 76 132

3 3

4 5 ~

~

~~~

~

"THF, Tetrahydrofolate; DHF, dihydrofolate; FA, folic acid; 6R,S signifies racemic (6ambo) form of tetrahydrofolate. *Apparent bioavailability- (percentage relative to FA) calculated as ratio of response to tested folate relative to the response of the folic acid control. Data from studies in which racemic (6R.S) folates were used were corrected for the presence of the inactive isomer. 'Reference: 1, Tamura and Stokstad (1973);2, Brown et al. (1973b); 3, Perry and Chanarin (1970); 4, Gregory et al. (1984b); 5 , Abad and Gregory (1988).

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

67

2. Nondietary Factors Affecting Folate Bioavailability

a . Altered Gastrointestinal Function and Folate Absorption. Various pathophysiological conditions in which the function of the digestive system is altered can have a profound effect on the bioavailability of ingested folate, as has been thoroughly reviewed (Butterworth, 1968; Bernstein et al., 1970; Rosenberg, 1976, 1981; Rosenberg et al., 1979; Audebert et al., 1978; Halsted, 1979, 1980). The malabsorption of nutrients that occurs in tropical and celiac sprue and a variety of other intestinal disorders frequently involves impaired absorption of both mono- and polyglutamyl folates (Hoffbrand et al., 1969; Corcino et al., 1976; Rosenberg, 1976; Halsted et al., 1978; Halsted, 1980; Botez and Bachevalier, 1981). It has been shown that patients with Crohn’s disease do not exhibit atypical folate absorption by apparently normal regions of the jejunum (Morgan et al., 1982). These observations suggest that, in affected areas of the intestine, a generalized disruption of folate absorption occurs rather than specific impairment of the deconjugation by brush border folate conjugase . Because of the influence of intestinal pH on folate absorption, factors that alter the pH of the intestinal contents frequently affect folate absorption. The effect of atrophic gastritis, which frequently causes reduced gastric acid secretion in the elderly, was studied by Russell et al. (1986). Patients with atrophic gastritis exhibited reduced absorption of [3H]folic acid relative to controls, although folate absorption could be enhanced by the concomitant administration of acid. Unexpectedly, the serum folate concentration was normal in the subjects examined, apparently due to a compensatory effect involving the synthesis of folate by intestinal bacteria. The resuIts of Markkanen (1968) were in contrast to these findings. On the basis of short-term plasma folate response, he reported that the absorption of polyglutamyl folate (from liver) by gastrectomized individuals was similar to that of normal controls; however, a high degree of intersubject variability was observed. An opposite effect of intestinal pH may also occur. Patients with pancreatic insufficiency exhibited greater folate absorption than normal controls because of their slightly acidic intestinal pH (nearer the optimum for folate absorption) because of reduced bicarbonate secretion (Russell et al., 1979). The oral administration of phosphate or bicarbonate buffering compounds normalized intestinal pH and reduced folate absorption to the normal range. Surgical resection of portions of the small intestine may reduce folate absorptive capacity, although compensatory increases in absorption of

68

JESSE F. GREGORY I11

other nutrients has been observed. Reisenauer et al. (1985) reported that the specific activity of jejunal brush border folate conjugase is increased in the remaining continuous intestinal tissue, which suggests a compensation that may enhance the utilization of dietary polyglutamyl folates. Similar increases in brush border conjugase activity also appear to occur in jejunum remaining following surgical resection (Halsted et al., 1986). The significance of these observations with respect to the bioavailability of dietary folate is unclear. b. Effects of Aging. The elderly are often cited as being prone to widespread inadequate folate nutnture, yet little evidence supports this hypothesis (Rosenberg et al., 1982; Senti and Pilch, 1985). However, the existence of marked folate deficiency in certain segments of the elderly population has been clearly documented (Bailey et al., 1979; Wagner et al., 1981). In addition to the age-related increased prevalence of gastrointestinal disease that may affect folate absorption, a variety of age-induced changes in absorptive function have been proposed to occur with respect to folate in the elderly. Baker et al. (1978) reported that elderly subjects absorbed folic acid as effectively as young adult controls, whereas elderly subjects exhibited marked reduction (-90%) in the bioavailability of polyglutamyl folate (fed in the form of 35 g of dried brewer’s yeast). This protocol may be criticized because the 35-g dose of yeast provided almost 2 mg folic acid equivalents of total folate, mostly polyglutamyl, which is greatly in excess of normal dietary levels. These results do, however, indicate a potential for reduced capacity to deconjugate dietary polyglutamy1 folate. Bailey et al. (1984) evaluated the proposed age-related changes in deconjugation and/or absorptive function in studies involving jejunal perfusion of radiolabeled mono- and polyglutamyl folates in the elderly and young adult controls. These studies showed that the elderly are equivalent to young adults with respect to their ability to deconjugate and absorb polyglutamyl folate, which indicates that brush border conjugase is not limiting in the elderly. In addition, the net absorption of mono- and polyglutamyl folates was comparable in each group of subjects. These findings were supported by in vitro studies by Said and Hollander (1984) that showed that aging did not cause altered K, or V,, of intestinal folate transport in rats. Kesavan and Noronha (1983) suggested that the conjugase activity of pancreatic fluid may be less in aged rats, although they did not perform direct analysis of pancreatic fluid. The relevance of this hypothesis to humans is presently unclear. It may be concluded from the results discussed above that the elderly

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

69

are not physiologically prone to folate deficiency, and there is little evidence suggesting an elevated folate requirement (Rosenberg et al., 1982). The existence of folate deficiency caused by inadequate dietary intake or as a result of antagonistic affects of various drugs should be investigated further. c. Antagonistic Effects of Ethanol and Other Drugs. The existence of folate deficiency in chronic alcoholics has been widely reported and thoroughly reviewed (Rosenberg, 1976, 1981; Rosenberg et al., 1979; Audebert et al., 1978; Bonjour, 1980; Halsted, 1980; Lindenbaum, 1980; Hillman and Steinberg, 1982). The antifolate effects of ethanol and a variety of other drugs could occur by impairment of intestinal deconjugation, absorption, or transport processes; impairment of metabolism; or enhanced catabolism or excretion of folates. Many of these factors have been examined experimentally in humans and animals. Chronic ingestion of ethanol has been shown to alter the excretion kinetics of folate in humans and animals. Tamura and Halsted (1983) observed that the kinetically rapid component of folate turnover was markedly enhanced in chronically alcoholic monkeys, while the slow phase was not affected. In contrast, Russell et al. (1983) reported that chronic ethanol ingestion in human alcoholics caused an increase in the t,,, of the slow elimination process as monitored by the specific radioactivity of urinary folate. Whether these conflicting findings are due to differences in species or the conditions of the experiments, the results indicate that ethanol alters the in vivo kinetics of folate. Lane et al. (1976) found no alcohol-related increase in urinary excretion of tritium following [3H]folate administration in humans. Additional studies with monkeys have indicated that alcohol increases the fecal excretion of tritium following administration of ['Hlfolate (Blocker and Thenen, 1987), which is in contrast to the findings of Tamura and Halsted (1983). Evidence of altered excretion kinetics in alcoholic rats has been reported by McMartin et al. (1983, 1986a,b), although there apparently is no alteration in the pattern of hepatic folates (Wilkinson and Shane, 1982; Keating et al., 1985). The influence of ethanol on the intestinal absorption of folate has been examined, with many contradictory results reported. Folate deficiency commonly associated with alcoholism can exacerbate the malabsorption of the vitamin (Klipstein et al., 1973). Ethanol does not significantly inhibit the activity of partially purified human jejunal brush border conjugase in vitro (Reisenauer and Halsted, 1981). Chronic alcohol ingestion in folate-adequate minipigs caused reduced specific activity of intestinal brush border conjugase and impaired the in vivo deconjugation and absorption of polyglutamyl folates (Naughton et al., 1988). Intestinal perfu-

70

JESSE F. GREGORY I11

sion studies of folate-deficient binge-drinking alcoholics using [3H]folic acid showed that folate absorption was impaired (Halsted et al., 1971), while similar findings have been reported in studies employing monkeys (Romero et al., 1981). The suitability of the rat as an animal model in alcohol research is unclear. In contrast to studies with other species, Racusen and Krawitt (1977) observed no inhibitory effect of alcohol when chronically administered to rats. Leichter and Lee (1984) reported that alcohol did not inhibit the absorption of mono- or polyglutamyl folates in vivo in pregnant rats. Said and Strum (1986), however, found that ethanol nonspecifically inhibited the absorption of 5-methyl-H4folateby everted rat intestine in vitro. The metabolism of ethanol may also impair the in vivo retention and coenzyme function of folate. Shaw et al. (1987) reported evidence that superoxide anion radicals formed during ethanol metabolism may cause cleavage of the C-+N-10 bond and, thus, accelerate the catabolism of folate. Few in vivo studies have been reported concerning the effect of alcohol on the bioavailability of dietary folate. Baker et al. (1975) examined the plasma folate response of chronic alcoholics and nonalcoholic controls to oral folk acid or brewer’s yeast. Although no difference was observed in the response to folic acid, the alcoholics exhibited a markedly lower response to the yeast. Whereas the design of this study may be criticized with respect to the large folate doses employed and the use of the plasma folate increment as the response criterion, these results indicate the potential for reduced bioavailability of dietary folate in alcoholics. It may be concluded from these diverse findings that chronic ethanol ingestion has the potential to impair the utilization of dietary folate by either interfering with absorption or increasing the rate of excretion. The extent to which these occur would be a function of the quantity and duration of ethanol consumption and the prior body stores of folate (Russell et al., 1983). Chronically poor diet is also a primary factor in the poor folate nutriture of many alcoholics (Halsted, 1980; Rosenberg, 1981). Several therapeutic drugs have been shown to be antagonists of folate absorption and metabolism. The antifolates methotrexate, trimethoprim, and primethamine are employed as dihydrofolate reductase inhibitors and, as such, could impair the utilization of dietary folates. These compounds, being folate analogs, also exert antifolate activity by inhibition of intestinal absorption (Selhub et al., 1983; Zimmerman et al., 1987) and presumably other aspects of folate transport (Wagner, 1985). A nonfolate drug that exhibits antifolate effects is salicylazosulfapyridine (Azulfidine, sulfasalazine), a compound used in inflammatory bowel disorders. The inhibitory properties of this compound with respect to folic acid absorption in humans were reported by Franklin and Rosenberg

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

71

(1973) and later by Halsted et al. (1981). Sulfasalazine inhibited folate absorption in rat jejunal rings in vitro by an apparently competitive mode (Strum, 198 1). Additional inhibitory effects on the utilization of dietary folate are mediated by the inhibition of human jejunal brush border conjugase (Reisenauer and Halsted, 1981). Sulfasalazine also has been shown to inhibit other enzymes involved in folate metabolism (Selhub et al., 1978).

Diphenylhydantoin (Dilantin) is an anticonvulsant drug that has been associated with folate deficiency. This compound has been shown to retard the intestinal absorption of folic acid (Gerson e f al., 1972; Botez and Bachevalier, 1981). The effect of diphenylhydantoin on the absorption of polyglutamyl folate has not been investigated directly, although Reisenauer and Halsted (1981) found that this drug did not inhibit human jejunal brush border conjugase in vitro. Nelson et al. (1975) reported that diphenylhydantoin did not inhibit the absorption of folate from orange juice, which exists mainly as 5-methyl-H4folatesof various chain lengths. The conclusions of this study are ambiguous, however, in view of questions regarding experimental design (Colman and Herbert, 1976). Krumdieck et al. (1978) examined the influence of diphenylhydantoin administration on the kinetics of folate excretion and on the pattern of excreted folate catabolites and observed no effect of the drug. Phenobarbital, another anticonvulsant, may enhance the catabolism of folate by induction of mixed-function oxygenases. Several nonprescription drugs have the potential of reducing folate absorption. Chronic consumption of aspirin and other salicylates may exert an antagonistic effect on folate status (Alter et al., 1971) by retarding absorption (Zimmerman et al., 1985) or by enhancing excretion. However, chronic aspirin ingestion did not reduce the bioavailability of mono- or polyglutamyl folate in rat bioassays (Hoppner and Lampi, 1986). Various antacids could reduce folate absorption by increasing the pH of the upper intestine. Russell et al. (1979) demonstrated that antacid consumption inhibits the absorption of simultaneously ingested folate in normal humans. Chronic bicarbonate consumption in a rat bioassay did not, however, reduce folate bioavailability (Hoppner and Lampi, 1988). The extent of other nutritionally significant folate-drug interactions is unclear at this time. 3. Bioavailability of Folates from Foods and the influence of Dietary Components on Folate Absorption and Utilization

Of the various factors affecting folate nutriture in humans, the bioavailability of folate is the factor that is least understood. Reviews concerning folate bioavailability have been published by Rodriquez (1978), Anderson

72

JESSE F. GREGORY Ill

and Talbot (1981),Sauberlich (1985),and Bailey (1988). In spite of considerable investigation, the bioavailability of dietary folate still cannot be predicted with the accuracy and precision needed for adequate dietary assessment. a. General Aspects of Folate Bioavailability. Bioassays with human subjects have indicated that foods differ widely with respect to the bioavailability of folate (Table X;Retief, 1969; Tamura and Stokstad, 1973; Babu and Srikantia, 1976; Stokstad et al., 1977). Limitations of these studies include low precision and the need for administration of unphysiologically large quantities of the materials tested. Consequently, the applicability of these results is uncertain. A general conclusion of these and animal studies is that the bioavailability of endogenous folates in animalderived foods (e.g., liver) is generally high, while folate bioavailability is variable in other foods (Retief, 1969; Tamura and Stokstad, 1973; Ristow et al., 1982b; Abad and Gregory, 1987; Babu and Lakshmaiah, 1987) A series of experiments by Colman and associates (Colman et al., 1975a,b; Margo et al., 1975; Colman, 1982) provided important informaTABLE X APPARENT BIOAVAILABILLTY OF ENDOGENOUS FOLATE IN SELECTED FOODS AS DETERMINED BY BIOASSAYS WITH HUMAN SUBJECTS

Amount consumed Food Banana Lima beans, dry, cooked Lima beans, frozen, cooked Liver, cooked, beef Cabbage, cooked Cabbage, raw Egg yolk, hard cooked Orange juice Tomato Spinach Banana Egg, whole Liver, goat

Apparent bioavailability (%) Mean

Range

Reference"

890

82

0- 148

1

560

70

0-138

1

360 63-94 500-700 500

96 50 47 47

48-181 22-103 0-127 0-93

I

250

82 35 37 63 46 72 70

61-100 29-4 1 24-7 1 26-99 0-148 35-137 9-125

I I 2 2 2 2 2

(g)

600

lo00 200 533-700 300-500 100

"Reference: 1, Tamura and Stokstad (1973); 2, Babu and Srikantia (1976).

1 1 1

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

73

tion concerning the bioavailability of folic acid in supplemental form and as used in food fortification. Short-term studies with human subjects were performed to examine the relative bioavailability (i.e., rate or extent of absorption) as evaluated by the 1-2 hr plasma folate response. The apparent bioavailability of folic acid in fortified maize and rice was 50-60% of that of aqueous folk acid, while the apparent bioavailability of fortified bread was approximately 30-40% in the same study (Colman et al., 1975a). Chronic administration of maize meal or bread fortified with folk acid substantiated these findings (Colman et al. 1975b; Margo et al., 1975). Bread fortified with folk acid to provide 900 pg per day yielded erythrocyte folate values similar to those obtained with 300 pg of folic acid in tablet form, which indicated approximately 33% bioavailability of the folate in bread. Similarly, maize meal providing 500 pg added folic acid per day yielded comparable erythrocyte folate values to those obtained with 300 pg folic acid tablets, i.e., 60% bioavailability. The factors responsible for the effect of these foods on the absorption and/or utilization of the added folic acid are unclear. In spite of the incomplete response, however, folic acid fortification was highly effective in correcting folate deficiency in the subjects examined (Colman, 1982). The results of Colman and associates suggested that interactions of the added folk acid with food components, possibly during cooking, caused impaired absorption. To investigate this hypothesis, Ristow et al. (1982b) prepared liquid model food systems (similar in compositions to infant formulas) which were fortified with folk acid or 5-methyl-H4folateand subjected them to retort processing (121°C for 20 min). After freeze drying, the solids from heated and unprocessed model food systems were analyzed by chick bioassay to determine the concentration of available folate. These studies indicated that the concentration of available folate was equivalent to total folate determined by L. casei and HPLC assays, which indicated no adverse effect of heat on the bioavailability of the remaining folates. The results of chick bioassays of various fortified foods have suggested the dietary components may affect the bioavailability of the added folk acid (Graham et al., 1980). The following discussion examines several topics concerning the bioavailability of dietary folates. b. Bioavailability of Folate in Yeast. The bioavailability of naturally occurring polyglutamyl folates in yeast has been the subject of extensive and often conflicting research (Schertel et al., 1965; Tamura and Stokstad, 1973; Grossowicz et al., 1975; Babu and Srikantia, 1976; Hoppner and Lampi, 1986b). Studies by Grossowicz et al. (1975) suggest that much of the data indicating poor utilization of yeast folate in humans was the

74

JESSE F. GREGORY 111

result of excessively large doses which may have exceeded the capacity of intestinal deconjugation and/or absorption processes. The existence of conjugase inhibitors, possibly nucleic acids, in yeast has been proposed to be responsible for the low bioavailability of yeast folate (Swenseid et al., 1947; Rosenberg and Godwin, 1971b).The significance of these inhibitors, when present at nutritionally relevant levels, and their effect on the brush border conjugase are unclear. Hoppner and Lampi (1986b) demonstrated by rat bioassay that the bioavailability of yeast folate was equivalent to that of folk acid. As indicated previously, the low bioavailability of yeast folates has been reported in chronic alcoholics and the elderly (Baker et al., 1975, 1978).

c. Bioavailability of Folate in Legumes. The bioavailability of folate in legumes has been the subject of extensive investigation. A component of legumes which is heat-activated and localized in the skin of the seed has been found to inhibit various folate conjugases (Krumdieck et al., 1973; Butterworth et al., 1974). Tamura and Stokstad (1973) and Babu and Srikantia (1976) reported generally high bioavailability of folate in several legumes, although the precision of the assays was extremely low. Similar results were also observed by Devadas et al. (1979). Keagy et al. (1988) evaluated the effect of white beans fed in a formula meal with added folk acid or PG-7 on plasma and urinary folate excretion of human subjects. The presence of the beans had little effect on the apparent bioavailability of the added mono- or polyglutamyl folate. Similar studies were performed using [3H]folatesin rats (Abad and Gregory, 1988). The presence of cooked red kidney beans caused a small (-20%) reduction in the bioavailability of both mono- and polyglutamyl folates. These results which suggested that beans, like several other dietary components (cabbage, orangejuice, wheat bran, and pectin), exert a weak inhibitory effect primarily on the absorption, rather than deconjugation, of folates in this animal model. d . Bioavailability of Folate in Orange Juice. Orange juice and other citrus products represent good sources of dietary folate. The folate in orange juice exists as 5-methyl-H, folate of varying polyglutamyl chain length (Tamura et al., 1976). Questions concerning the bioavailability of naturally occurring folate in orange juice arose following the report by Tamura and Stokstad (1973), which indicated a mean bioavailability of 3 1% in human subjects. Subsequent experiments indicated that the acidity of the large dose administered was responsible for the low response, as neutralization increased the bioavailability of orange juice folate or

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

75

added PG-7 (Tamura et al.. 1976). Presumably, the bioavailability of smaller, more nutritionally relevant, amounts of orange juice would be much greater. This was actually found by Rhode et al. (1983), who reported that orange juice folate was equivalent to folic acid supplements as a source of available folate in humans. Similarly, the apparent bioavailability of orange juice folate has been found to be greater than that of folic acid in rat bioassays (Abad and Gregory, 1987), along with no inhibitory effect on the utilization of added polyglutamyl folate. However, weak inhibitory effects were observed when [3H]folicacid or polyglutamyl folates were administered in the presence of orange juice solids (Abad and Gregory, 1988). The overall conclusion of these studies is that the bioavailability of orange juice folates is high in humans and experimental animals, and that orange juice components would not cause important reductions of the bioavailability of other foods in mixed diets. e . Bioavailability of Folate in Vegetables. While many plant-derived foods have been examined (Table X),cabbage is perhaps the most thoroughly studied. Tamura and Stokstad (1973)reported a bioavailability of endogenous folate in cooked cabbage of 47% (range 0-97%) for humans. A similar mean value was observed in chick bioassays, which indicated approximately 40% bioavailability of the folate in cooked cabbage (comprised mainly polyglutamyl folate) (Ristow et al., 1982b). By contrast, the folate of raw cabbage that had undergone deconjugation during diet preparation exhibited 100% bioavailability for chicks. Rat bioassays of available folate in blanched cabbage indicated 68% bioavailability of the endogenous, largely polyglutamyl folate (Abad and Gregory, 1987). In further studies, the presence of blanched cabbage in rat diets reduced the bioavailability of exogenous tritiated mono- and polyglutamyl folates (Abad and Gregory, 1988).These studies provided evidence for an inhibitory effect of cabbage components on the bioavailability of mono- and polyglutamyl folate in the rat. The specific factors involved and extent to which other vegetables act in a similar manner have not been determined.

f. Bioavailability of Folate in Milk. Folate in the milk of all species examined is largely associated with folate-binding proteins (Ghitis, 1966; Wagner, 1982, 1985). The function of folate-binding proteins in milk has not been conclusively determined, although it has been proposed that they are involved in ( 1 ) concentration and/or secretion of folate in the mammary gland (Metz et al., 1968; Selhub et al., 1984a);( 2 ) sequestration of folate from intestinal bacteria, thus promoting its absorption by the infant (Ford, 1974; Tani and Iwai, 1984);and (3) enhancement of absorp-

76

JESSE F. GREGORY 111

tion by interacting with mucosal cells (Colman et al., 1981). The latter function was proposed following the observation that folate-binding proteins enhanced the uptake of folate by isolated rat intestinal mucosal cells (Colman et al., 1981), which was confirmed in subsequent studies with intestinal brush border membrane vesicles (Salter and Blakeborough, 1988). This does not, however, appear to be a significant physiological function in whole intestine. Several studies have indicated that folatebinding proteins actually reduce the rate of folate absorption (Tani et al., 1983; Tani and Iwai, 1984; Said et al., 1986). The physiological implications of these findings are unclear (Anonymous, 1988). Mason and Selhub (1988) recently demonstrated that folate bound to folate-binding proteins is absorbed by a mechanism that is distinctly different from that responsible for the absorption of free folate. Swiatlo and Picciano (1988) recently examined by rat bioassay the bioavailability of exogenous folic acid added to diets containing milk from cows, goats, and humans. The diets containing goat’s milk exhibited the lowest bioavailability of the added folic acid, which may be a function of the greater folate binding capacity of goat’s milk. It is unlikely that such an effect would be nutritionally significant in humans consuming a typical mixed diet. Of additional interest is the evidence reported by Smith et al. (1985) that human milk or milk protein-based formula was the major contributing factor affecting the folate nutriture of infants, even after the initiation of solid foods. These findings suggest that the bioavailability of folate in human milk and milk-based infant formulas is higher than that of folate from other foods consumed. Thermal denaturation eliminates the binding activity of folate-binding proteins. Gregory (1982) reported that typical pasteurization of milk products has little effect on the folate binding capacity, although minor alterations of binding characteristics may occur. Sightly more severe pasteurization conditions or thermal sterilization eliminates folate binding activity (Ghitis, 1967). In view of the capability of humans and other mammals to absorb folate well in the absence of folate-binding protein, it appears that denaturation of the folate-binding protein would have little adverse effect on the absorption of the vitamin. g. Effects of Dietary Fiber on Folate Bioavailability. Because of the reported trend toward lower bioavailability of folate in certain plant-derived foods, the specific effects of dietary fiber have been the subject of several studies. Ristow et al. (1982a) reported that diets containing various forms of dietary fiber (cellulose, pectin, lignin, sodium alginate, and wheat bran) exhibited equivalent bioavailability of added folic acid in

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

77

chick bioassays. Similar conclusions were reached by Keagy and Oace (1985) using a rat bioassay. Equilibrium dialysis experiments yielded no evidence of folic acid binding by these forms of dietary fiber (Ristow et a / . , 1982a). These findings were in contrast to a report by Luther et al. (1965) that the insoluble residue of various diets, along with filter paper and cotton, could bind low concentrations of monoglutamyl folate. The nutritional significance of the data of Luther et al. is questionable in view of the low extraction efficiency observed by Engelhardt (1988). Russell et al. (1976) examined the effects of fiber in humans and reported that bread of high or low fiber content had little or no influence on the absorption of [3H]folicacid, relative to fasting controls. Keagy et al. (1979, 1988) observed that wheat bran delayed slightly the absorption of PG-7 relative to monoglutamyl folic acid. Wheat bran also reduced the response to added PG-7 in a rat bioassay (Keagy, 1985b). In contrast, Bailey et al. (1988) found in studies of the plasma folate kinetics in humans that bran cereal retarded the absorption of PG-7 but had no effect on the absorption of folic acid. The presence of pectin and wheat bran in hydrated gelled diets weakly retarded the utilization of mono- and polyglutamyl [-'H]folates in rats (Abad and Gregory, 1988). The potential of various forms of dietary fiber to interfere with the enzymatic deconjugation of polyglutamyl folates by humans has been examined in vitro (Gregory et al., 1987). In these studies, various ionic polysaccharides, including neutralized forms of pectin, poly-galacturonic acid, heparin, and sodium alginate, did not inhibit the activity of folate conjugase in isolated brush border membrane vesicles from human and pig jejunum. It was concluded that these polyanionic compounds, which are somewhat analogous to the polyanionic polyglutamyl folates, probably would not directly retard the action of brush border conjugase in vivo. The human intestinal brush border conjugase is zinc-dependent (Chandler et al., 1986a), which raises the possibility that dietary components such as ethylenediaminetetraacetic acid (EDTA) or anionic polysaccharides could inhibit the enzyme. Anionic polysaccharides have little ability to sequester Zn2+from the human or pig brush border enzyme, although EDTA inhibits enzyme activity in vitro (Gregory et al., 1987). It is unclear how dietary fiber exerts the inhibitory effects reported in several studies in view of data indicating little or no binding of folate by dietary fiber and a lack of effect on brush border conjugase activity. The observation that certain forms of fiber inhibit the absorption of both mono- and polyglutamyl folates in the rat (Abad and Gregory, 1988) suggests that the antagonistic effect may involve impaired diffusion of folates to the absorptive mucosal surface. In this context, the influence of food

78

JESSE F. GREGORY I11

composition on the diffusion of nutrients through the unstirred water layer and/or mucosal layer (Smithson et al., 1981) appears to be an important area for future investigation. h. Potential Effects of Dietary Antifolates. Although the inhibitory effects of folate analogs have been well characterized and serve as the basis for chemotherapeutic applications, relatively little information is available concerning the existence of antifolates in foods. Inhibitors of folate metabolism could impair the bioavailability of dietary folate by inhibiting the coenzymatic function and/or interconversions of the vitamin. An interesting example was reported by Butterworth et al. (1986) involving anemia caused by abnormal folate metabolism in pond-reared catfish. The etiology appeared to be related to bacterial cleavage of folate in the catfish ration to produce pteroic acid. As reviewed by Butterworth et al., pteroic acid can exhibit antifolate effects as an inhibitor of dihydrofoM ; Bertino et al., 1965) and by displacing late reductase (Ki = 8 x folate from tissue stores. Although small amounts of pteroic acid are present in certain foods, its antifolate properties do not appear to be significant with respect to human nutrition. The production of 10-formyl-folate and 5-methyl-5,6-H2folate,as discussed previously, has been examined as a potential source of dietary antifolate compounds. These investigations were conducted because of the probable existence of these compounds in foods as oxidation products of 10-formyl-H,folate and 5-methyl-H4folate,respectively. These folates are not substrates for mammalian or avian dihydrofolate reductase (Donaldson and Keresztesy, 1962; Bertino et al., 1965). Any inhibitory effect of 5-methyl-5,6-H2folatein vitro would not be relevant in vivo because of its rapid nonenzymatic reduction by ascorbate and thiols (Donaldson and Keresztesy, 1962), which apparently accounts for its in vivo metabolic utilization (Gregory et al., 1984b). The potency of 10-formyl-folateas an inhibitor of dihydrofolate reductase is illustrated by its Ki of 6 x M for the enzyme from Ehrlich ascites carcinoma cells (Bertino et al., 1965). Blair and associates have reported conflicting data concerning the nutritional properties of 10-formyl-folate in the rat (Beavon and Blair, 1975; Connor and Blair, 1979); the former study indicated that the compound is not metabolized, while the latter indicated that it is metabolized in a fashion similar to folic acid. Effective metabolic utilization of 10-formyl-folatewas observed in the rat and chick by Gregory et al. (1984b). These observations indicate that, although 10-formyl-folate may be a poor substrate and a potent in vitro inhibitor of dihydrofolate reductase, in vivo reduction does occur either before or after loss of the 10-formyl group. Saleh et al. (1982) reported

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

79

that a large doses of I0-formyl-folate (5 mg) administered to humans were excreted in urine largely unchanged. This suggests that the capacity of the subjects to metabolize the compound may have been exceeded by the large dose. This study also yielded evidence that a large dose of 10-formyl-folate interferes with the metabolic utilization of simultaneously administered folic acid. Such an antagonistic effect appears unlikely at nutritionally relevant doses such as those used by Gregory et al. (1984b). i. Effect of Nutrient Interactions on Folate Bioavailability. Several influences of other micronutrients have been reported that suggest that these factors may affect the bioavailability of folate. Severe folate deficiency, such as in sprue or chronic alcoholism, has been shown to cause structural changes in the intestinal mucosa that can impair the absorption of ingested folate (Klipstein et al., 1973). In addition, vitamin B12 deficiency can indirectly reduce folate bioavailabiiity (Das and Herbert, 1976). In vitamin B,, deficiency, 5-methyl-H4folateaccumulates due to impaired B,,-dependent transfer of methyl groups by methionine synthetase, the primary means of conversion of 5-methyl-H4folateto other folate coenzymes (Green et al., 1988). The reported interactions of zinc and folate have been a topic of recent interest. With respect to the bioavailability of polyglutamyl folates, human intestinal brush border conjugase is a zinc-dependent enzyme (Chandler et al., 1986a). Experimentally induced zinc deficiency in humans causes malabsorption of polyglutamyl but not monoglutamyl folates (Tamura et al., 1978). The extent to which variation in zinc nutriture affect the bioavailability of dietary polyglutamyl folates in free-living humans has not been determined. A mutual inhibitory effect on intestinal absorption has been reported between supplemental folic acid and trace metals, although considerable controversy exists. Milne et al. (1984)observed that 400 p.g/day of supplemental folic acid significantly increased fecal zinc in humans consuming low-zinc diets, although no effect was seen during high zinc intake. In vitro and in vivo transport studies in the rat did not support the proposed formation of a zinc-folate chelate under physiological conditions but did provide evidence of a mutually inhibitory action at the intestinal transport site (Ghishan et a f . , 1986). In direct contrast, Butterworth et a f . (1988) reported that the concentration of zinc in plasma and erythrocytes did not change in either folate-supplemented (10 mg folk acid/day) or placebo groups over a 4-month controlled trial. These researchers concluded that folate supplementation of that type did not pose a risk to zinc nutriture. Similarly, Keating et al. (1987) showed that 10-mg folic acid supplements in humans did not affect the kinetics of zinc absorption as evaluated by

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plasma zinc measurements following a concurrent 25-mg dose of zinc (as ZnSO,). While these conflicting results indicate the potential for zinc-folate interactions, the nutritional significance of mutually antagonistic effects appears to be minimal in most cases.

VI.

CONCLUSION

As discussed in this chapter, significant recent advances have been made in the development of methods for the determination of folate in foods and other biological materials and in isotopic procedures for the investigation of the bioavailability of the vitamin. The application of these methods in the near future will provide significant new information with respect to the identity, content, and bioavailability of folates in foods. Although the chemical behavior of folates in foods is reasonably well characterized, common methods of food preparation and processing often yield inevitable losses of folate by oxidative or other modes of degradation or by aqueous extraction. The fortification of staple foods with folic acid, a highly stable folate, has been shown to be a highly effective intervention tool in meeting the needs of deficient individuals. Additional study of the factors affecting folate bioavailability must involve further clarification of biochemical and physiological processes in the intestinal deconjugation and absorption of dietary folates, the chemical identity and interactions of ingested folates and other dietary components, and the metabolic and kinetic fate of folates following absorption.

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spectra of fluorinated derivatives of para-aminobenzoyl glutamic acid. Biomed. Environ. Mass Spectrom. 17, 73-79. U.S. Dept. of Agriculture 1978. “Composition of Foods. Baby Foods. Raw, Processed, Prepared.” Agriculture Handbook No. 8-3, Science and Education Administration, USDA. Washington, D.C. Usdin, E. 1959. Blood folk acid studies. V1. Chromatographic resolution of folic acid-active substances obtained from blood. J. Biol. Chem. 234,2373-2376. Usdin, E., and Porath, J. 1957. Separation of folic acid and derivatives by electrophoresis and anion exchange chromatography. Ark. Kem. 1 1 , 4 1 4 . Uyeda, K., and Rabinowitz, J. C. 1963. Fluorescence properties of tetrahydrofolate and related compounds. Anal. Biochem. 6, 100-108. Vonderschmitt, D. J., and Scrimgeour, K. G. 1967. Reaction of Cu” and Fe” with tetrahydropteridines. Biochem. Biophys. Res. Commun. 28, 302-308. Wagner, C. 1982. Cellular folate binding proteins; function and significance. Annu. Rev. Nutr. 2, 229-248. Wagner, C. 1985. Folate-binding proteins. Nutr. Rev. 43, 293-299. Wagner, P. A., Bailey, L. B., Krista, M. L., Jernigan, J. A., Robinson, J. D.. and Cerda, J. J. 1981. Comparison of zinc and folacin status in elderly women from different socioeconomic backgrounds. Nutr. Res. ( N . Y.) 1,565-569. Wang, T. T. Y., Reisenauer, A. M., and Halsted, C. H. 1985. Comparison of folate conjugase activities in human, pig, rat and monkey intestine. J. Nutr. 115, 814-819. Wang, T. T. Y., Chandler, C. J., and Halsted, C. H. 1986. lntracellular pteroylpolyglutamate hydrolase from human jejunal mucosa. J. Biol. Chem. 261, 13551-13555. Wilkinson, J. A., and Shane, B. 1982. Folate metabolism in the ethanol-fed rat. J. Nutr. 112,604-609. Wilson, S . D.. and Home, D. W. 1982. Use of glycerol-cryoprotected Lactobacillus casei for microbiological assay of folic acid. Clin. Chem. 28, 1198-1200. Wilson, S. D., and Home, D. W. 1983. Evaluation of ascorbic acid in protecting labile folic acid derivatives. Proc. Natl. Acad. Sci. U.S.A. 80, 6500-6504. Wilson, S. D., and Home, D. W. 1984. High-performance liquid chromatographic determination of the distribution of naturally occurring folk acid derivatives in rat liver. Anal. Biochem. 142,529-535. Wilson, S . D., and Home, D. W. 1986. High-performance liquid chromatographic separation of the naturally occurring folic acid derivatives. I n “Methods in Enzymology” (F. Chytil and D. B. McCormick, eds.), Vol. 122, pp. 269-273. Academic Press, Orlando, Florida. Wittenberg, J. B., Noronha, J. M., and Silverman, M. 1962. Folic acid derivatives of the gas gland of Physalia physalis L. Biochem. J . 85, 9-15. Wogan, G.N., Paglialunga, S., Archer, M. C., and Tannenbaum. S. R. 1975. Carcinogenicity of nitrosation products of ephedrine, sarcosine, folic acid, and creatine. Cancer Res. 35, 1981-1984. Zakrzewski, S . F. 1966. On the mechanism of chemical and enzymatic reduction of folate and dihydrofolate. J. Biol. Chem. 241, 2962-2967. Zakrzewski, S. F., and Sansone, A. M. 1971. A new preparation of dihydrofolic acid. I n “Methods in Enzymology” (D. B. McCormick and L. D. Wright, eds.), Vol. 18B, pp. 726-727. Academic Press, New York. Zakrzewski, S. F., Evans, E. A., and Phillips, R. F. 1970. On the specificity of labeling in tritiated folic acid. Anal. Biochem. 36, 197-206. Zimmerman, J., Selhub, J., and Rosenberg, 1. H. 1985. Drug-folate interactions in intestinal folate absorption: Comparison of sulfasalazine, phenytoin and salicylates. Gastroenterology 88, 1643 (abstr.).

CHEMICAL AND NUTRITIONAL ASPECTS OF FOLATE

101

Zimmerman, J . , Selhub, J., and Rosenberg, I. H. 1987. Competitive inhibition of folate absorption by dihydrofolate reductase inhibitors, trimethoprim and pyrimethamine. Am. J . Clin. Nurr. 46,518-522. Zorzopulos,J., Raj, B., and Kozloff, L. M. 1983. An electrophoretic method for the identification of poly-y-glutamyl chain lengths in folates and related compounds. Anal. Biochem. 135, 128-133.

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ADVANCES IN FOOD AND NUTRITION RESEARCH, VOL.

33

CALCIUM IN THE DIET: FOOD SOURCES, RECOMMENDED INTAKES, AND NUTRITIONAL BIOAVAILABILITY DENNIS D. MILLER Institute of Food Science Cornell University lthaca, New York 14853

I. 11. 111. IV.

V.

V1.

VII. VIII.

Introduction Calcium Intakes and Food Sources Recommended Dietary Allowances Calcium Homeostasis Intestinal Absorption of Calcium A. Calcium Absorption: Mechanisms and Regulation B. Calcium Absorption: Changes during Development C. Implications for Dietary Requirements Calcium Bioavailability A. Definition of Bioavailability B. Methods for Measuring Calcium Bioavailability C. Calcium Bioavailability: Dairy Products D. Calcium Bioavailability: Effects of Plant Foods E. Calcium Bioavailability: Protein and Phosphate Effects F. Calcium Bioavailability: Calcium Supplements Summary and Conclusions Research Needs References

1.

INTRODUCTION

Interest in dietary calcium has intensified in recent years as a result of scientific evidence linking it to osteoporosis, hypertension, and cancer. While the etiologies of all of these diseases are multifactorial and poorly understood, there is evidence to support the hypothesis that increased calcium intakes will reduce risk for each of the diseases (Marcus, 1982; Weaver, 1988; Newmark et al., 1984). Mechanisms for calcium’s role in 103 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

104

DENNIS D. MILLER

protecting against these diseases are also unclear or unknown but presumably are related to overall calcium nutritional status. It has been suggested, for example, that osteoporosis is a form of calcium deficiency (Nordin et af., 1987). However, it is important to emphasize that the evidence in all three cases is insufficient to establish clearly that diet is a major factor in the development of these diseases. Recently, a scientific workshop sponsored by the National Institutes of Health (NIH) and the National Osteoporosis Foundation was convened for the purpose of assessing the current state of knowledge in the area of osteoporosis and identifying directions for future research. In their report (Peck et al., 1988), they listed risk factors for osteoporosis and made an assessment of the strength of the evidence for each factor. Their assessment, summarized in Table I, was that the evidence relating low dietary intakes of calcium to osteoporosis risk is only moderate. There is much less evidence for the role of dietary calcium in hypertension and cancer. Nevertheless, many groups and organizations are recommending that dietary intakes of calcium be increased in some population groups. For example, the recent Surgeon General's Report on Nutrition and Health recommends that females, especially adolescents and young adults, increase their calcium intakes (Nutrition Policy Board, 1988). A Consensus Development Conference convened by the National Institutes of Health in 1984 concluded that calcium intakes of 1000 to 1500 mg per day beginning well

TABLE I SCIENTIFIC VALIDITY OF RISK FACTORS FOR OSTEOPOROSISa

Well establishedb

Moderate evidence

Obesity ( - )

Alcohol ( + )

Black ethnicity ( - ) Age ( + ) Premenopausal oophorectomy ( + ) Consumption of corticosteroids (+ ) Estrogen use ( - ) Extreme immobility ( + )

Cigarette smoking ( + ) Heavy exercise ( - ) Low dietary calcium ( + )

Drinking water fluoride

"Reproduced with permission from Peck el a / . (1988). + ), Increased risk; (-), decreased risk.

b(

Inconclusive or inadequate evidence Moderate physical activity Asian ethnicity Parity Diabetes Thiazide diuretic use Progestin use Caffeine use

CALCIUM IN THE DIET

105

before menopause would likely reduce the incidence of osteoporosis in postmenopausal women (NIH, 1984). If we accept the hypothesis that calcium nutritional status is suboptimal in a significant proportion of the population, then the obvious question is how can calcium status be improved? The answer depends on an understanding of human calcium requirements and factors which affect calcium absorption and utilization. The objective of this chapter is t o review the literature dealing with calcium requirements, calcium absorption, and calcium retention.

II. CALCIUM INTAKES AND FOOD SOURCES

Pa0 and Mickle (1981) classified three mineral elements (calcium, iron, and magnesium) as problem nutrients in the United States based on results from United States Department of Agriculture’s (USDA) 1977-78 Nationwide Food Consumption Survey (NFCS). Nutrients were classitied as problem nutrients if mean intakes were below 70% of the Recommended Dietary Allowance (RDA) for several of the age and sex categories listed in the 1980 edition of the Recommended Dietary Allowances. It should be noted that other mineral nutrients may also have been classified as problem nutrients had there been sufficient food composition data available to permit a thorough evaluation of intakes. Data in Table I1 show that, when expressed as a percentage of the RDA, calcium intakes in the United States are quite low, especially for females. More than half of all females over the age of 12 consume less than 70% of their RDA for calcium. Concern about the adequacy of U.S. diets for calcium has also been expressed by the U.S. Department of Agriculture/U.S. Department of Health and Human Services Joint Nutrition Monitoring Evaluation Committee (USDNUSDHHS, 1986). The JNMEC classified vitamin C, calcium, iron, and fluoride as nutrients warranting monitoring because of low intakes by some segments of the population. The following quote from the JNMEC report summarizes the committee’s concern with regard to calcium: “Because calcium deficiency has been implicated as a contributor to the prevalence of osteoporosis among postmenopausal white women, the relatively low intake of calcium among women is a cause of concern.” The wide distribution in calcium intakes within groups of individuals (see Table 11) may be attributed to the fact that Americans get most of their calcium from dairy products. Disappearance data from 1985 indicate that dairy products contribute 76% of the total calcium in the U.S. food

106

DENNIS D. MILLER

TABLE I1 CALCIUM INTAKES IN THE UNITED STATES"

Mean intake per individual (% of RDA)

Percentage distribution of intakes

> 100% of RDA

<70% of RDA

Age

Males

Females

Males

Females

Males

Females

>I 1-2 3-5 6-8 9-1 I 12-14 15-18 19-22 23-34 35-50 51-64 65-74 75 and over

173 102 95 1I4 104 91 98 1 I8 I07 94 93 88 89

173 102 95 I I4 96 71 62 76 74 66 69 70 74

82 47 41 61 49 35 43 53 47 35 36 32 35

82 47 41 61 42

4 26 30 14 21 32 29 24 28 36 36 38 38

4 26 30 14 25

15

13 25 23 16 18 18 21

51

62 51

55 61 57 57 51

"Data are from the 1977-78 USDA Nationwide Food Consumption Survey as reported by Pa0 and Mickle (1981).

supply (Marston and Raper, 1987). Contributions from other food groups are as follows: fruits and vegetables, 8%; meat, poultry, and fish, 4%; flour and cereal products, 4%; legumes, nuts, and soy, 3%; eggs, 2%; and miscellaneous, 2%. Since many dairy products are high in calcium, a few servings each day will meet the RDA (1 cup of skim milk contains 300 mg of calcium). On the other hand, persons who do not consume dairy products will tend to have low calcium intakes since most nondairy foods are low in calcium. The calcium contents of selected foods are listed in Table 111. It is clear TABLE I11 CALCIUM CONTENT OF SELECTED FOODS"

Food item Dairy Products-Milk Canned evaporated milk Skim milk Low-fat (I%) milk

Serving size

5 cup 1 cup

I cup

Calcium content (mglserving) 330 300 300 (continued)

107

CALCIUM IN T H E DIET

TABLE 111 (Continued) Food item Lactose-reduced milk Low-fat (2%) milk Whole milk Buttermilk Chocolate milk Dry skim milk powder Dairy Products-Cheese Ricotta, part skim Swiss Ricotta, whole milk Monterey jack Mozzarella, part skim, low moisture Muenster Cheddar American Mozzarella, whole milk, low moisture Parmesan, grated Cottage cheese (1% fat) Cottage cheese (4% fat) Brie Cream cheese Dairy Products-Other Yogurt, low-fat, plain Yogurt, low-fat, fruit Yogurt, whole milk Ice cream Ice milk Sherbet Cream (half and half) Sour cream Vegetables Spinach, cooked Turnip greens, cooked Broccoli, cooked Bok choy, cooked Collard greens, cooked Mustard greens, cooked Kale, cooked Other Foods Sardines, with bones Salmon, canned, with bonies Tofu Orange Bread, various types

Serving size 1 cup

1 cup 1 cup 1 cup I cup I J cup

5 cup I

02

;cup I oz I oz 1 02

Calcium content (mdserving) 300 295 290 285 280 280 335 270 255 2 10

1 02 1 oz

205 205 205 175

I 02 I Tbsp tI cup F cup 1 oz I 02

165 70 70 65 50 25

1 cup

415 345 275 90 90 50 15 15

I cup 1 cup I I cup I i CUP 5 cup 1 Tbsp I Tbsp

5 cup

85

cup t cup f cup z cup I i cup I i cup

100

3 02 3 02 4 02 I medium 1 slice

370 I65 I10 50 1540

I

i

"Reproduced with permission from Stark (1987).

90 80 75 50 45

108

DENNIS D. MILLER

that most dairy products are excellent sources of calcium. While there are alternatives to dairy products, consumption of these foods by the U . S . population is low, and consequently they do not contribute substantially to average calcium consumption.

Ill.

RECOMMENDED DIETARY ALLOWANCES

Daily dietary allowances for calcium recommended by expert groups from four countries and the World Health Organization are shown in Table IV (Committee on Dietary Allowances, 1980; Bloch and Shils, 1988). The large discrepancies in the recommendations (for some age groups, there are nearly twofold differences) underscore the difficulties and controversies associated with establishing values for human calcium requirements. Dietary allowances for calcium are based primarily on balance data (Committee on Dietary Allowances, 1980). Balance measurements are relatively simple to make but are susceptible to rather large errors since balance is calculated (in the case of calcium) as the difference between two relatively large values, neither of which can be obtained with high precision. Calcium balance is defined by the following relationship: Balance = Cai - (Ca, + Ca,

+ Cad)

where Ca, is the calcium intake, Ca, is fecal calcium, Ca, is urinary calcium, and Cad is dermal losses. There is a tendency in balance studies to overestimate intake and underestimate excretion. This tends to result in balance estimates that are more positive than true values (Hegsted, 1973). Another problem with using balance as the basis for calcium RDAs is that people apparently can adapt to a fairly wide range of calcium intakes. Efficiency of calcium absorption increases when subjects consume a low-calcium diet and decreases with high-calcium diets (Committee on Dietary Allowances, 1980). Thus, measurement of calcium balance in subjects that have not been adapted to the level of calcium in the experimental diet may give misleading results. Hegsted (1973) has argued that, since this adaptation is highly effective, calcium balance data should not be used to establish requirements. Nordin et al. (1987), on the other hand, contend that the balance technique is the only method available for determining calcium requirements. The 1984 publication of a National Institutes of Health consensus state-

TABLE IV RECOMMENDED DIETARY ALLOWANCES FOR CALCIUM FROM FOUR COUNTRIES AND THE WORLD HEALTH ORGANIZATION"

110

DENNIS D. MILLER

ment on osteoporosis (NIH, 1984) has spurred renewed interest in the question of calcium RDAs. The Consensus Development Panel stated in its report that the daily requirement for calcium should be about 1000 mgl day for premenopausal and estrogen-treated postmenopausal women and I500 mg/day for postmenopausal women. These recommendations were apparently based largely on balance studies conducted by Heaney er al. (1977, 1978). A discussion of these and other balance studies designed to determine adequate intake levels for calcium follows. Heaney and his associates (1977, 1978) conducted their balance studies on two rather large groups of perimenopausal women (130 and 168, respectively). In an attempt to overcome the confounding effects of adaptation, they adjusted calcium intakes during the balance study period to the usual intake level of each subject. Results from the second study (Heaney er al., 1978) are summarized in Fig. 1. The regression lines indicate that 0.989 g/day is the mean intake required for calcium balance in the premenopausal and treated postmenopausal group while a mean of 1.504 g/ day is required for balance in untreated postmenopausal women. These studies show that calcium requirements for perimenopausal women may

0.08

! 0

I

0.2

I

0.4

I

I

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Ca INTAKE (g/day) FIG. 1. Regression of calcium balance on calcium intake for two groups of women. (a) Premenopausal plus treated postmenopausal; (b) untreated postmenopausal. Crossbars indicate the mean k SEM. [Reproduced from Heaney et a / . (1978), with permission.]

111

CALCIUM IN THE DIET

be substantially higher that current recommended dietary allowances. Moreover, the significant positive correlation between usual calcium intakes and calcium balance suggests that women with higher intakes should have less bone loss. Spencer and Kramer (1986) have reported the results of a large calcium balance study in males. They performed a total of 181 “studies” (a study was 1 subject) in adult men ranging in age from 34 to 71 years. Study duration ranged from 29 to 38 days. Calcium intakes ranged from 234 to 2320 mg/day. The mean calcium balances at the various calcium intakes (intakes shown in parentheses) were as follows: -95 (234 mg), +22 (804). + 10 @lo), + 106 (1230), 106 (1248), + 104 (1431), + 147 (1467), 147 (2021), and + 139 (2320). Two sources of calcium were used, calcium gluconate and milk, but source of calcium did not appear to affect balance. These data suggest that positive balances may be achieved at about 800 mg but that higher intakes result in more positive balances. Since there was an apparent plateauing at about 1200 mg, the authors suggest that 1200 mg would be preferable to the current 800 mg RDA for adult men. To further support their argument that 800 mg is too low, the authors reported on the percentage of subjects at each level of intake who were in negative calcium balance. All of the subjects consuming the 234 mg Ca diet were in negative balance and they did not appear to adapt to the low calcium intake. At the 800 mg level, 34 and 41% (for calcium gluconate and milk, respectively) of the subjects were in negative balance. At intakes of 1200 and above, the percentage of subjects in each group who were in negative balance was much lower although, even at calcium intakes of 2021 and 2320 mg, two subjects (one in each group) were in negative calcium balance. Nordin et al. (1987) used a slightly different approach to determine calcium requirement. They used data from the literature to calculate the net amount of absorbed calcium necessary to balance urinary losses. Net absorption was defined as the difference between total absorption and endogenous fecal excretion of calcium, Endogenous fecal calcium is total fecal calcium minus unabsorbed dietary calcium. It includes calcium from gastrointestinal secretions and sloughed intestinal cells. They estimated that for “young normal individuals” a net absorption of 150 mg was required to match urinary excretion, on average, and that a mean intake of 540 mg was required to achieve a net absorption of 150 mg. They suggest that an allowance of at least 800 mg should be recommended since 540 mg represents the mean requirement and allowances higher than the mean requirement are necessary to meet the needs of most individuals in a population. The discrepancy between this last study and the first two may be re-

+

+

112

DENNIS D. MILLER

lated to the age of the subjects; it is well established that the efficiency of calcium absorption declines with age (Eastell and Riggs, 1987). Also, zero calcium balance may not be adequate for young adults because bone mass does not peak until the middle of the fourth decade of life. Recent advances in methodologies for measuring bone density and bone mass have yielded data on the effects of calcium intakes over the long term. This approach may be useful for validating allowances established with balance studies since it provides a direct measure of calcium status (bone mass or bone density), and the impact of a given calcium intake over the long term can be evaluated. Unfortunately, results are conflicting, with some studies suggesting that higher calcium intakes result in higher peak bone mass and reduced rates of bone loss while others show no relationship between calcium intake and skeletal integrety (Marcus, 1987). A possible reason for the conflicting data is the difficulty in determining nutrient intakes, especially retrospective intakes, in free-living populations. The balance studies by Heaney , Spencer, and their co-workers were well designed and carefully done. The results suggest that current RDAs may be too low for some population groups in the United States, a situation made all the more alarming by the fact that current calcium intakes by large numbers of people are well below the RDA. IV. CALCIUM HOMEOSTASIS

The difficulties associated with establishing calcium recommended allowances are due, in part, to the elaborate homeostatic systems that maintain calcium levels within narrow ranges inside cells and in extracellular fluid. Calcium concentrations in intra- and extracellular fluids are under precise homeostatic control. Cytosolic free calcium concentrations are very low, about lo-’ M (Wasserman, 1988). Total calcium concentrations in blood plasma are between 2.25 and 2.75 mM (9.0-11 mg/dl) of which 47.5% is free, 6.5% is complexed with low-molecular-weight ligands, and 46% is protein bound (Avioli, 1988). It is striking to note that the concentration of free calcium in the plasma ( 1 mM is 10,000 times greater than the cytosolic concentration of free calcium (Carafoli and Penniston, 1985). This tremendous gradient underscores the remarkable ability of cells to regulate calcium fluxes and concentrations. Free calcium concentrations within cells are regulated by calcium-binding proteins in cell membranes (plasma, endoplasmic reticulum, and rnito-

113

CALCIUM IN THE DIET

chondria membranes) (Carafoli and Penniston, 1985) and, ultimately, by control of the flux of calcium into and out of the cell (Wasserman, 1988). Plasma calcium concentration is regulated by the vitamin D-parathyroid hormone (PTH) system. When plasma calcium falls, the parathyroid gland is stimulated to secrete PTH. PTH stimulates the conversion of 25-OH-D, to 1,25-(OH),-D, in the kidney. 1,25-(OH),-D, stimulates Ca,

\\

Bone

c GI Tract

-

I

Ca (80-250mg)

Ca, (includes 100-15OmgCa,)

FIG. 2. Calcium metabolism. GI. Gastrointestinal tract; Ca, calcium intake; Ca,, fecal calcium: Ca,, absorbed calcium; Ca., endogenous calcium in intestinal secretions: Cad, dermal loss: C h , urinary calcium; PTG, parathyroid gland: PTH, parathyroid hormone: CT, calcitonin. Whole-body calcium homeostasis is maintained when calcium losses from the body (Ca, + Ca, + Cad)are balanced by Ca,. When plasma calcium levels fall, the PTG is stimulated to produce PTH. PTH stimulates the production of I ,25-(OH),-D, by the kidney. I ,25(OH),D, stimulates absorption of calcium in the intestine and, together with PTH, stimulates mobilization of calcium from bone. When plasma calcium levels rise, the “C” cells of the thyroid release CT which blocks bone resorption. (Adapted from DeLuca, 1980; Hillman rt a/. 1988.)

114

DENNIS D. MILLER

enhanced absorption of calcium from the intestine and, acting in conjunction with PTH, causes increased bone resorption and reabsorption of calcium in the kidney tubules (DeLuca, 1988). A schematic diagram of the homeostatic mechanisms governing the concentration of calcium in the extracellular fluid (represented by plasma) is shown in Fig. 2.

V.

INTESTINAL ABSORPTION OF CALCIUM

Calcium absorption by the intestine has been the subject of intense investigation for many years and several excellent reviews on the topic are available (Bronner, 1987; Bronner ef a!., 1986; Allen, 1982; Wasserman and Fulmer, 1983). A brief overview of calcium absorption with emphasis on those aspects that may impact on bioavailability will be presented here. A. CALCIUM ABSORPTION: MECHANISMS AND REGULATION It is well established that calcium absorption involves at least two mechanisms: passive diffusion and active transport (Bronner, 1987). In theory, passive diffusion occurs down an electrochemical gradient, with calcium flux being directly proportional to luminal calcium concentration. Moreover, passive diffusion should be nonsaturable. Active transport, on the other hand, can go against an electrochemical gradient and the process should be saturable. Evidence that both mechanisms operate in calcium absorption may be obtained by measuring calcium absorption at different luminal calcium concentrations. Figure 3 (from Papworth and Patrick, 1970) illustrates the effect of calcium concentration on calcium uptake by intestinal slices from rats. Curves similar to the upper curve in Fig. 3 have been observed when plots of calcium absorption by humans as a function of dietary calcium intake were made (Heaney ef af., 1975). The curvilinear nature of the curves suggests an active process since it shows that calcium absorption is not a constant proportion of intraluminal calcium concentration. If active transport were the only mechanism operating, the curves should plateau at the higher concentrations. The fact the curves have a constant positive slope at the higher concentrations is strong evidence that passive diffusion becomes the predominant mechanism for calcium flux at these concentrations. Moreover, when DNP, a metabolic inhibitor, was added to the incubation fluid in the in vitro study mentioned above (Papworth and Patrick, 1970), calcium uptake was not abolished but became a linear function of calcium concentration (Fig. 3).

115

CALCIUM IN THE DIET

0

CO Concentration (mM)

FIG. 3. Effect of calcium concentration on calcium uptake by slices of rat small intestine. Slices were incubated at 37°C in oxygenated buffer containing varying levels of calcium (0). The addition of 0. I mM dinitrophenol (DNP) lo the buffer abolished the saturable component ( 0 ) [Reproduced . with permission from Papworth and Patrick (1970).]

It is also well established that calcium absorption is a regulated process that responds to the calcium status of the animal. When animals are put on a low-calcium diet, the percentage of luminal calcium that is absorbed increases. Animals on a high-calcium diet absorb a lower percentage of ingested calcium. This regulation is mediated by vitamin D as noted above. As stated above, intestinal calcium absorption occurs by both active transport and passive diffusion. The active process is saturable, requires vitamin D, and occurs transcellularily, i.e., calcium enters the mucosal cell through the brush border membrane and exits via the basal-lateral membranes (Wasserman and Fulmer, 1983). The passive process is nonsaturable and probably is paracellular, i.e., calcium is transported from the mucosal to the serosal side by diffusing through the tight junctions between enterocytes (Bronner, 1987). The relative contribution of each process to total absorption depends on luminal calcium concentration and plasma levels of 1,25-(OH),-D,. At high luminal calcium concentrations, the diffusional process would predominate. Allen (1982) estimated that, in normal human adults, 95% of the calcium in a low-calcium meal and 80% of the calcium in a high-calcium meal would be absorbed by the

116

DENNIS D. MILLER

active process, indicating that vitamin D-regulated absorption predominates. The vitamin D-regulated process is influenced by vitamin D status, calcium status, and age (Bronner, 1987; Armbrecht et al., 1979). Subjects with poor vitamin D status resulting either from low intakes, lack of exposure to the sun or inability to convert vitamin D to its active form absorb calcium poorly. Sheikh et al. (1988a) used patients with endstage kidney disease and normal controls to study the effect of vitamin D status on the relative importance of the vitamin D-dependent and vitamin D-independent processes. Calcium absorption was determined by a I-day intestinal lavage procedure in which the gastrointestinal tract is cleansed by lavage, a single meal is given, and 12 hrs later, the gastrointestinal tract is flushed again with a lavage solution. True net calcium absorption is calculated as calcium load to the gut (from food and digestive secretions) minus fecal output. Calcium absorption was linearly correlated to serum concentrations of I ,25-(OH),-D,. By extrapolating to zero concentration of 1,25(OH),-D,, the authors were able to estimate the contribution of the vitamin D-independent process to total calcium absorption. Their results are summarized in Fig. 4 and suggest that calcium absorption by the vitamin D-dependent process is nearly independent of calcium intake, that the vitamin D-independent process is relatively more important when vitamin

B

A

C

150r

a -25 u -50

U 120mg

300mg

Ca

Ca Meal

Meal

i2Omg Co Meal

U

300mg Ca Meal

120mg Ca Meal

300mg Ca Meal

FIG. 4. Effects of vitamin D status and calcium intake on calcium absorption by vitamin D-independent (0)and vitamin D-dependent (a)processes. (A) Untreated dialysis patients: serum concentration of I ,25-(OH)2-D3is 8 pg/ml; (B)normal subjects: serum concentration of I,25-(OH)2-D, is 48 pg/ml; (C) dialysis patients treated with 1,25-(OH),-D,; serum concentration is 75 pg/ml [Reproduced with permission from Sheikh ef a / . (1988a).]

117

CALCIUM IN THE DIET

D status is impaired, and that the increase in calcium absorption that results when calcium intakes are increased is due primarily to the vitamin D-independent process. B. CALCIUM ABSORPTION: CHANGES DURING DEVELOPMENT Marked changes in the relative importance of the two routes of calcium absorption occur during early development. In rats, calcium absorption during the time immediately after birth until about 2-3 weeks of age proceeds almost exclusively by a passive, vitamin D-independent process (Toverud and Dostal, 1986; Dostal and Toverud, 1984; Pansu et al., 1983a). About the time when the pups begin consuming solid food, the mechanism for active, vitamin D-dependent calcium uptake develops rapidly and the passive process becomes relatively less important. However, in the rat, the saturable process peaks rather early, declining rapidly after about age 40 days (Pansu et al., 1983a). These changes are illustrated graphically in Fig. 5 (Toverud and Dostal, 1986). In agreement with the results of Pansu et al. (1983a) and Toverud and Dostal(1986), Armbrecht et al. (1979) showed, using an everted gut sac technique, that active transport of calcium in the rat duodenum was maximal at 3 weeks of age and declined to nondetectable levels by 3 months of age. It has also been

60

-

b, t I I

\

\

60

8

i . c M

40

-

\

40

-0

U

E

20

-

I

'\\

20

'\

\

I I I

b

t

n

LO

00

20

40

60

80 100 120

Age. days

saturable process (JWaJin rat FIG. 5. Effect of age on calcium absorption by the (0) duodenum. ( 0 )CaBP is intestinal calcium-binding protein. [Reproduced with permission from Toverud and Dostal ( 1986).]

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shown in the rat (Armbrecht et al,. 1979) that, when the capacity for active transport of calcium declines, so does the ability to adapt to a lowcalcium diet. Armbrecht et al. (1979) found only a marginal increase in calcium active transport in 12-month-old rats fed a low-calcium diet whereas, in younger rats, the low-calcium diet resulted in a substantial increase in calcium active transport. The effects of aging on calcium absorption in humans appear to be similar to the rat although, perhaps, less dramatic. It has been clearly demonstrated that calcium absorption in humans declines with age (Ireland and Fordtran, 1973; Gallagher et al., 1979). Furthermore, adaptation to low-calcium diets is less effective in the old compared to the young (Ireland and Fordtran, 1973; Gallagher et al., 1979). It has been proposed that these effects of aging are due to a decrease in the capacity of the kidney to convert 25-(OH)-D3to 1,25-(OH),-D, in the older animals and humans (Armbrecht et al., 1980; Gallagher et al., 1979; Tsai et al., 1984). Calcium absorption by the saturable process correlates strongly with the concentrations of mucosal calcium-binding protein (Pansu et al., 1983b) and serum 1,25-(OH),-D3(Sheikh et al., 1988a). C. IMPLICATIONS FOR DIETARY REQUIREMENTS While rates of both absorptive processes are responsive to luminal calcium concentration, the predominance of the active, regulated transport route suggests that calcium intake and thereby luminal calcium concentration should not be the limiting factor in calcium balance. This is provided, of course, intake is above some threshold level and vitamin D status is adequate. When intakes fall, percentage absorption should increase to compensate and vice versa. Unfortunately, the balance data discussed above suggest that this adaptation may not fully compensate for low intakes. Likewise, the high prevalence of osteoporosis in the United States suggests that calcium intakes may be too low for some population groups. Recent evidence reported by Cauley et al. (1988) shows that calcium intakes over the long term do affect bone density. They showed a positive association between high milk consumption throughout life and bone density in postmenopausal women. Halioua and Anderson (1989), in a similar study with premenopausal women, concluded that lifetime calcium intake is positively associated with bone mineral content and bone density. It is clear that increased levels of calcium in the diet will result in increased calcium absorption and vice versa, even though adaptive mechanisms partially blunt the effects of increases or decreases in intake.

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Whether increased absorption translates into improved skeletal integrity has not been conclusively established but several recent studies seem to suggest that it may. VI . CALC IUM BIOAVAl LAB1LlTY Nutritionists have long recognized that the quantity of a nutrient in the diet is not necessarily the best indicator of nutrient adequacy. This is because nutrients from different sources are absorbed with different efficiencies and, once absorbed, the utilization of nutrients may be influenced by other components in the food or diet, nutritional status, o r the chemical form of the nutrient. The literature related to calcium bioavailability is vast and, in many cases, conflicting. The topic has been reviewed admirably by a number of authors (Allen, 1982; Greger, 1988; Heaney, 1986; Kies, 1985; Hazell, 1985). In this section, I will attempt to focus on recent work but will also refer to some of the older papers that are pertinent. A.

DEFINITION OF BIOAVAILABILITY

There is no clear, widely accepted definition of the term bioavailability as it applies to dietary minerals. The term is used rather loosely and, in many cases, is used interchangeably with “availability,” “absorbability,’’ “absorption,” o r “retention.” A confounding factor, particularly in the case of calcium and iron, is the physiological need of the animal or human used in bioavailability measurements. Absorption of calcium (and iron) from a given diet will be markedly higher in deficiency than in repletion (Allen, 1982; Hallberg, 1981). Does this mean that the bioavailability of calcium from milk is higher for growing children or pregnant women than for adult men? The former will absorb a higher percentage of the calcium from their diets. Welch and House (1984) have proposed a definition for bioavailability that is presumably independent of the physiological status of the consuming organism: “Bioavailability to organisms of a mineral element is that proportion of an element in a nutrient medium which is potentially absorbable in a form which is metabolically active.” This is a reasonable definition and would allow for comparisons between studies. However, in practice it is difficult to determine potential absorbability since so many physiological factors are involved in the saturable component of calcium absorption. Furthermore, this definition raises questions about choice of physiological status of animals or humans used

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in mineral bioavailability studies. Should they be calcium deficient so they will absorb a high percentage of ingested calcium? If so, how severe should the depletion be and how long should the depletion period last? Can we extrapolate from conclusions based on bioavailability measurements in depleted animals or humans to populations consuming their usual diets which are probably not markedly deficient in calcium? With iron, nutritional status may influence results in bioavailability measurements. We found that citrate had no effect on iron absorption in irondeficient rats but depressed absorption when iron-adequate rats were used (Berner et al., 1985). Whether calcium status of subjects is a significant factor in studies designed to compare relative bioavailabilities of different sources of calcium is not known, but it may be one of the factors responsible for the large number of conflicting reports in the literature. Another potentially confounding factor in calcium bioavailability studies is the effect of diet on urinary excretion of calcium. As shown in Fig. 2, calcium loss is mainly via endogenous fecal and urinary excretion. Endogenous fecal excretion apparently is not a factor in the homeostatic regulation of body calcium (Avioli, 1988) and is not affected by physiological state (Heaney et al., 1978). Urinary excretion, on the other hand, is affected by physiological state (Heaney et al., 1978) and diet. Increased intakes of protein can have a marked calciuretic effect while elevations in phosphorus intake have a hypocalciuretic effect (Zemel, 1988). In view of the problems discussed above, bioavailability should be viewed as a relative rather than an absolute quantity. When animals are used, allocation of animals to treatment groups should be done to ensure that the nutritional status of each group is similar. With human studies, crossover designs should be employed whenever possible so that each subject can serve as her/his own control. A working definition of bioavailability, then, is the efficiency of utilization of a nutrient in a food or diet relative to an appropriate standard. Utilization may be quantified by measuring absorption, whole-body retention (balance), or other appropriate response parameter such as tissue concentration, bone mass, enzyme activity, etc. B. METHODS FOR MEASURING CALCIUM BIOAVAILABILITY Greger (1988) has summarized the methodologies that have been used in mineral bioavailability studies. The lack of sensitive indicators of calcium status and the precise homeostatic regulation of cytosolic and extracellular calcium concentrations limit the number of methods available for studying calcium bioavailability. For example, changes in the concentra-

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tion of plasma calcium following an oral calcium load are only a very poor indicator of calcium absorption because homeostatic processes maintain it at a nearly constant level even when large oral loads are given. I . Balance Methods The classical balance technique, with or without the use of isotopic tracers, remains the most widely used technique for studying calcium bioavailability in humans. Calcium balance is defined as Balance

=

Cai

-

(Ca,+ Ca,,

+ Cad)

where Ca, is calcium intake, Ca,is fecal calcium, Ca, is urinary calcium, and Cad is dermal losses (perspiration and sloughed skin). Dermal losses are usually ignored because they are small compared to fecal and urinary losses and because they are hard to measure. Apparent absorption, calculated as the difference between intake and fecal excretion, may also be used as an indicator of bioavailability. Apparent absorption measurements underestimate true absorption because fecal calcium includes substantial amounts of endogenous calcium in addition to unabsorbed dietary calcium. In theory, balance should be a good indicator of bioavailability because diets or foods which produce more positive balances are necessarily contributing more utilizable calcium than those which result in less positive balances. In practice, balance measurements are tedious and time consuming. Moreover, they are subject to rather large errors since precise measurement of intakes and losses is difficult and timing of collections may be off. Qualitative fecal markers such as Brilliant Blue dye may be used as an indicator for separating fecal collections into periods (Slavin and Marlett, 1980). Quantitative markers such as polyethylene glycol or Cr-mordant may be used to correct for incomplete fecal collections (Schwartz et al., 1986). Fordtran and his associates have devised a modified balance approach which overcomes many of the problems associated with long-term measurement of intakes and excretions (Bo-Linn et al., 1984). Subjects are given a lavage solution to wash out their entire gastrointestinal tract completely. Then they are given a meal and, 12 hr later, the washout is repeated. The entire procedure is repeated on a separate day substituting water for the meal. The entire rectal effluent following the meal (or the water) is collected and “net calcium absorption” is calculated as follows:

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Net Ca absorption = Ingested Ca - (Effluent Ca after meal - Effluent Ca after water) This approach has the advantage of being rapid and, therefore, less expensive than the traditional balance method. Absorption, not actual balance, is measured since an accurate assessment of urinary excretion cannot be made in this short time period. The authors claim that absorption by the small intestine is not altered by the procedure. It is likely that absorption by the colon would be altered since colon bacteria would be mostly absent and transit through the colon would be much faster than normal. Whether this will have a major effect on net absorption will depend on the extent to which calcium is absorbed in the colon. 2 . Isotopic Tracer Techniques

Heaney and his co-workers have made balance measurements by labeling dietary calcium with radioisotopic tracers (Heaney et al., 1975, 1977). An oral tracer, e.g., 47Ca,is mixed with the diet and given in a single meal. Two hours after the meal, a second tracer, e.g., 4’Ca, is given intravenously. Fractional or true calcium absorption may then be calculated as the ratio of the two tracers in blood or urine samples taken at some time after the isotopes are administered (DeGrazia et al., 1965): Fractional absorption =

Fraction of oral tracer Fraction of iv tracer

This approach has the advantage that calcium absorption may be estimated from a single blood or urine sample, an obvious advantage when using absorption as an indicator of bioavailability. Total absorbed calcium may then be calculated by multiplying calcium intake by fractional absorption. Endogenous fecal calcium is determined from the fecal content of the iv tracer. Calcium balance may then be calculated as follows: Ca balance = Absorbed Ca

-

(Endogenous fecal Ca

+ Urine Ca)

Whole-body counting following oral administration of 47Cais another approach for estimating calcium bioavailability . Whole-body radioactivity is measured shortly after the dose is given and again 7 days later (Shipp et al., 1987). Retention at 7 days is expressed as a percentage of the initial dose. While this method will underestimate true absorption since some excretion of the absorbed tracer will have occurred by the seventh day, it is an excellent method for comparing bioavailability between test meals.

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Unfortunately, whole-body counters large enough for humans are expensive and not widely available. When whole-body counting equipment is not available, the method may be modified to give an estimate of relative absorption or retention. Schuette and Knowles (1988) used a large-volume gamma counter to measure forearm radioactivity in a study designed to compare intestinal absorption of calcium from two calcium salts. 3. Isotopic Labeling of Foods

The validity of isotope tracer procedures for measuring calcium bioavailability depends on the assumption that the tracer is absorbed and utilized with the same efficiency as the intrinsic calcium in the food. Foods may be labeled either extrinsically or intrinsically. Extrinsic labeling is accomplished by simply mixing a solution of the tracer with the food prior to giving it to the subject. Intrinsic labeling is achieved by biological incorporation of the tracer into the food. In the case of animal products, intrinsic tracers are introduced by iv injection of a solution of the tracer. With plants, tracers are added to nutrient solutions and the plants are grown hydroponically. Extrinsic labeling in very simple but intrinsic labeling is tedious and expensive. It may be assumed with confidence that calcium tracers in intrinsically labeled foods are chemically identical with the stable element in the food. Extrinsically added tracers, on the other hand, may not fully equilibrate with the stable element in the food and, consequently, may have a different bioavailability. We have compared intrinsic and extrinsic tracer methods for estimating calcium bioavailability from dairy foods (Buchowski et al., 1989). Intrinsically labeled milk was prepared by injecting a lactating goat with 45Ca. Various products were prepared from the intrinsically labeled milk and, following extrinsic labeling with 47Ca,were given orally to rats. Ratios ("Ca : 45Ca)of the two isotopes in rat tibias were used to compare the absorption of the tracers. Ratios were not different from 1 in rats dosed with either milk or yogurt but, in the case of fresh cheese curd, the ratio was slightly greater than 1, indicating more efficient absorption of the extrinsic tracer. However, correlations between tibia 47Caand tibia 45Ca were high for all foods suggesting, in the case of dairy products, that extrinsic labeling is valid. Unfortunately, these results cannot be generalized to all foods. Weaver et al. (1987) compared calcium absorption from intrinsically and extrinsically labeled spinach and kale. Absorption of the two tracers by rats was similar for kale but absorption of the extrinsic tracer from the spinach was twofold greater than the intrinsic tracer. Another disadvantage of radioactive tracers for human studies is concern over the potential hazards of ionizing radiation, especially when ad-

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ministered to children and pregnant or lactating women. Technology is now available for measuring stable isotopic tracers in biological matrices (Hillman et al., 1988; Yergey et al., 1987; Weaver et al., 1988). Hopefully, the use of stable isotopes in calcium bioavailability research will become more widespread in the future. 4. Animal Models

The methods described above can also be used in animal models. In addition, a variety of other procedures in animals have been used to great advantage in the study of calcium bioavailability. Poneros and Erdman (1988) have used total tibia calcium in rats following 27 days on experimental diets to compare calcium bioavailability from several test foods. As discussed below, uptake of tracers by ligated intestinal segments, everted gut sacs, and intestinal slices has been used extensively to study calcium absorption. C. CALCIUM BIOAVAILABILITY: DAIRY PRODUCTS It is widely held that the calcium in dairy products is readily absorbed and utilized. This view was confirmed recently by Recker et al. (1988). Using an extrinsic tracer technique, they compared the fractional absorption of calcium from several milk products (whole milk, chocolate milk, yogurt, imitation milk, and cheese) and calcium carbonate in postmenopausal women. They found no significant differences between any of the products or the calcium carbonate, a readily absorbable calcium source. In a subsequent study (Heaney et al., 1988), they compared calcium absorption from spinach and milk and found calcium absorption from the milk to be 28% while in the spinach it was only 5%. It appears, therefore, that dairy products in a variety of forms are good sources of highly bioavailable calcium. Is there something unique to milk that enhances the availability of it’s calcium? This question has intrigued researchers for a long time and many have hypothesized that the lactose in milk enhances calcium absorption. A review of the evidence on the so-called “lactose effect” follows. Lactose Effect

The enhancing effect of lactose on calcium absorption was demonstrated as early as 1926 when Bergeim (1926) compared calcium absorp-

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tion by rats from diets containing 25% of either lactose, glucose, sucrose, maltose, or starch to a control diet. Lactose increased calcium absorption but the other carbohydrates did not. Since then, numerous investigators have studied the lactose effect in a variety of experimental animals and in humans. While the data are not entirely consistent, it is likely that, at least under some conditions, lactose does increase calcium bioavailability. a . Lactose Efiect in Animals. In a study that conclusively showed that lactose enhances calcium absorption in the rat, Wasserman et al. (1956) compared several organic substances for their effect on calcium absorption. Fasted rats were given 45CaCI,by stomach tube. The 4sCaCI, was given alone (control) or with the various substances. Appearance of 45 Ca in the femur 24 hr after dosing was used to compare treatment effects. Both lactose and amino acids increased calcium absorption but lactose had the most potent effect (a 2.5-fold increase over the control). The mechanism for the effect of lactose on calcium absorption has not been conclusively identified, although several hypotheses have been advanced. Lengemann (1959) confirmed that lactose given orally enhanced the absorption of simultaneously administered calcium (as CaCl,) and showed that lactose injected intraperitoneally had no effect on calcium absorption. He also showed, using a ligated segment technique, that lactose had to be in the same segment as the calcium to exert its effect-when lactose and CaCI, were injected into adjacent segments, no effect was observed. These results suggest that lactose exerts its effect in the intestine and not via bone or blood or some other tissue. Wasserman and Lengemann (1960) showed that lactose does not increase the in vitro solubility of calcium from CaHPO,, suggesting that lactose does not act by increasing the luminal concentration of soluble calcium. It is possible, however, that enhanced calcium solubility in the intestinal lumen is a factor in the lactose effect since luminal conditions are undoubtedly different from the in vitro conditions Wasserman and Lengemann used in their in vitro study. Wasserman and Lengemann (1960) also showed that antibiotics did not alter the lactose effect, indicating that fermentation of lactose by intestinal bacteria is not involved. (It has been proposed that the lactose effect is the result of acidification of the intestinal luminal contents caused by the rapid fermentation of lactose to lactic acid by intestinal microflora; Bergeim, 1926.) In a definitive study using rat models, Lengemann et al. (1959) provided answers to several questions pertinent to the lactose effect. Absorption was assessed by measuring the appearance of an isotope in bone following oral or ligated segment administration of various doses. Both

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45

Ca and 85Srwere used as tracers. The absorption of strontium, also an alkaline earth element, is qualitatively but not quantitatively similar to that of calcium, i.e., percentage absorption of strontium is usually lower but factors which enhance calcium absorption also enhance strontium absorption. 85Srwas used because it is a gamma emitter and is therefore easier to assay than "Ca. Lengemann et al. (1959) showed, using vitamin D-deficient rats, that lactose enhanced strontium absorption in vitamin D deficiency. They also showed, using ligated segments, that while lactose enhanced lactose absorption in all regions of the small intestine, the effect was by far most pronounced in the ileum (increases were as follows: duodenum, 42%; jejunum, 31%; and ileum, 320%). Armbrecht and Wasserman (1976) employed an everted gut sac technique to study further the mechanism for the lactose effect. They found that gut sacs preincubated in a lactose-containing medium took up more calcium than sacs preincubated without lactose. These results show that lactose does not have to be present during actual calcium absorption. Rather, lactose apparently conditions the mucosa to increase calcium uptake. This mucosal conditioning, however, appears to be short-lived. Lengemann (1959) found that rats fed a diet containing 10% lactose, fasted for 24 hr, and then dosed with 45Cadid not,absorb more calcium than controls. Armbrecht and Wasserman also measured lactose uptake by intestinal segments and found no evidence for cotransport of calcium and lactose. Armbrecht and Wasserman (1976) suggest that the lactose may enhance calcium absorption by increasing the permeability of the brush border membrane to calcium. A summary of key findings from studies with rat models where calcium was administered orally or directly into ligated intestinal segments in fasted animals or into the medium bathing everted gut segments follows. 1 , Lactose consistently increases the percentage of calcium absorption from an oral dose by about twofold. 2. The effect is largest in the ileum but has also been demonstrated in the duodenum and the jejunum. 3. Lactose enhances calcium absorption in vitamin D-deficient as well as in vitamin D-adequate animals. 4. Lactose need not be present in the lumen simultaneously with the calcium to exert its effect, but the effect appears to operate at the level of the intestine and not at some other site.

While the above mentioned studies do not clearly identify a mechanism for the lactose effect, they consistently and conclusively show that lactose increases calcium absorption in rats when given to fasted intact ani-

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mals. In vitro studies with isolated intestinal segments confirm the lactose effect and shed light on possible mechanisms that may be operating. Even though the animal studies discussed above clearly show a lactose effect, the nutritional significance of the effect is much less clear. Is the effect unique to lactose or will other sugars or complex carbohydrates also enhance calcium absorption? Will the effect be important when calcium is ingested with food and not just as a solution of a simple calcium salt? Both questions have been addressed. The answer to the first seems to be no, i.e., other sugars also enhance calcium absorption. Lengemann and Comar (1961)found that lactose, lysine, and glucose all increased the absorption of calcium injected into ligated segments of rat ilium. Armbrecht and Wasserman (1976) showed that lactose, fructose, mannitol, cellobiose, and sucrose but not glucose, galactose, or choline increased calcium uptake by everted segments of rat ilium. However, when compared directly with glucose, lactose does appear to have a greater effect. Lengemann (1959) prefed rats diets containing either 10% glucose or 10% lactose and then, following a 24-hr fast, dosed them orally with solutions containing 4'Ca and either glucose or lactose. Absorption from doses containing lactose were nearly twofold greater than doses containing glucose. In another comparison of glucose and lactose, Schaafsma and Visser (1980) raised rats on diets containing either 15% lactose or 15% glucose. They reported that calcium absorption, measured by a balance technique, was significantly higher in rats fed the lactose-containing diet. Regarding the second question, there is evidence that lactose does exert its effect even in the presence of a complex meal. Wasserman and Lengemann (1960) reported that rats fed a ration containing 30% lactose absorb and retain more "Sr than rats fed diets containing 30% sucrose. Pansu et al. (1979) fed weanling rats nutritionally adequate diets containing either 67% starch or 37% starch + 30% lactose. Calcium absorption and duodenal calcium-bindingprotein content were assayed at 2,3,4, and 5 months of age. Calcium absorption was measured using in situ ligated duodenal segments: disappearance of 45CaC1, from the segment over a 1-hr period was determined. Lactose-fed rats absorbed significantly less calcium and had lower duodenal calcium-binding protein compared with the controls. These results would be expected in rats fed a high-calcium diet, and suggest that feeding lactose has the same effect on long-term calcium status as feeding a higher level of calcium, convincing evidence that lactose can increase calcium absorption over the long term. These same investigators in a subsequent study (Pansu et al., 1981)fed rats diets containing 0.44% calcium and either 64% dextrose or 34.4% dextrose + 30% lactose. Their results were similar to their first study and they con-

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cluded that adding the lactose to the 0.44% calcium diet had an effect equivalent to increasing the calcium to 0.7%. This study is also noteworthy because it shows that lactose affects calcium absorption differently than dextrose, another sugar. The potential nutritional significance of the lactose effect was demonstrated by Miller, S . C. et al. (1988). Miller and co-workers fed vitamin D-deficient rats vitamin ‘D-deficient diets containing either sucrose or lactose as the primary carbohydrate source. The rats on the lactose diet gained faster and had improved skeletal growth and bone development. 6 . Lactose Effect in Humans. While the evidence supporting a lactose effect in experimental animals is quite consistent, the situation in humans is less clear. Mills et al. (1940) found that calcium balances in young boys were improved when 36 g of lactose per day was added to their diets. This study needs to be interpreted with caution, however, because the data were not analyzed statistically and the magnitude of the effect varied markedly among the subjects (the average increase in calcium retention was 33% but in three of the five the increase was only about 10% while in one it was 89% and in another 51%). In a frequently cited paper, Condon et al. (1970) conclude that when persons with “normal lactose tolerance” ingested lactose with their meals, there was a fall in both fecal and urinary calcium resulting in a “striking improvement in calcium and phosphorus balance.” This study, however, should be viewed with caution for several reasons. First, three of the four subjects were partially immobilized because of fractures and the fourth had poliomyelitis. It has been shown that persons immobilized in casts rapidly develop negative calcium balances (Whedon et al., 1957) and that polio patients suffer higher than normal calcium losses (Whedon and Schorr, 1957). Condon et al. (1970) measured calcium balances without lactose in the first 3 weeks of their study and balances with lactose in the last 3 to 4 weeks. It is possible, at least with the three subjects with fractures, that mobility improved during the course of the study and increased mobility rather than that lactose was responsible for the improvement in calcium balance. This would explain the fall in urinary calcium during the latter part of the study, a finding that does not fit with increased calcium absorption since urinary calcium would be expected to increase, not decrease, with increased calcium absorption (Avioli, 1988). Kocian et al. (1973) fed fasting subjects (one group was lactose tolerant, the other lactose intolerant) milk or lactose-free milk labeled with 47 Ca. They found that maximum plasma levels of 47Cafollowing ingestion

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of the milks were higher with the milk than with the lactose-free milk in every case in the lactose-tolerant group. The opposite was true with the lactose-intolerant group. However, retention of 47Ca(calculated from the difference between the ingested isotope and the isotope recovered in urine and feces) over a 7-day period was the same for all groups. In a carefully designed experiment, Cochet et al. (1983) studied the effect of lactose on calcium absorption by normal and lactase-deficient human adults. A double-isotope technique was used to measure calcium absorption. The calcium (CaCI, in 450 ml water) was given (with or without 50 g of lactose) to the subjects after an overnight fast and no food was allowed until 4 hr later. Lactose increased fractional calcium absorption in every one of the normal-lactase subjects and decreased it in every one of the lactase-deficient subjects. Lactose caused a delay in peak absorption rate in the normal-lactase group but not in the lactase-deficient group. The authors attributed the higher overall absorption in the normallactase group following the lactose load to a prolonged maximal absorption rate. It is interesting to note that the enhancement of calcium absorption in the normal-lactase group occurred only after the first hour. The authors suggest this apparent delay was caused by delayed stomach emptying. It is possible, however, that lactose exerts its greatest effect in the ileum and therefore calcium absorption does not begin increasing until the calcium and the lactose reach the ileum. Lengemann et al. (1959) have shown that, in the rat, lactose is most effective in the ileum. As the authors acknowledge, the effects they observed may or may not exist when calcium is ingested in the form of dairy products. It should also be noted that 50 g is a very large lactose load; one cup of milk contains only about 1 I g of lactose. Using a study design similar to that of Cochet et al. (1983), Trernaine et al. (1986) evaluated the effect of lactose in milk on calcium absorption. Lactase-sufficient and lactase-deficient adults were given either lactose hydrolyzed milk or unhydrolyzed milk after an overnight fast. Calcium absorption was determined by a double-isotope deconvolution method. Contrary to the results of Cochet et al. (1983), the presence or absence of lactose in the milk did not affect calcium absorption. The lactase-deficient subjects did absorb more calcium from both milks than the lactase-sufficient subjects, reflecting, perhaps, higher usual calcium intakes in the lactase-sufficient group. The contradictory findings from these two studies could be due to several factors. First, the lactose load in the Cochet study was 50 g compared to about 1 I g in the Tremaine study. Second, the products of lactose hydrolysis, glucose and galactose, were present in the Tremaine study but not in the Cochet study. Under some conditions, glucose has been shown

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to enhance calcium absorption (Norman et al., 1980; Knowles et al., 1988).Third, the form of calcium in the two studies differed. Most of the calcium in milk is colloidal while virtually all of the calcium in the Cochet study would have been in a soluble, ionic form. Lengemann et al. (1959) has shown that, in rats, EDTA, a calcium chelator, suppresses the lactose effect. It is possible, therefore, that digestion products of milk chelate the calcium and suppress the lactose effect. The question of whether lactose impairs calcium absorption in lactasedeficient humans is important since, if it does, lactose-containing dairy products may do this group more harm than good. Unfortunately, the data are conflicting. Condon et al. (1970)found that calcium balance became more negative in one lactase-deficient subject following the addition of 20 g lactose to each meal. As already mentioned, Cochet et al. (1983) clearly demonstrated a depressive effect of lactose on calcium absorption in lactase-deficient subjects but they used an unphysiologically large lactose load (50 8). Tremaine et al. (1986) showed that calcium absorption from milk was not affected by the enzymatic hydrolysis of the milk lactose in either lactase-sufficient or deficient adults. If lactose were a significant factor for calcium absorption, calcium availability from fermented dairy products should be different from that in milk since fermenting organisms contain @-galactosidaseswhich hydrolyze lactose. Smith et al. (1985) compared calcium absorption from milk and yogurt in lactase-deficient and sufficient subjects. The milk and yogurt were extrinsically labeled with 45Caand given to humans following an overnight fast. Blood samples were taken serially over a 24-hr period and assayed for 4SCa. The areas under curves constructed by plotting serum 45Caconcentration versus time were used to quantify relative calcium absorption. Calcium absorption from the milk and yogurt were the same in both groups of subjects suggesting, in agreement with Tremaine et al. (1986), that lactose does not affect calcium absorption from dairy foods. It should be noted that, in this study, the lactose contents of the milk and yogurt were quite similar-7.5 to 10.3 g/dase for the milk and 5.5 g/dose for the yogurt. The relatively high level of lactose in the yogurt is a result of the common practice of adding milk solids to fluid milk prior to the yogurt fermentation so, even though lactose is hydrolyzed during the fermentation, significant amounts remain in the final product. Nevertheless, lactase-deficient persons generally tolerate yogurt better than milk because the P-galactosidase produced by the fermenting organisms remains active and resumes digestion of lactose once the yogurt reaches the small intestine of the person consuming it (Kolars et al., 1984). A possible explanation for the conflicting reports noted above is that carbohydrate in general enhances calcium absorption, particularly if there

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is a delay in the digestion and absorption of the carbohydrate. Kelly et al. (1984) tested this hypothesis by comparing calcium absorption from water and three meals. Meal calcium was labeled with 47Caand fractional calcium absorption was determined by a double-isotope technique. A single test meal was given after an overnight fast. Calcium absorption by the human subjects was measured following ingestion of the following meals: A, water; B, a breakfast meal (whole wheat bread, an egg substitute, orange juice, nondairy creamer, and sugar; C, Ensure (a liquid formula containing a glucose polymer); and D, Frodex-I5 (a glucose polymer) in water. The respective carbohydrate contents of the four meals were A, 0; B, 77 g; C, 54 g; and D, 40 g. Fractional absorption of calcium by normal adult subjects was A, 32.9%; B, 23.3%; C, 44.7%; and D, 52%. The authors report that absorption from C was significantly greater than from B but not from A. Apparently, results from meal D were not evaluated since only two subjects were used. The authors conclude that the glucose polymer, whether in the formula or by itself, enhances calcium absorption. These results should be interpreted with caution, however, because carbohydrate was not the only variable and other factors could have been operating. In a more clear-cut study, Knowles et al. (1988) administered 47 Ca along with varying amounts of glucose to fasted women. Fractional calcium absorption was determined by measuring radioactivity in the arm. They found that 222 mmol glucose increased calcium absorption by 49%. Likewise, Heaney et al. (1989) showed that coingestion of a meal with various calcium sources increased calcium absorption by 10-30%. In the latter study, the meal consisted of white toasted bread, butter or margarine, and coffee in trials with human subjects or of a semipurified rodent diet in experiments with rats. These results were not confirmed in a recent study by Sheikh et al. (1988b). They compared calcium absorption from CaCO, given to humans either with water, a steak and potatoes meal, or a steak meal plus 50 g of glucose polymer powder. Calcium absorption was measured by a single day balance technique in which the gastrointestinal tract was flushed by lavage before and 10 hr after ingestion of the meal. Calcium absorption did not differ between the three test meals, suggesting that neither starch (from the potato) nor the glucose polymer affects calcium absorption in human adults. Another possible explanation for the divergent data relating to the lactose effect may be that the effect is too small to measure when the predominant route of calcium absorption is by active, vitamin D-mediated transport. In adults adapted t o a high calcium intake, circulating 1,25(OH),-D, would be low and, therefore, active transport would also be low, leaving passive diffusion as the predominant route for calcium absorption. In adults accustomed to a lower calcium intake, as might be the

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case with lactase-deficient subjects, I ,25-(OH),-D, would be higher and the predominant route for absorption would be active transport. As a result, a lactose effect might be observed in lactase-sufficient but not lactase-deficient subjects, as was the case in several of the studies discussed above. Such an explanation is consistent with the observation that, in vitamin D-deficient rats, lactose increased femur uptake of "Sr by 60% while in vitamin D-supplemented rats, lactose increased uptake by only 25% (Lengemann et al., 1959). It is also in agreement with an intriguing hypothesis proposed by Flatz and Rotthauree (1973). They observe that the only major population where lactose tolerance is prevalent is northern European. They suggest that the persistence of the ability to digest lactose in adulthood in this population resulted from natural selection, i.e., lactose provided a survival advantage by its ability to enhance calcium absorption in an environment of low dietary vitamin D and low exposure to ultraviolet radiation. It follows that lactose would not have the same advantage in areas where vitamin D intake is high or where sun exposure converts 7-dehydrocholesterol to vitamin D, in the skin, and therefore there would be no selection pressure for lactase persistence. Given the conflicting data described above, it is dimcult to draw any firm conclusions regarding the effect of lactose on calcium absorption from usual diets or even to provide an explanation for the divergent data. It is likely that lactose, at levels normally present in dairy products, does not have a significant effect on calcium absorption by healthy adults consuming normal diets. c . Lactose Effect in Human Infants. Lactose does appear to affect calcium absorption in infants. Kobayashi et al. (1975) studied calcium absorption in three groups of infants ranging in age from 2 to 8 months. They found that calcium absorption (defined as the difference between intake and fecal excretion) was highest in the group given a proprietary milk treated with lactase (72%), intermediate in the group given untreated proprietary milk (60%),and lowest in the group given a lactose-free milk (36%). While this study suggests a lactose effect, there may have been physiological differences among the groups that could have accounted for the observed effects. Ziegler and Foman (1983) compared calcium absorption from two soybased infant formulas. In one the carbohydrate source was lactose, in the other, a mixture of 56% starch hydrolysate and 44% sucrose. The subjects were six healthy infants. Metabolic balance was used to assess absorption. Mean calcium absorptions were significantly higher from the lactose-containing formula (48% versus 33%, p < 0.01). This study raises the question of whether the composition of infant formulas may be a factor

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in determining calcium status. Steichen and Tsang (1987) addressed this question in a long-term study. Thirty-six healthy term infants were fed either a soy-based, lactose-free formula (group 1) or a milk-based formula (group 2) during their first year of life. Bone mineral content at midshaft radius was measured several times during the study period. The bone mineral content was significantly higher in the infants fed the cow milkbased formula (Fig. 6). In this study calcium absorption was not measured 160

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130

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120

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5-

110

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loo

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70

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Age (Weeks)

FIG. 6. A comparison of bone mineral content at rnidshaft in infants fed either (cow’s) milk-based formula (0)or soy milk-based formula (m). Bars indicate mean 2 SEM. [Reproduced with permission from Steichen and Tsang (19871.1

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and the formulas differed in both protein source and carbohydrate source. Nevertheless, these results are consistent with the hypothesis that lactose enhances calcium absorption in the infant. The lactose effect observed in infants fits with the hypothesis that lactose is a significant factor in calcium absorption only when passive diffusion is the predominant route of absorption. Calcium absorption in preweanling rats is almost exclusively by an efficient nonsaturable (passive) process (Toverud and Dostal, 1986) and, although there are very few data, it has been suggested that calcium absorption by human infants is also by a passive process (Younoszai, 1981).

d . Lactose Effect in the Elderly. Most studies of lactose effects on calcium absorption have been carried out with young animals or with infants or young to middle-aged human adults. To my knowledge, no one has systematically studied the lactose effect in elderly human subjects. There is a recent report describing the effect of age on the lactose effect in rats. Armbrecht (1987) measured calcium uptake by everted intestinal segments taken from rats aged 2-3 months, 12-14 months, and 22-24 months. Prior to measurement of calcium uptake, the intestinal segments were incubated in the presence or absence of lactose. Lactose caused a significant increase in calcium uptake in all three age groups and was independent of vitamin D and oxygen availability. The author suggests that lactose may be important in older mammals because it can enhance calcium absorption at a time when the vitamin D-mediated mechanisms for increasing calcium absorption are blunted. D. CALCIUM BIOAVAILABILITY: EFFECTS OF PLANT FOODS Recommendations calling for increased consumption of dietary fiber and complex carbohydrates translate into diets with a higher proportion of plant foods. This has spurred renewed interest in possible adverse effects this may have on mineral utilization. Perhaps the most likely adverse consequence of increased consumption of plant foods is impairment of mineral absorption. There have been hundreds of papers published on the topic and several excellent reviews are available (Southgate, 1987; Allen, 1982; Kelsay, 1986; Walker, 1985; James, 1980). I . Fiber and Phytate

As might be expected, the answer to the question of whether fiber adversely affects calcium absorption is far from clear-cut. Conflicting data

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in the literature have led to conflicting conclusions by reviewers of the topic. The following quotes taken from summaries of recent reviews illustrate the controversial nature of the topic. “The impairment of calcium absorption by fiber components is nutritionally significant. The studies discussed here show that the addition of reasonable amounts of whole wheat, cellulose, fruits, and vegetables to normal diets put subjects consistently into negative calcium balance, despite adequate calcium intakes” (Allen, 1982). “The evidence presented in this chapter suggests that fiber-rich diets can induce marked malabsorption of nutritionally important minerals, but it is still not clear that the malabsorption relates to the fiber content of the food” (James, 1980). “Results of the balance studies reviewed here and of others previously reported indicate that intakes of about 25 g NDF/day may be consumed without adverse effects on mineral balances. Higher levels, however, may not be advisable” (Kelsay, 1986). “The recommendations for increasing dietary fiber intakes by the order of 50-100% in Western communities would not be expected to have any significant adverse effects on mineral absorption, provided that adequate intakes of protein and minerals in question are maintained” (Southgate, 1987). There are many possible reasons for the discrepancies in the literature. A major one may be the poor sensitivity of the methodology. Chemical balance has been the most commonly used method and, as discussed earlier, it is subject to considerable error. Another confounding factor that was pointed out by Southgate (1987) is the change in the overall composition of the diet that results when fiber intake is altered by substituting natural foods with a higher (or lower) fiber content. High-fiber foods frequently contain phytate or oxalic acid, substances known to interact with calcium. Therefore, observed effects may be due to factors other than fiber. Also, high-fiber diets normally contain more starch, less sugar, and less fat than low-fiber diets (Southgate, 1987). All three of these components may affect calcium absorption. a. Mechanism of the Fiber Effect. Several mechanisms have been proposed to explain observed effects of dietary fiber on nutrient absorption. In vitro studies have shown that fiber can bind cations, presumably via carboxylic acid groups on uronic acid residues (James et al., 1978). This binding could result in reduced absorption since fiber is largely indigestible in the stomach and small intestine, and nutrients bound to it would be carried through the gut without being absorbed. However, substantial fiber digestion can occur in the colon and, if calcium can be absorbed from the colon, the calcium released during fiber digestion could still be absorbed. Partridge (1978a) has shown that calcium is absorbed in

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the colon of the pig and there is evidence that calcium may be absorbed by the rat cecum (Favus, 1985). Sandstrom et al. (1986) measured colonic calcium absorption in humans by instilling a solution of 47Cainto the colon with a colonoscope and following retention with whole-body counting. They estimated that the mean absorption of the calcium tracer from the colon was 14.1%, a surprisingly high value in view of the fact that absorption of orally ingested calcium is in the range of 20 to 30%. However, the isotope was instilled following 2 days on a clear liquid diet and bowel cleansing with laxatives and enemas. It is likely that absorption would be lower under more normal conditions due to more rapid transit times and calcium binding to colon contents. Calcium binding by fiber is strongly pH dependent. For example, James et al. (1978) showed that carrot fiber bound 45% of the calcium in a solution at pH 6.9 but only 15% at pH 2.5. Therefore, in the duodenum, where pHs may be in the range of 3.0 to 5.0, fiber binding of calcium may be low. Fiber may alter rates of transit of gastrointestinal contents, thereby affecting absorption. Fibers which increase the viscosity of luminal contents may slow gastric emptying. A slower rate of delivery of stomach contents to the small intestine may increase nutrient absorption. Fiber may also affect rates of passage through the small intestine. An increase in the rate could reduce nutrient absorption simply by allowing less time for nutrients to diffuse to the mucosal surface to be absorbed. Another possible mechanism for a fiber effect is related to the water content of the digesta. Fiber increases the water content of digesta, resulting in dilution of nutrient concentrations in the gut lumen and lower concentration and electrochemical gradients across the mucosa (Partridge, 1978b). Alternatively, it may be that components associated with fiber in foods rather than fiber itself are responsible for altering nutrient absorption. Phytate is the most likely candidate for this possibility. 6 . Effects of Cereals on Calcium Bioavailability. To put this topic into perspective, it is useful to describe a classical study by McCance and Widdowson (1942). The objectives of the study were to compare calcium absorption from diets containing white bread (made from 69% extraction flour) or brown bread (made from 92% extraction flour) and to determine whether any effect on calcium absorption was due to phytate. Bread was a major component of both diets, contributing 40-50% of total calories. Ten subjects (five men and five women) participated. The balance approach was used and balance periods for each diet lasted for at least 14

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days, usually longer. Food intakes were carefully measured and aliquots of the food were taken for chemical analysis. During the balance periods, complete urine and fecal collections were made. Calcium absorption (defined as the difference between calcium intake and fecal calcium) from the diets containing brown bread was lower than from diets containing white bread. On the white bread diet, seven of the nine subjects were in satisfactory calcium balance (mean calcium intake for the group was 5 I 1 mg). In contrast, eight of the nine subjects consuming the brown bread diet were in negative calcium balance on a mean intake of 558 mg. Addition of sodium phytate had a marked inhibitory effect on calcium absorption to the extent that fecal excretion of calcium was higher than calcium intake for many subjects. Addition of calcium, as either carbonate or phosphate salts, to the bread at the rate of 100 mg calcium/l00 g bread restored most subjects consuming the brown bread diet to calcium balance. The addition of calcium to the bread brought calcium intakes up to greater than I g/day for most subjects. Even though this extensive study may be criticized for its methodology, relatively short duration, small number of subjects, and lack of statistical evaluation of data, the results were quite dramatic. It is remarkable, perhaps, that numerous studies since the one by McCance and Widdowson have not given us a basis for a more definitive answer to the question. The important question appears to be not whether cereals may depress calcium absorption but whether the effect is nutritionally significant. This will depend on two variables: the amount of fiber in the diet and the amount of calcium in the diet. Although McCance and Widdowson did not analyze their diets for fiber, fiber intakes of those on the brown bread diets were no doubt quite high since subjects were getting between 40 and 50% of their calories from the bread. Even so, when calcium intakes were raised above lo00 mg/day, most subjects were in positive calcium balance. Unfortunately, because of different methodologies used to measure fiber, it is difficult at this time to provide a quantitative assessment of the extent to which increasing fiber intake may increase calcium requirements. However, two recent studies may shed some light on the question. Sandberg et al. (1982) compared calcium absorption by ileostomy patients from a low-fiber diet with and without added bran. Addition of 15 g of bradday had no effect on calcium absorption as measured by a balance technique. Balasubramanian et al. (1987), also using a balance technique, studied the effect of wheat bran on calcium absorption in older adults in a 33-day study divided into three periods. In the first period, subjects consumed their usual diets. In the subsequent two periods, the subjects

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continued with their usual diets but a daily wheat bran supplement (30 g) was included. Apparent calcium absorption was 22.1, 21.6, and 8.6% for the three periods, respectively. These data suggest that fiber does not affect calcium absorption over the short term but does reduce absorption when consumed for longer periods. This is a curious finding because it is well established that the efficiency of calcium absorption is enhanced when animals or humans are maintained on a low-calcium diet (Pansu et al., 1981; Ireland and Fordtran, 1973). If wheat bran reduces calcium absorption by lowering luminal concentrations of soluble calcium, a shortterm fall in calcium absorption followed by a gradual increase as subjects adapted would be expected. The age of the subjects may have been a factor: older subjects do not increase their plasma 1,25-(OH),-D, levels in the face of dietary calcium depravation to the same extent as younger subjects (Prince et al., 1988). Others have also found that wheat bran decreases calcium absorption in short-term balance studies. Reinhold et al. (1976) compared calcium balances in two subjects fed diets containing either white bread or whole wheat bread. Fiber intakes from the diet containing whole wheat bread were about 30 g/day compared with intakes of about 22 g/day for the white bread. Calcium balances were negative during the whole wheat period. One subject was in positive balance when consuming white bread, the other showed a negative balance of 40 mg/day, but this was substantially higher than the negative balance of 122 mg/day for this subject during the whole wheat period. In a similar study with four subjects, Cummings et al. (1979) adjusted the fiber content in their diets by substituting a wheat bran cereal for corn flakes and whole wheat bread for white bread. The fiber content of the low-fiber diet was 22 g/day, for the higher-fiber diet it was 53 g/day. Calcium balances on the low-fiber diet averaged + 32 mg/ day with a mean calcium intake of 960 mg, compared with -77 mg/day on the high-fiber diet at an intake of 1302 mg. In contrast, van Dokkum et al. (1982) found no significant differences in calcium balance among diets containing bread baked from white flour, white flour + added wheat bran, or whole wheat flour. The factor or factors in cereal brans responsible for the impairment of calcium absorption has not been identified with certainty but fiber and/or phytate are the most likely culprits. It is difficult to separate clearly the effects of the two because their concentrations in cereal products are correlated. McCance and Widdowson (1942) clearly showed that the addition of purified phytate to diets reduced calcium absorption. Phytic acid has a high affinity for calcium and may form insoluble complexes with calcium under gastrointestinal conditions (Graf, 1983). Nolan et al. (1987) also showed that phytate precipitates calcium in vitro. They observed,

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however, that, at pHs below 5.5, calcium was 100% soluble over a range of calcium-to-phytate ratios, while at pH 6 and 7, solubility fell to 20% when the calcium-to-phytate molar ratio was varied between 0.1 and 1 .O. It should be noted that purified phytate and naturally occurring phytate may affect mineral bioavailability differently since the phytate native to cereals is presumably already complexed with minerals (Erdman, 198 1). Moreover, partial hydrolysis of phytate (inositol hexaphosphate) may occur during food processing and in the gastrointestinal tract. Lonnerdal et al. (1989) have shown that the inhibitory effect of phytate on calcium and zinc absorption is abolished when two or more of the phosphate groups are removed by hydrolysis. This may explain some of the conflicting data in the literature, since many methods for measuring phytate do not distinguish between phytate and some of its hydrolysis products (Lonnerdal et al., 1989). On the other hand, purified fiber components with presumably low phytate contents may depress calcium absorption (see Section I1,D,1,d). While it is clear that increased intakes of unrefined cereals or cereal brans reduce calcium absorption in the short term, long-term effects have not been systematically examined. Robertson et al. (1981) have suggested that the increase in the prevalence of rickets in Dublin in 1942 was due to a change of the extraction rate of the national flour from 70 to 100%. They base their suggestion on an analysis of food consumption data and rickets surveys collected in Ireland during World War 11. These data, they note, show that there was a marked increase in rickets when the extraction rate of flour was increased and a fall in incidence when extraction rates were again lowered in 1942-1943. They further argue that these changes in the incidence of rickets cannot be accounted for by vitamin D intakes and, although they did not evaluate calcium intakes, they stated that “the Irish diet was in other respects entirely adequate.” c. Effects of Fiber from Fruits and Vegetables. Kelsay and her associates have studied the effects of fruit and vegetable fiber on mineral utilization. In their first study (Kelsay et al., 1979), diets containing either fruit and vegetable juices (low fiber) or fruits and vegetables (high fiber) were fed to 12 adult male subjects. Spinach was included as a vegetable every other day. Balances were determined during the last 7 days of each diet period, which lasted 26 days. Calcium balance was +72 mg/day on the low-fiber diet and - 122 mg/day on the high-fiber diet, a significant difference. In a second study of similar design (Kelsay et al., 1981) but with cauliflower replacing the spinach, increased levels of fiber did not affect calcium balance, suggesting that in the first study it was a component in spinach and not fiber that produced the negative calcium balances

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on the high-fiber diet. This led them to a third study in which three diets were compared: low fiber with spinach, higher fiber with spinach, and higher fiber without spinach (Kelsay and Prather, 1983). The diets were fed for 4 weeks each and balances were determined during weeks 3 and 4 for each diet. Calcium balances were positive during week 3 on all three diets. Only during week 4 on the higher-fiber diet with spinach was calcium balance negative and significantly different from balances for the other diets. When balances for weeks 3 and 4 were combined, calcium balances were not significantly different among the three diets. The authors concluded that fiber did not affect mineral balance and suggested that the negative calcium balance during week 4 of the higher-fiber with spinach diet may have been due to a fiber-mineral-oxalate complex. A surprising finding in this study was the difference in calcium balance on the higher-fiber with spinach diet between week 3 (+60) and week 4 (-73). As noted above, Balasubramanian et al. (1987) found a similar pattern with wheat bran: wheat bran did not affect apparent calcium absorption during the first 10 days it was fed but significantly lowered absorption during the second 10-day period. These findings suggest either that subjects are unable to adapt to factors which may lower calcium absorbability or that week-to-week variability within subjects may invalidate conclusions based on short-term balance studies. In an attempt to evaluate the problem of weekly variability, Kelsay et al. (1988a) conducted a fourth balance study on the effects of vegetable fiber on mineral balances. This time they fed each diet, one low fiber and one high fiber, for 6 weeks. Calcium balances were positive on both diets and did not differ significantly between diets when the analysis was based on the means for the entire 6-week period. However, there was significant weekto-week variability of calcium balance. The authors concluded that balance data for l to 2 weeks is insufficient for detecting diet effects on mineral balances. Schwartz et al. (1986) reached a similar conclusion based on a 7-week balance study employing young adult males as subjects. They found that two to three consecutive balances periods of 1 week each were necessary to obtain reliable balance data. Kelsay et al. (1988b) have also compared calcium utilization among vegetarian and nonvegetarian adults consuming their usual diets. Asian Indian and American vegetarians (all living in the Washington D.C. area) and American nonvegetarians were asked to record their dietary intakes carefully for 14 consecutive days. During the study period, subjects were asked to consume their usual diets and to prepare duplicate portions of all food and beverages consumed. Mineral balances were measured over a 1-week period. While fiber intakes were higher for the American vegetarians, there were no significant differences in calcium balance among

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the groups, suggesting that fiber intakes do not affect calcium balance when people are maintained on their usual diets. Mean balances for all groups were negative. The authors speculate that this may have been the result of lower intakes during the collection week. Calcium intakes during the collection week were 10% lower than intakes determined from the diet records.

d . Ejfects of Purified Fibers. Since the substitution of a high-fiber food into a diet will invariably change more than just the fiber content, the use of purified fibers may be useful for studying the fiber effect. Several groups have recently tried this approach. Slavin and Marlett (1980) added 16 g of refined cellulose to a control diet composed of normal foods. The addition increased the neutral detergent fiber content of the diet from 9.5 to 23.5 g. Healthy young female subjects were maintained on each diet for approximately 1 month and calcium balances were calculated from 5-day composites of feces and urine. Calcium intake on the control diet averaged 585 g/day compared to 600 g/day on the experimental diet. Calcium balances were - 16 mg/day without cellulose and - 199 mg/day with cellulose, a significant difference. In contrast, Behall et al. (1987) found no significant effect of cellulose on calcium balance. They substituted either refined cellulose, carboxymethylcellulose, karaya gum, or locust bean gum to a basal diet composed of normal foods. The diets were fed to men for 4 weeks each. Each experimental diet contained 7.5 g of refined fiber per 1000 kcal; actual intakes of added fiber ranged from 19.1 to 27 g/day. Calcium intakes were about 700 mg/day except for the karaya gum diet, where the intake averaged 1046 g/day. Balances were measured during the last 8 days of each period. None of the fibers significantly altered calcium balance; however, mean apparent retentions of calcium were lower than the basal diet for all the fibers except for the karaya gum. It is possible that the higher calcium intakes in the Behall study were high enough to offset an effect due to the fiber while, in the Slavin study, the marginal calcium intake became even more marginal in the presence of the fiber. In another study using a refined fiber source, Sandberg et al. (1983) added citrus pectin to a low-fiber diet given to ileostomy patients. Apparent absorption was calculated as the difference between intake and excretion in the ileostomy fluid. The addition of 15 g pectin to the daily diet had n o effect on apparent calcium absorption. Calcium intakes were, however, extremely high, averaging over 2000 mg/day. Taken together, these studies indicate that fiber per se does not have a marked effect on calcium bioavailability, especially when incorporated into diets adequate in calcium. With the exception of cellulose, the re-

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fined fibers were all “soluble” or gel forming. Behall et a!. (1987) speculate that gel-forming fibers have less effect on mineral absorption than particulate fibers, which are the predominant fibers in cereals. This may explain why whole-grain cereals appear to have a greater effect on mineral bioavailability than fiber-containing fruits and vegetables.

2. Oxalic Acid The extremely poor solubility of calcium oxalate suggests that oxalatecontaining foods will adversely affect calcium absorption. There are two important questions in this regard: What is the availability of the calcium in the oxalate-containing food? and Do high-oxalate foods impair calcium absorption from other sources when the two are consumed together in the same mehl or diet? The first question has been addressed recently by Weaver et al. (1987). They fed rats diets that contained various sources of intrinsically labeled calcium and assessed calcium absorption by measuring femur radioactivity. Their results clearly show that calcium absorption from calcium oxalate and spinach, a high-oxalate vegetable, was much lower than absorption from other calcium sources. In a follow-up study using humans (Heaney et al., 19881, the same dramatic effect was confirmed. In this study, 13 human adults were fed a standard breakfast in which the calcium source was either intrinsically labeled spinach or extrinsically labeled milk. The mean fractional calcium absorption from milk was 27.5% compared to 5.1% from the spinach. These studies provide strong evidence that leafy vegetables that are high in oxalate are not good calcium sources. The answer to the second question is less clear. In a recent review, Kelsay (1985) pointed out that most of the work related to this question was conducted prior to 1950. Whether oxalate affects calcium from other sources in the same meal may be related to the calcium-to-oxalate ratio in the food. Presumably, if all the oxalate were present as calcium oxalate, then there would be no free oxalate to combine with calcium from other sources. It turns out, however, that some leafy vegetables contain substantial amounts of soluble oxalate. Prenen et al. (1984) reported the oxalate content of spinach to be 3.5 mmo1/100 g wet weight of which 55% was soluble and 45% was insoluble. Pingle and Ramasastri (1978) analyzed Amaranthus spp. leaves and found very high oxalate levels (24 mmoV100 g, 32% soluble and 68% insoluble). They designed a study to determine whether the water-soluble oxalates would affect calcium absorption from milk. Diets containing milk and cooked Amaranthus leaves (with or without the cooking water) were

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fed to adult male subjects. Urinary excretion of calcium was used as an indicator of calcium absorption. They found that, when the cooking water was ingested with the meal, Amaranthirs depressed calcium absorption from milk but when the water was discarded, no effect was seen, suggesting that only the soluble oxalate depresses calcium absorption from other sources. Landis et al. (1987) compared calcium utilization from spinach and cheese in a balance study using seven premenopausal women as subjects. The subjects were fed a diet composed of normal foods but the cheese or spinach contributed about 77% of the total calcium intake. Mean calcium balances were -55 and - 168 mg/day on the cheese and spinach diets, respectively, but the differences were not statistically significant. Urinary calcium, however, was significantly higher on the cheese diet suggesting, possibly, greater calcium absorption. These studies and the fact that a significant proportion of the oxalate in vegetables is soluble suggest that foods high in oxalate can reduce calcium absorption from other sources in the same meal.

E. CALCIUM BIOAVAILABILITY: PROTEIN AND PHOSPHATE EFFECTS It is well established that dietary protein and phosphate can markedly affect calcium balance. These effects have been thoroughly reviewed (Zemel, 1985; Linkswiler et al., 1981; Zemel, 1988), so only a brief summary will be presented here. Under normal dietary conditions, protein and phosphates appear to have little effect on calcium absorption. For example, when dietary phosphate levels were held constant, increasing the protein intake from 50 to 150 g/day in a diet providing 500 mg calcium per day did not significantly affect calcium absorption by adult males (Hegsted et al., 1981). Whether protein affects calcium absorption may depend on calcium intakes, however. Linkswiler et al. (1974) found that protein increased calcium absorption when calcium intakes were 800 and 1400 mg but not when they were 500 mg. Likewise, phosphates do not appear to affect calcium absorption markedly. Spencer et a f . (1978) showed that the addition of 1200 mg phosphorus per day in the form of sodium glycerophosphate did not affect calcium absorption by men. Zemel and Linkswiler (1981) found that orthophosphate did not affect apparent calcium absorption by men but that polyphosphates did decrease calcium absorption slightly. Protein and phosphate do, however, have marked effects on renal handling of calcium. Increases in dietary protein increase urinary calcium significantly (Linkswiler et al., 1981). Zemel (1988) has estimated that a doubling of protein intake will increase urinary calcium by 50% when cal-

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cium and phosphorus intakes are held constant. Increases in dietary phosphate have the opposite effect on urinary calcium, i.e., they cause a fall in urinary calcium excretion (Spencer er al,, 1978; Zemel, 1985). Fortunately, foods high in protein are usually also good sources of phosphate, so the net effect on calcium balance of increased dietary protein is much less than would be expected from studies where purified proteins have been used to manipulate protein intakes. In the case of dairy products, protein is associated with calcium as well as phosphorus, suggesting that an increase in dairy product consumption may have a net positive effect on calcium balance even though dairy products are rich in protein. Recker and Heaney (1985) have conducted a study to determine whether an increase is milk intake would affect calcium balance. They divided 22 postmenopausal women into two groups. One group continued to consume their usual diet and the other was instructed to consume the equivalent of 24 ounces of milk per day in addition to their usual diet. Calcium absorption and calcium balances were measured at the beginning and after I year. Intakes of calcium, phosphorus, and nitrogen in the milksupplemented group increased by 0.792, 0.423, and 3.006 glday, respectively. In the milk-supplemented group there were significant increases, compared with the control group, in absorbed calcium, endogenous fecal calcium, and urinary calcium. Computed calcium balance increased 0.044 g/per day but the increase was not significant. Nevertheless, the authors concluded that dairy products can be recommended as sources of calcium since, while not quite statistically significant, the improvement in calcium balance was probably real and the milk did not appear to suppress bone remodeling as much as calcium supplements do. Moreover, if milk were substituted for other high-protein foods rather than added to them, the increase in protein intake would be less and therefore would not result in increased urinary losses of calcium. In light of the calciuretic effect of protein, the high-protein diets common in the United States may be a factor in bone health over the long term. Protein supplements made from purified protein and food ingredients such as protein-based fat substitutes should be evaluated for their potential impact on calcium balance.

F. CALCIUM BIOAVAILABILITY: CALCIUM SUPPLEMENTS In spite of conflicting evidence from studies designed to evaluate the effect of calcium supplements on loss of bone mineral, sales of calcium supplements have skyrocketed in recent years (Riggs er al., 1987; Ettinger er al., 1987; Riis er al., 1987). This trend is due, at least in part, to the recommendation of the NIH Consensus Development Conference on

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Osteoporosis that women unable to consume diets containing 1000 to 1500 mg calcium per day take a calcium tablet (NIH, 1984). Food companies have also capitalized on consumers’ hunger for calcium by fortifying foods with calcium. Calcium is being added to flour, orange juice, readyto-eat breakfast cereals, and even some dairy products. Several studies have been published recently comparing calcium bioavailability from the various salts that have been used in the formulation of calcium tablets or for addition to foods. An important factor to consider when choosing a salt for use in tablets in the percentage of calcium in the salt. This is important because tablets made from salts with low calcium content may be too large for easy swallowing. The calcium content of several salts in common use is given in Table V. Bioavailability from tablets depends on the solubility of the calcium in the gastrointestinal tract (Schuette and Knowles, 1988; Carr and Shangraw, 1987). Solubility of calcium salts is a function primarily of pH and the anion. For example, calcium carbonate is quite insoluble in water at neutral pH but readily dissolves in acidic solutions. Calcium lactate is quite soluble over a wide pH range. Bioavailability of calcium from tablets may also be influenced by the rate of tablet disintegration in the gastrointestinal tract (Blanchard, 1989). Disintegration is affected by the nature of binders used in the formulation and by the amount of compression used to form the tablets (Carr and Shangraw, 1987). Overcompression and formulation without starch may cause slow disintegration (Carr and Shangraw, 1987). The bioavailability of calcium from calcium salts appears to be influenced by gastric acid secretion and by simultaneous ingestion of food. Sheikh et al. (1987) compared calcium absorption from various calcium salts and milk in humans using their intestinal lavage method. The suppleTABLE V WATER SOLUBILITY AND CALCIUM CONTENT OF SELECTED CALCIUM SALTS“

Salt

Water solubility (g/lOO ml cold water)

Calcium content

1.5 x lo-?

40 21 9 13 25

Calcium carbonate Calcium citrate . 4 H 2 0 Calcium gluconate * H,O Calcium lactate . 5H,O Calcium acetate “Values are from Weast (1979).

0.85 3.3 3.1 31

(%)

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ments and milk were given to fasting subjects. Absorption from the various doses was as follows: calcium acetate, 32%; calcium lactate, 32%; calcium gluconate, 27%; calcium citrate, 30%; calcium carbonate, 39%; and milk, 31%. Differences between treatments were not significant. These results seen to indicate that water solubility is not a good predictor of calcium bioavailability. The authors measured solubility in a pH 2.5 buffer and found that 100% of the calcium in all the salts had dissolved after 1 hr. Recker (1985) compared calcium absorption from calcium carbonate and calcium citrate in normal humans and humans with achlorhydria. He found that fasted normal subjects absorbed similar amounts of calcium from the two salts: 24% from the citrate and 22% from the carbonate. Fasted achlorhydric subjects, on the other hand, absorbed 45% of the calcium from the citrate salt but only 4% from the carbonate. When the calcium carbonate was given to the achlorhydric subjects with breakfast, absorption was normal. This suggests that salts that require acid for solubilization may be poor sources of calcium for persons with impaired gastric acid secretion unless ingested with a meal. Solubilization in the acidic stomach, however, does not guarantee efficient absorption. Schuette and Knowles (1988) compared the absorption of calcium by fasted humans from two soluble salts: calcium citrate and Ca(H,PO,),. Solutions of the salts were labeled with ,'Ca and fractional calcium absorption was calculated from forearm activity measurements following oral and iv administration of the isotope. Calcium absorption from the citrate salt (18.2%) was more than twice the absorption from the phosphate salt (7.6%). The authors speculate that the explanation for the difference was due to the formation of insoluble phosphate salts in the small intestine. As the pH rises, HP0:- and PO:- will form from H,PO;. It is likely, however, that these differences would not have been observed had the salts been given with a meal. As noted above, the effect of phosphate on calcium absorption from foods appears to be minimal. Complexation of calcium by ligands yielding soluble complexes may also affect calcium absorption. Rumenapf and Schwille (1988) compared absorption by fasted humans following oral administration of either calcium chloride or calcium chloride plus hexapotassium hexasodium pentacitrate. The molar ratio of citrate to calcium in the second preparation was approximately 4 : 1 . Precipitation did not occur in either preparation. Absorption was calculated by deconvolution from blood radioactivities of oral and iv tracers. The citrate significantly reduced calcium absorption. These data suggest that complexed calcium may be less available than free ionic calcium even though the complex is soluble in the intestinal lumen. It is notable that this inhibitory effect of citrate occurs only

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when citrate-to-calcium ratios are high. Both Sheikh et al. (1987) and Recker (1985) found the calcium in calcium citrate to be highly available. Recently, a new salt for food fortification has been developed by Procter & Gamble Co. (Smith et al., 1987). The salt called CCM is prepared by dissolving calcium carbonate in an aqueous solution of citric and malic acids. The final ratio of calcium to citrate to malate is 6 : 2 : 3. The mixed salt is much more soluble than either calcium citrate or calcium malate. Procter & Gamble Co. is currently using CCM to fortify one of its orange juices. Smith et al. (1987) compared the absorption of calcium from CCM with absorption from calcium carbonate or milk. The labeled calcium sources were given to humans with a breakfast meal following an overnight fast. Absorption of calcium from a CCM tablet was significantly higher than from a calcium carbonate tablet (37.3 versus 29.6%) Likewise, calcium absorption from CCM mixed with orange juice was higher than calcium absorption from milk (38.3 versus 29.4%). In a similar study, Miller, J. Z. et al. (1988) compared calcium absorption from calcium carbonate and CCM. The two calcium salts were administered in tablet form with breakfast to adolescent subjects. Fractional calcium absorption from the CCM was significantly higher than from calcium carbonate (36.2 versus 26.4%). While these differences are not large, it is remarkable that they are measurable even when the salts are given with a meal. VII. SUMMARY AND CONCLUSIONS

Calcium nutritional status among some groups in the United States is suboptimal when judged by calcium intakes and the high prevalence of osteoporosis. Unfortunately, however, it is not clear that increases in calcium intake will have a significant impact on osteoporosis or other chronic diseases that have been linked to calcium nutriture. There is still considerable controversy surrounding the issue of calcium RDAs. The body’s ability to adapt to varying levels of calcium intakes, the lack of sensitive indicators of calcium status, and the complexity and slow progression of chronic diseases such as osteoporosis make it very difficult to establish the role of diet in this regard. Great progress has been made in the study of calcium absorption. Much is known about the mechanisms involved in calcium absorption and its regulation. Thus, a rapidly advancing field and further developments will be invaluable to our understanding of the role of diet in calcium nutrition. Calcium bioavailability is affected by diet composition and the chemical form of calcium in foods. The calcium in dairy products is readily

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absorbed in the intestine. Lactose enhances calcium absorption efficiency under some conditions. Components of plants such as fiber, phytate, and oxalic acid may depress calcium absorption. High intakes of protein increase urinary losses of calcium but this effect may be partially offset by the phosphate association with most high-protein foods. Calcium absorption from salts used in supplement tablets is generally good. Absorption from salts such as calcium carbonate which require acid for dissolution may be poor in persons with achlorhydria unless the tablets are consumed with a meal. The practical significance of factors that may alter calcium bioavailability in normal mixed diets is difficult to assess. It may be a significant factor when calcium intakes are marginal or when absorption by the active transport, vitamin D-dependent process is impaired or not fully developed, i.e., it may be significant when vitamin D status is poor, in the elderly, and in young infants. VIII.

RESEARCH NEEDS

There is a great need for research that will answer questions regarding the calcium requirement issue. We need a better understanding of all the factors that are involved in the development of osteoporosis so that the role of calcium can be more clearly defined. If it turns out that calcium intakes are too low in a significant proportion of the population, research is needed to determine the most effective approaches for increasing calcium intakes. Should nutrition education be used to get people to alter their food selections? Should calcium fortification of foods be increased? If so, what form of calcium should be used and what vehicle should be chosen? Should people be encouraged to take calcium supplements? Given the large number of people that are currently consuming calcium supplements, research is needed to identify possible adverse effects of high calcium intakes. It has been shown, for example, that calcium supplements taken with a meal can markedly reduce iron absorption from the meal (Dawson-Hughes ef al., 1986). Research is needed to identify chemical forms of calcium that are available for absorption from the gastrointestinal tract. Is only free ionized calcium absorbable or can soluble calcium complexes cross the intestinal barrier intact? Does the route of absorption (active transport or passive diffusion) make a difference in this regard? The roles of age and vitamin D status in calcium absorption need to be further investigated. Are diets containing inhibitors of calcium absorption more of a problem when calcium is absorbed primarily by the passive diffusion route as is apparently the case in young infants and persons with

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impaired vitamin D status? A great deal needs to be learned about effects of food processing on calcium bioavailability. For example, does cheese ripening affect bioavailability?

ACKNOWLEDGMENT This work was supported by a grant from the Wisconsin Milk Marketing Board.

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Shipp, C. C.. Maletskos, C. J., and Dawson-Hughes, B. 1987. Measurement of 47Caretention with a whole-body counter. Culcif. Tissue Int. 41, 307-312. Slavin, J. L., and Marlett, J. A. 1980. Influence of refined cellulose on human bowel function and calcium and magnesium balance. Am. J. Clin. Nurr. 33, 1932-1939. Smith, T. M., Kolars, J. C., Savaiano, D. A., and Levitt, M. D. 1985. Absorption of calcium from milk and yogurt. Am. J. Clin. Nutr. 42, 1197-1200.

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Smith, K. T., Heaney, R. P., Flora, L., and Hinders, S. M. 1987. Calcium absorption from a new calcium delivery system (CCM). Culcif. Tissue Inr. 41, 351-352. Southgate, D. A. T. 1987. Minerals, trace elements, and potential hazards. A m . J. Clin. Nutr. 45, 1256-1266. Spencer, H., and Kramer. L. 1986. The calcium requirement and factors causing calcium loss. Fed. Proc., Fed. A m . SOC.Exp. Biol. 45, 2758-2762. Spencer, H., Kramer, L., Osis, D., and Nonis, C. 1978. Effect of phosphorus on the absorption of calcium and on the calcium balance in man. J. Nurr. 108, 447-457. Stark, C. 1987. A woman’s guide to calcium. “Food for Health.” Division of Nutritional Sciences, Cornell University, Ithaca, New York. Stasse-Wolthius, M.,Albers, A. F. F., van Jeveren, J . G. C., de Jong, J. W., Hautvast, J. C. A. J., Hermus, R. J. J., Katan, M. B., Brydon, W. G., and Eastwood, M. A. 1980. Influence of dietary fiber from vegetables and fruits, bran or citrus pectin on serum lipids, fecal lipids, and colonic functions. A m . J. Clin. Nutr. 33, 1745-1756. Steichen, J. J., and Tsang, R. C. 1987. Bone mineralization and growth in term infants fed soy-based or cow milk-based formula. J. Pediatr. 110, 687-692. Toverud, S. U., and Dostal, L. A. 1986. Calcium absorption during development: Experimental studies of the rat small intestine. J. Pediutr. Gastroenterol. Nutr. 5 , 688695. Tremaine, W. J., Newcomer, A. D., Riggs, B. L., and McGill, D. B. 1986. Calcium absorption from milk in lactase-deficient and lactase-sufficient adults. Dig. Dis. Sci. 31, 376378. Truesdell, D. D., Whitney, E. N., and Acosta, P. B. 1984. Nutrients in vegetarian foods. J. A m . Diet. Assoc. &I, 28-35. Tsai, K.-S., Heath, H., Kumar, R., and Riggs, B. L. 1984. Impaired vitamin D metabolism with aging in women, possible role in pathogenesis of senile osteoporosis. J . Clin. Invest. 73, 1668-1672. U.S. Department of Agricu1tureAJ.S. Department of Health and Human Services 1986. “Nutrition Monitoring in the United States: A Progress Report from the Joint Nutrition Monitoring Evaluation Committee.” DHHS Publication No. (PHS) 86-1255. Washington D.C., U.S. Govt. Printing Office. Van Dokkum, W.. Wesstra, A., and Schippers, F. A. 1982. Physiological effects of fibrerich types of bread. I. The effect of dietary fibre from bread on the mineral balance of young men. Br. J. Nutr. 47, 451-460. Walker, A. R. P. 1985. Mineral metabolism. In “Dietary Fibre, Fibre-Depleted Foods and Disease” (H. Trowell, D. Burkitt, and K. W. Heaton, eds.), pp. 361-375. Academic Press, Orlando, Florida. Wasserman, R. H. 1988. Calcium and phosphate entry into cells: A brief overview. Prog. Clin. Biol. Res. 252, 3-13. Wasserman, R. H., and Fullmer, C. S. 1983. Calcium transport proteins, calcium absorption, and vitamin D. Annu. Rev. Physio/. 45, 375-90. Wasserman, R. H . , and Lengemann, F. W. 1960. Further observations on lactose stimulation of the gastrointestinal absorption of calcium and strontium in the rat. J . Nutr. 70, 377-384. Wasserman, R. H., Comar, C. L., and Nold, M. M. 1956. The influence of amino acids and other organic compounds on the gastrointestinal absorption of calcium and strontium in the rat. J. Nutr. 59, 371-383. Weast, R. C. 1979. “Handbook of Chemistry and Physics.” CRC Press, Boca Raton, Florida. Weaver, C. M. 1988. Calcium and hypertension. Cereal Foods World 33, 792-794.

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ADVANCES IN FOOD A N D N U T R I T I O N RESEARCH, VOL.

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DIETARY AND BIOCHEMICAL ASPECTS OF VITAMIN E ROBERT S. PARKER Division of Nutritional Sciences and Department of Food Science Cornell University Ithucu. New York 14853

1. 11.

Ill.

IV. V.

VI.

VII.

VIII.

Introduction Dietary Sources, Stability, and Intake of Tocopherols A. Tocopherols in Fats and Oils B. Influence of Food Processing and Preparation on Tocopherols C. Dietary Intake of Tocopherols Intestinal Absorption of Tocopherols A. Mechanism and Efficiency of Tocopherol Absorption B. Effect of Diet Composition on Bioavailability of Tocopherols Transport of Tocopherols in Blood Tocopherols in Tissues A. Tocopherol Levels in Human Tissues B. Tocopherols in Human Milk C. Tocopherols in Ocular Tissues D. Mechanisms of Tissue Uptake of Tocopherols E. Tissue Mobilization and Turnover of Tocopherols F. Assessment of Vitamin E Status in Humans Tocopherol Binding and Transfer Factors A. Tocopherol Binding Factors B. Tocopherol Transfer Factors C. Membrane Binding of Tocopherols Tocopherol Metabolism A. Metabolism of a-Tocopherol B. Metabolism of Other Tocopherols C. Regulation of Tocopherol Metabolism Functional Aspects of Tocopherols in Biomembranes A. Antioxidant Function in Membranes B. Regeneration of a-Tocopherol from a-Tocopheroxyl Radical C. Concentration and Mobility of a-Tocopherol in Membranes D. Physical Effects of Tocopherols in Membranes

157 Copyright 0 1989 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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1X. Future Research Directions References

1.

INTRODUCTION

It is the purpose of this article to review various aspects of the nutrition, physiology, and biochemistry of vitamin E. While the importance of the tocopherols in human nutrition has been recognized for several decades, only recently have many of the details of the metabolism and function of these compounds become known with any degree of certainty. The application of newer techniques of analytical chemistry has allowed the investigation of a number of phenomena associated with the tocopherols which have hitherto been impossible, yielding results which in some cases have resolved long-standing inconsistencies in the chemistry or biology of vitamin E. In addition, the recent interest in promoting relatively large alterations in dietary fat intake and the development of fat substitutes warrants renewed scrutiny of the important dietary sources of tocopherols in human diets and continued estimation of vitamin E intakes in various population groups. It is not the intent of this review to address all areas of vitamin E nutrition, nor to include all published studies within a specific topic. Rather, it is intended to update past reviews of selected aspects of vitamin E and its function, and to discuss in depth subject areas not previously addressed in detail. The emphasis will be placed on the nonpharmacological aspects of the vitamin in normal individuals, as several recent excellent reviews of the clinical consequences of vitamin E deficiency or supplementation are available (Lubin and Machlin, 1982; Porter and Whelan, 1983; Hayaishi and Mino, 1987). As far as possible, this review emphasizes vitamin E nutrition in humans, although reliance on animal models has often been necessary. Since the interpretation of many of the cited studies depends on the employed methodology, an attempt has been made to include sufficient methodological detail to permit comparison of results between laboratories, especially where conflicting results have been reported. Finally, suggestions are included for future research directions in the area of vitamin E nutrition and biochemistry. It is abundantly clear that many details of the metabolism and function of this vitamin in humans remain unknown, and clarification of these may have implications regarding both recommended intakes in normal individuals and clinical applications in specific disease states.

DIETARY AND BIOCHEMICAL ASPECTS OF VITAMIN E

11.

159

DIETARY SOURCES, STABILITY, AND INTAKE OF TOCOPHEROLS A. TOCOPHEROLS IN FATS AND OILS

Vegetable oils and oil products are among the major contributors of dietary tocopherols, either directly (e.g., as salad oil) or indirectly as ingredients in prepared or processed foods. The predominant tocopherols in vegetable oils are a-,y-, and &tocopherol, occurring almost exclusively in the RRR form (Fig. I). While p-tocopherol is found in low concentrations in many common vegetable oils, only cottonseed oil, with 40

B

C HO

FIG. I . Structures of commonly occurring tocopherols. (A) R,R,R-a-Tocopherol (D-atocopherol). (B) R.R,R-y-tocopherol, and (C) R.R,R-&tocopherol.

160

ROBERT S. PARKER

mg/kg @-tocopherol,contains appreciable amounts of this vitamer (Carpenter, 1979). Tocotrienols, which contain three double bonds in the phytyl side chain, are also found in most vegetable oils, but generally in concentrations much lower than the tocopherols, and, consequently, their practical contribution to total dietary vitamin E equivalents is negligible. One exception is palm oil, which has been reported to contain high levels of tocotrienols (Syvaoja, 1986). The concentrations of total and individual tocopherols in several vegetable oils as determined by high-pressure liquid chromatography (HPLC) or gas-liquid chromatography are shown in Table I. These data illustrate several points. First, some common vegetable oils are rich sources of tocopherols in general while others, such as olive and coconut oils, are relatively poor sources. Second, oils of different types can have widely differing tocopherol compositions. For example, corn and soybean oils contain predominantly y-tocopherol, while safflower and sunflower oils contain mostly a-tocopherol. Third, different brands of the same oil type can differ in their tocopherol concentrations. This latter variation may result from varietal differences, different stages of seed maturity at harvest, or from geographical and climatic conditions under which the oilseed is grown (Bauernfeind, 1980). Since vegetable oils are among the richest sources of dietary tocopherols, alteration of tocopherol content during oil production and processing is of interest. Bauernfeind (1977) has summarized earlier studies on this subject, concluding that a number of oil processing steps result in tocopherol losses. Recently, Wong et al. (1988) estimated total tocopherol losses resulting from the various unit processes involved in the fractionation and refining of palm oil. Palm oil typically contains in excess of 800 ppm total tocopherol (approximately 50% as a-tocopherol), making it one of the richest tocopherol sources, roughly equivalent to that of soybean oil. Detergent fractionation caused no tocopherol loss, simply a redistribution between the olein and stearin fractions such that the olein fraction contained the greater proportion of tocopherols. Degumming (0.04-0.07% phosphoric acid, 85OC) and bleaching (1-2% earth, 1lOOC) resulted in 2-4% loss of tocopherols. Steam refining and deodorization (270°C) resulted in similar minor losses. The overall loss of tocopherols amounted to approximately 8%, with 62% of the starting tocopherols retained in the RBD palm olein fraction and 30% in the fatty acid steam distillate. Therefore, while processing from crude palm oil to refined palm olein involves an apparent reduction in tocopherol content of 30%, this loss is strictly related to redistribution during steam refining, rather than tocopherol destruction. Significant losses of tocopherols resulting from steam refining of soybean oil were reported by Jawad et al. (1984), who compared the effects

TABLE I REPORTED RANGES IN CONCENTRATION OF TOCOPHEROL VITAMERS IN COMMERCIAL VEGETABLE OILS

a-Tocopherol"

y-Tocopherol"

%Tocopherol"

Referenceb

Referenceb

Referenceb

Oil type'

1

2

3

1

2

3

1

2

Corn oil Soybean oil Safflower oil Sunflower oil Olive oil Peanut oil

91-209 31-139 248404 401-578 75-109 130-154

247-371 91-1 18 575 387-967 102-263

120-220

180-584 183-875 11-23 39-94 10-20 100-121

630-890 -795 10-22 NAd-38 7-13 -

460-750 420-1020 NA~ NAd

3-10 17-97 NAd NAd-I0 N A ~

15

NAd-5

33-59 325-406 N A ~ NAd-16 N A ~ -

-

P

50-140

480-600 14 21

-

"Units are in milligrams per kilogram of oil. bReference:1, R. S. Parker (unpublished data); 2, Speek ef al. (1985); 3, Carpenter ef al. (1976). T h e various samples of the same oil type were of different brands and were obtained from local retail stores. %A, Not available. e-, Not reported.

3 ~ ~ ~ - 5 0

110-370 N A ~

-

NAd NA~

162

ROBERT S. PARKER

of various processes on nonsaponifiable compounds of soybean oil. Their data showed that phosphoric acid degumming and light bleaching resulted in loss of only 12% of total tocopherols, with the greatest percentage loss occurring in 6-tocopherol, followed by y-tocopherol and a-tocopherol (the latter is a minor tocopherol in soybean oil). Further losses (disappearance) from the oil due to steam refining were dependent on both time and temperature of the refining process. Refining at 280°C for 0.5, 1 , and 3 hr resulted in reductions in total tocopherol concentration of 45,64, and 97%, respectively. No consistent trend in the relative stability of the major tocopherol vitamers was evident, with all forms subject to substantial loss. Hernandez and Riera (1987) reported tocopherol losses of up to 50% in sunflower oil and olive oil, with most of the loss occurring during deodorization. In contrast, Ludwicki et al. (1986) reported that the greatest loss of tocopherols in a sunflowedrapeseed oil blend took place during the acidification-neutralization process, with only minor losses due to decolorization or deodorization. Overall losses approached 59% for atocopherol. However, since no analyses of the steam-stripped fractions were reported in these studies, the mechanism of loss (physical destruction versus removal) cannot be ascertained. In light of the data of Wong et al. (1988), partition of the tocopherols into the stripped fraction must be considered a likely effect of steam refining. Regardless, the result of this type of refining is to yield oils of generally lower tocopherol content, rendering the product more susceptible to oxidative degradation and decreasing its vitamin E value. The variation in tocopherol content or composition of commercial oils of the same type does not appear to be related to the method of oil extraction. For example, cold-pressed oils do not consistently exhibit higher tocopherol levels than conventionally processed oils (Carpenter et al., 1976; Speek et al., 1985). Antioxidants or other additives also show no consistent influence on tocopherol content, at least in comparison of different brands of the same oil type (Carpenter er al., 1976). Oil products, such as mayonnaise, salad dressings, margarines, and shortenings also generally contain high concentrations of tocopherols. The tocopherol composition of these products will reflect that of their constituent oils. The tocopherol concentration is generally a function of the constituent oil and the type or extent of processing. The occurrence of tocopherols and tocotrienols in various Finnish oils and oil products has been reported by Syvaoja (1986). Analysis of fats and oils showed that the tocotrienol concentration was small in most of the oils and margarines sampled. The exceptions were palm oil, in which a-,y-, and 8-tocotrienol together accounted for approximately 80% of total vitamin E vitamers (27 mg/100 g), and hydrogenated coconut oil, in

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163

which tocotrienols accounted for about 50% of the 3.9 mg vitamin E vitamers per 100 g lipid. In all other oils and margarines, tocotrienols contributed less than 5% of total vitamin E vitamers and were undetectable in many products. Differences between crude and refined rapeseed, soybean, sunflower, and palm oils were also determined. These losses amounted to 10-33% of a-tocopherol, 20-33% of the other tocopherol isomers, and 43-48% of the tocotrienols. The influence of specific refining steps on tocopherol losses was not reported. Also noted were differences between margarine hardness and tocopherol content, with the softer margarines containing substantially more a-tocopherol than semisoft or hard margarines (means of 24, 13, and 7 mg a-tocopherol per 100 g margarine, respectively). The relative contributions of hydrogenation versus oil composition were not addressed. Hydrogenated vegetable oils of many types are common constituents of margarines, spreads, salad dressings, and shortening agents. However, there are few reports on the influence of hydrogenation on the tocopherol content or composition of food oils. Hernandez and Riera (1987) found that hydrogenation decreased the tocopherol content of sunflower oil, and that the loss of tocopherols followed first-order kinetics with time. However, comparison of the tocopherol content of margarines made at least partially with hydrogenated vegetable oil with that of the nonhydrogenated oil (US.Department of Agriculture, 1979) yields inconsistent results after allowing for the increased water content of the margarines. For example, margarines made from hydrogenated safflower oil or corn oil contained substantially less a-tocopherol (or total tocopherol) than nonhydrogenated safflower or corn oil. But margarines made from hydrogenated soybean oil did not consistently show reduced tocopherol levels when compared to the parent oil. In comparing the tocopherol content of retail margarines with that of the starting oil mixture prior to margarine production, Lambertsen et al. (1964) concluded that hydrogenation does not result in destruction of a significant proportion of margarine tocopherols. This is in contrast to an earlier report of Ward (1958), who found that hydrogenation of groundnut or palm oils resulted in the loss of about 60 and 66%, respectively, of total tocopherols without substantially altering the tocopherol composition of these oils. Dicks (1965) summarized reports indicating that hydrogenation could result in tocopherol losses of 5-70%. Storage of oil or oil products has been shown to result in significant losses of tocopherols, and storage time may be an important contributing factor to the observed variability in tocopherol content of commercial oils of the same type. Storage losses are a function of the oil type, time, temperature, and concentration of endogenous and added antioxidants,

164

ROBERT S. PARKER

as reviewed by Bauernfeind (1977). For example, safflower oil lost 45% of its tocopherols during storage for 6 months at 10°C and 55% after storage at room temperature for 3 months. Lambertsen et al. (1964) reported average losses of 20 and 14% of a- and y-tocopherol, respectively, in margarines stored for 7 months at 5°C. Tocopherol concentrations in animal fats are generally much lower than in vegetable oils and fats, even after refining and hydrogenation of the latter. Kanematsu et al. (1983) reported the total tocopherol content of body fat of beef, lamb, pork, and chicken to be 6.4, 8.6, and 4.5 mgl kg, respectively. a-Tocopherol represented 90.7,94.6, and 69.8% of total tocopherols in these fats, respectively. Chicken fat had a higher tocopherol level (10.7 mg/kg), but a lower percentage as a-tocopherol (58%). The U.S.Department of Agriculture (1979) Handbook 8-4 lists the total tocopherol concentration of beef tallow at 2.7 mg/kg, butter at 1.58 mgl kg (essentially all a-tocopherol), pork fat at 1.3 mg/kg (1.2 mg/kg a-tocopherol), and chicken fat at 2.7 mglkg. Thus animal fats are generally two to three orders of magnitude less concentrated in tocopherols than vegetable oils. Many fish and fish oils are also reasonably good sources of tocopherols, mostly a-tocopherol, as recently reviewed by Kinsella (1987). For example, salmon and cod liver oils contain up to 200 mg tocopherol/kg oil, which is comparable to many peanut and olive oils. Fish oil supplements usually contain added tocopherols in order to increase the stability of these highly refined, unsaturated oil concentrates.

B. INFLUENCE OF FOOD PROCESSING AND PREPARATION ON TOCOPHEROLS Several unit processes involved in food processing may influence tocopherol levels, as reviewed by Bauernfeind (1977). Such processes include canning, dehydrating, and freezing, and tocopherol losses resulting from these treatments may approach 50% or higher. Bunnell et al. (1965) reported a 96% loss of tocopherols in canned peas relative to fresh peas. Frozen vegetables have been reported to have a lower a-tocopherol content compared to their fresh counterparts (Leth and Anderson, 1982), with losses reaching 75%. The time course of tocopherol loss has not been well established, but it is likely that longer frozen storage times are associated with lower tocopherol content. Carlson and Tabacchi (1986) examined the effects of heating on the tocopherol content of frying oils and french fries during simulated and actual foodservice frying operations. Losses of a-tocopherol after heating at 177°C for 8 hr ranged from 30 to 41%, and were linearly related to

DIETARY AND BIOCHEMICAL ASPECTS OF VITAMIN E

165

peroxide value. Total losses resulting from initial heating, sustained heating, cooling, and reheating for an additional 8 hr amounted to 41-73%. These losses appeared dependent on the fatty acid composition, initial tocopherol content, and presence of additives such as tert-butyl hydroquinone (TBHQ), citric acid, and silicone derivatives. For example, total a-tocopherol percentage loss in corn oil (initial a-tocopherol, 7.9 mg/100 g oil) with added TBHQ, citric acid, and dimethylsiloxane, partially hydrogenated soybean oil with similar additives (initial a-tocopherol, 4.3 mg/100 g oil), and semisolid hydrogenated soybean and palm oil shortening with added mono- and diglycerides (initial a-tocopherol, 4.2 mg/100 g oil) were 53, 41, 48%, respectively. A decrease in concentration of tocopherols in partially hydrogenated soybean oil with added TBHQ, citric acid, and dimethylsiloxane during actual foodservice frying operation occurred only during the initial day of oil use, probably due to the customary addition of fresh oil during the subsequent 3 days to replace oil absorbed by the food. A 40% decrease in concentration of both a- and y-tocopherol occurred over the entire 4-day operation. The stability of a-tocopherol at various water activities (A,), temperatures, and oxygen tensions in a model food system containing 1% methyl linoleate was described by Widicus and Kirk (1981). Loss of a-tocopherol over a 3-month period followed zero-order kinetics, regardless of A, or temperature. The degradation rate of a-tocopherol occurred as a nonlinear function of A,, being minimal at A, = 0.23, the monomolecular A, for the model system. Degradation rate increased at A, of 0. I 1 and 0.42. The observed effect of A, on a-tocopherol loss was similar to the relationship between A, and fatty acid oxidation described by Labuza (1980). In a fat-free model system, a-tocopherol loss increased with A, from 0.10 to 0.65 (Widicus ef al., 1980). Therefore the kinetics of a-tocopherol loss, at least in these model systems, appears to be closely linked with oxidation of unsaturated fatty acids. At a headspace oxygen : tocopherol molar ratio of 12 : 1, a-tocopherol (125 pg/g) was completely destroyed in 70 days at 30°C. However, at a ratio of 0.13 : 1, a steady-state a-tocopherol concentration of 20 pg/g was attained at 50 days. Thus only small amounts of oxygen can catalyze the destruction of a substantial quantity of a-tocopherol, and likely reflects the involvement of fatty acid free radical intermediates in the tocopherol oxidation process. Further evidence for the involvement of fatty acid oxidation products in the loss of a-tocopherol is provided by the observation of Widicus and Kirk (1981) that a-tocopherol loss rate is increased at higher initial a-tocopherol concentrations, a result discordant with zero-order kinetic theory. First-order decreases in concentration of a-and y-tocopherol in heated soybean oil have also been reported by Gertig and Duda (1984). a-To-

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ROBERT S. PARKER

copherol exhibited a more marked decline in reciprocal reaction rate, compared to y-tocopherol, as the temperature was increased from 130” to 180°C.

Domestic food preparation procedures may result in further losses of tocopherols, depending on the time, temperature, and heating method employed. DeRitter et al. (1974) measured a-tocopherol in 14 prepared frozen dinners before and after heating and observed a 15% reduction in a-tocopherol content in the cooked dinners. Booth and Bradford (1963) reported that boiling for 30 min resulted in the loss of 1-8% of tocopherols in various vegetables (brussels sprouts, cabbage, carrot). The influence of food preparation procedures on the stability of tocopherols in a variety of foods was reported by Piironen et al. (1987). Baking resulted in losses of tocopherols in bread dough of up to 50%. Stewing and roasting procedures resulted in relatively minor reductions in tocopherol content of meat and fish.

C. DIETARY INTAKE OF TOCOPHEROLS The current (1980) recommended daily intake of vitamin E is 10 International Units (IU) for adult males and 8 IU for adult females. Unfortunately, precise estimates of daily intake of tocopherols of individuals are difficult to obtain, particularly if the intakes are calculated from food composition tables as opposed to being measured directly in the prepared meals. As suggested by Bauernfeind (1977), the use of tabular values is perhaps of greatest value when applied to estimation of average daily intakes by relatively large populations. Regardless of the technique used to assess intake, considerable variation exists in reported values of individual daily intake of vitamin E, which is usually expressed as milligrams a-tocopherol, milligrams total tocopherol, or International Units per day. Smith et al. (1971) measured the a-tocopherol content of foods consumed by 50 individuals, and estimated that a-tocopherol intake varied from 1 to 10 mg per day, with 36 subjects consuming less than 5 mg per day. Bunnell et al. (1965), also employed tocopherol analysis but constructed “typical” U.S. meals, and estimated a-tocopherol intake to be between 2.6 and 15.4 mg per day, with an overall average of 7.4 mg per day. Bieri and Poukka-Evarts (1973, 1974) reported average U.S. intake to be 9 mg a-tocopherol per day using tocopherol analysis of a variety of investigator-selected cafeteria meals, with a range of 4.4 to 12.7 mg/day. The latter study also reported y-tocopherol intakes of about 2.5 times that of a-tocopherol. The USDA Continuing Survey of Food Intake of Individuals (1985) estimated daily intake of vitamin E of women and children over 4 consecu-

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tive days, using food records and food composition data banks. Mean daily intakes at the 50th percentile were (in a-tocopherol equivalents) 4.7 for children aged 1-5, and 6.2 for women aged 19-50. Intake at the 10th percentile were 2.9 for children and 3.0 for women. The 90th percentile intakes were estimated at 8.1 for children and 11.2 for women. About 10% of children and 20% of women had intakes below 50% of the RDA, while 37% of children and 30% of women had intakes of at least 100% of the recommended daily allowance (RDA). The contribution of specific foods to vitamin E intake depends not only on the tocopherol concentration and composition of the individual foods, but on the amount and frequency of consumption. Haile et al. (1986) attempted to identify important tocopherol sources using data obtained from a detailed food frequency questionnaire. The questionnaire included 429 foods, selected to contribute either 0.5 mg a-tocopherol equivalents, I g dietary fiber, 4 g total fat, 250 IU total vitamin A, or 3 mg vitamin C. The population polled was 60% female with a mean age of 59 years. The top vitamin E contributors were found to be peanuts or cashew nuts, almonds, raw or baked apples, lettuce plus dressing, sunflower seeds, oranges, peanut butter and peanut butter cookies, raw cabbage, tomatoes, oatmeal cookies, bananas, avocados, carrots, and brownies. The mean vitamin E intake was 6.7 mg per person per day. For several of these items (peanut butter and oatmeal cookies, brownies) invisible vegetable oil is the indirect contributor. The cabbage entry is surprising due to the reported low tocopherol content of this vegetable (Lehmann et al., 1986). Lehmann et al. (1986) described the important dietary a-tocopherol contributors in a typical diet designed to contain 2400 kcal and 30 g linoleic acid. The 10 top food tocopherol sources were, in order of their contribution, baked haddock prepared with 10 g safflower oil, stewed tomatoes, cooked carrots, margarine, asparagus, mayonnaise, brownies, shortbread cookies, grapefruit segments, and orange juice. The total a-tocopherol content of the entire menu was 13.8 mg. This same menu contained 37.5 mg y-tocopherol, supplied primarily from margarine, mayonnaise, brownies, and cookies. It has generally been assumed that human diets higher in polyunsaturated fats (from linoleic acid) will also be higher in a-tocopherol equivalents, such that the a-tocopherol : linoleic acid ratio will remain within an adequate range. This position is based on the assumption that the most important sources of linoleic acid (vegetable oils) are also the best sources of a-tocopherol. While for some oils this is true, many of the common vegetable oils supply mostly y- or &tocopherol. Thus, in diets high in polyunsaturated corn or soybean oils, the increase in linoleic acid content may not be paralleled by an equivalent increase in dietary a-tocopherol

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equivalents. Evidence for such a nonparallel intake of a-tocopherol and polyunsaturated fatty acids was recently reported by Lehmann et al. (1986). Tocopherol and fatty acid evaluation of typical diets containing either 10 g or 30 g linoleic acid indicated that, in diets supplying 3600 or 4000 kcal energy, the vitamin E contents of the low- and high-linoleic acid diets were nearly the same. This occurred because the most common source of linoleic acid in the high-linoleic acid diet was soybean oil, or margarine and mayonnaise made from soybean oil in which y-tocopherol is the major tocopherol isomer. In contrast, in diets containing 2400 kcal and either 10 or 30 g linoleic acid, the vitamin E intake increased from 8.8 mg to 17.5 mg per day, respectively. The authors also noted that, in diets low in total calories or fat calories, foods that are not usually considered significant sources of vitamin E, such as various fruits and vegetables, become quantitatively important. Ill. INTESTINAL ABSORPTION OF TOCOPHEROLS

A. MECHANISM AND EFFICIENCY OF TOCOPHEROL ABSORPTION There are few reported studies concerning the efficiency of intestinal absorption of tocopherols in humans consuming typical diets devoid of vitamin E supplements. The ability of an individual to absorb tocopherols is linked to the ability to absorb dietary triglycerides. That is, individuals with impaired fat absorption, either due to intestinal pathology or resection, or to genetic defects, typically exhibit low tocopherol utilization. Inherited disorders expressing dependency on supplemental vitamin E were recently reviewed by Elsas and McCormick (1986). Studies through 1975 of tocopherol absorption efficiency in humans and experimental animals were reviewed by Bieri and Farrell (1976), and indicated that the predominant route of absorption is via the lymphatic system. Blomstrand and Forsgren (1968) administered either dl-a-tocopheryl3,4-I4C2acetate or dl-[a-5-meth~l-~H] C tocopherol to two human female subjects (51 and 66 years of age) with malignant disease but no signs of malabsorption, and fitted with thoracic duct cannulae. The tocopherols were administered orally in 5 g olive oil, followed by 200-300 ml skimmed milk and routine hospital diets. In one subject in which lymph was collected for a total of 6 hr, 25% of the administered radioactivity from a-tocopheryl acetate was recovered. In the second subject, lymph was collected for 8 days, during which time absorption of four different tocopherols or analogs was examined in separate experiments, with lymph

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collection times of 16-24 hr each. Recovery of a-tocopheryl acetate was 28% and recovery of a-tocopherol was 21%. Interestingly, recovery of N-dl- [y-methyl-’Hltocopheraminewas 91%, and that of dl-a-tocopheramine-3,4-I4C, was 63%. All compounds were associated with chylomicrons in the lymph, and the variation in recovery was not associated with any alteration in lymph flow. Kelleher and Losowsky (1970) estimated the absorption of dl-[a-5methyl-3H]tocopherolin 50 human subjects by measuring unabsorbed fecal radioactivity over 12 days postdosing. In normal subjects, apparent absorption ranged from 51 to 86% of the dose, while in those with steatorrhea, absorption ranged from 3 1 to 83%. Thin-layer chromatographic analysis of fecal extracts showed that in all subjects more than 70% of fecal radioactivity was unchanged to a-tocopherol, and 10-15% migrated with a-tocopheryl quinone. MacMahon and Neale (1970) also used fecal excretion of radioactivity to estimate the a-tocopherol absorption in humans to be 55-79%, in close agreement with the data of Kelleher and Losowsky (1970). Thus it is apparent that high efficiency of absorption of fats does not necessarily result in similarly high efficiency of absorption of a-tocopherol. For example, Bieri and Tolliver (1982) observed up to 40% fecal excretion of a-tocopherol in rats in which efficiency of fat absorption approached 95%. (In rats fed retinoic acid in place of retinyl palmitate, fecal tocopherol excretion increased to nearly 70% with no reduction in fat absorption efficiency.) These results confirmed and expanded upon an earlier study (Bieri et al., 1981) in which a-tocopherol absorption in rats fed a normal diet was only 18% of a dose of radiolabeled a-tocopherol emulsion introduced directly into the duodenum. Most of the rats had a substantial proportion of the dose remaining in the cecum and large intestine. The reason for the relatively low efficiency of intestinal absorption of a-tocopherol (or other tocopherols) in rats or humans is at present unclear. There is evidence from human studies that absorption efficiency of @-carotene,thought to be absorbed in a manner similar to tocopherols, is also relatively poor. For example, Goodman et al. (1966) measured the intestinal absorption of [ 15, 15’-’H]@-carotenein two humans by cannulation of the thoracic duct and complete lymph collection following oral dosing. Only 9% recovery of radioactivity (sum of all forms, including retinyl esters and other metabolites) was observed in one subject and 17% in the other subject. The contribution of gastric or intestinal destruction of tocopherols to their low apparent absorption efficiency relative to that of dietary fats has not been clearly defined. In rats fed retinyl palmitate and fitted with

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mesenteric lymph duct cannulae, Bieri et al. (1981) recovered only 66% of a dose of radiolabeled a-tocopherol introduced directly into the upper duodenum. Recovery increased to 82% in rats fed 4 mg/kg retinoic acid in place of retinyl palmitate. The authors suggested that perhaps 8% of the dose may have been excreted in feces (not collected in the experiment). Thus actual recovery, including that in feces, may have approached 75-90%. If intestinal destruction of a-tocopherol were of quantitative significance, then it might be expected that (1) substantial amounts of tocopheryl quinone would be routinely found in feces, (2) fecal recovery of the free alcohol form would be less than the acetate form, and (3) increased amounts of tocopheryl quinone would be recovered in lymph from rats fed the free alcohol form compared to those fed the acetate form. Bieri and Tolliver (1982) did report less fecal recovery of atocopherol compared to a-tocopheryl acetate (30 versus 37% of dose) but the difference was not statistically significant. Regarding the second possibility, Bieri et al. (1981) reported that the quinone form accounted for less than 3% of total lymph tocopherols following an intraduodenal dose of [a-3H]tocopherol.Since the acetate form is more stable than the free alcohol to oxidative degradation, lymph recovery of quinone from the former would be expected to be even less, although such a comparison has not been reported. Thus it appears that in rats fed semipurified diets, intestinal destruction probably does not account for the relatively low absorption efficiency of a-tocopherol. The predominance of a-tocopherol over other tocopherol isomers in human and animal tissues does not appear to be largely the result of high selectivity of intestinal absorption for a-tocopherol isomers. Peake et al. (1972) compared the absorption and lymphatic and plasma transport of aand y-tocopherol in the rat. Recovery of more than 90% of duodenally infused tocopherols in lymph, feces, and intestine indicated that nonlymphatic absorption played at best a minor role. y-Tocopherol appeared to be absorbed at 85% the extent of a-tocopherol, a minor difference compared to the predominance of the a-form in plasma and tissues. In addition, clear differences in lymph or plasma transport, or in tissue uptake of the two isomers were lacking. It was apparent, however, that y-tocopherol was lost more rapidly than a-tocopherol from plasma and tissues, and thus differences in clearance rates seem to account for much of the difference in biopotency of these two common dietary tocopherols. Behrens and Mad&re(1983) presented evidence suggestive of an effect of dietary a-tocopherol on the absorption or retention of y-tocopherol. In separate groups of rats fed a tocopherol-deficient diet for 3 months, plasma y-tocopherol concentration rose 10.8 k 3.8 pg/ml following a dose of 50 mg a-tocopherol. However, when 50 mg of each vitamer was given

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simultaneously, the 24-hr y-tocopherol level was only 1.2 2 0.3 pg/ml while the a-tocopherol level was unaffected at 9.5 2 1.5 pg/ml. These data indicate that the two vitamers are probably absorbed and transported with equal efficiency when only one vitamer is present. However, simultaneous administration (at least at relatively high levels) seems to result in preferential absorption of the a-isomer or an accelerated rate of elimination of the y-isomer. Since only 24-hr blood samples were analyzed, discrimination between these two possibilities was not possible. The data of Behrens and Madbre (1983) also indicated that previous tocopherol intake may influence absorption (or elimination) rates. Rats previously fed for 3 months a diet containing 938 mg/kg a-tocopherol and 32 mg/kg ytocopherol, then maintained on a tocopherol-free diet for 3 days prior to intragastric administration of 50 mg y-tocopherol exhibited a 24-hr plasma y-tocopherol level of only 4.6 & 2.0 Fg/ml, half that of rats maintained on a low-tocopherol diet for the entire period. Rats maintained on a diet containing 208 mg/kg a-tocopherol and 32 mg/kg y-tocopherol until 3 days prior to y-tocopherol administration exhibited a 24-hr plasma y-tocopherol value of 5.9 ? 2.0 pg/ml, intermediate between the other two treatments. Assuming that the 3-day washout period was sufficient to empty the intestine of tocopherols, it appears that some aspect of y-tocopherol absorption and/or metabolism was influenced by prior a-tocopherol intake. In adult men fed stipulated diets containing a daily average of either (1) 9.7 mg a-tocopherol, 10.5 mg y-tocopherol, and 10 g linoleate, or (2) 13.9 mg a-tocopherol, 42.4 mg y-tocopherol, and 30 g linoleate (Lehmann et af., 1984), plasma a-tocopherol levels after 6 weeks were similar between the two diets (10.1 and 9.8 pg/ml for diets 1 and 2, respectively). However, plasma y-tocopherol levels of diet 2 were nearly double those of diet I (2-3 versus 1.5 Kg/ml). Erythrocyte and platelets showed similar effects, demonstrating that, on average, a fourfold increase in dietary ytocopherol without a substantial corresponding increase in a-tocopherol results in a doubling of circulating levels of the y-isomer.

B. EFFECT OF DIET COMPOSITION ON BIOAVAILABILITY OF TOCOPHEROLS Several studies concerning the effect of dietary fibers on tocopherol bioavailability have been reported in recent years, prompted at least in part by the observed reduction in cholesterol absorption by these dietary constituents. DeLumen et af. (1982) observed that young rats fed 40 IU/ kg a-tocopherol and 10% pectin exhibited a 50% reduction in plasma tocopherol levels and a 40% reduction in erythrocyte tocopherol levels

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compared to pair-fed controls, with the majority of the decline occurring during the initial 2 weeks. However, since plasma cholesterol levels were also reduced by nearly 50% in the pectin group at 28 days, it is impossible to tell whether the drop in plasma and erythrocyte tocopherol levels was due to impairment of tocopherol absorption or a reduction in the blood transport capacity. The fact that tissue levels (liver, lung, heart) also exhibited large reductions in tocopherol content with pectin feeding indicates that impaired absorption was the likelier of the two possibilities. Adipose tissue tocopherols were not measured. More recently, Schaus el al. (1985)examined this pectin effect more closely, feeding 40 IU/kg vitamin E and 0,3,6, or 8% citrus pectin to rats over a similar 8-week period. Three percent pectin had no effect on plasma, heart, or liver tocopherol levels. Six percent pectin significantly reduced liver tocopherols by approximately 50%, increased erythrocyte hemolysis three-fold, and reduced plasma tocopherol by 30%, although the latter was not statistically significant. Eight percent pectin significantly reduced tocopherol levels in plasma and liver, and increased erythrocyte hemolysis to the same extent as 6% pectin by 8 weeks. Thus modest amounts of dietary pectin have no effect on vitamin E status in rats over a 2-month period, while higher levels reduce the bioavailability of this vitamin. No studies on the influence of pectin on the biopotency of tocopherols (using models of reversal of vitamin E deficiency syndromes) have been reported. The mechanism of the decrease in tocopherol bioavailability at high pectin levels is unknown. Schaus e f al. (1985) observed no significant increase in fecal fat of rats fed 6 or 8% pectin, and demonstrated earlier (Omaye et al., 1983) that pectin does not bind vitamin E in vitro. Wheat bran has also been shown to influence plasma tocopherol levels in rats. Omaye and Chow (1984a) reported that rats fed 60 mg/kg a-tocopherol and 20% wheat bran exhibited plasma tocopherol levels 28% lower than rats fed 5% wheat bran. A possible time-dependent nature of this fiber effect was shown in a subsequent study (Omaye and Chow, 1984b) in which 20% wheat bran (relative to 5 or 0% wheat bran) decreased plasma a-tocopherol levels of rats at 35 days, but not at 56 days of elevated fiber intake. These authors suggested a compensatory mechanism of adaptation which reversed a temporary adverse effect of wheat bran on a-tocopherol bioavailability . More recently, Mongeau et al. (1986)fed rats diets containing a higher level of a-tocopherol (200 mg/kg) and either 0,2,4,6,8, or 10% by weight fiber from wheat bran for a total of 42 days. (Wheat bran is approximately 50% fiber.) Plasma, white adipose (site unspecified), and pancreas a-tocopherol levels were all essentially unaffected by increasing levels of bran. All bran fiber levels decreased liver a-tocopherol content by about

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25% relative to a fiber-free diet. Therefore, there was no dose-related wheat bran effect on any parameter of a-tocopherol status in these rats as the dietary fiber content was varied from 2 to 10%. However, these results must be viewed in the context of a relatively high dietary a-tocopherol level, and a relatively long duration of dietary treatment. As pointed out by Omaye and Chow (1984b), wheat bran may exert only a temporary decrease in a-tocopherol bioavailability, such that compensation (or 'loss of effect) had occurred by the end of the 42-day period employed by Mongeau et al. (1986). Interestingly, Mongeau et al. (1986) observed a dose-related increase in white adipose y-tocopherol concentration with increasing wheat bran intake, such that the concentration of this vitamer was nearly doubled in rats consuming 10% wheat bran relative to the fiber-free diet. Since y-tocopherol intake (essentially all from corn oil) was similar among all groups, wheat bran may have increased the efficiency of absorption of y-tocopherol. The lack of an effect of wheat bran on y-tocopherol levels in either plasma or liver may reflect efficient metabolism and clearance of this vitamer except in those tissues with low tocopherol turnover rate (e.g., white adipose tissue). A transient decrease in plasma a-tocopherol levels in rats fed 40 mg/kg a-tocopherol and 10% fiber (from cellulose or wheat bran), relative to 5% cellulose fiber, was also reported by Kahlon et al. (1986). This decrease had disappeared by 3 weeks. Liver a-tocopherol levels in rats fed 10% fiber from coarse wheat bran (2 mm) were significantly lower than those of rats fed 10% fine (0.5 mm) wheat bran fiber, 10% cellulose, or 5% cellulose for 6 weeks. The authors suggested this apparent decrease in bioavailability of a-tocopherol in the coarse bran diet may have resulted in alterations in gut morphology or in physiological processes of the intestinal lumen. Their concurrent observation that all the 10% fiber groups exhibited significantly higher liver vitamin A levels suggests a specific effect on absorption efficiency of tocopherols, rather than a general effect on all dietary lipids. Two reports have indicated that dietary fish oils may reduce the bioavailability of tocopherols. Mackenzie et af. (1941) reported that dietary vitamin E supplementation could not reverse this effect in rabbits. More recently, Meydani et al. (1987) found that mice fed 5% fish oil maintained lower plasma and tissue a-tocopherol levels than mice fed corn or coconut oils. This effect occurred at 30, 100, and 500 mg/kg (diet) a-tocopherol. In addition, the plasma and liver a-tocopherol content of the fish oil-fed mice did not increase significantly as the dietary a-tocopherol level was increased from 30 to 500 mg/kg. The authors suggested fish oil may have interfered with the absorption of a-tocopherol or increased its postabsorptive utilization.

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IV. TRANSPORT OF TOCOPHEROLS IN BLOOD

Bjornson et al. (1976) reported the amounts and relative proportions of a-tocopherol in fasting plasma lipoproteins in three normal and four hypertriglyceridemic subjects. In normal subjects, low-density lipoproteins (LDL) contained the majority of a-tocopherol in two subjects, while in one subject high-density lipoprotein (HDL) was highest. In hyperlipidemic subjects total plasma a-tocopherol was significantly elevated, with the very-low-density lipoprotein (VLDL) fraction (not separated from chylomicrons) invariably containing the greatest proportion of total a-tocopherol. The concentration of a-tocopherol in the individual lipoprotein classes was not determined, but the results likely reflect the increased number of particles in the VLDL fraction in these subjects. In another subject, who received 2 g of a-tocopheryl acetate daily for 3 weeks, total plasma tocopherol more than doubled but the relative distribution among lipoproteins was unchanged. Behrens et al. (1982) also studied the distribution of a-tocopherol in lipoproteins of normal human plasma collected in the fasting state. This study employed gel filtration separation of lipoprotein classes, HPLC analysis of a-tocopherol (in contrast to the spectrophotometric method employed by Bjornson et al., 1976) and reported estimates of tocopherol concentration in each lipoprotein fraction on a protein basis. In males (n = 6) most of the total plasma a-tocopherol was associated with the LDL fraction (59 2 3%), with 33 4% associated with HDL and 8 5 1% with VLDL. In females (n = 6), the majority of plasma a-tocopherol was associated with HDL (56 5 3%), with 42 3 and 2 0.3% associated with LDL and VLDL, respectively. However, on a protein basis in males, VLDL exhibited the highest concentration of a-tocopherol (7 pg/mg protein), followed by LDL (4 pglmg), and HDL (1.5 pg/mg). In females, LDL showed the highest concentration (4.7 pg/mg), followed by VLDL (3.9 pg/mg) and HDL (2. I pg/mg). The difference in the proportion of atocopherol carried by LDL and"HDL between males and females could be accounted for by a higher level of HDL-protein in females. In contrast to the situation with LDL, the mass of a-tocopherol in HDL was highly correlated with the amount of HDL-protein (r = 0.93), causing the authors to speculate on a more specific manner of binding of a-tocopherol to HDL than to LDL. It is now established that tocopherol can exchange between various lipoprotein classes. The kinetics of transfer of a-tocopherol between model and native lipoproteins was reported by Massey (1984). Transfer of [3H]a-tocopherolfrom apolipoprotein AI- I-palmitoyl-2-oleoylphospha-

*

*

*

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tidylcholine (POPC) complexes to POPC-ganglioside vesicles showed that a-tocopherol mass spontaneously transferred with a half-time of 65 min at 37°C. about three times slower than cholesterol transfer. The transfer of a-tocopherol between apolipoprotein AII-dimyristorylphosphatidylcholine vesicles occurred with a half-time of 40 min. Transfer of a[3H]tocopherolfrom HDL to LDL or VLDL was also studied. At equilibrium (60 min at 37"C), 75% of HDL radioactivity transferred to VLDL, and 25% to LDL. A transfer of 83% to VLDL was predicted according to the total lipid mass of HDL and VLDL. The time required for equilibration of a-tocopherol between lipoproteins is thus much shorter than the lifetimes of lipoproteins in the circulation, and these data corroborate the data of Bjornson et al. (1976), which showed that the relative tocopherol content in plasma lipoproteins is best correlated with the total lipid content of each class. Reversible mass transfer of triglyceride and cholesteryl ester between VLDL and LDL or HDL in plasma has been shown to be facilitated by a neutral lipid transfer protein (Nichols and Smith, 1965; Quarfordt et al., 1971 ; Deckelbaum et al., 1986). This protein exchanges triglyceride from VLDL for cholesterol ester from either LDL or HDL, in a mass ratio of I : 1. Evidence that this protein is not involved in the transfer of tocopherol between lipoproteins was recently presented by Granot et al. (1988). Using lipoproteins labeled in vitro with either ['4C]trioleinor d-[a-5-methyl-3H]tocopherol,tocopherol transfer from one lipoprotein to another was not correlated with transfer of neutral lipid over a 10-fold range of the latter. Rather, transfer from VLDL to HDL was dependent on the ratio of the concentration of the lipoproteins in the incubation system. Approximately 30% of VLDL radioactivity transferred to HDL at an HDL: VLDL protein ratio of I .O, whereas 50% transferred at a particle protein ratio of 10. Contrary to suggestions of earlier studies of tocopherol distribution between fasting plasma lipoproteins, transfer was not strictly related to particle lipid content. For example, using an LDL : HDL protein ratio of 2 : I , at which the corresponding lipid ratio is approximately 8 : I , a-tocopherol transfer from LDL to HDL approached 60% of the starting LDL label. Similar experiments using in vivo-labeled lipoproteins have not been reported. Bjgirneboe P t al. (1987) described the appearance of [a-3H]tocopherolin rat plasma HDL following intravenous injection of in vivo-labeled lymph chylomicrons. Ten minutes after injection most (91%) of the serum radioactivity was associated with chylomicrons, and only 8% with HDL. After 2 hr, 35% of serum radioactivity was in chylomicrons and 51% with HDL. (It should be noted that HDL are the major circulating lipoproteins in the rat, in contrast to humans.) The authors

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suggested that redistribution of a-tocopherol into HDL followed liver secretion of VLDL, since chylomicron-to-HDL transfer of a-tocopherol is slow. Due to obvious diurnal and possibly seasonal differences in dietary tocopherol intake in most humans, fluctuations in plasma tocopherol levels over similar time frames might be expected. However, Niernberg and Stukel (1986)reported that no signifcant diurnal or seasonal variations in plasma a-tocopherol concentration were observed in 15 normal adult volunteers. No estimates of tocopherol intake were obtained. The absence of measurable diurnal fluctuations might be due to a combination of slow sustained release of tocopherol from lymphatics to general circulation, efficient postprandial clearance of tocopherol from chylomicrons and uptake of chylomicron remnants by liver, and the presence of a relatively large exchangeable storage pool which would serve to buffer temporal differences in intake. While large fluctuations in total plasma a-tocopherol levels may not occur, postprandial changes in tocopherol content of specific lipoprotein fractions apparently do take place, as described by Meydani et al. (1985). Blood from seven female and four male subjects was analyzed after a 12hr fast and at 0, 3, 6,9,and 12 hr after consumption of 0.02% of RDNkg of a-tocopherol in a defined meal. In fasting blood, 27% of the total plasma a-tocopherol was associated with the chylomicron- + VLDL fraction (d < 1.006 g/ml), 35% with LDL (1.019-1.063g/ml), and 38% with HDL (1.063-1.210g/ml). Only trace amounts were found in intermediatedensity lipoprotein (IDL). Postprandial a-tocopherol levels peaked at 6 hr after a 49% increase in the amount of a-tocopherol in the chylomicron-VLDL fraction. The authors estimated that half of this increase was due to new intestinal input and half resulted from transfer from LDL and HDL, although the means of calculating these relative contributions was not detailed. The changes in total plasma tocopherol concentration were not reported following intake of this small dose, which would average approximately 100 pg per subject. Recently, Traber et al. (1988)used deuterated d-a-tocopheryl acetate to follow the incorporation of ingested tocopherol into various lipoprotein classes over time in a small number of humans. The use of isotopically labeled a-tocopherol permits the distinction between preexisting endogenous a-tocopherol and newly absorbed a-tocopherol. This is necessary since the postabsorptive change in total mass of tocopherols in the various lipoprotein and cellular pools of tocopherol in the blood may be small, and exchange between pools is known to occur. Label from a dose of 15 mg deuterated 2R,4'R,8'R-a-5,7-[C2H,],-tocopheryl acetate appeared

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most rapidly in the chylomicron fraction of the plasma, peaking at 5 hr, with a second small chylomicron peak at 12 hr which corresponded with the peak in total plasma deuterium. Only up to 10% of the chylomicron a-tocopherol, and up to 4% of the total plasma a-tocopherol consisted of the labeled a-tocopherol with this dose, which was consumed with a normal breakfast following an overnight fast. In time course studies with larger doses (140 mg) of deuterated a-tocopherol, the order of lipoprotein labeling observed was chylomicrons first, followed by VLDL, then LDL and HDL, the latter two exhibiting similar kinetics of labeling. Deuterated a-tocopherol appeared in erythrocytes only after its appearance in LDL and HDL. At 3 hr, the chylomicron fraction contained nearly 40% of the deuterated a-tocopherol present in plasma, with VLDL containing nearly 50%. Interestingly, the chylomicron data suggested that the intestine continued to secrete chylomicrons containing deuterated a-tocopherol up to 24 hr following ingestion of the dose. In another subject given 60 mg deuterated a-tocopherol, 56% of the plasma deuterated tocopherol was present in HDL at 3.5 hr, while only about 8% was in the chylomicron fraction. The authors concluded that the results support the notion that tocopherol absorption occurs primarily though a process involving chylomicron formation, and that newly ingested a-tocopherol equilibrates between LDL and HDL. The results do not permit determination of whether the majority of a-tocopherol in postabsorptive plasma arises from the catabolism of VLDL to LDL or from the transfer of tocopherol from one lipoprotein particle to another. However, these studies do illustrate the value of deuterated tocopherols in the study of tocopherol physiology and metabolism in humans. The formed elements of the blood, which include red blood cells, white blood cells, and platelets, also contain tocopherols. Presumably, tocopherols become associated with these fractions both during their formation (e.g., in bone marrow for erythrocytes) and during their circulating phase via passive diffusion or facilitated transfer from the various lipoproteins. Reported ranges of concentration of a- and y-tocopherol in the various cellular components of the blood in relation to plasma tocopherol concentrations have recently been summarized by Chow (1985). Unfortunately, most measurements have been expressed on a cell number or blood volume basis, which can be misleading. For example, leukocytes have been reported to contain more a-tocopherol than erythrocytes on a per cell basis, but since the former contain more total membrane mass (phospholipid), the cellular membrane concentrations are likely to be more similar than would otherwise be indicated.

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V. TOCOPHEROLS IN TISSUES A. TOCOPHEROL LEVELS IN HUMAN TISSUES

There is a large body of information concerning the relationship between dietary level and tissue concentration of a-tocopherol in experimental animals. In general, the tissue concentration in adult animals is related to the log of dietary intake (Bieri, 1972). Thus a 10-fold increase in dietary intake is needed to achieve a doubling in the concentration of a-tocopherol in most tissues. The exception is adipose tissue, in which the tocopherol concentration appears to increase with age at constant dietary intake (Bieri, 1972). Tissue levels of a-tocopherol seem difficult to saturate, as Machlin and Gabriel ( 1982) found tissue concentrations continue to increase through 20 weeks of feeding 10.000 ppm a-tocopherol to mature rats. However, the cellular and subcellular disposition of tissue tocopherols in animals fed such high levels of a-tocopherol has not been determined; thus, the relationship between dietary level and concentration in specific subcellular membranes over a wide range in dietary levels is largely unknown. Such information would be desirable since cellular membranes are the presumed site of a-tocopherol function. Since many tissues contain adipocytes or other fat-storing cells, and since mobilization from adipocytes may either not occur or occur slowly, whole-organ a-tocopherol levels may not be representative of all cell types of that organ. Since different cell types are likely to exhibit different rates of tocopherol uptake and turnover, substantial differences in steady-state atocopherol content of different cell types within the same organ are also likely. Much of the available information concerning the tocopherol content of human tissues has been summarized by Gallo-Torres (1980). Values for only a relatively small number of tissues have been obtained, and many of the analyses have utilized nonspecific methods of tocopherol quantitation. However, it is evident that interindividual differences (unrelated to age) in organ or tissue tocopherol content are considerable. Liver and adipose tissues have been the most widely studied in this regard in the adult. Recently, Kayden and Traber (1987) reported adult liver tocopherol concentrations to range from 5.6 to 23.5 mg/g wet weight in three normal subjects, and from 1.1 to 19.3 in subjects with a P-lipoproteinemia. Kayden (1983) reported the total tocopherol content of needle biopsy samples taken from four adults to be 262 33 pg/g tissue. A much larger variation in tocopherol concentrations in deep abdominal adipose tissue was reported by Parker (1988), with a-tocopherol values ranging from 61 to 811 pg/g tissue. This study also reported concentrations of other

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tocopherol vitamers. y-Tocopherol and 8-tocopherol concentrations ranged from 17 to 203 Fg/g and 1.0 to 15.0 Fg/g, respectively. A correlation of 0.84 between a-tocopherol and &tocopherol concentrations in these subjects was observed. a-Tocopherol represented an average of 81% of total adipose tissue tocopherols, with a range of 70-95%. In general, the tocopherol composition in adipose tissue appears to be similar to that reported for adult human plasma by Piironen et al. (1984) using similar analytical techniques. Adipose tissue tocopherol appears to be located in the bulk lipid phase of this tissue, and may represent up to 9% of total body tocopherol in the human (Traber and Kayden, 1987). Little information on the a-tocopherol content of human tissues other than liver and adipose tissue is available. Such data, obtained in conjunction with the analogous plasma tocopherol values from the same donor individuals, would help to clarify both the range and frequency distribution of tissue tocopherol levels in humans, and also the value of plasma levels in predicting those in solid tissues. 9. TOCOPHEROLS IN HUMAN MILK

A study of the tocopherol composition of human milk by Syvaoja et al. (1985) found that the total tocopherol concentration in milk decreased

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with progressively later stages (2.06 f I .73,0.74 & 0.25, and 0.56 0.18 mg/100 g in colostral, transitional, and mature milk, respectively). a-Tocopherol was the major isomer present at all stages, making up 92, 88, and 85% of total tocopherols in colostral, transitional, and mature milk, respectively. The small decrease in a-tocopherol contribution with stage was accompanied by a rise in the y-tocopherol proportion of total tocopherols (5, 9, and 12% in colostral, transitional, and mature milk, respectively). Substantial interindividual differences in colostral a-tocopherol content were found, ranging from 0.74 to 6.26 mg/100 g milk. The proportion of total tocopherols as a-tocopherol was higher than the values reported by Kobayashi et al. (1975) and Jansson et al. (1981), and the 6tocopherol proportion (trace) lower than in previous reports (Kobayashi et al., 1975; Bell, 1980). The temporal changes in tocopherol concentration or composition could not be accounted for by differences in total lipid content of the milks (3.43 k 1.27, 3.90 f 1.03, and 4.47 f 1.06 g/100 g for colostral, transitional, and mature milk, respectively). Total lipid and a-tocopherol contents were correlated within transitional ( r = 0.75) and mature ( r = 0.70) milk samples, but not in colostral samples. The changes in tocopherol content were also not correlated with linoleate content of the milk, which remained relatively constant at 0.25 d100 g milk.

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C. TOCOPHEROLS IN OCULAR TISSUES The occurrence of tocopherols in human ocular tissues has been the subject of several recent investigations due to interest in the use of atocopherol to prevent retinopathy of prematurity in preterm infants or age-related macular degeneration. Premature and low-birth-weight infants are susceptible to vitamin E deficiency, since placental transfer is poor and there is limited adipose tissue for storage (Winnick, 1980). Supplemental vitamin E has been used in these situations to ameliorate or prevent retrolental fibroplasia, hemolytic anemia, and neonatal hyperbilirubinemia in infants who receive oxygen (Bieri et al., 1983). Using eye bank donor tissues, Organisciak ef al. (1987) observed the a-tocopherol concentration in retinal pigment epithelium to be 4-7 times higher than that of the retina. Similar intertissue differences were reported earlier by Stephens ef al. (1986). The concentration in both tissue types was age-dependent, with retinal pigment epithelium tocopherol levels 3-4 times higher by 80 years than at 20 years of age. Substantial variation in tissue a-tocopherol content was apparent even with the same age group, presumably due to differences in dietary intake. The influence of a-tocopherol supplementation on the tocopherol content of ocular tissues was studied in newborn kittens by Bhat et al. (1987). Administration of a single dose of 100 mg dl-a-tocopherol resulted in a 5fold increase in retinal a-tocopherol. Interestingly, retinal a-tocopherol levels remained elevated even when the plasma levels were declining, and retinal levels did not reflect plasma levels at any time. a-Tocopherol was also observed in choroid and vitreous tissues, although the levels were much lower than those in retinal tissue. Also noted were significant differences in tocopherol content between eyes of the same animal. Such intraindividual differences have not been studied in humans, nor has the plasma-ocular tissue tocopherol relationship.

D. MECHANISMS OF TISSUE UPTAKE OF TOCOPHEROLS The mechanism by which tocopherols are taken up by tissues from the bloodstream is incompletely understood at present. However, two recent studies have shown that receptor-dependent LDL uptake is probably one important aspect of this process. Traber and Kayden (1984) incubated normal and LDL-receptor-deficient human fibroblasts with LDL isolated from normal human serum. Normal fibroblasts exhibited a doubling of cellular a-tocopherol in 4 hr, while receptor-deficientfibroblasts (originating from a subject with homozygous familial hypercholesterolemia) showed no change in tocopherol content when both cell types were incu-

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bated with 100 pg LDL protein per milliliter. Normal and receptor-deficient cells showed 5-fold and 2-fold increases, respectively, in cellular tocopherol when incubated for 24 hr with 100 pg LDL protein milliliter, and higher LDL concentrations had no additional effect. All cells were routinely preincubated with lipoprotein-deficientserum for 24 hr to stimulate LDL receptor synthesis (in normal fibroblasts only). No data were provided on tocopherol uptake in nonpreincubated cells. Interestingly, both types of cells exhibited the same initial cellular tocopherol content following the preincubation but not exposed to LDL, and the presence of LDL in the medium resulted in an increase in cellular tocopherol in receptor-deficient cells after 24 hr incubation. This suggested additional mechanism(s) of tocopherol uptake, perhaps either via receptor-independent endocytosis or by internalization-independenttransfer of a-tocopherol from LDL to the fibroblast plasma membrane. As discussed in the preceding section, tocopherol can readily transfer between lipoproteins. As noted by Traber and Kayden (1984), the existence of other mechanisms is consistent with the observation that patients with homozygous hypercholesterolemia do not necessarily become vitamin E deficient. Thellman and Shireman (1985) extended the studies of Traber and Kayden (1984) by using LDL labeled with RRR-[a-5-methyl-3H]tocopherol and "'I. Confluent cultures of normal and LDL-receptor-deficient human fibroblasts (both different from the cell lines used by Traber and Kayden, 1984) were preincubated for 24 hr in lipoprotein-deficientserum to induce receptor synthesis, followed by incubation with labeled LDL at varying concentrations and durations. Incubation at 4°C allows LDL binding to coated pits but inhibits internalization of the LDL particle. Uptake of 3H by receptor-deficient fibroblasts was approximately 35% that of normal cells, while uptake of '"I by the deficient cells was approximately 45% that of the normal cells when incubated at 37°C for 2 hr. Incubation of normal fibroblasts at 4°C completely blocked "'1 uptake, and reduced [a-3H]tocopheroluptake (2 hr) to 25% of that at 37°C. Also, uptake of a-[3H]tocopherolfrom methylated LDL by normal fibroblasts was approximately half the uptake from native LDL. These data confirm those of Traber and Kayden (1984) that receptor-mediated LDL internalization is an important mechanism of uptake of a-tocopherol by cultured human fibroblasts. They also indicate that 25 to 50% of total tocopherol uptake by fibroblasts takes place by mechanism(s) other than internalization of receptor-bound LDL. Uptake at 4°C or from methylated LDL may indicate diffusion of a-tocopherol from receptor-bound LDL to plasma membrane. Thellman and Shireman (1985) have pointed out that accurate determination of uptake via specific (receptor-dependent) binding, which normally requires the use of excess unlabeled LDL, is made difficult by

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the fact that [a-3H]tocopherolcan facilely transfer between LDL particles, unlike 1251-labeled apoprotein. The relative importance of the LDL receptor-mediated pathway of tocopherol uptake also cannot be estimated at this time since both Traber and Kayden (1984) and Thellman and Shireman (1985) employed “normal” fibroblasts with induced levels of LDL receptor activity. In noninduced cells, or in other cell types with lower LDL receptor activity, LDL receptor-dependent uptake may be quantitatively less important. Clearly, cell types with differing LDL internalization capacities need to be studied in this regard. If LDL receptor-mediated uptake is a quantitatively important determinant of tissue tocopherol levels, then one might expect that those tissues with the highest rates of LDL clearance would have higher tissue tocopherol levels. Indeed, several rat tissues that typically exhibit relatively low tocopherol contents, such as muscle, pancreas, and nervous tissue, have been found by Spady et al. (1985) to have low (or negligible) LDL receptor activity. Conversely, some tissues with high a-tocopherol content (e.g., adrenal gland) exhibit relatively high rates of LDL clearance in vivo. However, other tissues with low LDL receptor activity such as heart and adipose tissue exhibit relatively high tocopherol contents (Machlin, 1984). These tissues apparently rely on other mechanisms to obtain tocopherol, unless of course the rate of utilization of a-tocopherol in these tissues is correspondingly low. Unfortunately, there are no quantitative data on the comparative rates of in vivo utilization of a-tocopherol in different tissues. A quantitatively important role for the LDL receptor pathway in tissue acquisition of a-tocopherol may also mean that conditions which downregulate cell surface LDL receptor activity also decrease the rate of uptake of tocopherol from the bloodstream. The observation that hypercholesterolemic individuals have higher circulating levels of a-tocopherol (Bjornson et al., 1976) supports this suggestion. On the other hand, the apparent existence of receptor-independent tocopherol uptake suggests a means of compensation in instances of low receptor activity in order to maintain tissue tocopherol levels. While LDL-mediated tissue a-tocopherol uptake is clearly indicated, roles for other tocopherol-transporting plasma lipoproteins have not been investigated. These include HDL, VLDL, and chylomicrons. HDL-specific receptors have been identified in many tissues, and HDL has been observed to transport a greater proportion of total plasma tocopherol than LDL in some individuals (Behrens et al., 1982). Kayden and Bjornson (1972) and Bjornson et a / . (1975) reported that spontaneous transfer of atocopherol to erythrocytes occurred more rapidly in vitro from HDL than

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from VLDL or LDL. Bjornson et af. (1975) suggested that this difference may be due to differential molecular positioning of the tocopherol in the various lipoproteins, in that HDL tocopherol may be more peripherally located than LDL tocopherol. Human studies have also indicated the possibility of an important role for HDL in tissue uptake or release of atocopherol. Aftergood et d. ( 1975) reported that contraceptive administration concomitantly lowered both HDL and total plasma tocopherol in female subjects. Takahashi et al. (1978) found that pregnancy resulted in increased plasma tocopherol which was paralleled by an increase in plasma HDL. Indirect transfer of VLDL a-tocopherol to tissues can take place via VLDL conversion to LDL by lipoprotein lipase (LPL), followed by receptor-mediated LDL uptake. However, transfer of a-tocopherol directly from VLDL to tissues by passive diffusion cannot be ruled out as of yet. Another potentially important mechanism is chylomicron-mediated tocopherol transfer. The liver actively takes up chylomicron remnants from the bloodstream subsequent to removal of a significant proportion of chylomicron triglyceride by tissue LPL. This mechanism likely accounts for a significant proportion of clearance of plasma tocopherol following a meal. Consistent with this suggestion is the observation of Bjdrneboe et a/. (l987), who found that, following an intravenous (femoral vein) injection of a physiological amount of lymph chylomicrons containing labeled a-tocopherol, the plasma half-life of the label was 12 min, roughly equivalent to the half-life of chylomicrons in the rat. Evidence for LPL-mediated tocopherol transfer in vitro has been presented by Traber et al. (1985). Using a system containing chylomicrons (from a Type I hypertriglyceridemic)or lipid emulsion as the tocopherol donor, and LPL from bovine milk, these investigators found LPL to increase tocopherol content of human erythrocytes by 60% and of fibroblasts by 35-fold over a I-hr period. Fibroblasts incubated with chylomicrons in the absence of LPL showed no change in cellular tocopherol content. Coincubation of fibroblasts with both chylomicrons (containing 33 nmol a-tocopherol) and lipid emulsion (containing 100 nmol y-tocopherol) resulted in the transfer of equal amounts of both types of tocopherols over a I-hr period in the presence of 8 pg LPL. Simultaneous addition of both LPL and apolipoprotein CII (an activator of LPL) resulted in a nearly 6-fold increase in y-tocopherol transfer from lipid emulsion to fibroblasts compared to transfer with LPL alone. Addition of small amounts of heparin to the incubation medium inhibited tocopherol uptake during LPL-stimulated triglyceride hydrolysis, indicating that tocopherol transfer involves obligatory binding of LPL to the cell membrane. Tocopherol transfer from lipid emulsion to normal fibroblasts and LDL re-

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ceptor-deficient fibroblasts was similar, indicating a minor role for receptor-mediated lipoprotein uptake in this lipoprotein-free system. The authors suggested that this LPL-dependent pathway of tocopherol transfer from plasma to tissues may explain the low adipose tissue tocopherol level and elevated plasma tocopherol level in a patient with LPL deficiency, and the normal adipose tissue tocopherol level in a patient with LDL receptor deficiency. Since practically all published studies to date concerning blood tocopherol transport have utilized postabsorptive plasma, the roles of both chylomicrons and VLDL in distributing tocopherols in the absorptive state in normal individuals remain to be determined. Also, the relative importance of the LPL-mediated and LDL receptor-mediated tocopherol transfer to tissues of normal individuals is unknown at present. Conceivably, tissue uptake in the absorptive state involves primarily the LPL mechanism, while uptake in the fasting state is more dependent on receptor-mediated lipoprotein endocytosis. However, this hypothesis has yet to be tested in humans or other primates. Gardner et al. (1986) recently reported a biphasic pattern of disappearance of [a-3H]tocopherolfrom the rat bloodstream in vivo. Serum was labeled with radioactive a-tocopherol in vitro and injected back into the animals. The fast and slow decays exhibited half-lives of 2-4 and 289 min, respectively. Hepatectomy resulted in a monophasic decay curve with a half-life intermediate between the intact rat fast and slow decay rates. Both the rate of decay in the intact rat and the influence of hepatectomy were different for a-tocopherol when compared to triolein and cholesterol. These data suggest an important role for the liver in the clearance of a-tocopherol from blood, although in vitro labeling of serum with tocopherol has not been validated with respect to the molecular positioning of a-tocopherol in the various lipoprotein fractions relative to that in vivo. Tissue uptake of [a-3H]tocopherolin rats following injection of physiological amounts of in vivo-labeled lymph chylomicrons was reported by Bjgrneboe et af. (1987). Liver contained over 50% of the injected radioactivity after 60 min, most of which (85%) was associated with the parenchymal cell fraction. Of the nonhepatic tissues examined, muscle accumulated the highest proportion of injected radioactivity (4%), followed by adipose tissue (3%), lung (I%), and kidney (0.5%). However, extrapolation of these quantitative results to the human may not be valid since receptor-mediated LDL uptake is likely a major mechanism of tissue uptake of a-tocopherol, and rat serum LDL levels are considerably lower than those found in humans. The data of Bjgrneboe et af. (1987) regarding muscle uptake of 3H following [a-3H]tocopherolinjection are of interest since they indicated a biphasic pattern of accumulation of labeled a-tocopherol: an initial rapid uptake and plateau by 30 min following injec-

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tion, and a latter uptake between 2 and 4 hr. This may indicate an early, lipoprotein receptor-independent mechanism of uptake (possibly via LPL catabolism of chylomicrons) and a later phase, possibly reflecting receptor-mediated LDL- or HDL-tocopherol uptake. The role of plasma lipoproteins in the removal of a-tocopherol from tissues has not been investigated. If passive diffusion from membraneassociated lipoprotein particles to tissues does in fact occur, then diffusion in the opposite direction may also occur, with the rate constants for forward and reverse transfer dependent on the a-tocopherol concentrations in the two compartments. Such concentration-dependent transfer has been demonstrated for the exchange of a-tocopherol between lipoproteins and erythrocytes, with HDL being a more efficient effector of this passive transfer than LDL or VLDL (Kayden and Bjornson, 1972). Even in the rat, in which HDL carriers the majority of plasma tocopherol, exchange between erythrocytes and lipoproteins occurred most rapidly with HDL (Bjornson et al., 1975). In addition to lipoproteins, transfer of tocopherols from blood to tissues may involve donation from the formed elements of the blood which include erythrocytes, monocytes, lymphocytes, granulocytes, and platelets. Since, as discussed above, tocopherol exchange takes place between lipoproteins and erythrocytes (which lack LDL or HDL receptors), it is conceivable that exchange also takes place between blood cells and tissue cells. In fact, if it is true that lipoprotein tocopherol is located exclusively in the core lipids and not exposed on the exterior of the lipoprotein particle, then tocopherol in the outer bilayer leaflet of blood cell membranes may be more easily transferred to tissue cells (by receptor-independent means) than lipoprotein tocopherol. Also, since contact between donor and recipient cells is probably required for this type of transfer, the kinetics and duration of contact, and the surface morphology of donor and recipient cells may be crucial factors in determining the probability of a transfer event. Transfer of a-tocopherol between tocopherol-deficient and tocopherol-sufficient red blood cells was examined by Tanaka and Mino (1986) using indirect means. Comixing of the two populations decreased dialuric acid-induced hemolysis of the deficient cells. Inhibition of hemolysis by comixing was reduced by increasing the viscosity of the medium, and enhanced by treating the cells with protease, indicating that cell collision is necessary for intermembrane tocopherol transfer to occur. Both the intracellular fate and lifetime of newly acquired cellular tocopherol may depend on its mode of entry. Internalized lipoprotein particles are transported in vesicles to the lysosomes for degradation of the lipoprotein particle and redistribution of its constituent lipids and proteins. Subsequent binding and transfer factors would be needed for lipo-

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protein-associated a-tocopherol to find its way to intracellular membranes, either directly to existing membranes or to membrane segments under construction in the Golgi. The evidence for such intracellular factors is reviewed below. On the other hand, a-tocopherol incorporated by diffusion from impinging lipoproteins (bound nonspecifically or by receptors but not internalized) into the plasma membrane may exhibit kinetics of intracellular distribution and clearance different from that requiring export from lysosomes. In addition to the various mechanisms described above, it is likely that several other factors influence the rate of uptake of tocopherols by tissues from the bloodstream. These may include the degree of vascularity and total tissue blood flow. A more complete understanding of the determinants of tocopherol uptake, beyond adding to our knowledge of the metabolism and physiology of tocopherols, will be of value in devising means to enhance rates of tissue accumulation of this vitamin for applications such as decreasing sensitivity to ionizing radiation or organ-specific drug toxicities. E. TISSUE MOBILIZATION AND TURNOVER OF TOCOPHEROLS As discussed above, it is likely that a substantial fraction of fasting plasma a-tocopherol is derived from the liver. Bjarneboe et al. (1987) reported a relatively rapid clearance from liver tissue of newly taken up [a-3H]tocopherolfollowing intravenous injection of in vivo-labeled lymph chylomicrons in vitamin E-sufficient rats. The liver contained 50% of the injected dose 60 min after injection, but only 11% of the dose 23 hr later, with little further decrease between 24 and 48 hr. Approximately 15% of the injected a-tocopherol was excreted in bile in 24 hr. It thus appears that, of the labeled a-tocopherol taken up by the liver in chylomicron remnants, 20% remained in the liver, 30% was excreted in bile [mostly as non-a-tocopherol product(s)], and the remainder was exported, probably associated with VLDL. In the rat, therefore, the liver is of central importance in the determination of retention and blood pharmacokinetics of newly absorbed a-tocopherol. The rate of depletion of a-tocopherol in various rat tissues following complete withdrawal of dietary a-tocopherol has been described by Bieri (1972). Clearly, different tissues exhibit widely varying rates of disappearance of a-tocopherol under these circumstances. However, it is not known what proportion of the cleared tocopherol was as unchanged atocopherol. There is comparatively little information regarding the mobilization of a-tocopherol from tissues in a-tocopherol-replete animals.

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Machlin and Gabriel ( 1982) followed the disappearance of a-tocopherol from various tissues of mature rats initially fed 65 IU/kg diet, then fed 1,000 or 10,000 IU/kg for 14 weeks, then returned to the 65 IU/kg diet for 4 weeks. During the latter 4-week depletion period, rapid depletion rates were seen in liver, lung, and brain, and slow rates in muscle and adipose tissue. However, elevated plasma levels were not maintained, and dropped about 50%, although the levels at the end of the experiment were still approximately 3-fold higher than the starting (65 IU/kg) level of 0.5 mg/ml. In general, the return of plasma a-tocopherol levels to presupplementation levels lagged behind those of the tissues (with the exception of adipose tissue), possibly indicating mobilization of tissue a-tocopherol into the plasma. However, as mentioned above, it is not possible to determine what proportion of disappearing tissue a-tocopherol was due to export of unchanged a-tocopherol as opposed to tissue consumption and metabolism of the vitamin. It is also impossible to determine if the cleared a-tocopherol was originally associated with cellular membranes or in storage sites such as lipid droplets. While the phytyl tail of a-tocopherol serves only to position the vitamer in the membrane phospholipid bilayer, its structural configuration influences its tissues uptake or retention, and therefore its biopotency. The tail contains three chiral carbons (2,4’, 8’), each of which can exist in the S or R configuration. Naturally occurring a-tocopherol is predominantly in the RRR configuration, whereas synthetic a-tocopherol is an equimolar mixture of all eight possible racemates (all-rac-a-tocopherol). The RRR form is the most biopotent stereoisomer in the rat fetal gestation-resorption assay (Weiser and Vecchi, 1982), and in general the chirality at carbon 2 appears to be the determining factor in the relative biopotencies of the isomers (Machlin et al., 1982). Recently, Ingold et al. (1987) studied the tissue uptake and retention of RRR- and SRR-a-tocopherol in rats fed the deuterated tocopherol acetates (36 mg/kg diet), singly or in combination, following a long period of consumption of an equivalent level of nondeuterated RRR-a-tocopheryl acetate. The results clearly demonstrated that different tissues exhibited different rates of a-tocopherol turnover (replacement of nondeuterated RRR-a-tocopherol by the deuterated form), and different selectivities for the RRR form relative to the SRR form (rates of enrichment with SRR over time with combination feeding). Lung and liver exhibited the fastest turnover rates (7-10 days) while spinal cord had the slowest rate (76 days). Brain showed the highest selectivity for the RRR form. The red blood cells also showed a marked preference for the RRR form, which was subsequently (Cheng et al., 1987) shown to be due to a selective retention of this form over the SRR form, rather than to differential uptake of the natural (RRR) stereoisomer.

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The preferential retention of the RRR form was attributed to a “solvent effect” of the chiral erythrocyte membrane constituents (phospholipids, cholesterol, proteins) such that the “compatibility” of RRR-a-tocopherol with the membrane exceeded that of the SRR isomer, thus leading to an enrichment with the natural stereoisomer over time. In both humans and rodents, adipose tissue is a quantitatively important site of deposition of tocopherols, based on their relatively high concentration in this tissue. However, the extent to which this tocopherol pool can be mobilized has not been clarified, particularly in the human. Machlin et al. (1979) reported that the rate of total tocopherol depletion from adipose tissue (combined mesenteric, perirenal, epididymal) of mature guinea pigs fed a tocopherol-devoid diet for 4 months was extremely slow. Adipose tissue contained only slightly reduced tocopherol levels when plasma tocopherol levels were well below 50 pg/dl and cardiac myopathic lesions were evident. Fasting guinea pigs for 4 days resulted in a 25% decrease in deep fat depot mass, while the tocopherol content of these depots decreased by 11%, and tocopherol concentration (per gram fat) increased 20%. These results indicate that in the rodent, adipose tissue a-tocopherol cannot be mobilized at a rate sufficient to maintain tissue or plasma tocopherol levels and prevent vitamin E deficiency pathologies. Very little direct information on the mobilization of tocopherols from human tissues is available. Schaefer et al. (1983) reported the effect of weight reduction on adipose tissue triglyceride, cholesterol, and a-tocopherol levels in a single human subject. Following a weight reduction from 147 to 99 kg, adipose tissue tocopherol concentration (on a lipid basis) increased, although no data were given. No significant change in tocopherol content was seen when expressed on a cell basis. While these data suggest that tocopherols were not mobilized under these conditions, the subject was apparently supplemented with 75 mg a-tocopherol per day, leaving open the possibility that mobilization of tissue tocopherol was counterbalanced by tissue uptake. Plasma tocopherol levels prior to or following the weight reduction were not assessed. The reason(s) for the apparent slow rate of release of a-tocopherol from adipose tissue is at present unknown. However, one factor may be that most (99?4~)of adipose tissue tocopherol resides in the bulk lipid droplets of this tissue (Traber and Kayden, 1987), and the high solubility of tocopherol in this lipid may retard its exit. Other possibilities include (1) lack of a specific intracellular mechanism to transfer a-tocopherol from bulk lipid to the cell surface, and (2) lack of a suitable extracellular receptor for a-tocopherol. Fatty acids mobilized by adipose tissue lipase are bound at the extracellular surface by serum albumin for transport away

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from the cell surface. While albumin has been reported to bind tocopherols (Voth and Miller, 1958), its potential role in adipose tocopherol mobilization has not been examined. If adipose tissue tocopherols are mobilizable in humans, then it might be expected that plasma tocopherol levels would increase under conditions of adipose triglyceride mobilization such as weight loss. Vandewoude et al. (1987) reported a significant rise in plasma a-tocopherol levels of nearly 25% in obese females having completed a 5-week weight reduction program, in which approximately 10% of initial body weight was lost. Weight reduction was achieved by use of a protein-sparing modified fast diet supplying 700 kcal (42% fat, 40% protein) and 8 mg a-tocopherol daily. Short-term increases in plasma a-tocopherol concentration have also been shown to occur in normal humans undergoing strenuous exercise (Pincemail et al., 1986). Treadmill or bicycle exercise resulted in approximately 25% increases of plasma a-tocopherol at the halftime of maximal exercise on the two apparatus, respectively. Interestingly, plasma tocopherol levels at the time of exhaustion on each apparatus were lower than those at the exercise halftime, although both were significantly elevated above initial levels. After 10 min of rest following exercise, plasma levels had returned to the initial levels. The rapid, short-term, and nonsustainable nature of the rise in plasma a-tocopherol levels may indicate hormonal involvement in the mobilization of the vitamin during exercise. It remains to be determined, however, if the rise in serum a-tocopherol following exercise is actually due to mobilization from tissue or to shortterm suppression of tissue uptake.

F. ASSESSMENT OF VITAMIN E STATUS IN HUMANS Several methods have been used for the assessment of vitamin E status in humans. These include measurement of a-tocopherol (or total tocopherol) concentration in plasma, other blood constituents (red blood cells, lymphocytes, platelets), and adipose tissue biopsies, or the use of in vitro erythrocyte hemolysis in the presence of hydrogen peroxide or dialuric acid. The drawbacks of the erythrocyte hemolysis assay have been discussed by Chow (1985). The use of adipose tissue tocopherol levels may also not accurately reflect status since, as discussed above, this tocopherol pool may not be bioavailable, i.e., in equilibrium with plasma, and thus may be more reflective of long-term dietary absorptive history than current status. Recent reports have indicated that platelet a-tocopherol content may be a more reliable indicator of vitamin E status than total plasma a-to-

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copherol levels. Vatassery et al. (1983) found that, unlike plasma a- and y-tocopherol, platelet tocopherol levels in adult males were not significantly correlated with plasma total lipid, cholesterol, or triglyceride concentrations. Consequently, platelet tocopherol levels are not as likely to be susceptible to fluctuations in blood lipids as are plasma tocopherol levels. The relationship between serum tocopherol and lipid levels in relation to vitamin E status has been discussed previously by Horwitt et al. (1972). The sensitivity of tocopherol concentrations of various blood components to changes in dietary a-tocopherol intake in adult humans has been recently described by Lehmann et al. (1988). Platelet a-tocopherol levels were most reflective of a-tocopherol intake, followed by red blood cells, lymphocytes, and plasma. Accurate assessment of vitamin E status is of particular interest in cases of imposition of low-fat, low-cholesterol diets. While the long-term effects of such diets on vitamin E status remain the subject of investigation, Haddad et al. (1985) reported that 4 weeks of such a diet did not influence vitamin E status in adult hyperlipidemic men. While plasma atocopherol was reduced in conjunction with plasma cholesterol levels, the a-tocopherol content of erythrocytes increased. These findings again illustrate the disadvantage of using total plasma a-tocopherol levels as the sole indicator of vitamin E status, particularly if not corrected for plasma lipid levels. Regardless of the blood parameter used, the underlying assumption involved is that the component level of a-tocopherol will reflect that of critical tissue sites. This assumption has been largely untested in humans, although animal data would suggest a good correlation between plasma (or other blood component) and tissue levels of a-tocopherol. However, the use of specific blood component a-tocopherol concentrations to predict actual concentrations in a variety of solid tissues, or in subcellular membrane fractions of those tissues, remains to be validated in normal humans of various ages. Several studies have been reported concerning vitamin E status in elderly populations relative to younger age groups, on the basis of plasma tocopherol levels. Kelleher and Losowsky (1978) reported a summary of such studies published between 1964 and 1977. With one exception, serum tocopherol concentrations generally increased through age 70. These authors also reported original data that supported the notion that, in general, serum tocopherol levels do not decrease with age. A substantial percentage (65%) of a study population of 70 acutely ill elderly had serum tocopherol levels below 450 pg/dl, well below the normal range for healthy younger controls. These low values were not related to serum

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LDL levels, but did seem to reflect poor diets based on the findings of low serum p-carotene and leukocyte vitamin C. The observed relationship between depressed status of both vitamin C and vitamin E is interesting in light of the proposed functional interaction between a-tocopherol and ascorbic acid discussed below. Recently, the measurement of hydrocarbon gases in expired breath has been proposed as an index of vitamin E status in humans. Lemoyne et a / . (1987) found that breath pentane, thought to form from peroxidation of n-6 fatty acids, was significantly elevated in individuals deficient in vitamin E as indicated by low plasma a-tocopherol levels. Pentane output was negatively correlated with plasma a-tocopherol levels ( r = -0.66). In normal subjects supplemented with vitamin E (735 mg d-a-tocopherol per day for 10 days), breath pentane output decreased 35% as plasma atocopherol levels doubled. Thus pentane expiration may provide an additional, and noninvasive, tool to assess vitamin E status in humans. Other aspects of this technique have been reviewed recently by Pincemail et al. (1987). VI. TOCOPHEROL BINDING AND TRANSFER FACTORS

Evidence has been reviewed above that a-tocopherol is probably taken up by cells by at least the processes of receptor-mediated internalization of lipoproteins and lipoprotein lipase-dependent transfer. Tocopherols so internalized would probably initially reside in the lysosome or plasma membrane, and thus would require additional mechanism(s) for redistribution into the other various subcellular membrane fractions of the cell. In liver, a mechanism is needed to account for the transfer of newly internalized tocopherols from chylomicron remnants to VLDL for subsequent export into the plasma. Such mechanisms are unlikely to include passive diffusion through the aqueous compartment due to the low water solubility of the tocopherols. Therefore, some form of facilitated transfer seems indicated, and the evidence for specific factors associated with binding and transfer of tocopherols is reviewed below. A. TOCOPHEROL BINDING FACTORS

Catagnani (1975) first demonstrated that 105,000 g supernatant from livers of rats fed a tocopherol-deficient diet contained a protein that bound RRR-[a-3H]tocophero1with high affinity and specificity. This protein sedimented in the 3 s region on sucrose gradients, and exhibited an approximate apparent molecular weight of 3 1,000 by Sephadex G-100 gel

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filtration, which separated the tocopherol-binding protein from both free a-tocopherol and lipoprotein-associated tocopherol. The binding was inhibited by an excess of unlabeled a-tocopherol and by treatment with pronase. A subsequent study (Catagnani and Bieri, 1977) reported that incubation of RRR-[a-3H]tocophero1 with 105,000 g supernatants from rat liver, lung, heart, testes, intestinal mucosa, muscle, brain, serum, and erythrocyte hemolysate showed the presence of this tocopherol-binding protein only in the liver supernatant. Serum exhibited a-tocopherol binding in the 4.6s region by sucrose gradient centrifugation and was attributed to serum albumin, which has been reported to bind tocopherol (Voth and Miller, 1958). Maximal a-tocopherol binding by the liver supernatant factor was maximal after 4 hr at 26"C, and saturation of binding was achieved at 90-1 10 nM a-tocopherol. Bound tocopherol represented 33-50% of total added tocopherol under the conditions employed. The liver content of this protein was estimated to be 5-9 p,g per gram of tocopherol-depleted liver. While a 400-fold excess of a-tocopherol reduced binding of [a-3H]tocopherol by 98%, incubation with a similar excess of &tocopherol or a-tocotrienol reduced binding by 60 and 70%, respectively, indicating preferential binding of a-tocopherol but at least some affinity for other tocopherol compounds. A wide variety of other lipid-soluble compounds, including a-tocopheryl quinone, a-tocopheryl acetate, retinol, retinoic acid, 1,25dihydroxycholecalciferol, cholesterol, oleic acid, vitamin K, , coenzyme Q9, dexamethasone, and diphenylphenylenediamine were without effect on binding of [a-3H]tocopherol. High-affinity a-tocopherol binding was also found in liver supernatants of mouse, guinea pig, hamster, rabbit, and chicken. Murphy and Mavis (1981a) compared the ability of Sephadex G-75 gel filtration fractions of 100,000 g supernatants from vitamin E-sufficient rat liver, lung, heart, and brain to bind [a-3H]tocopherol and to facilitate its transfer from egg phosphatidylcholine liposomes to rat liver microsomes. Liver exhibited two tocopherol-binding fractions, one eluting with an apparent molecular weight of 34,000 and a second eluting with the void volume. This latter peak was attributed to a lipoprotein, similar in size to plasma VLDL, whose a-tocopherol is not exchangeable with liver microsomes (Rajaram et al., 1973). Removal of this high-molecular-weight binding activity by pH reduction to 5.1 did not result in any loss of liver supernatant transfer activity. Void volume a-tocopherol binding was also observed in brain and heart supernatants, while such binding was undetectable in lung supernatant.

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Interestingly, the relative extent of void volume tocopherol binding (brain > liver > heart > lung) was inversely proportional to the relative tissue microsomal a-tocopherol concentration in vitamin E-sufficient rats (lung > heart > liver > brain) reported in an earlier study (Kornburst and Mavis, 1980). This may indicate that the high-molecular-weight binding activity reflects competition from endogenous a-tocopherol already bound to the factor in supernatants prepared from tissues of a-tocopherol-sufficient rats. Sklan and Halevy (1982) studied the binding of [a-14C]to~opherol by chick liver cytosol following either gastric or intraperitoneal administration of the labeled tocopherol. Sepharose 6B gel filtration fractionation of 105,000 g supernatants showed that over 80% of the I4C eluted close to the void volume, estimated at a molecular weight of 2,000,000, with a secondary peak of radioactivity eluting at a molecular weight of 200,000300,000. The high-molecular-weight fraction (2,000,000 MW) was approximately 50% lipid, and is likely the very-low-density-like lipoprotein reported by Rajaram ef al. (1973). Specificity of this binding fraction was not studied, nor was its potential to transfer tocopherol to or from cell membranes. Guarnieri et al. (1980) examined the ability of a high-molecular-weight factor from a-tocopherol-deficient rabbit heart 105,000 g supernatant to bind and transfer a-tocopherol to heart nuclei. This factor eluted near the void volume on Sephadex G- 100 and probably corresponds to the highmolecular-weight lipoprotein a-tocopherol binding factor reported by Rajaram et al. (1973) in rat liver cytosol. No evidence of binding of [~x-~H]tocopherol by a 32,000 MW factor was observed in heart 105,000g supernatant. Guarnieri et al. (1980) also found that d-[a-5-methyl-3H]tocopherol complexed in vitro by this factor was more efficiently bound by nuclear acidic protein than free d-[a-3H]tocopherolwhen incubated with isolated rabbit heart nuclei. These authors concluded that a high-molecular-weight cytosolic tocopherol-protein complex was necessary for tocopherol binding by chromatin protein in intact nuclei. In light of the observations of others (Patnaik and Nair, 1975, 1977; Nair et al., 1978) that labeled atocopherol becomes associated with rat liver nuclei, and the report of Patnaik (1978) that a-tocopherol stimulates RNA synthesis in rat liver nuclei, these authors speculated that a-tocopherol may be involved in the regulation of gene activity. The authors did not determine whether the high-molecular-weight tocopherol-protein complex entered the nucleus intact, or if the a-tocopherol was first incorporated into the nuclear envelope. If the high-molecular-weight complex reported by Guarnieri et al. (1980) is in fact identical to that reported by Rajaram et al. (1973), it must

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be noted that the latter found no ability of this complex to transfer atocopherol to rat liver microsomes. A gene regulation role for a-tocopherol remains highly speculative. The studies described above demonstrated in vitro the existence of a soluble a-tocopherol binding protein of 32,000-34,000 molecular weight. More recent evidence of this factor in vivo has also been provided. Behrens and Madkre (1982a) gavaged Wistar rats with d-[a-5-methyl-3H]tocopherol and subjected the 24-hr 100,OOO g liver supernatants to Sephadex (3-100 exclusion chromatography. Two peaks of radioactivity were found, one with an apparent molecular weight in excess of 15,000,000 and a second of approximately 32,000, consistent with the in vitro observations of both Catagnani (1975) and Murphy and Mavis (1981a). The 32,000 MW protein was detectable in livers of rats fed low, adequate, or high atocopherol diets. Dietary 6-tocopherol bound the high-molecular-weight (lipoprotein) fraction but not the 32,000 MW factor to any great extent. Further, dietary 6-tocopherol did not appear to influence the binding of a-tocopherol to this factor, consistent with the specificity of this protein for a-tocopherol reported by Catagnani and Bieri (1977). The potential role of interstitial retinol-binding protein (IRBP) in binding and intraocular transport of tocopherols in the human eye was studied by Alvarez et al. (1987). The primary function of this glycoprotein is believed to be retinol transport. Purified IRBP from bovine eyes bound d[a-5-methyl-3H]tocopherol, and could be displaced by unlabeled retinol with a K , of approximately M. Much higher concentrations of atocopherol were required for retinol displacement, although a K, could not be determined due to limitations of a-tocopherol solubility. No endogenous IRBP-bound tocopherol could be found, and, in conjunction with the binding data, the findings cast doubt on a physiologically important role of this protein in tocopherol transport.

B. TOCOPHEROL TRANSFER FACTORS Murphy and Mavis (1981a), in addition to measuring rat liver cytosolic tocopherol binding activity, also studied the ability of cytosol to transfer a-tocopherol from phosphatidylcholine liposomes to rat liver microsomes. Liver supernatant exhibited a single peak of a-tocopherol transfer activity on Sephadex G-100 which coincided with the binding activity, and the authors proposed that a single protein was responsible for both phenomena. Supernatants from lung, heart, and brain exhibited no detectable transfer activity. If the transfer and binding activities are attributable to the same protein, then this result is consistent with the lack of

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32,000-34,OOO MW binding activity observed in extrahepatic tissues both in this study and by Catagnani and Bieri (1977). Transfer of a-tocopherol between dipalmitoylphosphatidylcholine liposomes and rat liver mitochondria was examined by Mowri et al. (1981). Liposomes containing trace masses of d-[a-5-methyl-3H]tocopherol and gly~erotri['~C]oleate were incubated at 37°C with mitochondria in the presence or absence of the 105,000 g supernatant. In the absence of supernatant, 10% of the liposomal 'H was transferred to mitochondria in 30 min, while 30% transferred in the same period in the presence of supernatant (0.5 mg protein). At 1.0 mg supernatant protein, transfer was maximal, and the transfer rate was 12 times higher than in the absence of supernatant. Further characterization of the supernatant revealed that the transfer activity was nondialyzable, (NH,),SO,-precipitable, heat labile, and trypsin labile. N-Ethylmaleimide did not influence the transfer-stimulating activity of the supernatant. Increasing the liposomal content of unlabeled a-tocopherol reduced transfer, indicating a limited capacity of the transfer factor. &Tocopherol significantly reduced a-tocopherol transfer, although not as effectively as excess a-tocopherol. Sephadex G-75 chromatography indicated the transfer factor apparent molecular weight to be 30,000 and that the transfer factor was distinct from phosphatidylcholine transfer activity. When liposomes were incubated with 105,000 g supernatant only, all a-tocopherol recovered in the supernatant was associated with a 30,000 MW factor, and this complexed tocopherol could subsequently be transferred to mitochondria. These data suggested that the binding and transfer activities were the same protein. Interestingly, while specific binding activity was 4-fold higher in 105,000 g liver supernatant from a-tocopherol-deficient rats, transfer activity was unchanged. In addition, transfer activity was also found in 105,000 g supernatant from heart, spleen, and lung, all of similar magnitudes to the liver activity. Kidney and brain supernatants exhibited much lower transfer activity. Unfortunately, binding activity was not measured in these other tissues. The absence of binding activity, reported by other investigators, in the presence of transfer activity would suggest that distinct proteins may perform these functions. More recently, Verdon and Blumberg (1986) reported an a-tocopherol transfer activity isolated from rat liver which facilitated the transfer of d[a-3H]tocopherol from liposomes to human erythrocyte ghosts. This activity was prepared by ammonium sulfate precipitation of high-speed supernatant without subsequent chromatography. Incubation of ghosts with increasing liposomal [a-3H]tocopherol concentrations resulted in saturation of transfer activity. Subsequent studies (Verdon and Blumberg, 1988)

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have shown that this transfer activity coincided with a-tocopherol binding activity and exhibited an apparent molecular weight of 39,000. The factor transferred a-tocopherol from egg phosphatidylcholine liposomes to tocopherol-sufficient rat erythrocyte ghosts at a rate equal to that transferred to rat liver mitochondria when rates were normalized for acceptor membrane lipid phosphorus. This suggests that the rate of tocopherol transfer mediated by this factor is relatively insensitive to acceptor membrane characteristics other than phospholipid content. This observation also suggests that the rate of transfer by this factor to inside-out and rightside-out erythrocyte membrane vesicles would be similar, although such an experiment has not been reported. (Presumably, the erythrocyte ghosts, employed by Verdon and Blumberg, were right-side-out.) Appreciable transfer occurred from liposomes containing only 0.3 mol % a-tocopherol (relative to phospholipid) to erythrocyte ghosts containing 0.12 mol % endogenous a-tocopherol, indicating that this factor is likely to operate at physiological concentrations of membrane a-tocopherol. C. MEMBRANE BINDING OF TOCOPHEROLS Transfer of a-tocopherol from one compartment to another requires not only the presence of a binding and transport factor to overcome solubility problems, but a receptor mechanism in the recipient compartment. If the recipient is a membrane the receptor mechanism may be nonspecific, such as diffusion of the tocopherol ligand from transport protein to the bulk lipid domain of the membrane, or specific, such as interaction of the ligand or protein transporter with a surface protein of the membrane. As reviewed below, evidence has been reported for both types of mechanisms, using a wide variety of delivery modes, including the 32,000 MW cytosolic factor described above. Behrens and Madere (1982b) studied the transfer of d-[a-5-methyl3H]tocopherolfrom an in vivo-labeled 32,000 MW liver cytosol tocopherol binding fraction to rat liver microsomes. In addition to ammonium sulfate fractionation and Sephadex G-100 chromatography, the 32,000 MW fraction was further purified by DEAE-Sephadex ion-exchange chromatography, in which the labeled tocopherol eluted in a single peak at 0.14 M NaCI. Incubation with microsomes indicated that equilibrium was reached at 3 hr. Microsomal binding increased linearly with increasing concentration of a-tocopherol (protein-bound),indicating nonsaturability of the microsomes over this range. However, coincubation of microsomes with [a-3H]tocopherol-proteincomplex and increasing amounts of unlabeled a-tocopherol-protein complex (isolated from vitamin E-sufficient rats) showed that unlabeled complex competed with labeled com-

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plex for [a-3H]tocopherol binding, indicating a saturable binding process. The authors suggested that the data confirmed a transfer role for the 32,000 MW fraction and that microsomal binding involves a microsomal protein receptor rather than solubilization of the tocopherol in microsoma1 phospholipid. The binding of bovine serum albumin-complexed d-[a-5-methyL3H]tocopherol to tocopherol-depleted microsomes from rat liver, lung, heart, and brain was studied by Murphy and Mavis (1981b). Increasing concentrations of a-tocopherol from 27 to 1279 (*A4 resulted in increased amounts of the vitamin bound to microsomes from all tissues, as well as the time required to reach equilibrium. The total amount bound at equilibrium per milligram microsomal protein was linearly proportional to the final concentration of unbound tocopherol, indicating nonsaturability of the binding process over a range which exceeds normal physiological levels of membrane tocopherol. Murphy and Mavis note these findings as consistent with the intercalation of a-tocopherol into the phospholipid domain of the membrane, rather than interaction with a limited number of specific protein receptors. The functionality of the transferred tocopherol was demonstrated by the ability of the added tocopherol to inhibit induced lipid peroxidation in the recipient microsomes, indicating that the new membrane tocopherol had indeed become incorporated into the phospholipid domain. The apparent affinity of microsomes from the four tissues for a-tocopherol (albumin complex) was negatively correlated with normal in vivo levels of a-tocopherol in these membranes. Lung microsomes exhibited the highest in vivo levels but the lowest apparent affinity. Thus, in vivo differences in membrane a-tocopherol content could not be explained on the basis of a significantly differential affinity of the membranes for a-tocopherol, at least when presented as an albumin conjugate. In addition, since tocopherol transfer activity by the soluble 32,000 MW factor described above seems to be present only in liver, differential tissue microsomal tocopherol contents also cannot be explained on the basis of differential activity of this factor. The internal inconsistency in the data of Behrens and M a d b e (1982b) regarding saturability of microsomal binding of a-tocopherol delivered by the 32,000 MW factor, and the data of Murphy and Mavis (1981b) showing nonsaturable binding of albumin-complexed a-tocopherol to rat liver microsomes indicate a need for further investigation of this question. However, the possibility of the existence of a microsomal receptor for a tocopherol-binding protein, but not for a-tocopherol itself, needs consideration. This would indicate the experimental importance of the form in which the tocopherol is presented to the membrane in interpretation of the kinetics of the binding process.

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Evidence that human erythrocyte membranes have specific, saturable binding sites for a-tocopherol itself was presented by Kitabchi and Wimalasena (1982).d-[a-3H]Tocopherol in ethanol was incubated with washed erythrocytes, and specific binding reached saturation at 4 hr at 37°C over an a-tocopherol concentration range of 6-24 nM. Nonspecific binding (binding in the presence of 110 pM unlabeled a-tocopherol) represented less than 20% of total binding. Kinetics of specific binding indicated the presence of two independent binding sites. For the high-affinity site K, was 2.64 f 0.4 x lo7 M - ' , with 7,000-8,OOO sites per cell, and for the low-affinity site K , was 1.24 ? 0.3 x 10"M - ' , with 140,000-160,000sites per cell. Trypsin treatment greatly decreased binding, although a differential trypsin effect on the high- versus low-affinity sites was not investigated. Reincubation with N-ethylmaleimide reduced binding by 50%, suggesting the involvement of functional disulfide bonds in the binding process. d-y-Tocopherol successfully competed with d-a-tocopherol for specific binding in a concentration-dependent fashion, while Mocopherol, a-tocopheryl quinone, and a variety of tocopheryl esters had no effect on binding. Neither polymorphonuclear cells nor platelets showed any specific binding of a-tocopherol, and possibly acquire their a-tocopherol via a different mechanism. An important question is whether a-tocopherol bound by the red blood cell receptors under these conditions is functional, i.e., able to inhibit peroxidative membrane damage. Kitabchi and Wimalasena (1982)demonstrated that preincubation of erythrocytes with 1.1 p M d-a-tocopherol significantly reduced subsequent hemolysis by H,O,. It is not known, however, whether this protection was afforded by tocopherol bound by specific (receptor) or nonspecific means, or if the low- or high-affinity specific binding is more important in this regard. Whether receptor-bound a-tocopherol is directly functional, or whether initially bound tocopherol is subsequently transferred to the bulk phospholipid of the erythrocyte membrane remains to be clarified. Interestingly, d-y-tocopherol at 1.1 pM was 50% as effective as d-a-tocopherol at preventing H,O,-induced hemolysis, and at 11 p M was equally effective as d-a-tocopherol at 1.1 p M . The localization of erythrocyte d-a-tocopherol-specific binding in the plasma membrane was also reported by Wimalasena et al. (1982).Washed erythrocyte ghosts exhibited two specific binding sites, with K , values of 3.3 x lo' M-' and 1.5 x 10" M-'.As in intact erythrocytes, only ytocopherol was able to compete with a-tocopherol for binding (although much less effective), and N-ethylmaleimide pretreatment inhibited binding by 60%. Attempts at solubilization of the ligand-free binding sites by a variety of means were unsuccessful. Solubilization of [a-3H]tocopherolbinding site complexes was accomplished using Triton X-100,and Sepharose 6B gel chromatography indicated the presence of two labeled species

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with M , values of 65,000 and 125,000. The larger species could be resolved into the smaller species upon rechromatography. A more complete structural characterization of these complexes is clearly needed. Specific receptor sites for a-tocopherol in purified cell membranes isolated from adrenocortical cells were demonstrated by Kitabchi et al. (1980). The adrenal is an interesting tissue due to its relatively high content of a-tocopherol and polyunsaturated fatty acids (Kitabchi and Williams, 1968). Incubation of adrenocortical membranes with d-[~x-~H]tocopherol (in ethanol) in the presence of various concentrations of unlabeled a-tocopherol indicated two binding sites with apparent K , values of 7 x lo6M - ' and 0.4 x lo6 M - ' . The binding capacities for these sites were estimated at 12 and 120 pmol/mg protein, respectively. Preincubation at 65°C (or with trypin at 37°C) for 30 min abolished all specific binding, suggesting a protein nature of the binding site. The authors observed that the apparent K , values agree with the typical serum a-tocopherol concentration of 23 pl4, suggesting physiological relevance of this receptor. It must also be noted, however, that serum a-tocopherol is lipoprotein-bound, and thus association constants derived from administration of a-tocopherol in ethanol may not accurately reflect those in vivo. VII. TOCOPHEROL METABOLISM

Most studies concerning metabolism of tocopherols have focused on aspects of the pharmacokinetics and biotransformation of a-tocopherol, with relatively few dealing with the other, less active tocopherol vitamers, despite their widespread occurrence in human foods. Many details of tocopherol metabolism remain unclarified, particularly those enzymes which are involved in tocopherol biotransformation and their regulation. Relatively few metabolites of the tocopherols have been purified from human or animal tissues and excreta and rigorously identified using modern methods of structural analysis such as mass spectrometry and nuclear magnetic resonance spectroscopy. As is the case with other oxidizable analytes, the study of tocopherol metabolism is complicated by the need to avoid the formation of artifacts during extraction, purification, and analysis of these compounds. A.

METABOLISM OF a-TOCOPHEROL

Several studies have demonstrated the formation of a-tocopheryl quinone from a-tocopherol in vivo using radiolabeled a-tocopherol, although reported concentrations of this metabolite vary widely. The quinone derivative of a-tocopherol apparently arises in tissues via the nonenzymatic

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oxidation of a-tocopherol, presumably as a result of its antioxidant activity, as discussed later in this review. McCay ef al. (1971) found that atocopheryl quinone formed readily from a-tocopherol during NADPHstimulated lipid peroxidation in rat liver microsomes. Its formation is apparently irreversible, as Chow et al. (1967) observed no reduction of a-tocopheryl quinone to a-tocopherol in rat liver. Csallany et al. (1962) injected 2 mg d-[a-5-methyl-'4C]tocopherol into adult vitamin E-deficient rats, and recovered 9% of the dose in an acetone liver extract 48 hr later. Of the recovered liver radioactivity, 25% was unchanged a-tocopherol, and 19% was a-tocopheryl quinone. The latter was identified by cochromatography with synthetic a-tocopheryl quinone using several methods and solvent systems, and by recrystallization with camer a-tocopheryl quinone to constant specific radioactivity. No quinone formation from [a-'4C]to~~pher~1 was reported to result from the isolation or chromatographic procedures employed. However, it is unclear if the animals were adequately prevented from reconsumption of fecal products. Other studies have reported widely varying concentrations of a-tocopheryl quinone in tissues of rats administered [a-I4C]tocopherol by various routes, as summarized by Gallo-Torres (1980). Saifutdinov (1986) reported the occurrence of a-tocopheryl quinone in human plasma and erythrocyte lipids as indicated by electron paramagentic resonance, and found the levels to increase in subjects given intramuscular injections of 0.6 g a-tocopherol in oil twice daily for 3 days. However, absolute concentrations of the quinone compound were not estimated. Prevention of coprophagy in experimental animals administered radiolabeled tocopherols is important since a-tocopheryl quinone, conjugated tocopherol metabolites, and possibly unchanged a-tocopherol are apparently secreted into bile and eliminated with feces. Schmandke and Pro11 (1964) reported that bile of adult rats fed 8 mg a-tocopherol per kilogram body weight per day contained 0.4-1.3 p,g/ml a-tocopherol, a concentration likely to be much lower than the corresponding plasma concentration of the vitamer in these animals. MacMahon et al. (1971) administered radiolabeled a-tocopherol into the duodenum of bile duct-cannulated rats and recovered 8% of the dose in bile in 24 hr. However, nearly all of the bile radioactivity was contained in compound(s) more polar than atocopherol, with less than 2% migrating with standard a-tocopherol on paper chromatography. This contrasts with an earlier study by Krishnamurthy and Bieri (1963), who found that, in rats given a single oral dose of 0.5mg [a-5-methyl-'4C]tocopherol, all the fecal radioactivity recovered through 21 days was accounted for by unchanged a-tocopherol. More recently, Ingold e f al. (1987) found that in rats switched from consuming

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20 1

nondeuterated a-tocopherol to [a-5-CD3]tocopherol,the nondeuterated form in fecal material was detectable at appreciable levels for up to 4 weeks. These observations together suggest that in rats a portion of orally administered a-tocopherol may persist in the gastrointestinal tract for long periods of time, or that small quantities of a-tocopherol are secreted into the intestine via the bile. Chow et al. (1967) examined the metabolism of a-tocopheryl quinone and a-tocopheryl hydroquinone in rats using radiolabeled compounds. Forty-eight hours following a 1-mg intraperitoneal injection, fecal excretory products accounted for 3-10% of the injected dose, with 30-70% of fecal radioactivity extractable by petroleum ether (PE). Small quantities of a-tocopheryl quinone were observed in this fraction, but most PE-extractable label was more polar than the quinone, but not hydrolyzed by P-glucuronidase. Reactivity toward sulfatase was not examined. HCI hydrolysis of PE-insoluble fecal material converted 50% to PE-soluble forms, of which a-tocopheryl quinone was a minor constituent, the remainder being more polar and uncharacterized. The proportion of HCI hydrolysate not extractable by PE also was not characterized. The use of fecal analyses to study biliary secretion of tocopherol metabolites is made tenuous by several factors, including further metabolism of secreted compounds by gut microflora and incomplete absorption of dietary tocopherols in the case of oral administration. Gallo-Torres (1 980) compared the biliary secretion of label from a-tocopheryl acetate and atocopheryl nicotinate in bile duct-cannulated rats, using intragastric administration of 1-2 mg of the tritiated compounds. The rate of biliary label excretion was 4-fold higher in rats given a-tocopheryl acetate than those given a-tocopheryl nicotinate. In agreement with MacMahon et al. (1971), little or no a-tocopherol was identified in the bile using thin-layer chromatography. Most of the secreted radioactivity was slightly more polar than synthetic a-tocopheryl glucuronide. After treatment with a combination of (3-glucuronidase and sulfatase, the product was slightly more polar than a-tocopheronic acid (see Fig. 2), but less polar than a-tocopheryl glucuronide. Complete structural identification of this rat biliary product, either in conjugated or nonconjugated form, has apparently not been described to date. More recently, Bjqmeboe et al. (1987) also studied tocopherol metabolism using bile duct-cannulated rats. In animals given a single intravenous dose of 15-30 nmol a-tocopherol (injected into the femoral vein as in vivolabeled lymph chylomicrons), approximately 14% of the dose was excreted in bile within 24 hr. A significant proportion (13.5%) of the bile radioactivity coeluted with a-tocopherol on normal phase HPLC, with the remainder uncharacterized. Thus it would appear that biliary secre-

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tion is a quantitatively important route of elimination of a-tocopherol and its metabolites. The finding of apparently unchanged a-tocopherol in bile is intriguing, especially in light of the small dose levels and a physiologically relevant means of introduction of the tocopherol into the bloodstream (i.e., associated with lymph chylomicrons) employed in this study. If confirmed, the observation of unchanged tocopherol in feces or intestinal contents of rats given small oral a-tocopherol may not be entirely due to incomplete absorption, but also to biliary secretion. Evidence indicating that a major proportion of biliary (and urinary, as discussed below) metabolites of tocopherols are conjugates suggests that there may be substantial interspecies variation in rates of tocopherol elimination due to differences in hepatic or kidney conjugation capabilities. Sklan et al. (1982) suggested that the lower tissue concentration of atocopherol in turkeys relative to that of chickens fed an equivalent dietary level of a-tocopherol is due to greater biliary secretion of tocopheryl glucuronides in the turkey. While bile glucuronide concentrations per se were not measured, turkeys exhibited higher levels of duodenal and ileal glucuronidase-releasableradioactivity than chickens following dietary administration of [a-5-methyl-3H]tocopherolfor 4 days. Unfortunately, no mention of coprophagy prevention was made, particularly important in this case due to the relatively long period of labeled tocopherol feeding. Kayden and Traber (1987) have stated that the tocopherol concentration in human bile is the same as that in plasma, although no data were presented. If true, then the biliary pathway would represent a major excretory route for tocopherols in humans. The nature and concentration of the tocopherols in human bile needs further clarification. The identification of urinary metabolites of a-tocopherol was first examined in detail in rabbits and humans by Simon et al. (1965a,b). These investigations indicated the presence of two polar metabolites in the urine of humans ingesting large oral doses (3-5 g per day) of dl-a-tocopherol. The compounds were purified by column chromatography on Florex or silicic acid-Celite. Both compunds, one neutral and one acidic (pK, 4.9), exhibited ultraviolet absorption spectra identical to that of a-tocopheryl quinone but were considerably more polar. The neutral and acidic compounds had estimated molecular weights (Rast method) of 267 and 315, respectively. Infrared spectral analysis and hydroxamic acid reactivity suggested the post-P-glucuronidase treatment structures as 2-(3-hydroxy3-methyl-5-carboxypentyl)-3,5,6-trimethylbenzoquinone (tocopheronic acid) and its y-lactone (Fig. 2). The structures of the conjugates as they existed in urine were not determined. The authors suggested that the partial hydrolysis of the lactone, artifactual or otherwise, may have accounted for the hydroxy acid metabolite. The data indicated that the uri-

DIETARY AND BIOCHEMICAL ASPECTS OF VITAMIN E

A

203

6

C 7

FIG. 2. Metabolites of u-tocopherol. (A) Tocopheronic acid, (B) tocopheronolactone, and (C) tocopheronolactone glucuronide. 0,Glucuronic acid.

nary metabolites had undergone ring opening of the chroman moiety, shortening of the phytyl side chain from 16 carbons to 3 carbons, and conjugation with glucuronic acid. While many investigators have subsequently relied on the structures assigned to the urinary metabolites by Simon et al. (1956b), confirmation of their structures in either the conjugated or nonconjugated forms by nuclear magnetic or mass spectral techniques has not been reported. The importance of urinary excretion in the elimination of tocopherol metabolites is unclear, as there are several conflicting reports on this subject. The metabolites of Simon et al. (1956b) were collected from urine of individuals consuming massive amounts of a-tocopherol, while in rabbits given a 10-15 mg oral dose of radioactive a-tocopherol only traces of the label appeared in the urine (Simon et af., 1956a). Krishnamurthy and Bieri (1963)found only about 1% of label from a 0.5-mg intragastric dose of [a5-methyl-'4C]toc~pher~l in urine of rats through 2 1 days postdosing, while 13% was recovered in feces. Weber and Wiss (1963) recovered 4-6% of acetate in the urine of a 2-mg oral dose of dl-[cll-8-methyl-'4C]toc~pheryl tocopherol-deficient rats.

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Chow et al. (1967) recovered l0-20% of label from a l-mg intraperitoneal dose of radioactive a-tocopherol in the urine of rats 48 hr after injection, Nearly all the urinary metabolites were petroleum ether (PE)-insoluble, but about half of the label was converted to PE-soluble material following HCl hydrolysis. This material migrated with tocopheronic acid on paper and thin-layer chromatography. P-Glucuronidase treatment released only a small portion of the PE-insoluble urinary radioactivity, and the released material appeared to be tocopheronic acid. Sulfatase treatment failed to release any PE-soluble label. The identity of the PE-insoluble material following HC1 or P-glucuronidase treatment was not determined, but clearly represented a major proportion of the total urinary matabolites. Fischer and Whanger (1977) reported that about 20% of an unspecified intragastric dose of [a-5-methyl-3H]tocopheryl acetate was excreted in urine in 48 hr, a proportion which increased in selenium-deficient rats. The metabolites were water soluble but not characterized. Chiku et al. (1984) reported that urinary metabolites accounted for the excretion of 20% of an intravenous dose of 3 mg radiolabeled a-tocopherol through 8 days postdosing. Due to the restrictions on the use of radioactive tocopherols in humans, few studies are available concerning urinary and biliary excretion of tocopherol metabolites. Kelleher and Lowsowsky (1970) found less than 6% of orally administered dl-[a-5-rnethyl-’H]tocopherolexcreted in the urine of 50 patients studied for 7 days. Thus it would appear that the quantitative importance of the urinary route, relative to the biliary route, may depend not only on dose but on route of administration. Intravenous administration generally results in increased urinary excretion of tocopherol metabolites compared to oral (intragastric) administration. However, it is likely that at least small amounts of a-tocopherol are eliminated via both routes under normal conditions. While the total number and complete identity of the rat urine and fecal a-tocopherol metabolites remain undetermined, it appears that at least half exist as conjugates hydrolyzable by HCl. Conjugates of tocopheronic acid appear to predominate in rat urine, although glucuronic acid or sulfate conjugates may represent only a small proportion of total metabolites. Chow et al. (1967) proposed fecal elimination of the glucuronide conjugate of a-tocopheryl hydroquinone, although they reported no direct evidence to support this suggestion. However, their finding of small amounts of free and HCl-releasable a-tocopherol quinone in feces of rats injected with this substance indicates that tocopherol metabolites with intact phytyl side chains may be eliminated via the bile. There is no evidence suggesting urinary excretion of any tocopherol metabolites containing intact phytyl side chains. Chow et al. (1967) sug-

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205

gested that P-oxidation of the side chain in the kidney may account for the lack of side chain-containing metabolites in urine, but kidney-specific tocopherol metabolism studies have not been reported. It is possible that side chain oxidation, with or without subsequent conjugation, takes place primarily in the liver, with the resulting tocopheronic acid (or lactone) and its conjugates sufficiently water soluble to be released into the bloodstream and filtered by the kidney. Several earlier studies have reported or denied the occurrence of dimers or trimers of a-tocopherol in animal tissues following administration of radiolabeled a-tocopherol, as reviewed extensively by Gallo-Torres (1980). Strauch et al. (1969) reexamined this issue using both rats and mice. No polymeric forms were found in rat liver 48 hr following a 60-wg oral dose of [a-5-methyl-'4C]tocopherol, whereas approximately 2.5% of an intravenous dose was recoverd from liver as apparent dimers after a similar time period. These authors suggested that polymerization (oxidative condensation) of a-tocopherol occurs in vivo only when nonphysiological amounts of a-tocopherol are administered by nonphysiological routes. In vitro studies of a-tocopherol oxidation have suggested the formation of other tocopherol metabolites in tissues. For example, the formation of a-tocopherol-linoleic acid hydroperoxide adducts in an anaerobic, nonbiological system employing photo- or iron-catalysis was reported by Gardner et al. (1972). Air prevented the reaction, with a-tocopheryl quinone the major product in the aerobic system. No evidence for tocopherol-fatty acid addition products has been found in animal tissues. B. METABOLISM OF OTHER TOCOPHEROLS Little detailed information is available concerning the metabolism of the non-a-tocopherol vitamers. Peake et al. (1972) and Peake and Bieri (1971) found that y-tocopherol disappeared more rapidly than a-tocopherol from rat plasma and tissues, a phenomenon which probably explains in large measure the predominance of the a-vitamer in animal tissues. The reason(s) for the higher rate of clearance of non-a forms may include the specificity of tissue receptors for a-tocopherol, but also likely involves differential rates of conversion to excretable forms in the liver and kidney. Chiku et al. (1984) studied aspects of the metabolism of isotopically labeled a-and &tocopherol in rats. Intravenous injection of 10 mg a- or &tocopherol resulted in fecal excretion of 20-30% of the dose, regardless of isomeric form. Urinary excretion, however, accounted for 50% of the dose of &tocopherol but only 20% of the dose of a-tocopherol. Approximately 92% of the labeled urinary 6-tocoperol metabolites were accounted for by a sulfate conjugate. The derivative of this conjugate was

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released by sulfatase treatment, purified by thin-layer chromatography, and subjected to infrared, nuclear magnetic resonance, and mass spectral analyses. The desulfated structure was determined to be 2,8-dimethyl-2(2'-carboxyethyl)-6-chromanol(Fig. 3). The proposed structure was confirmed by analytical comparison with a synthetically derived standard. The authors proposed that the biosynthesis of this metabolite involved side-chain shortening to three carbons by f3-oxidation, sulfation of the phenolic hydroxyl, but no opening of the chroman ring. This latter feature appears to be in contrast to the metabolism of a-tocopherol, in which chroman ring opening, followed by glucuronidation or sulfation, appears to constitute the major pathway of urinary excretion as discussed above. To date, no quantitative studies of the comparative rates of metabolism of a-,y-, and &tocopherol simultaneously in the same system, including polar metabolite identification, have been reported.

C. REGULATION OF TOCOPHEROL METABOLISM The factors which regulate tocopherol metabolism and excretion are poorly understood at present. A recent report by Behrens and Madere (1987) suggests that a-tocopherol status may influence the metabolism or disposition of y-tocopherol in rats. They demonstrated that tissue and plasma levels of y-tocopherol, 24 hr following an intragastric dose (20 mg), were inversely related to the previous dietary level of a-tocopherol. That is, organ and plasma y-tocopherol levels were substantially higher in rats previously fed a a-tocopherol-devoid diet for 2 months than in rats fed a-tocopherol-sufficient diets. However, a-tocopherol-sufficient rats fed the tocopherol-devoid diet for 3 days prior to y-tocopherol dosing exhibited subsequent tissue y-tocopherol levels intermediate to the a-tocopherol-deficient and -sufficient groups. Similarly, rats fed a high a-tocopherol diet for 2 months then fed a tocopherol-devoid diet for 3 days prior to y-tocopherol dosing exhibited tissue y-tocopherol levels intermediate to rats chronically fed the sufficient or high a-tocopherol levels without the 3-day washout period. These data indicated that a period of

FIG. 3. 2,8-Dimethyl-2-(2'-carboxyethyl)-6-chromanol sulfate, a urinary metabolite of 6tocopherol (Chiku ef al. 1984).

DIETARY AND BIOCHEMICAL ASPECTS OF VITAMIN E

207

dietary tocopherol withdrawal sufficient to clear the gastrointestinal tract of tocopherols was sufficient to enhance 24-hr tissue levels of the y-vitamer. Since rats dosed simultaneously with a- and y-tocopherol had tissue y-tocopherol levels substantially lower than those given an equivalent amount of y-tocopherol, the authors suggested that a-tocopherol inhibits absorption of y-tocopherol when both are present in the intestine. However, they also present plasma y-tocopherol versus time curves which show no effect of dietary a-tocopherol intake on plasma y-tocopherol kinetics from 0 to 4 hr, and which diverge only after this point to reflect inversely the dietary (and tissue) levels by 24 hr. This, along with the finding that the 3-day washout period did not yield the same effect on tissue y-tocopherol levels 24 hr post-y-tocopherol dosing as the chronic a-tocopherol-devoid diet, seems to indicate a postabsorptive effect of atocopherol status on tissue y-tocopherol levels. Behrens and Madke (1987) showed that 6 5 4 5 % of tissue y-tocopherol was lost between 24 and 72 hr post-y-tocopherol dosing (a-tocopherol-devoid diet) except in the heart, in which the y-vitamer was retained. The effect of varying dietary a-tocopherol levels on the rate of loss of tissue y-tocopherol remains to be determined. The interaction between a- and y-tocopherol may occur largely, though not exclusively, in the liver. It is tempting to speculate that the liver content of a-tocopherol (or a specific hepatic atocopherol pool) may influence the proportion of new hepatic (chylomicron remnant) y-tocopherol which is metabolized and secreted into the bile. Alternatively, the relative proportions of the two vitamers in the chylomicron remnant may affect the proportion of y-tocopherol which survives bile secretion and is subsequently exported (via VLDL) to peripheral tissues. This latter scenario is also supported by the data of Behrens and Madkre (1987), who showed that tissue y-tocopherol levels were inversely proportional to plasma a-tocopherol concentration, regardless of dietary treatment. A central role for the liver in regulating y-tocopherol metabolism and distribution would also be consistent with the results of Bjomeboe et al. (1987) regarding a-tocopherol disposition. VIII.

FUNCTIONAL ASPECTS OF TOCOPHEROLS IN BIOMEMBRANES

A. ANTIOXIDANT FUNCTION IN MEMBRANES The primary function of the tocopherols, and that most substantiated, is as antioxidants in hydrophobic environments. All of the four tocopherol vitamers (a-, p-, y-, and &tocopherol) are effective chain-breaking antioxidants in bulk unsaturated lipids. However, since a-tocopherol is

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the predominant tocopherol found in human and animal biomembranes, the present discussion focuses on this vitamer. While many of the precise details of the membrane antioxidant mechanism of a-tocopherol remain under investigation, its chemistry is sufficiently understood to make knowledgeable inferences regarding the sequence of events involved in its function. a-Tocopherol is currently believed to act as a quencher of oxygen- or carbon-centered fatty acyl radicals or active oxygen via donation of a hydrogen atom from the phenyl hydroxyl of the chromanol ring. The phytyl side chain serves to anchor the vitamer in the phospholipid bilayer, and to position the tocopherol molecule perpendicular to the plane of the bilayer. Evidence that the phytyl group lies parallel with the acyl chains and that the chrornanol moiety is located near the membrane surface was provided by Perly et al. (1985). The rate of reaction of a-tocopherol and other phenols with peroxyl radicals of styrene under conditions of controlled peroxyl radical formation has been reported by Burton and Ingold (1981, 1986) and Burton et al. (1985). Their results demonstrate that the radical quenching (hydrogen donating) capability of a-tocopherol exceeds that of the other tocopherols and synthetic phenolic antioxidants, and thus may explain in part the superior vitamin E activity of the a-vitamer relative to the p-, y-, and 6forms. Apparently, a-tocopherol is a more effective hydrogen donor than the other tocopherols since electron-releasing methyl groups ortho to the phenolic hydroxyl, lacking in the other vitamers, stabilize the developing phenoxyl radical and increase the rate of the reaction: ROO.

+ ArOH + ROOH + ArO.

The number of alkyl radicals with which each phenoxyl group reacts is approximately two, as determined by Burton and Ingold (1981), using the inhibited oxidation of styrene (10s)method. As shown in Fig. 4, hydrogen abstraction from the phenolic hydroxyl results in formation of the phenoxyl (chromanoxyl) radical of a-tocopherol. Subsequent removal of a second electron yields a carbonium ion, which can react with water to give a-tocopheryl quinone. As in synthetic phenolic antioxidants such as butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT), the phenoxyl radical is resonance stabilized, an essential characteristic central to the antioxidant capabilities of these compounds. Burton and Ingold (1986) have clarified the stereoelectronics of the resonance stabilization of the tocopheroxyl radical, assigning an important role to the oxygen atom para to the phenyl hydroxyl group. They suggest this oxygen atom stabilizes the radical by

DIETARY AND BIOCHEMICAL ASPECTS OF VITAMIN E

CH 3

3

CH3

CH3

1 a-TOCOPHERYL

QUINONE

FIG. 4. Oxidation of a-tocopherol.

209

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ROBERT S . PARKER

conjugative electron delocalization permitted by the overlap of its p-type lone-pair orbital with the semioccupied molecular orbital of the developing phenoxyl radical. To be maximally effective, antioxidant radicals such as the chrornanoxyl radical should be relatively unreactive with molecular oxygen. Burton et al. (1985) have shown that the rate constant for decay of the chromanoxyl radical in oxygen-saturated solutions is not significantly different from that in oxygen-free solution, thus indicating its low reactivity with molecular oxygen. The chromanoxyl radical also should ideally be relatively unreactive toward organic compounds, such as unsaturated fatty acids. Accordingly, Burton and Ingold (1981) provided evidence that the chain transfer (propagation) reaction, ArO.

+ RH + ArOH + R.

is retarded when the phenoxyl oxygen is sterically protected by two ortho alkyl groups, as is the case with a- tocopherol. In addition to its proposed role in quenching oxygen- or carbon-centered fatty acyl radicals of membrane phospholipids, a-tocopherol may also function to inhibit the initiation of membrane peroxidation by quenching reactive oxygen species. Membrane-bound a-tocopherol has HO,and HO,. radicals (Fukuzawa and been reported to react with 02*, Gebicki, 1983). The positioning of the phenoxyl group at the surface of the membrane (Perly et al., 1985) makes feasible the notion that the vitamer may intercept these radical species prior to their interaction with allylic carbons of polyunsaturated fatty acids, which would reside deeper in the membrane. At the present time, it is not possible to estimate the relative importance (or frequency) of chain-breaking events versus initiation-preventing events in the inhibition of lipid peroxidation by cx-tocopherol in actual biomembranes. a-Tocopherol is also an extremely efficient quencher of singlet oxygen. Fahrenholtz et al. (1974) and Foote et al. (1978) reported its rate constant of singlet oxygen quenching in pyridine to be 2.5 x 10' mol-' sec-', and a rate constant for chemical reaction of a-tocopherol with 0, of 2 x lo6 mol- * sec- I , indicating that a-tocopherol could quench about 120 singlet oxygen molecules before itself being irreversibly oxidized. In addition to quenching peroxyl radicals of polyunsaturated fatty acids in biomembranes, a-tocopherol may also function to protect or repair membrane proteins. Bisby et al. (1984) found that Trolox C (an a-tocopherol analog made water soluble by removal of the phytyl side chain) could rapidly and efficiently repair (quench) radicals of several amino acids and of lysozyme in N,O-saturated aqueous solution. The high rate constants

DIETARY AND BIOCHEMICAL ASPECTS OF VITAMIN E

21 1

observed (ranging from 5 x lo7 M - ' sec-' for tryptophan and lysozyme to 8 x 10' M - ' sec-' for histidine) suggest a physiological role for atocopherol in repairing radical species of integral membrane proteins. More recently, Dean and Cheeseman (1987)reported a protective effect of a-tocopherol against radiolysis-induced fragmentation of mitochondria1 monoamine oxidase.

B. REGENERATION OF a-TOCOPHEROL FROM a-TOCOPHEROXYL RADICAL The efficient antioxidant activity of a-tocopherol despite its low concentration in biological membranes relative to that of polyunsaturated fatty acids suggests that the effective half-life of the vitamin in membranes is rather long. Several studies have indicated that the chromanoxyl radical of a-tocopherol may be reduced to regenerate a-tocopherol by ascorbic acid or glutathione. The interactions between ascorbic acid and a-tocopherol have been reviewed recently by McCay (1989,and only selected studies are described here. That ascorbic acid could quench the chromanoxyl radical of a-tocopherol was demonstrated by Packer et al. (1979)using pulse radiolysis. The rate of reaction (in water-isopropanol-acetone mixtures) was estimated at 1.55 x lo6 M-'sec-'. A somewhat lower rate of reaction of ascorbate with a-tocopheroxyl radicals in phosphatidylcholine vesicles (2 x lo5 M - ' sec-') was reported by Scarpa et al. (1984),a not unexpected finding due to the partitioning of the reactants between two phases and potential steric effects at the membrane interface. More recently, Mukai et al. (1987)measured the second-order rate constant of reaction of ascorbic acid with 5,7-diisopropyl-tocopheroxy1radical in benzene-ethanol at 5.5 x lo2 M - ' sec-I, considerably lower than that reported previously. The reason for this discrepancy is not apparent, although solvent effects or lower reactivity of the diisopropyl analog are possibilities. Leung et al. (1981)found that ascorbic acid lengthened the induction time of Fe +2-inducedperoxidation in rat liver microsomes. In liposomes composed of bovine phosphatidylcholine and phosphatidylethanolarnine ( I : I), ascorbic acid alone delayed peroxidation, and appeared to act synergistically with a-tocopherol at longer incubation times. Doba et al. (1985)reported a synergistic effect of ascorbic acid in a-tocopherol inhibition of peroxidation of dilinoleoylphosphatidylcholine vesicles induced by a lipid-soluble initiator. In this model ascorbate itself had no antioxidant effect, but doubled the induction period caused by a-tocopherol. This model had the advantage of employing oxygen uptake as the indicator of lipid peroxidation, thus avoiding the complications of using the thi-

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obarbituric acid (TBA) value, which depends on production of secondary products of fatty acid peroxide breakdown. However, the induction periods observed by Doba et al. (1985)are likely to be much longer than those of natural biomembranes, which contain high proportions of polyunsaturated fatty acids more readily peroxidizable than linoleate. In egg phosphatidylcholine liposomes, ascorbic acid (1 mM) has been reported to decrease both the threshold a-tocopherol concentration needed to prevent peroxidation induced by xanthine-xanthine oxidase-FeCl,-ADP, and the rate of consumption of the antioxidant (Fukuzawa et al., 1985). While a direct interaction between ascorbate and atocopherol may not be necessary to produce this effect, the data indicated a sparing of a-tocopherol by ascorbate in this system. Glutathione may also play a role in the reduction of intermediate oxidation products of a-tocopherol. Niki et al. (1982)observed decay of the electron spin resonance (ESR) signal associated with the a-tocopheroxyl radical in the presence of either ascorbic acid or glutathione (in ethanol : water, 5: 1 vh). The comparative rate of reduction by the two watersoluble compounds was not presented. The addition of glutathione to atocopherol-deficient or -sufficient rat liver microsomes increases the lag time prior to maximal rate of lipid peroxidation (TBA value) by about 2.8-fold (Hill and Burk, 1984). Since little or no evidence of glutathione oxidation was found, and trypsin treatment completely abolished the lag in lipid peroxidation, these authors proposed the existence of a glutathione-dependent microsomal enzyme which complements the action of atocopherol without directly affecting its metabolism.

C. CONCENTRATION AND MOBILITY OF a-TOCOPHEROL IN MEMBRANES Estimates of the concentration of a-tocopherol in specific membrane fractions or organelles vary considerably, which is not surprising, due to the correspondingvariation in tissue fractionation, extraction, and analytical techniques employed. McMurchie and Mclntosh (1986)have summarized reported membrane a-tocopherol concentrations, using published values recalculated as mole percent of membrane phospholipid. These include 0.05-0.13 rnol % a-tocopherol for rat liver mitochondria, 1.O rnol % for rat liver microsomes, 0.6 rnol % for rat platelets, 0.1-0.6 rnol % for human platelets, and 10 rnol % for bovine retinal outer rod segments. Verdon and Blumberg (1988)measured 0.12 rnol % a-tocopherol in rat erythrocyte ghosts. Values ranging from 0.2 to 5.4 rnol % for human adipocyte membranes were observed by Traber and Kayden (1987).Kornburst and Mavis (1980)reported the concentration of a-tocopherol in mi-

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213

crosomes from several rat tissues relative to fatty acids containing three or more double bonds. Assuming most phospholipids contain one such fatty acid (some phospholipids will contain linoleate at the sn-2 position), these values will approximate (though slightly overestimate) concentrations as mole percent of phospholipid. Lung, heart, liver, kidney, testes, and brain microsomes contained means of 3.3, 1.4, 0.55, 0.52, 0.51, and 0.38 mol % a-tocopherol, respectively. These data confirmed the earlier findings of Taylor et al. (1976) of higher concentrations of a-tocopherol in microsomes of lung and heart relative to liver. The effectiveness of a-tocopherol in preventing or limiting membrane lipid peroxidation should depend, at least in part, on its concentration relative to that of fatty acids susceptible to peroxidation. Consistent with this hypothesis is the finding of Kornburst and Mavis (1980) that the rate of ascorbate/Fe*+-inducedlipid peroxidation in microsomes from various rat tissues was inversely related to the membrane concentration of a-tocopherol. Lung and heart microsomes, which contained the highest atocopherol : peroxidizable fatty acid ratio, exhibited the slowest rates of peroxidation. Fukuzawa el a / . (1985) reported that the concentration of a-tocopherol needed to inhibit completely the peroxidation (TBA) of egg phosphatidylcholine liposomes initiated by a xanthine-xanthine oxidase-FeC1,-ADP system was dependent on the concentration of added EDTA-Fe3'. At 0 and 0.2 mM EDTA-Fe3', the required a-tocopherol concentrations were approximately 0.25 and 1.5 mol % (of phospholipid), respectively. Therefore, since the phospholipid contained only 3 mol % arachidonate (the only fatty acid present with three or more double bonds, which are considered to be the readily peroxidizable fatty acids), the minimum arachidonate : tocopherol ratio required to prevent peroxidation (0 mM EDTA-Fe3') in this system was about 24 : 1. This ratio is much lower than that found in natural biomembranes, which typically contain over 25% fatty acids with three or more double bonds. Fukuzawa et a / . (1985) found that the amount of a-tocopherol lost during 30 min incubation in liposomes with a-tocopherol concentrations exceeding that required to prevent the formation of TBA-reactive substances was a constant, regardless of the initial a-tocopherol concentration or amount of EDTA-Fe3' added. The authors suggested that a-tocopherol was lost primarily through reaction with fatty acyl peroxyl (ROO-) or carbon-centered (R-)radicals, and above the threshold a-tocopherol concentration, when the concentration of ROO. is minimal, the destruction of a-tocopherol is a function of the (constant) rate of formation of the initial radical, Re. The effectiveness of a-tocopherol in quenching alkyl peroxyl radicals produced in membrane phospholipid bilayers will clearly depend in large

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ROBERT S. PARKER

part on the mobility of a-tocopherol, i.e., the time required to come within effective quenching range of the radical species. To be effective, this period must be short compared to the half-life of the peroxyl radical, specifically the time before such a radical attacks other membrane fatty acids or proteins. Mobility of a-tocopherol in a membrane includes both lateral mobility in one monolayer of the bilayer, and flip-flop across the bilayer to the opposite monolayer. The rate of lateral (translational) mobility of a-tocopherol in either model or biological membranes has not been estimated. Lateral translation of phospholipids has been studied by ESR and NMR techniques in liquid crystalline systems, and calculated to be on the order of I pm/sec in unobstructed bilayers (Thompson and Huang, 1980). It seems reasonable that, in the absence of tight association of a-tocopherol with a membrane protein (receptor), the rate of lateral translation of a-tocopherol will be similar to that of phospholipids. The rate of exchange, or flip-flop, of a-tocopherol across the bilayer from one monolayer to the other is currently not known with precision, but has been estimated to occur with half-life of between and lo3 sec (Cheng et al., 1987). This rate is substantially faster than the rate of flipflop of cholesterol or phospholipids, estimated in a variety of experimental systems, which are generally on the order of several hours or days (Thompson and Huang, 1980). Thus, it is not surprising that a-tocopherol resides in both monolayers of membrane bilayers. Perly et al. (1985) reported equal concentrations of a-tocopherol in inner and outer monolayers of egg phosphatidylcholine unilamellar liposomes. D. PHYSICAL EFFECTS OF TOCOPHEROLS IN MEMBRANES In addition to its documented role in protecting cellular membranes from peroxidative damage, a role for a-tocopherol in influencing membrane physical state or lipid-lipid and lipid-protein interactions has been suggested (Lucy, 1978; Diplock, 1983). A physicochemical role for a-tocopherol in stabilizing biological membranes was suggested by Diplock and Lucy (1973), who, on theoretical grounds, proposed a specific complex between the phytyl side chain of a-tocopherol and arachidonic acid of membrane phospholipid. Such a complex was suggested to result from London-Van der Waals interactions between the phytyl methyl groups and pockets provided by the cis double bonds of the arachidonyl residue, and include polar interactions between the phospholipid polar head group and the chromanol hydroxyl at the surface of the membrane. The functional consequences of the tocopherol-arachidonyl interaction were proposed to include (1) protection of arachidonyl residues from oxidative damage, (2) reduction of perme-

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215

ability of membranes containing a high proportion of polyunsaturated fatty acids, and (3) inhibition of membrane phospholipase activity. The ability to carry out these functions at the typically dilute concentrations of a-tocopherol in biomembranes (arachidonic acid : tocopherol ratio of 500 : 1) was proposed to result from transbilayer asymmetry of phospholipids and (presumably) a-tocopherol. The Diplock-Lucy (1973) model has prompted several studies concerning either the molecular disposition of a-tocopherol in biomembranes or its effects on membrane physicochemical characteristics. Lucy (1978) reported the influence of a-tocopherol on the surface pressure of constant area phospholipid monolayers. Significant penetration of a-tocopherol into diarachidonylphosphatidylcholine monolayers occurred only above 0.3 mol a-tocopherol per mole phospholipid. At ratios of 0.5-2.0 rnol atocopherol per mole phospholipid, surface pressure was constant, at approximately 7-fold that at 0.2 mol a-tocopherol per mole phospholipid. The magnitude of the increase in surface pressure occurring upon penetration of a-tocopherol into phospholipid monolayers (a-tocopherol : phospholipid, 1 : 1) increased with unsaturation of the phospholipid, up to two double bonds per fatty acid. Dilinoleoyl- and diarachidonylphosphatidylcholine monolayers displayed similar responses to a-tocopherol. Increases in surface pressure equal to those found with monolayers of unsaturated phospholipid alone were obtained with mixed saturatedunsaturated monolayers containing only 20% unsaturated phospholipid. These results were interpreted to suggest that the interaction between atocopherol and polyunsaturated phospholipid is a dynamic one, such that a-tocopherol may interact with several phospholipid molecules in the bilayer, thus influencing bilayer properties at tocopherol : phospholipid molar ratios of less than one. a-Tocopherol has been shown to influence the thermotropic behavior of membranes. The effects of incorporation of a-tocopherol into saturated phospholipid bilayers at relatively high concentrations (1 mol % or greater) include reduction in transition enthalpy, elimination of the pretransition peak, broadening of the temperature range over which the transition takes place, and a slight decrease in the phase transition temperature (Massey et al., 1982; McMurchie and McIntosh, 1986). These results suggest that at these relatively high concentrations, a-tocopherol increases fluidity (disorder) in the hydrocarbon chain region of these saturated phospholipid bilayers, possibly due to the disruptive influence of the phytanoyl methyl groups. McMurchie and McIntosh (1986) also inferred a reduction in the dimensions of the cooperative unit of lipids participating in the acyl chain phase transition. Massey et a/. (1982) noted that the thermotropic data from saturated bilayers are consistent with the

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expected behavior of a substance which aligns itself primarily with the direction of the acyl chains of the bilayer leaflet. They also observed an increase in fluorescence polarization of diphenylhexatriene at 37°C with increasing concentrations of bilayer a-tocopherol. This indicated that atocopherol disturbs lipid packing even in the liquid-crystalline state, although the data of Massey et al. (1982) suggest a threshold concentration for this phenomenon of between 1 and 5 mol %. The findings of Massey et al. (1982) and McMurchie and McIntosh (1986) contrast with those of Ohki et al. (1984), who found that a-tocopherol actually increased the order parameter in rat liver phosphatidylcholine liposomes at concentrations exceeding 0.2 M. a-Tocopherol at this concentration had no effect on the order parameter of egg phosphatidylcholine vesicles. Thus it appears that the effect of a-tocopherol on acyl chain order of phospholipid bilayers is dependent both on the tocopherol : phospholipid ratio and on the acyl chain composition of the constituent phospholipids. In membranes containing significant amounts of polyunsaturated fatty acids, particularly those with four or more double bonds, a-tocopherol may increase order. In more saturated bilayers, a-tocopherol decreases order by interfering with acyl chain packing. However, as pointed out by McMurchie and McIntosh (1986), the concentrations of atocopherol necessary to effect such structural alteration are far in excess of that observed in most biological membranes, assuming homogeneous dispersion of a-tocopherol in the bilayer leaflets. Of course, a nonhomogeneous dispersion characterized by regions of high a-tocopherol concentration could result in local influences on phospholipid acyl order. Srivastana et al. (1983) compared partitioning of the spin label 2,2,6,6tetramethylpiperidine-N-oxyl into dipalmitoylphosphatidylcholine vesicles with or without a-tocopherol (phospholipid :tocopherol ratio of 5 : 1). Phase transition behavior as revealed by this technique consisted of a broadening of the typically sigmoidal phase transition of the pure phospholipid bilayers and elimination of the pretransition. These results are consistent with those of Massey et al. (1982), who employed differential scanning calorimetry. Srivastana et al. (1983) also used I3C-NMR to probe the interaction of a-tocopherol and dipalmitoylphosphatidylcholine in single bilayer vesicles. Spin lattice relaxation times, thought to reflect overall tumbling and segmental motion of nuclei, indicated that both a-tocopherol and the phospholipid lose mobility when they coexist in bilayers. By comparing the behavior of a-tocopherol with its acetate derivative, the authors concluded that a-tocopherol bound strongly to the saturated phospholipid through both hydrophobic bonding and hydrogen bonding with the polar

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head group. Urano et al. (1987) extended the use of [a-'3C]to~opherol to study its spin relaxation behavior in liposomes varying in fatty acid composition. These investigators found increasing segmental mobility of the tocopherol phytanoyl chain with increasing depth in the bilayer leaflet (increasing distance from the chromanol moiety). Segmental motion of the 6' methylene carbon, positioned at about the midpoint of the phytanoyl chain, was 2040% greater in unsaturated bilayers (made from egg or rat liver phosphatidylcholine) compared to dipalmitoylphosphatidylcholine bilayers. In addition, T , values of the methyl carbons of the side chain were not significantly different in bilayers of different arachidonic acid contents. These data seem to be at odds with the suggestion of Diplock and Lucy (1973) that the 4' and 8' methyl groups of the phytanol side chain bind to pockets of arachidonoyl groups of bilayer phospholipids. Interestingly, Urano et al. (1987) also found that T, values for the three methyl carbons attached to the aromatic ring (5a, 7a, 8b) of a-tocopherol in egg and liver phosphatidylcholine were decreased relative to the corresponding values in saturated phospholipid liposomes. However, it should be noted that the I3C-NMR studies of Urano et al. (1987) used bilayers with a phospholipid : tocopherol ratio of 2 : 1, at which tocopherol-tocopherol interactions may interfere with tocopherol-phospholipid interactions normally expressed at dilute tocopherol concentrations. While several studies have reported physical effects of a-tocopherol in phospholipid vesicles, few studies have employed actual membranes, in which protein contributes substantially to total membrane mass and which may modulate the influence of added lipids. In one such study, Steiner (1981) examined the effect of added a-tocopherol on fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatrienein normal human platelets in vitro. Above 27"C, fluorescence polarization increased with increasing atocopherol concentration. Liposomes prepared from platelet lipids also exhibited increased fluorescence polarization above 15°C when enriched with a-tocopherol. These results suggested that a-tocopherol exerts a fluidizing effect on platelet membranes. The results of studies of platelet membrane fluidity with in vitro enrichment of a-tocopherol contrast to similar studies using in vivo dietary depletion of a-tocopherol. For example, Whitin et al. (1982) compared the temperature dependence of fluorescence polarization of 1,6-diphenyI1,3,5-hexatrieneincorporated into intact platelets from a-tocopherol-sufficient or -deficient rats. Probe polarization behavior was identical in platelets from rats fed the diets for 6 to 10 weeks, at which time plasma atocopherol levels were below 1 & n l in the deficient group. While actual platelet concentrations of a-tocopherol were not measured, these results

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suggested that a-tocopherol, at normal platelet concentrations, exerts little effect on platelet membrane fluidity as reflected by fluorescence polarization. In most cellular membranes in animals, cholesterol concentrations generally exceed that of a-tocopherol. Cholesterol also influences the thermotropic properties of phospholipid bilayers, but Massey et al. (1982) reported a more influential effect of a-tocopherol when the two compounds were compared in saturated bilayers. The greater effectiveness of a-tocopherol was attributed to the configurational mobility of the phytanoyl side chain. The influence of a-tocopherol on membrane permeability to ions or small molecules has also been the subject of several studies. The influence of a-tocopherol on glucose and chromate permeability of phospholipid multilaminar vesicles at 30°C was studied by Diplock et al. (1977). In liposomes containing egg phosphatidylcholine (PC), phosphatidic acid, and a-tocopherol in a ratio of 6 : 1 : 1, tocopherol prevented the increase in leakage from vesicles containing increased amounts of arachidonic acid (5 versus < 1% arachidonic acid). Tocopherol had no effect in vesicles made with PC containing less than 1% arachidonate, even though the phospholipid contained 13% linoleate. In liposomes made with PC containing 8% arachidonate and a PC : phosphatidic acid :tocopherol ratio of 2 : I .5 : I , leakage was also significantly lower than in the corresponding tocopherol-devoid liposomes. Importantly, a-tocopherol had no effect on permeability in vesicles in which the phospholipid :tocopherol ratio was reduced to 20 : 1. Fukuzawa et al. (1979) also compared the influence of a-tocopherol on glucose permeability in multilamellar phosphatidylcholine (PC) vesicles of differing fatty acid composition. In saturated PC vesicles, increasing a-tocopherol concentrations from 0 to 20 mol % of phospholipid lowered the transition temperature for marked release of entrapped glucose. aTocopherol (phospholipid : tocopherol 5 : 1) decreased permeability of egg phosphatidylcholine liposomes at 5" and 40"C, but had no effect at the intermediate temperatures. The same level of a-tocopherol slightly reduced permeability of highly saturated soy phosphatidylcholine vesicles at all temperatures, but the effect was small compared to that of saturated phospholipid vesicles. Srivastana et al. (1983) studied the influence of a-tocopherol on ascorbate permeability of dipalmitoylphosphatidylcholinevesicles above the phase transition temperature by estimating rates of reduction of spin label derivatives of 5-ketostearic acid in the outer and inner bilayer leaflets. Incorporation of a-tocopherol into vesicles at a phospholipid :tocopherol

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ratio of 5 : 1 increased vesicle ascorbate permeability compared to tocopherol-devoid membranes. The effect of a-tocopherol on membrane permeability appears to be concentration dependent. As indicated above, a-tocopherol may have little effect at tocopherol : phospholipid ratios substantially less than 1 :20. Also, effects at low concentrations may differ from those at relatively high concentrations. For example, Fukuzawa et al. (1979) observed a biphasic effect of a-tocopherol on glucose permeability of saturated phospholipid vesicles at 37°C as a function of membrane a-tocopherol content. a-Tocopherol increased permeability when incorporated at 0 to 5 mol %, but decreased permeability from 5 to 20 mol %. In earlier studies, Fukuzawa et al. (1971) reported that lysosomes incubated with 1 x M atocopherol exhibited reduced rates of acid phosphatase release, but release was stimulated at 5 x M a-tocopherol. a-Tocopheronolactone (1 x M ) was more effective than a-tocopherol at inhibiting acid phosphatase release, and a-tocopheryl quinone stimulated release at all concentrations tested. Curiously, none of these tocopherol compounds had any effect on P-glucuronidase release. Since the observed effects on acid phosphatase release were not correlated with antioxidant capability, the authors postulated a role for these compounds in altering the structure of lysosomal membranes. However, interpretation of these data is made difficult by the fact that quantitative estimates of incorporation of these various tocopherols into lysosomal membranes were not made. Mino and Sugita (1978)found that a-tocopheryl acetate inhibited hemolysis in human erythrocytes subsequently incubated with retinol and hydrogen peroxide. a-Tocopherol inhibited hemolysis to the same extent as the acetate form. Since only about 1% of membrane-associated radioactivity from [a-3H]tocopherylacetate was recovered as a-tocopherol, the authors proposed that both a-tocopherol and its acetate form stabilized erythrocyte membranes by physical means, rather than via an antioxidant mechanism. The final membrane concentration of a-tocopheryl acetate (or total tocopherols) was not determined, and therefore it is difficult to compare these results with those described above, or interpret the results in terms of a potential function of a-tocopherol. Douglas et al. (1986) studied the influence of tocopherols on platelet phospholipase A, activity. Enzyme activity of vitamin E-deficient rats (plasma a-tocopherol, 0.23 0.06 mg/dl) was increased by 50% over that of platelets from rats fed 100 IU a-tocopherol per kilogram diet (plasma tocopherol, 2.36 2 0.02 mg/dl). However, there was no difference in enzyme activity in platelets of rats fed 100 IU/kg compared to those fed 1000 IU/kg. In vitro incubation of sonicated platelets from vitamin E-defi-

*

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cient rats with 23 p,M d-a-tocopherol reduced platelet phospholipase A, activity to that of the vitamin E-sufficient rats. Tocol was as effective as d-a-tocopherol in this respect. Platelet phospholipase A2 was partially purified by Sepharose 6B column chromatography and its activity assayed in the presence of different concentrations of various tocopherols. Activity was inhibited nearly 50% by 100 pA4 d-a-tocopherol, and 75% by 100 tocol. Since enzyme activity was assayed in the presence of phospholipid vesicles, the authors noted the inability to determine whether tocopherols influence phospholipase A2 by direct interaction with the enzyme, by interacting with the phospholipid substrate, or by altering the physical characteristics of the bilayer with which the enzyme interacts. IX. FUTURE RESEARCH DIRECTIONS

While clinical signs of vitamin E deficiency remain rare in the general population, continued assessment of tocopherol intake is needed. Such information will be more important for that segment of the population chronically consuming low-fat diets, or if the uses of fat substitutes become widespread. Under such conditions, the major dietary sources of vitamin E may shift away from vegetable oils and oil products to other dietary components. The current and future contribution of vitamin E supplements to tocopherol intake likewise is in need of more accurate assessment. The use of tocopherols labeled with stable isotopes in human studies should greatly facilitate the clarification of issues related to tocopherol absorption, pharmacokinetics, and excretion. Such issues include efficiency of absorption of a-,y-, and 8-tocopherols at various levels of intake, retention of various stereoisomers of a-tocopherol and its acetates, structural identification of biliary and urinary metabolites of the various tocopherol vitamers, and interactions between tocopherols which influence absorption and excretion. Stable isotopes may also prove useful in defining a-tocopherol requirements by humans as a function of age, gender, and health status. Deuterated a-tocopherol has been used to obtain the first estimates of a-tocopherol turnover in various organs in vitamin E-sufficient rats, as discussed above. Such studies need to be extended to include turnover rates in other nutritional states such as vitamin E deficiency (or excess intake), selenium deficiency, and aging. Also, the relative contributions to tissue turnover rates of a-tocopherol consumption versus mobilization need to be determined.

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The various factors contributing to the observed interindividual variation in serum a-tocopherol levels, and the factors regulating fasting plasma tocopherol levels in humans remain largely unknown. It is thought that diet is an important contributor to this variation, but other biochemical and physiological factors such as absorption efficiency, rate of utilization and excretion, efficiency of tissue uptake, and rate of tissue mobilization may also be involved. Clarification of this issue is important since plasma tocopherol levels are often used in drawing inferences regarding tocopherol intake or current status. A related unresolved question is the potential role of adipose tissue as a determinant of the bioavailability of a-tocopherol. If adipose tissue deposition is indeed irreversible in humans, then tocopherol bioavailability would be defined as not only that proportion of ingested tocopherol which is absorbed, but rather the proportion of absorbed tocopherol which escapes adipose tissue deposition. The relative importance of Lipoprotein-mediated (receptor dependent or independent) and lipoprotein lipase-mediated mechanisms of tocopherol uptake by various mammalian tissues remains to be determined, as do the potential interspecies differences in tissue uptake by these routes. In contrast to the human, rat plasma contains little LDL, seemingly due to incomplete conversion of VLDL to LDL and efficient clearance of partially lipolyzed VLDL from plasma (Eisenberg, 1979). Consequently, receptor-mediated LDL tocopherol uptake by extrahepatic tissues may be of lesser significance in the rat than in the human. In addition, the major mechanism of tissue tocopherol uptake may differ between tissues, depending on factors such as receptor density, lipoprotein lipase activity, and vascularity. The potential role of HDL in tissue uptake or clearance of tocopherols also needs clarification. The pathways of tocopherol biotransformation to polar metabolites by various tissues, particularly liver and kidney, and the regulation of these pathways need to be determined using appropriate model systems. Examples of such systems might include isolated or cultured cells, organ culture, or cell-free systems containing the enzymatic and nonenzymatic factors necessary for side-chain oxidation and conjugation. In addition, continued research concerning the role of ascorbic acid, glutathione, or other reducing substances in the sparing or regeneration of a-tocopherol in biological systems is needed. A number of experimental factors must be borne in mind when designing and implementing studies of tocopherol biochemistry and physiology. Animal models are often more convenient, and occasionally the only alternative, systems in which to study aspects of tocopherol metabolism, transport, or status. However, due to documented interspecies differences in lipid and lipoprotein metabolism, results from animal models may not be directly extrapolatable to humans

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and caution must be exercised in their interpretation. Particularly problematic are the areas of tocopherol absorption, transport, and tissue uptake, all processes which seem to be closely linked to lipid metabolism. Likewise, interspecies differences in conjugation capacity may have important impacts on both the quantitative and qualitative aspects of tocopherol metabolism and excretion. Detailed studies of the antioxidant role of a-tocopherol in biomembranes in vivo is hampered by the technical difficulties associated with the observation of membrane lipid peroxidation in intact cells or tissues. The intriguing question as to why a-tocopherol is apparently able to protect cellular membranes from peroxidative damage despite its low membrane concentration presupposes that such membranes are in fact under continual “attack” by active oxygen species or other substances capable of initiating lipid or protein oxidation. This, however, is largely conjecture and the development of more sophisticated techniques is needed before we can know with any certainty the extent to which a-tocopherol is called upon to prevent or limit peroxidative damage in intact cells. aTocopherol is but one of many antioxidant factors present in mammalian cells, and its quantitative importance in the prevention of oxidative membrane damage relative to these other factors is largely unknown. Explanation of its documented efficiency, even at similarly low concentrations, in model membrane systems remains elusive, and continued research in this area is needed.

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Behrens, W. A., and Madere, R. 1985. Transport of a- and delta-tocopherol in human plasma lipoproteins. Nurr. Res. ( N .Y . ) 5 , 167-174. Behrens, W. A., and Madtre, R. 1987. Mechanisms of absorption, transport and tissue uptake of RRR-a-tocopherol and d-y-tocopherol in the white rat. J. Nutr. 117, 1562- 1569.

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Bhat, R., Raju, T., Barrada, A., and Evans, M. 1987. Disposition of vitamin E in the eye. Pediatr. Res. 22, 16-20. Bieri, J. G. 1972. Kinetics of tissue a-tocopherol depletion and repletion. Ann. N . Y. Acad. Sci. 203, 181-191. Bieri, J. G . , and Farrell, P. M. 1976. Vitamin E. Vitam. Horm. 34,31-75. Bieri, J. G., and Poukka-Evarts, R. 1973. Tocopherols and fatty acids in American diets. J. Am. Diet. Assoc. 62, 147-151. Bieri, J. G . , and Poukka-Evarts. R. T. 1974. Gamma-tocopherol: Metabolism biological activity and significance in human vitamin E nutrition. Am. J . Clin. Nutr. 27,980-986. Bieri, J. G., and Tolliver T. J. 1982. Reversal by bile acid on the inhibition of a-tocopherol absorption by retinoic acid. J . Nurr. 112,401-403. Bieri, J. G., Wu,A-L., and Tolliver, T. J. 1981. Reduced intestinal absorption of vitamin E by low dietary levels of retinoic acid in rats. J. Nutr. 111, 458-467. Bieri, J. G., Corash, L., and Hubbard, V. S. 1983. Medical uses of vitamin E. N . Engl. J. Med. 308, 1063-1071. Bisby, R. H., Ahmed, S., and Cundall, R. B. 1984. Repair of amino acid radicals by a vitamin E analogue. Biochem. Eiophys. Res. Commun. 119, 245-251. Bj@rneboe,A., BjZrneboe, G-E. A., and Drevon, C. A. 1987. Serum half-life, distribution, hepatic uptake and biliary excretion of a-tocopherol in rats. Biochim. Eiophys. Acta 921, 175-181.

Bjornson, L. K., Gniewkowski, C., and Kayden, H. J. 1975. Comparison of exchange of atocopherol and free cholesterol between rat plasma lipoproteins and erythrocytes. J. Lipid Res. 16, 39-53. Bjornson, L. K., Kayden, H. J., Miller, E., and Moshell, A. N. 1976. The transport of atocopherol and p-carotene in human blood. J. Lipid Res. 17, 343-352. Blomstrand, R., and Forsgren, L. 1968. Labelled tocopherols in man. Int. Z. Vitam. Forsch. 38,328-344.

Booth, V. H . , and Bradford, M. P. 1963. The effect of cooking on a-tocopherol in vegetables. Inr. J. Vitam. Nirr. Res. 33, 276-278. Bunnell, R. H.. Keating, J. P., Quaresimo, A. J., and Parman, G. K. 1965. Alpha-tocopherol content of foods. A m . J. Clin. Nutr. 17, 1-10. Burton, G. W., and Ingold, K. U. 1981. Autoxidation of biological molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro. J. Am. Chem. Soc. 103,6472-6477. Burton, G. W., and Ingold, K.U. 1986. Vitamin E: Application of the principles of physical organic chemistry to the exploration of its structure and function. Ace. Chem. Res. 19, 194-201.

Burton, G. W., Doba, T., Gabe, E. J., Hughes, L., Lee, F. L., Prasad, L., and Ingold, K. U. 1985. Autoxidation of biological molecules. 4. Maximizing the antioxidant activity of phenols. J. Am. Chem. Soc. 107,7053-7065. Carlson, B. L., and Tabacchi, M. H.1986. Frying oil deterioration and vitamin loss during foodservice operation. J. Food Sci. 51, 218-221.

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Carpenter, A. P., Jr. 1979. Determination of tocopherols in vegetable oils. J. Am. Oil Chem. SOC.56,668-67 1. Carpenter, D. L., Lehmann, J., Mason, B. S., and Slover, H. T. 1976. Lipid composition of selected vegetable oils. J. Am. Oil Chem. SOC.53,713-718. Catagnani, G . L. 1975. An a-tocopherol binding protein in rat liver cytoplasm. Biochem. Biophys. Res. Commun. 67, 66-72. Catagnani, G. L., and Bieri, J. G. 1977. Rat liver a-tocopherol binding protein. Biochim. Biophys. Acta 497,349-357. Cheng, S. C., Burton, G. W.,Ingold, K. V., and Foster, D. 0. 1987. Chiral discrimination in the exchange of a-tocopherol stereoisomers between plasma and red blood cells. Lipids 22,469-73. Chiku, S., Hamamura, K., and Nakamura, T. 1984. Novel urinary metabolite of d-6-tocopherol in rats. J. Lipid Res. 25, 40-48. Chow, C. K. 1985. Vitamin E and blood. World Rev. Nutr. Diet. 45, 133-166. Chow, C. K., Draper, H. H., Csallany, A. S., and Chiu, M. 1967. The metabolism of CI4a-tocopheryl quinone and C"-a-tocopheryl hydroquinone. Lipids 2, 390-396. Csallany, A. S., Draper, H. H., and Shah, S. N. 1962. Conversion of d-a-tocopherol-CI4to tocopheryl-p-quinone in vivo. Arch. Biochem. Biophys. 98, 142-145. Dean, R. T., and Cheeseman, K. H. 1987. Vitamin E protects proteins against free radical damage in lipid environments. Biochem. Biophys. Res. Commun. 148, 1277-1282. Deckelbaum, R. J., Eisenberg, S., Oschry, Y.,Granot, E., Sharon, I., and Bengtsson-Olivecrona, G. 1986. Conversion of human plasma high density lipoprotein-2 to high density lipoprotein-3. Roles of neutral lipid exchange and triglyceride lipases. J. Biol. Chem. 261,5201-5208. DeLumen, B. 0.. Lubin, B., Chiu, D., Reyes, P. S., and Omaye, S. T. 1982. Bioavailability of vitamin E in rats fed diets containing pectin. Nutr. Res. ( N .Y.) 2, 73-83. DeRitter. E., Osadca. M., Scheiner, J., and Keating, J. 1974. Vitamins in frozen convenience dinners and pot pies. J. Am. Diet. Assoc. 64, 391-397. Dicks, M. W. 1965. Vitamin E content of foods and feeds for human and animal consumption. Agric. Exp. Sta. Bull. Univ. Wyoming, Laramie (435, Dec.). Diplock, A. T. 1983. The role of vitamin E in biological membranes. ClBA Found. Symp. 101.

Diplock, A. T., and Lucy, J. A. 1973. The biochemical modes of action of vitamin E and selenium: A hypothesis. FEBS Lett. 29, 205-210. Diplock, A. T., Lucy, J. A., Veninder, M.,and Zieleniewski, A. 1977. a-Tocopherol and the permeability to glucose and chromate of unsaturated liposomes. FEBS Lett. 82, 341-344. Doba, T., Burton, G. W., and Ingpld, K. U. 1985. Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes. Biochim. Biophys. Acta 835, 298-303. Douglas, C. E., Chan, A. C., and Choy, P. C. 1986. Vitamin E inhibits platelet phospholipase A,. Biochim. Biophys. Acta. 876, 639-645. Eisenberg, S. 1979. Very-low-density lipoprotein metabolism. Prog. Biochem. Pharmacol. 15, 139-165. Elsas, L. J., and McCormick, D. B. 1986. Genetic defects in vitamin utilization. Part 1: General aspects and fat-soluble vitamins. Vitam. Horm. 43, 103-144. Fahrenholtz, S. R., Doleiden, F. H., Trozzolo, A. M.,and Lamola, A. A. 1974. On the quenching of singlet oxygen by a-tocopherol. Photochem. Photobiol. 20, SO5-509. Fischer, W. C., and Whanger P. D. 1977. Effects in selenium deficiency on vitamin E metabolism in rats. J. Nutr. Sci. Vitaminol. 23, 273-280.

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Foote, C. S., Clough, R. L., and Yee, B. G. 1978. Photooxidation of tocopherols. I n “Tocopherol, Oxygen and Biomembranes” (C. deDuve and 0. Hayaishi, eds.). Elsevier, Amsterdam. Fukuzawa, K., and Gebicki, J. M. 1983. Oxidation of a-tocopherol in micelles and liposomes by the hydroxyl, perhydroxyl, and superoxide free radicals. Arch. Biochem. Biophys. 226,242-25 I . Fukuzawa, K., Suzuki, Y., and Uchiyama, M. 1971. Modified lipophilic vitamin-IV. Stabilizing effect of a-tocopherol and tocopheronolactone on mouse liver lysosomes in vivo and in vitro. Biochem. Pharmacol. 20, 279-287. Fukuzawa, K., ikeno, H., Tokumura, A., and Tsukatani, H. 1979. Effect of a-tocopherol incorporation on glucose permeability and phase transition of lecithin liposomes. Chem. Phys. Lipids 23, 13-22. Fukuzawa, K., Takase, S., and Tsukatani, H. 1985. The effect of concentration on the antioxidant effectiveness of a-tocopherol in lipid peroxidation induced by superoxide free radicals. Arch. Biochem. Eiophys. 240, 117-120. Gallo-Tomes, H. E. 1980. Absorption, metabolism and transport. In “Vitamin E. A Comprehensive Treatise” (L. J. Machlin, ed.), pp. 170-267. Dekker, New York. Gardner, H. K., Vang, M. J., and Mavis, R. D. 1986. Removal of a-tocopherol from blood and its comparison with other lipids. Fed. Proc., Fed. Am. SOC.Exp. Biol. 45, 841. Gardner, H. W., Eskins. K., Grams, G. W., and Inglett, G. E. 1972. Radical addition of linoleic hydroperoxides to a-tocopherol or the analogous h ydroxychroman. Lipids 7, 324-334.

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Haddad, E., Blankenship, J. W., and Register, U. D. 1985. Short term effect of a low fat diet on plasma retinol and a-tocopherol and red cell a-tocopherol levels in hyperlipidemic men. A m . J . Clin. Nutr. 41, 599-604. Haile, R. W. C., Hunt, I. F., Buckley, J., Browdy, B. L., Murphy, N. J., and Alpers, D. 1986. Identifying a limited number of foods important in supplying selected dietary nutrients. J . Am. Diet. Assoc. 86, 61 1-616. Hauswirth, J. W.,and Nair, P. P. 1972. Ann. N . Y . Acad. Sci. 203, 111-121. Hayaishi, O., and Mino, M., eds. 1987. “Clinical and Nutritional Aspects of Vitamin E.” Elsevier, Amsterdam. Hernandez Rabascall, N., and Riera, J. B. 1987. Changes in tocopherol and tocotrienol content during the extraction, refining and hydrogenation of edible oils. Grusas Aceites (Seville) 38, 145-148. Hill, K. E., and Burk, R. F. 1984. Influence of vitamin E and selenium on glutathionedependent protection against microsomal lipid peroxidation. Biochem. Pharmacol. 33, 1065- 1068.

Honvitt, M. K., Harvey, C. C.. Dahm, C. H., Jr., and Searcy, M. T. 1972. Relationship between tocopherol and serum lipid levels for determination of nutritional adequacy. Ann. N . Y. Acad. Sci. 203, 223-236. Ingold, K. U., Burton, G. W., Foster, D. O., Hughes, L., Lindsay, D. A., and Webb, A.

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OXIDATION OF POLYUNSATURATED FATTY ACIDS: MECHANISMS, PRODUCTS, AND INHIBITION WITH EMPHASIS ON FISH R. J. HSIEH Campbell Institute for Research and Technology Campbell Soup Company Camden, New Jersey 08103

J. E. KINSELLA Institute of Food Science Cornell University Ithaca, New York 148S3

I.

Introduction Mechanisms of Oxidation of Polyunsaturated Fatty Acids A. Oxygen Activation B. Free Radical Oxidation C. Photosensitized Singlet Oxygen Oxidation D. Enzyme-Initiated Lipid Oxidation 111. Products of Lipid Oxidation Decomposition of Acyl Hydroperoxides IV. Other Effects of Lipid Oxidation I n Vivo Effects of Peroxidation V. Lipid Oxidation in Fish Factors Affecting Oxidation VI. Control of Lipid Oxidation A. Reactants and Inhibitors B. Oxidative Enzymes C. Antioxidants: Flavonoids VII. Conclusions References 11.

233 Copyright 0 1989 by Academic Press, Inc. All righls of reproduction in any form reserved.

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1.

INTRODUCTION

Numerous chemical and biochemical reactions affect the quality attributes (color, odor, flavor, texture), nutritional value, safety, and physiological effects of food components and no component epitomizes this more than the oxidation of unsaturated fatty acids. The oxidative degradation of the unsaturated fatty acid components of food lipids may be beneficial in some foods in generating low levels of desirable flavorful carbonyl compounds. However, in general, oxidation causes deleterious changes in flavor, taste, color, texture, and possibly safety of foods (Lundberg, 1962; Simic and Karel, 1980; Chan, 1987). The polyunsaturated fatty acids (PUFA), particularly the trienoic, pentaenoic, and hexaenoic PUFA commonly found in oilseeds and seafoods, render these foods particularly sensitive to oxidative changes which limit their selflife. The growing interest in the possible beneficial effects of increasing dietary intake of polyenoic fatty acids, particularly the n-3 PUFA species (Kinsella, 1987), has dramatized the need for improving procedures for controlling lipid oxidation. This has underscored the need for more fundamental information concerning the mechanisms of lipid oxidation in foods because an understanding of mechanisms (chemistry, kinetics, and thermodynamics) should facilitate the development of more effective control measures. Theoretically, molecular oxygen and PUFA cannot interact because of thermodynamic constraints; however, numerous agencies can initiate peroxidation which, via subsequent free radical mechanisms, can result in rampant autoxidation. Thus, several enzymes, photosensitizers, and transition metals can directly and indirectly facilitate oxidative reactions. In the following sections, some of these mechanisms are reviewed, particularly with regard to the oxidation of the polyunsaturated fatty acids in fish. II. MECHANISMS OF OXIDATION OF POLYUNSATURATED FATTY ACIDS

A. OXYGEN ACTIVATION

The interaction of oxygen with an unsaturated fatty acid is an important reaction occurring under a wide range of conditions; however, direct interaction is extremely slow because the initiation of the oxidation of unsaturated fatty acids requires an active form of oxygen. The ground state of unsaturated fatty acids corresponds to the singlet state which

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235

is diamagnetic, i.e., there are two paired electrons in the outer electronic shell. In contrast, ground state oxygen is in a triplet state (paramagnetic) with two unpaired electrons that have the same spin but are in different orbitals. Hence the reactivity of triplet oxygen with unsaturated fats is forbidden because of the spin restriction imposed by these spin states (Lowry and Richardson, 1981; Kanner e t al., 1987). This spin restriction of molecular oxygen can be overcome by any of the following four initiation mechanisms: (1) singlet oxygen; (2) partially reduced or activated oxygen species such as hydrogen peroxide, superoxide anion, or hydroxyl radicals; (3) active oxygen-iron complexes (ferry1 iron); and (4) iron-mediated homolytic cleavage of the hydroperoxides, which generate organic free radicals (Kanner e t al., 1987).

1 . Singlet Oxygen Singlet oxygen can exist in two forms characterized by different energy states. In one form which is 37 kcal above ground state, the two unpaired electrons in separate orbitals spin in opposite directions. In the other form, which is 22 kcal above ground state, the outer shell electrons occupy the same orbital and have opposite spins. Because reactions of oxygen with organic molecules must proceed via transfer of pairs of electrons (which must have opposite spins), singlet oxygen is biologically more reactive than ground state oxygen (Singh and Petkau, 1978). Fridovich (1977) reported that '0, reacts with histidine at a rate of 6 x lo7 mol does not react at all at this sec-' at 20°C whereas triplet oxygen (30,) temperature. In biological systems, active oxygen species, such as singlet oxygen (lo,),hydroxyl radicals (OH.), superoxide anion (0;), and hydrogen peroxide (H,O,), can be generated via nonenzymatic and enzymatic mechanisms (Fridovich, 1976). Natural pigments such as hematoporphyrin and flavins may serve as sensitizers to yield '0, in the presence of 0, and visible light. Enzymes such as microsomal oxidases, lipoxygenase, and prostaglandin synthase may directly or indirectly generate '0, (KoryckaDahl and Richardson, 1980). Deterioration of biological tissues by peroxidation initiated by '0, mechanisms can be minimized by removal of natural pigments and peroxides. The inclusion of singlet oxygen scavengers, such as (3-carotene and a-tocopherol, can inhibit '0, mechanisms and reduce oxidation (Krinsky, 1981).

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R. J. HSIEH AND J. E. KINSELLA

2. Hydroxyl Radical

The addition of a third electron to H202forms a hydroxyl radical (.OH), a most potent reactive oxygen species. In vivo sources of the hydroxyl radical include homolytic fission of the bond in H,02 which is catalyzed by heat, radiation, and the Fenton-Haber-Weiss reaction (Kanner et al., 1987): Fe3' + O2+ Fez' + O2 Fez' + H202 + Fe3++OH-

+ .OH

These may also be generated by microsomal electron transfer (Cohen and Cederbaum, 1979), enzymatic oxidations (Porter et al., 1980a), activated polymorphonuclear leukocytes (Walling, 1982), and lipoxygenase or cyclooxygenase (Hammond et al., 1985). 3. Superoxide Anion

The acceptance of one electron by '02produces a superoxide anion radical (O;,). Superoxide radicals are formed in almost all aerobic cells (Halliwell, 1978b; Bast and Haenen, 1984). Neutrophils, monocytes, macrophages, and eosinphils all produce 0;.(Misra and Fridovich, 1972a). Several enzymatic oxidations such as xanthine oxidase, cytochrome P450 oxidases, aldehyde oxidase (Fridovich, 1978; Misra and Fridovich, 1972b), and the mitochondria1 electron transport chain (Freeman and Crapo, 1982) can generate 0;-. The autoxidation of catecholamines, hydroquinones, reduced ferredoxin, hemoglobin, and myoglobin can also produce 0;.(Krinsky, 1981; Sagone, 1981; Williams and Chance, 1983). Superoxide anion dismutates spontaneously or in the presence of the enzyme superoxide dismutase to yield hydrogen peroxide (Fridovich, 1976). Several enzymes D-amino-acid oxidase, uricase, glucose oxidase, and xanthine oxidase, may also produce hydrogen peroxide in biological tissues (Babior and Peters, 1981). Hydrogen peroxide may interact with superoxide anion via the iron-catalyzed Haber-Weiss reaction to generate hydroxyl radical (Fridovich, 1976). The superoxide anion radical is not the major cytotoxic reactive oxygen species. The importance of 0;.in the pathobiology of cells is its role as a precursor to the perhydroxyl radical and its dismutation to hydrogen peroxide and dioxygen (Gabig and Babior, 1981). Superoxide anion can be quenched by superoxide dismutase or by compounds which easily react with it such as vitamin C, thiols, hydroquinones, and catechols.

OXIDATION OF POLYUNSATURATED FATTY ACIDS

237

4 . Hydrogen Peroxide

The addition of a second electron to the superoxide anion hydrogen peroxide radicals forms a nonradical peroxide ion (0’;).At physiological pH values, the peroxide ion immediately protonates to yield hydrogen peroxide. Hydrogen peroxide can result from the spontaneous dismutation of superoxide or it can be generated enzymatically by superoxide dismutase, xanthine oxidase, glucose oxidase as well as peroxisoma1 enzymes (Babior and Peters, 1981). Other sources include the release of H,O, from polymorphonuclear leukocytes and granulocytes and liver when activated by a variety of stimuli (Walling, 1982; Hammond et al., 1985; Singh and Sagone, 1981). Hydrogen peroxide alone is not detrimental to biochemical compounds; however, via the Fenton-Haber-Weiss reaction it can yield hydroxyl radicals (Gebicki and Bielski, 1981). Superoxide and hydrogen peroxide are relatively stable reduced oxygen species. Lipid membranes are readily permeable to hydrogen peroxide (Root et al., 1978; Fridovich, 1978). Neither superoxide nor hydrogen peroxide is thought to cause lipid peroxidation directly, which requires a more reactive oxygen species such as the hydroxyl radical (OH-).

5 . Perhydroxyl Radical The perhydroxyl radical (HO,.) is the conjugate acid of 0;. and is a stronger oxidant than superoxide. The pK, of O;/HO,* is pH 4.8; thus, at physiological pH, approximately 1% of any 0; formed is protonated (Hamberg and Samuelsson, 1974; Fridovich, 1978). Moreover, it is well established that a drop in pH occurs near the membrane of cells and subcellular organelles. Hence, the formation of HO; is favored in membranes of cells where the potential for oxidative damage is greater. The HO,. radical can directly initiate lipid peroxidation in cell membranes where 0;. will not because it cannot penetrate the membrane (Nugteren, 1975). In this respect, O;., the less reactive radical, may diffuse over a larger area of the cell. When 0;- approaches a proton-rich solvent shell, which surrounds the negatively charged membrane surface, it becomes protonated to H02*with increased reactivity.

6 . Metal-Oxygen Complexes Although the Haber-Weiss reaction is thermodynamically exothermic, a transition metal (Fenton reagent), such as Fe or Cu, is needed for

238

R. J. HSIEH AND J. E. KINSELLA

catalysis (Mason, 1982), otherwise the reaction is too slow to account for the formation of oxidizing agents (Czapski and Ilan, 1978). Iron is most often considered in this regard because it is widely available in biological systems. The general scheme for the Fenton reagent-mediated production of a hydroxyl radical is shown below O;.+M"

HzOz + M"-'

+ O,+M"-' + OH*+OH-

+ M"

It should be noted that the need for superoxide as a reductant can be circumvented by direct reduction of the metal. The requirement for metal ions in the conversion of 0; and H,O, to HO* by the FentonHaber-Weiss reaction has caused much controversy (Kanner et al., 1987). The formation of HO*radicals in vitro when metal ions are added to the system have been reported; however, attempts to form HO*radicals without added metal ions have failed (Misra and Fridovich, 1972b). I n vivo, most metal ions are bound to proteins or chelated to cellular constituents such as citrate and phosphate-ester-containing compounds (ADP, ATP, etc.), but nevertheless some may be available to react in Fenton-type reactions. Consequently, metal-bound active oxygen species have been described to explain complex biochemical reactions (Kanner et al., 1986). Transition metals may initiate lipid oxidation by several mechanisms. They may, via a single-electron transfer or hydrogen abstraction, generate a free radical of unsaturated fatty acid and thus remove the spin restriction between the substrate unsaturated fatty acid and triplet oxygen as exemplified by lipoxygenase (Schewe et al., 1986). They may interact directly with triplet oxygen to generate superoxide radicals, which can generate more reactive oxygen species via the Haber-Weiss reaction (Fridovich, and Porter, 1980). Transition metals may also be indirectly involved in the generation of oxygen species by oxidizing flavin cofactors, e.g., cytochrome P-450oxidoreductase (Tien and Aust, 1982). Transition metals interact with peroxides and, by forming an oxene, raise the formal metal oxidation state from + 3 to + 5 (Rutter and Hager, 1982). These numerous reactions which activate oxygen occur in vivo and obviously must be rigorously controlled to minimize fatty acid oxidation. However, in tissues postmortem (meat, muscle, fish, etc.) these reactions may be uncontrolled and thereby accelerate oxidative deterioration via various free radical mechanisms.

OXIDATION OF POLYUNSATURATED FATTY ACIDS

B.

239

FREE RADICAL OXIDATION

I . Mechanism of Autoxidation The reaction of unsaturated lipids (RH) with oxygen to form hydroperoxides is generally a free radical process that involves three basic steps (Fig. 1). The formation of free radicals (R-) in the initiation step (I) can occur by the reactions outlined in Section II,A by thermal or lightinduced decomposition of peroxides or hydroperoxides, or by ultraviolet irradiation. The reaction of R- with oxygen in the propagation step (2) leads to peroxy radicals (ROO-) which react with unsaturated fatty acids to form hydroperoxides (ROOH). The formation of nonradical products can occur by the interaction of R* and ROO*i.e., a termination step (3). The propagation step (2a) is very fast in the presence of air (Ingold, 1973). The free radical cycle closes when a peroxy radical ROO- abstracts a hydrogen from another unsaturated fat molecule by step (2b), which is the slowest and rate-limiting step. The attack of free radical on unsaturated fatty acid results in the abstraction of an allelic hydrogen. The fatty acid radical formed subsequently rearranges to a cis, trans-diene with the incorporation of oxygen to yield a hydroperoxy radical (Aust and Svingen, 1982, Pryor, 1973). The abstraction of an allelic hydrogen is energetically preferred over other methylene hydrogen because of a lower bond energy (86 versus 96 kcal, respectively) and the resonance stabilization of the radical intermediate. The newly formed hydroperoxy radical can itself abstract hydrogen from another fatty acid propagating the reaction. It has been estimated that such a reaction sequence goes through 8 to 14 propagation cycles before termination (Wu et a f . , 1978). In the presence of air, termination step (3c) is the most important. Termination steps RH R* + O2

ROO.

+ RH

Initiator fast slow

(1

w

ROO*

( 2a

+

ROOH

(2b)

R * + R * LR R R * + ROO* ROO-

+ ROO*

1

R*

w

ROOR

w

ROOR

( 3a

1 1

(3b)

+

(3c)

RH : Lipid

FIG. I .

General scheme of lipid oxidation. [From Frankel (1985b).]

240

R. J . HSIEH AND J. E. KINSELLA

(3a)and (3b)become more important when theoxygenconcentration islow, e.g., away from the surface of a fat system. Termination can also occur by antioxidants (AH) that interrupt the free radical chain by interfering with either initiation or propagation (Scott, 1965). Oxidation inhibitors that interfere with initiation include metal chelators, such as citric and phosphoric acids, and UV deactivators, such as phenyl salicylates and ahydroxybenzophenone. Peroxide destroyer's, such as sulfur compounds and phosphines, reduce hydroperoxides to more stable alcohols (Scott, 1965). Because the propagation step (2b) is slow, hydrogen abstraction from unsaturated fats becomes selective for the most weakly bound hydrogen. The ease of hydroperoxidation thus depends on the number of double bonds present. The relative rate of autoxidation of oleate, linoleate, and linolenate was reported to be on the order of 1 : 40-50 : 100 on the basis of oxygen uptake and on the order of 1 : 12 :25 on the basis of peroxide development (Gunstone and Hildtitch, 1945; Lea, 1952). More recently, it was reported that, based on oxygen uptake, the initial rate of oxidation of linolenate in solution was twice that of linoleate (Yamamoto et al., 1982). The presence of a doubly allelic methylene in linoleate, - C H = CH-CH,-CH=CH-, explains its much higher reactivity than oleate. The single allelic methylene in oleate, -CH=CH-CH,-, has a bond strength estimated to be 77 kcal/mol compared to 52 kcallmol for the doubly allelic methylene in linoleate (Sehon and Swarc, 1950). Linolenate has two doubly allelic methylenes, - C H = C H - C H , - C H = C H - C H , CH=CH-, but each is not activated by the other, and the rate is therefore only about twice that of linoleate. The ratio of peroxide value to oxygen uptake was 1.0 for linoleate and only 0.5 for linolenate (Yamamot0 et al., 1982). Polyunsaturated fatty acids, such as arachidonic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, containing 3 , 4 , and 5 doubly allelic methylenes, respectively, are much less stable than linoleic and linolenic acids. Arachidonic acid was reported to be oxidized T.9 times faster than linoleic acid (Porter ef al., 1981). Ethyl esters of EPA and DHA were autoxidized rapidly even at 5°C in the dark after an induction period of 3-4 days, whereas the induction periods of linoleate and linolenate were 20 and 60 days, respectively (Cho et a[., 1987a). Oxygen uptake of EPA and DHA esters after the induction periods was 5.2 and 8.5 times faster than that of ethyl linolenate (Cho et al., 1987a). Under light irradiation, the respective rates of oxygen uptake by esters of EPA and DHA were about 7 and 10 times higher than that of linolenate (Cho et al., 1987b). The simple kinetic picture of autoxidation involving steps (I), (2), and (3) becomes more complicated in later stages of autoxidation because of the

OXIDATION OF POLYUNSATURATED FATTY ACIDS

24 1

decomposition of hydroperoxides. On standing, hydroperoxides are readily decomposed thermally or in the presence of metal ions (Frankel, 1983). Linolenate hydroperoxides are particularly unstable and the decomposition pathways become much more complex with linolenate than with either oleate or linoleate.

2 . Oleic Acid The accepted mechanism of autoxidation of oleic acid involves hydrogen abstraction on allelic C-8 and C-11 to produce two three-carbon allelic radicals. Oxygen attack at the end-carbon position of these intermediates produces a mixture of four allelic hydroperoxides with oxygen on C-8,

A

Oieate

10-Hydroperoxide

9-Hydroperoxide

8-Hydroperoxide

11-Hydroperoxide

+

+

FIG. 2. An outline of the mechanism of autoxidation of oleate. [From Frankei (1980).]

242

R. J. HSIEH AND J. E. KINSELLA

C-9, C-10, and C-11 positions (Chan and Levett, 1977; Frankel er al., 1979) (Fig. 2). According to this mechanism, the formation of these isomeric allelic hydroperoxides would be expected in equal quantities. However, studies have shown higher amounts of the 8- and l l- isomers than of the 9- and 10- isomers (Chan and Levett, 1977; Frankel et al., 1979). Stereochemical studies showed a mixture of eight cis and trans allelic hydroperoxides. With increase in temperature, the relative proportions of cis-8 and cis-I1 isomers decreased, those of trans-8, trans-11, cis-9, and cis-I0 isomers increased, while those of trans-9 and trans-I0 isomers did not change. These results reflect the greater reactivity of the free radicals on terminal C-8 and C-I0 with molecular oxygen. 3. Linoleic Acid

Hydrogen abstraction at the doubly allelic C-11 position of linoleate produces a pentadienyl radical. Reaction of this intermediate with oxygen 12

9

Linoleate

O + 2\*

13-Hydroperoxide

c J

+

O2

9-Hydroperoxide

FIG. 3. An outline of the mechanism of autoxidation of linoleate. [From Frankel (1980).]

OXIDATION OF POLYUNSATURATED FATTY ACIDS

243

at the end-carbon positions produces a mixture of conjugated diene 9- and 13-hydroperoxides (Fig. 3). The formation of equal proportions of these two isomers occurred at a wide range of oxidation temperatures. Stereochemical studies showed a mixture of four cis, trans- and trans, transconjugated diene hydroperoxides (Fig. 4). The distribution of truns,cistruns,truns-hydroperoxidesdepended on the ability of cosubstrate to donate hydrogen (i.e., K , value, the rate constant of the abstraction of hydrogen) to the linoleate peroxy group (Beckwith and Ingold, 1980) and the rate of p-scission (Kbrthe rate constant of fragmentation of peroxy group) fragmentation of C - 0 bond of peroxy radical which leads to the trans,trans-product (Fig. 4). The rate of p-scission for diene peroxy radicals is relatively independent of the position and substitution in the fatty acid chain. The rate of self-propagation of linoleate oxidation is 62 M - ' sec-' (Howard and Ingold, 1967). 4 . Linolenic Acid

Hydrogen abstraction at doubly allelic C-1 1 and C-14 of linolenate produces two pentadienyl radicals. Reaction with oxygen at the end-carbon produces a mixture of four conjugated diene hydroperoxides containing

AA Llnoleate

trans, cis-Hydroperoxide HOO

I

LH: Lipid trans, trans-Hydroperoxide

FIG. 4. Formation of truns,cis- and rruns,truns-hydroperoxidesof linoleate. [From Porter et a/. (1981).]

244

R. J. HSIEH AND J. E. KINSELLA

a third isolated double bond. The significantly greater concentrations of the external 9- and 16-hydroperoxidesthan the internal 12- and 13-hydroperoxides have been confirmed (Chan and Levett, 1977; Frankel et al., 1980) (Fig. 5). This uneven distribution of isomeric hydroperoxides has been attributed to the relative facile 1,3-cyclizationof the internal 12- and 13-hydroperoxidesinto hydroperoxy cyclic peroxides (Fig. 5 ) (Coxon et al., 1981; Neff et al., 1981; Toyoda et al., 1982). Cyclization of the 12and 13-hydroperoxides was first supported by the indirect evidence for

Linoienate

HOO

I+

I+

H*

* 1 16-Hydroperoxide

9-Hydroperoxide

+

OOH

12-Hydroperoxide

HOO

v+-v-

13-Hydroperoxide

1,3-Cyclizetion

4

A

%I+

W

9

H*

1 L

L H*

H -

H

9-Hydroperoxy-l0,12-~ycloperoxide16-Hydroperoxy-13,15-cycloperoxide

FIG. 5. An outline of the mechanism of autoxidation of linolenate and its subsequent formation of hydroperoxy cyclic peroxide. [From Frankel (1985a).]

OXIDATION OF POLYUNSATURATED FATTY ACIDS

Linolenate

I

1

1,3-cycllzCltlon

'2

Bicycloendoperoxide

I

245

13-Hydroperoxide

+Ha

I

OOH

FIG. 6. Formation of bicycloendoperoxide of linolenate from its 13-hydroperoxide precursor. [From Frankel (1985a).]

the formation of 9,10,12- and 13, 15, 16-trihydroxystearatesby hydrogenation of the oxidation products of linolenate. Bicycloendoperoxides structurally related to prostaglandins were produced by autoxidation of the 13linolenate hydroperoxide prepared by lipoxygenase action (Fig. 6) (O'Connor et al., 1981). Cyclization of homoallylic hydroperoxides apparently accounts for the limited tendency of isomerization of cis,trunslinolenate hydroperoxides into tramtrans configuration and limited incorporation of molecular oxygen into monohydroperoxides (Terao et al., 1984; Porter et al., 1980a).

246

R. J. HSIEH AND J. E. KINSELLA

Hydrogen donors such as p-methoxyphenol (Porter et al., 1980b) and tocopherol (Burton and Ingold, 1986) inhibit cyclization and geometric isomerization of linoleate and linolenate hydroperoxides. From autoxidized methyl linolenate, significant amounts of 9,10,12- and 13,15,16-trihydroxy esters were taken as indirect evidence of the existence of hydroperoxy cyclic peroxides.. Dihydroperoxides were identified in smaller yields as a mixture of 9,12-, 9,16- and 13,16-isomers (Fig. 7). These dihydroperoxides were generated from the secondary oxidation of 9- and 16hydroperoxy linolenate, via hydrogen abstraction and formation of the second pentadienyl radical. Epoxy-hydroxy or epoxy-hydroperoxy dienes were formed via an oxy-radical intermediate and identified in minor amounts in autoxidized methyl linolenate (Toyoda et d.,1982; Terao et al., 1984).

do

H *

16- Hydroperoxide

H* and

9 -Hydroperoxide

+4

4+

and Ha

9,16-Dihydroperoxlde

13,16-Di hydroperoxide

9,12-Dlhydroperoxide

FIG. 7. Formation of dihydroperoxidesof linolenate from their hydroperoxide precursors. [From Neff et al. (19821.1

OXIDATION OF POLYUNSATURATED FATTY ACIDS

247

5 . Arachidonic Acid Hydrogen abstraction at the doubly allelic C-7, C-10, and C-13 positions of arachidonate produces three pentadienyl radicals, which then react with oxygen at the end-carbons, C-5 and C-9, C-8 and C-12, (2-11 and C-15, respectively (Fig. 8). As with lindenate, the external 5- and 15hydroperoxides are formed in larger amounts than the internal 8-, 9-, I I-, and 12-isomers (Terao and Matsushita, 1981b). All of these have trans,cis-conjugated diene stereochemistry (Porter et al., 1981). The hornoallylic structure of the internal hydroperoxides should allow cyclization and explain their relatively low concentrations compared to the external isomers. Cyclization reaction of arachidonate is in the exo mode (Terao and Matsushita, 1981b). Hydroperoxides of 8,l I, ICeicosatrienoic and 6,9,12-octadecatrienoic acids, which had a double bond at the P,aposition to the carbon-bearing peroxy group, can cyclize to form epidioxide. In the presence of I% of a-tocopherol, the isomeric composition of monohydroperoxides was apparently more homogeneous. Furthermore, there was no signifcant difference in the susceptibility to decomposition among the isomer (Yamagata et al., 1983). Complex isomeric mixtures of dihydroxy, trihydroxy,tetrahydroxy ,and pentahydroxy derivatives were OOH

11- or 15Hydroperoxide

I

f 5- or 9-

Hydroperoxide

8- or 12Hydroperoxide

FIG. 8. An outline of the mechanism of autoxidation of arachidonate. [From Frankel (1984).]

248

R. J. HSIEH AND J . E. KINSELLA

identified from the secondary autoxidation products of methyl arachidonate (Fig. 9). The diols were assumed to come from epoxide or dihydroperoxides; triols from epoxyhydroperoxides or hydroperoxy epidioxide; tetrahydroxy compounds from dihydroperoxy bicycloendoperoxide; pentahydroxy compounds from hydroperoxy epidioxide, bicycloendoperoxide, or hydroperoxy bis-epidioxide (Neff et al., 1982; Yamagata et al., 1984). 6 . Eicosapentaenoic and Docosahexaenoic Acids

-

Autoxidation (Yamauchi et al., 1983) and myoglobin-catalyzed peroxidation of eicosapentaenoic acid (Yamaguchi et al., 1985) produces eight monohydroperoxide isomers (5,8-, 9-, 1 I-, 12-, 14-, 1 5 , 18-hydroperoxides) (Fig. 10). The 5- and 18-isomersoccurred in higher yields than the inner 8-, 9-, I I-, 12-, 14, and 15-isomers; 8-, 9-, 1I-, 12-, 14-, and 15-peroxy COOH

Arachidonic Acid COOH

0

I

c

Dihydroxy and Trihydroxy Tetrahydroxy derivative Derivatives

r Dihydroxy derivatives

FIG. 9. Formation of di-, tri-, and tetrahydroxy derivatives of arachidonate from their hydroperoxide precursors. [From Yamagata et a/. (1984).1

249

OXIDATION OF POLYUNSATURATED FATTY ACIDS COOH

1

11- or 15Hydroperoxlde

14- or 18Hydroperoxlde

1

5-or 9Hydroperoxide

8- or 12Hydroperoxide

FIG. 10. An outline of the mechanism of autoxidation of eicosapentaenoate. [From Yamaguchi ef a / . (1983, 1985).]

radicals cyclized to form the hydroperoxy endoperoxide, or to form the prostaglandins-like hydroperoxy bicyclic endoperoxides. The autoxidation of docosahexaenoic acid produces 10 isomers of docosahexaenoic acid hydroperoxides (4-, 7-, 8-, lo-, l l - , 13-, 14-, 16-, 17-, and 20-hydroperoxideisomers) (Fig. 1 1 ) (Van Rollins and Murphy, 1984). The various hydroperoxides are cleaved or dismutated to give a wide range of products as discussed in Section 111.

C. PHOTOSENSITIZED SINGLET OXYGEN OXIDATION Allylic hydroperoxides of unsaturated fatty acids can also be formed following exposure to light in the presence of oxygen and a sensitizer which activates the oxygen. It is important to distinguish between sensitized photooxidation and photolytic autoxidation. The photolytic oxidation is initiated by UV-catalyzed decomposition of peroxides and hydroperoxides, and propagated by the resultant free radicals via reactions as

250

R. J. HSIEH AND J. E. KINSELLA

..

1

1.4-Pentadiene

1

16-or201O-W144.~8Hydroperoxide Hydroperoxid Hydroperoxide 7- or 1113-or 17Hydroperoxide Hydroperoxide

FIG. 1 I . An outline of the mechanism of autoxidation of docosahexaenoate. [From Van Rollins and Murphy (19841.1

described in the previous section. Hence the products of photolytic oxidation are the same as those generated by thermal autoxidation initiated in the dark. Photosensitized oxidation can be differentiated from normal free radical autoxidation (Korycka-Dahl and Richardson, 1980). Photosensitized oxidation generally involves light-induced excitation of a sensitizer (sens) to an excited singlet state followed by the excitation of ground triplet oxygen (Frankel, 1980). One pathway known as type I involves reaction of the triplet sensitizer with the acceptor (A), which then reacts with ground state triplet oxygen (30,) to form singlet oxygen. This type I mechanism (substrate-sensitizer) involves activation of the substrate and is exemplified by the riboflavin-sensitized photooxidation of unsaturated fatty esters. This reaction proceeds by hydrogen abstraction and produces the same conjugated diene hydroperoxides as free radical autoxidation (Chan, 1977). A different distribution of isomeric hydroperoxides may be generated from photosensitized oxidation. Another mechanism for photosensitized oxidation, known as type I1 (singlet oxygen), involves energy transfer from a singlet sensitizer to ground state triplet oxygen (30,) that becomes activated into the singlet state ('0,). The reaction between '0, with fatty acid (RH) yields a hydro-

OXIDATION OF POLYUNSATURATED FATTY ACIDS

25 1

peroxide (Frankel, 1985) (Fig. 12). One of the most important ways of generating ‘0,is by exposure to light in the presence of photosensitizers such as chlorophyll, certain heme compounds, methylene blue, fluorescein derivatives, erythrosine, and polycyclic aromatic hydrocarbons (Korycka-Dahl and Richardson, 1980). There is evidence that oxygen may be activated to the singlet state in the dark. Several chemical methods are known to produce singlet oxygen. One is the reaction of sodium hypochlorite with H,O, (Korycha-Dahland Richardson, 1980). Another possible source of singlet oxygen involves the decomposition of secondary h ydroperoxides, via the interaction of two peroxy radicals to form a tetraoxide intermediate, to produce a ketone, an alcohol, and singlet oxygen (Frankel, 1985a,b). ROO.

+ ROO.+

RCO

+

ROH + ‘0,

Finally, catalysis by metals and their complexes may activate oxygen to yield the singlet oxygen (Hawco et al., 1977). Products of Photooxidation of Fatty Acids

The reaction of singlet oxygen with unsaturated fats proceeds by a mechanism which is entirely different from free radical mechanism. Autoxidation via free radical mechanism yields only conjugated hydroperoxides, and no nonconjugated diene hydroperoxides (Chan, 1977; Frankel, 1980). Singlet oxygen can react with linoleate to yield a mixture of conjugated and nonconjugated diene hydroperoxides (Terao and Matsushita, 1977). Singlet oxygen reacts directly with carbon-carbon double bonds by a concerted “ene” addition (Fig. 13). Oxygen is thus inserted at either carbon of a cis double bond, which is shifted to yield an allylic hydroperoxide in the trans configuration because of its lowest thermal energy state and steric hindrance (Thomas and Pryor, 1980). Linoleate reacts with Ground State ( Chlorophyll )

Singlet Oxygen

Lipid

hv ( Chlorophyll )

FIG. 12. The simplified scheme of lipid oxidation catalyzed by singlet oxygen mechanism. [From Frankel (1980).]

252

R. J. HSIEH AND J. E. KINSELLA

A

Oieate

+

Singlet Oxygen

1°0H

9- Hydroperoxide

+ 1°0H

10-Hydroperoxide

FIG. 13. Oxidation of oleate by singlet oxygen mechanism. [From Frankel (1984).]

singlet oxygen at a rate at least 1500 times faster than with normal triplet oxygen (Korycha-Dahl and Richardson, 1980). Hydroperoxides formed by singlet oxygen mechanism can decompose thermally or in the presence of metal catalyst. The resulting alkoxy and peroxy radicals accelerate free radical autoxidation. The decomposition of linoleic acid hydroperoxides, catalyzed by metals, methemoglobin, or hematin, can concomitantly form singlet oxygen (Hawco et al., 1977). Hence, it is very difficult to distinguish between singlet oxidation and free radical autoxidation without careful analysis of distribution of isomeric hydroperoxides during the early stages of lipid oxidation. As expected from the concerted addition mechanism, the reactivities of oleate (Fig. 13), linoleate (Fig. 14), and Iinolenate with singlet oxygen are in the same order as the number of double bonds (Terao and Mat-

OXIDATION OF POLYUNSATURATED FATTY ACIDS

12

253

9

Linoleate

I 9- Hydroperoxide

12-Hydroperoxide

+

Singlet Oxygen

10-Hydroperoxide

-

11 Hydroperoxide

FIG. 14. Oxidation of linoleate by singlet oxygen mechanism. [From Frankel (1984~1

sushita, 1977). By this mechanism, hydroperoxides are formed at each unsaturated carbon of a fatty acid. Oleate thus forms two hydroperoxides isomers, the 9- and 10-hydroperoxides. Linoleate forms four hydroperoxides, two with conjugated dienes (9- and 13-hydroperoxides) and two with unconjugated dienes (10- and 12-hydroperoxides).Similarly, linolenate produces six hydroperoxide isomers (9-, lo-, 12-, 13-, 1 5 , and 16-00H) (Terao and Matsushita, 1977; Thomas and Pryor, 1980). The distribution of isomeric hydroperoxides from photooxidation is uneven in linoleate and linolenate. For linoleate the ratio of 9- and 13-hydroperoxides to 10- and 12- hydroperoxides was 2 : 1. In the case of linolenate, the 9- and 16-hydroperoxides were formed in larger proportions (20-25%) than the lo-, 12-, 13-, and 15-hydroperoxides (12-15% for each) (Terao and Matsushita, 1980). The relative distribution of isomeric hydroperoxides produced by autoxidation and photosensitized oxidation is summarized in Table I. The relatively lower concentrations of internal 10- and 12-hydroperoxides in

254

R. J. HSIEH AND J. E. KINSELLA

TABLE I THE RELATIVE DISTRIBUTION OF POSITIONAL ISOMERS OF HYDROPEROXIDES OF OLEATE, LINOLEATE, AND LINOLENATE FORMED BY FREE RADICAL AND PHOTOSENSITIZED OXIDATIONS‘

Distribution of isomeric hydroperoxidesb(%) Methyl oleate Free radical Photooxidation Methyl linoleate Free radical Photooxidation Methyl linolenate Free radical Photooxidation

9-00H 22-25 48-5 1 10-00H

10-00H 22-24 49-52 12-00H

16-17 10-00H 12-00H 8-13 13 12-14

17 13-00H 10-13 14-15

8-00H 26-28

-

9-00H 48-53 32 9-00H 28-35 20-23

-

-

I 1-00H 26-28

-

13-00H 48-53 34-35 I5-OOH 16-00H 41-52 12-13 25-26

“From Frankel (1985a,b). bOOH, Hydroperoxide of fatty acid.

linoleate (Mihelich, 1980; Frankel et al., 1982) and lo-, 12-, 13-, and 15hydroperoxides in linolenate (Neff et al., 1982) reflect their cyclization into hydroperoxy cyclic peroxides. Radical cyclization may occur if a remote double bond, such as p,?-double bond, is present in the peroxy radical fatty acid (Porter et al., 1976; Mihelich, 1980). This structure permits a facile 1,3-cyclization. Cyclization is a secondary free radical reaction in which a methylene-interrupted unsaturated hydroperoxide apparently lose a hydrogen radical. Monocyclic peroxides, bic yclic peroxides, and epoxy alcohols are products derived from radicals formed by cyclization (O’Connor et al., 1981). The relative distribution between trans,cis- and trans,trans-hydroperoxide and cyclic peroxy radical is determined by three factors, viz (1) K,, (2) K,, and (3) the cyclization rate constants ( K J Because the rate of constant for cyclization (about 800 sec- for hydroperoxide of arachidonic acid) is five times higher than that of 9-scission (about 140 sec-’), p-scission cannot effectively compete with cyclization. The ratio of trans,&- to trans,trans-hydroperoxidesvaried from 4.2 at 10°C to 0.23 at 50°C. Generally, in polyene oxidation, truns,trans-hydroperoxides makes up an insignificant portion of the product mixture. At higher autoxidation temperatures, autoxidation of linoleic acid generates more trans,trans- products and 9-scission pathways become competitive with hydrogen transfer reactions. Higher concentrations of linoleate give more trans,& products. Product distribution between trans,cis- and trans,trans products are independent of oxygen pressure between 10 and

’

OXIDATION OF POLYUNSATURATED FATTY ACIDS

255

1000 mm molecular oxygen (Haselbeck et al., 1983). The hydroperoxide with a conjugated diene structure may rearrange by a free radical mechanism, and atmospheric oxygen and hydroperoxide oxygen can exchange via p-scission processes (Chan et al., 1979; Porter et al., 1981; Haselbeck et al., 1983). For reaction mixtures containing hydrogen donors, such as vitamin E and thiols, p-scission is reduced. Following the autoxidation of methyl linoleate, hydroxy diene, keto diene, epoxy-hydroperoxy, epoxy-hydroxy monoene, keto-epoxy monoene, dihydroxy, and trihydroxy derivatives are formed as secondary oxidation products (Neff et al., 1983).The mechanism of formation of epoxy derivatives involves cyclization of an oxy-radical intermediate, followed by hydroxy, or hydroperoxy addition with or without double bond migration to form epoxy-hydroxy, epoxy-hydroperoxy compounds (Gardner et al., 1974).The formation of di- and trihydroxy esters from oxidized linoleate can also be explained by a similar mechanism involving either the allylic epoxy radical by hydration or dienoic alkyoxyl radical undergoing 1- or 3- addition of hydroxyl group (Frankel, 1980). Cyclization of homoallylic hydroperoxides of linolenate can be effectively inhibited by hydrogen donors such as p-methoxyphenol and a-tocopherol (Porter et al., 1980a,b; Peers et al., 1981). Similarly free radical quenchers, such as tert-butylhydroxytoluene can also prevent the cyclization of internal 10- and 12-hydroperoxides of linolenate to result in equal distribution of 9-, lo-, 12-, and 13-hydroperoxides (Peers et al., 1984). Singlet oxygen-catalyzed oxidation of arachidonic acid with methylene blue as sensitizer generated hydroperoxides isomers at positions 5-, 6-, 8-, 9-, 11-, 12-, 14-, and 15- (Terao and Matsushita, 1981a). The smaller amount of the nonconjugated diene 6-, 14-isomers may reflect selective attack by singlet oxygen. Singlet oxygen equally attacks the inner double bonds, e.g., at C-8 and C-11, in the tetraene structure of arachidonic acid. No difference was observed among the ratios of inner 8-, 9-, 1 I-, and 12isomers. The ratio difference between the outer 5- and 15-isomers suggests that singlet oxygen preferentially attacks toward the methyl rather than the carboxyl side. Selective attack of singlet oxygen may also be explained by electronic effect of olefinic double bonds or stearic hindrance of carboxyl group. The attack of the second oxygen atom on the monohydroperoxides of arachidonic acid occurs at the pentaenoic double bonds opposite the position binding the hydroperoxy group, that is, at C11, C-12, C-14, C-15 of the 5-, 6-, 8-, and 9-monohydroperoxidesisomers; and at C-5, C-6, C-8, C-9 of the 11-, 12-, 14-, and 15-monohydroperoxide isomers, to produce dihydroperoxides. Singlet oxidation of unsaturated fats can be inhibited by compounds, such as diphenylfuran, which react with singlet oxygen significantly faster

256

R. J. HSIEH AND J. E. KINSELLA

than the substrate, and by quenchers that deactivate singlet oxygen to the ground triplet state (Kellogg and Fridovich, 1975). p-Carotene and a-tocopherol are effective natural singlet oxygen quenchers (Logani and Davies, 1980). The inactivating effect of a-tocopherol involves both reaction with and quenching of singlet oxygen. The reactivity with and quenching of singlet oxygen vary with the different isomers of tocopherol in the relative order of a > p > y > +isomers (Burton and Ingold, 1986). D. ENZYME-INITIATED LIPID OXIDATION In biological tissues of plant animal or marine origin, there are numerous mechanisms by which lipid oxidation may be initiated and promoted (Kanner et al., 1987). Many enzyme systems may indirectly and/or directly initiate the oxidation of polyunsaturated fatty acids. Three major groups of enzymatic mechanisms can be involved, e.g., microsomal enzymes, peroxidase, and dioxygenase-type reactions.

I . Microsomal Enzymes The occurrence of enzymatically induced peroxidation of lipids in rat liver microsomes was reported by Hochstein and Ernster (1963). This enzymatic peroxidation involved microsomal flavoprotein as NADPHcytochrome P-450 reductase (Ernster et al., 1982; Oprian and Coon, 1982). The cytochrome P-450 enzymes are heme proteins that are widely distributed in animal tissues, plants, and microorganisms (Nebert et al., 1981; Coon et al., 1982). The cytochrome P-450 enzymes exhibit a wide substrate heterogeneity in the monooxygenation of lipophilic substances. The typical cytochrome P-450-catalyzed oxygenation reaction proceeds as follows: RH

+ 0,+ NAD(P)H + H + + ROH + H,O + NAD(P)'

The initiation of lipid oxidation by NADPH-dependent microsomal enzyme is via a free radical mechanism since BHT causes complete inhibition (Buege and Aust, 1978). Oxygen activation occurs by the formation of an iron-oxygen complex. This activated complex is generated by providing reducing equivalents for a system containing ADP-ferric ion complex and molecular oxygen. The reducing equivalent is supplied by the reduction of iron by NADP' with microsomal NADPH-cytochrome P450 reductase (Fig. 15) (Tien and Aust, 1982). The reaction is enhanced

257

)I

OXIDATION OF POLYUNSATURATED FATTY ACIDS

NADPH

x

NADPH

+

NADP

Cyt. P-450 reductasex O i Oxidized

ADP-Fe(lll)

Cyt. P-450 reductase Reduced

0,

Cyt. P-450 reductas Oxidized

EDTA-Fe(ll)

ADP-Fe( II)

L

Cyt. P-450 reductase )(EDTA-Fe(lll) Reduced

0: O2 I

FIG. IS. The simplified reaction scheme showing microsome-catalyzed lipid oxidation. [From Kanner

et al. (1987).]

by the addition of EDTA-Fe3' complex, but not by EDTA because it chelates available iron (Pederson and Aust, 1975). Perferryl ion-promoted initiation of NADPH-dependent microsomal lipid peroxidation was first proposed by Hochstein and Ernster (1963). The putative perferryl ion was proposed to be formed in two steps: (1) NADPH-dependent reduction of ADP-chelated ferric ion via a microsoma1 flavoprotein, and (2) the addition of dioxygen to the ADP-chelated ferrous ion complex (Tien and Aust, 1982). The proposed requirement for ADP involved the chelation of the ferric ion and maintaining the iron ion in solution at neutral pH. If not chelated, the ferric ion would precipitate as the hydroperoxide and the concentration of ferric ion would be reduced. Chelation affects the redox potential of iron. The redox potential of iron affects the stability of perferryl ion (Tien and Aust, 1982), and the chelated iron which participates in the reductive activation of hydroperoxides (Frankel, 1984). In addition, complexation of oxygen with transition metal imparts free radical characteristics to dioxygen. This free radical property of dioxygen circumvents the spin restriction that exists for the reaction of ground state dioxygen with organic molecules, and thereby allows the reaction with polyunsaturated fatty acid to occur.

258

R. J. HSIEH AND J. E. KINSELLA

The reduction state of the dioxygen-ferrous ion complex can be represented by the following structure: Fe"0,

+ (Fe3'0;)-

The perferryl ion is quite electronegative and is a strong oxidant. Other reactive oxygen species, such as HO-, H202,and superoxide, cannot promote and apparently do not participate in the lipid oxidation in microsomal enzymes (Pederson and Aust, 1975). H202is apparently not involved because catalase has no effect on microsomal lipid oxidation (Tien and Aust, 1982). Free hydroxyl radicals ( H a ) may not be formed by Fenton's reagent except under acidic conditions (Aust and Svingen, 1982). It is only under conditions of low pH that Fenton's reagent-promoted reactions give products with a distribution characteristic of a free radical oxidation mechanism. As the polarity and/or pH of the reaction mixture is altered, the reaction products are less characteristic of free radical mechanisms and show the increasing influence of the perferryl ion-mediated oxidation. In microsomal lipid oxidation reactions, the K, of NADPH was 0.55 p M while NADP+ (Ki= 7.2 p.M) was a competitive inhibitor of ADP-Fe3+-induced microsomal lipid oxidation. In the absence of ADP-Fe3+, the K , of NADPH was 100 pM and the reaction was not inhibited by NADP+. The molar ratio of 0, consumption/NADPH oxidation is approximately 6. Both the rate and extent of lipid peroxidation were proportional to the amount of microsomal protein (Ernster et al., 1982). Many drugs, chemicals, and endogenous compounds are oxygenated by cytochrome P-450 in mammalian tissues and, in some instances, potentially toxic or carcinogenic epoxides may be generated (Wislocki et al., 1980). Thus, in addition to metabolism by cyclooxygenase and lipoxygenase, naturally occurring olefins can also be oxygenated by microsomal cytochrome P-450 enzymes (Gibson et al., 1980; Wislocki et al., 1980). The oxidation of polyunsaturated fatty acids by microsomal enzymes can result in four distinct classes of oxidation, e.g., w-hydroxylation, (w - 1) - hydroxylation, epoxidation, and lipoxygenase-like hydroxylation. The w and w - 1 oxidation of arachidonic by cytochrome P-450 can be catalyzed by microsomal preparations of rabbit renal cortex (Morrison and Pascoe, 1981), purified renal cytochrome P-450 (Capdevila et al., 1981), and microsomal preparations of rabbit liver (Oliw et al., 1982). These metabolic transformations result in the formation of 19-oxoeicosatetraenoic acid (by w - 1 oxidation) and 20-OH-eicosatetraenoic and eicosatetraen-l,20-dioic acids (by w oxidation). These P-450 enzymes ap-

OXIDATION OF POLYUNSATURATED FAlTY ACIDS

259

pear to be inducible by p-napthoflavone (Oliw et al., 1982). The w and w - 1 oxidation products of docosahexaenoic acid formed by rat liver microsomes were 21- and 22-hydroxydocosahexaenoic acid (Van Rollins et al., 1984). Other arachidonate and docosahexaenoate metabolites are produced by phenobarbital-induced hepatic microsomal enzymes, including a series of products formed by epoxidation of each double bond of these fatty acids. The epoxides can then be further converted to vicinal diols by hepatic microsomal and cytosolic fractions or purified microsomal epoxide hydrolase (Morrison and Pascoe, 1981; Oliw et al., 1982).The metabolites were the 5,6-, 8,9-, 11,12-, and 14,15-diols of arachidonic acid and 7,8-, 10,ll-, 13,14-, 16,17-, and 19,20-diols of docosahexaenoic acid. In addition, these types of oxidation can occur concertedly to produce trihydroxy fatty acids, such as 11,12,20-, 11,12,19-, 14,15,20-, and 14,15,19trihydroxy derivatives of arachidonic acid (Oliw and Oates, 1981). Another pathway of microsomal oxidation, the lipoxygenase-like oxidation of polyunsaturated fatty acids, has been studied with arachidonic (Capdevila et al., 1982), and docosahexaenoic acids (Van Rollins et af., 1984). Under this reaction scheme, the fatty acids were oxidized to compounds containing conjugated double bonds with allylic hydroxyl groups. Generation of these hydroxy fatty acids requires the initial abstraction of the bisallylic hydrogen atom (Frankel, 1980). This lipoxygenase-like reaction is also mediated by a P-450 monooxygenase (Capdevila et al., 1981). The isomers formed are 8-, 9-, 11-, 12-, and 15-hydroxyderivatives ofarachidonic acid (Capdevilaetal., 1982);and7-, 8-, lo-, 11-, 13-, 14-, 16-, and 17- derivatives of docosahexaenoic acid (Van Rollins et af., 1984). The potential roles of microsomal enzymes in initiating and promoting lipid oxidation in muscle tissues are further discussed below (Section IV).

2. Peroxidase Reaction Peroxidase is a rather nonspecific enzyme that catalyzes the one-electron hydrogen peroxide oxidation of many substrates, including fatty acids (Kanner and Kinsella, 1983a,b). In animal tissues its function is often associated with antimicrobial function, e.g., phagocytosis and protein halogenation (Gabig and Babior, 1981). Its activity may also initiate lipid peroxidation and p-carotene oxidation (Kanner and Kinsella, 1983a,b). The “resting” ferric peroxidase enzyme are probably pentacoordinated. Various peroxides and hydroperoxides react with the native ferric form of peroxidase to form compound I (Fig. 16). If R is H or an ethyl group, there is no net uptake or release of hydrogen ion during this reac-

260

R. J. HSIEH AND J. E. KINSELLA

em

+ fi

L

fiH OH

( Comp. II )

( Comp. I )

FIG. 16. The simplified reaction scheme of peroxidase-catalyzed lipid oxidation. AH, Hydrogen donor in peroxidase; B, Schiff base in peroxidase; ROOH, lipid peroxidase; ROH, hydroxy lipid. [From Ishimaru, 1980).]

+

tion. The compound I contains iron in the 4 oxidation state and a porphyrin cation radical. The 4 + iron is probably oxygenated to form a ferry1 ion, FeO". The porphyrin cation radical structure of compound I has been confirmed by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Compound I then oxidizes the substrate, a hydrogen donor, in a one-electron step to produce compound 11, which has four oxidizing equivalents. Compound I1 may oxidize the substrate in another one-electron step to regenerate the native ferric enzyme (Ishimaru, 1980). At low pH values, the kinetics of the reaction through compounds I and I1 become a one-step, two-electron process. For one isozyme of peroxidase, this change in mechanism occurs at pH 4.5; for another isozyme the change occurs around pH 7.7 (Gabig and Babior, 1981). The reaction of dioxygen with reduced peroxidase forms compound 111or oxyperoxidase. Compound 111can also be formed from compound I1 and hydrogen peroxide. Peroxidase activities are present in various animal and plant tissues. Neutrophils and monocytes contain myeloperoxidase activity. The phagocytizing potential of these cells has been attributed in part to their myeloperoxidase activity (Gabig and Babior, 1981). Polymorphonuclear

OXIDATION OF POLYUNSATURATED FATTY ACIDS

26 1

leukocytes play an important role in host defense by virtue of their myeloperoxidase actions (Gabig and Babior, 1981). Myeloperoxidase is a heme enzyme, a constituent of a microbiocidal system in which the other components are H,O, and C1-. These serve as substrates for the peroxidase, which catalyzes their conversion to hypochlorous acid (HOCl), the microbial agent in these systems, and water (Harrison and Shultz, 1976). Oxidative degradation of biological substrates by HOCl has been examined under reaction conditions similar to those found in active phagosomes. Iron sulfur proteins are bleached extremely rapidly, followed in decreasing order by p-carotene, nucleotides, porphyrins, and heme proteins (Albrich e f al., 1981). Rapid irreversible oxidation of cytochromes, adenine nucleotides, and p-carotene pigments occurs when bacterial cells are exposed to exogenous HOCl. In v i m bleaching of p-carotene was also observed by a lactoperoxidase/H,O,/halide system (Kanner and Kinsella, 1983a). Chlorine, or activated chlorine generated during the reaction of a peroxidase/H,O,/Cl- system, was suggested to be accompanied by lipid oxidation (Harrison and Shultz, 1976; Libby e f al., 1981). During phagocytosis, lipid peroxidation occurs possibly via a free radical mechanism induced by the peroxidase/H,O,/halides system (Shohet e f al., 1974; Stossel e f al., 1974). Free radical mechanism was also suggested in the peroxidation of linoleate by a peroxidase/H,O,/halides system. The reactive halide species generated in this reaction can abstract allylic hydrogen and form conjugated diene intermediates (Kanner and Kinsella, 1983a,b). The radical intermediate of linoleate subsequently interacts with oxygen to form hydroperoxides. The product patters of peroxidase-catalyzed oxidation of polyunsaturated fatty acids are generally similar to those generated by free radical mechanisms. Peroxidase is found abundantly in the roots and sprouts of higher plants, such as horseradish (Hayashi and Yamazaki, 1979), cauliflower (Lee and Pennesi, 1984), and turnip (Job and Ricard, 1975). Peroxidase appears to be one of the most heat-stable enzymes in fruits and vegetables (Burnette, 1977) and is used as a primary index of vegetable blanching prior to canning and freezing. Its activity has an adverse effect on flavor and color changes of raw and under-blanched vegetables (Burnette, 1977). The potential roles of peroxidase in initiating and promoting lipid oxidation in muscle tissues are discussed in Section IV. 3. Lipoxygenase

Lipoxygenases of different molecular weights and isoelectric points and with different specificities are present in various animal and plant tissues (Table 11). They occur in the cytosol (Nugteren, 1975; Siege1 et

262

R. J. HSIEH AND J. E. KINSELLA

TABLE I1 THE POSITIONAL SPECIFICITY OF LIPOXYGENASES FROM PLANT AND ANIMAL TISSUES

Sources of lipoxygenase Soybean-I Soybean-2 Potato Corn Tomato Wheat Human neutrophil Rabbit reticulocyte Bovine blood Guinea pig skin Human platelet Human lung Human eosinophil Fish gill

Position oxygenated Substrate

(%I

18:2 n-6 18:2 n-6 20:4 n-6, 20:3 n-6 18:2 n-6 18:2 n-6 18:2 n-6 20:4 n-6 18:2 n-6 20:4 n-6 20:4 n-6 20:4 n-6 20:4 n-6 2 0 4 n-6 20:4 n-6 225; 22:6 n-3

n-6(77%); n-10(23%) n-6(25%); n- l0(75%) n-16 and n-13 n-6(5%); n-10(95%) n-6(4%); n-10(96%) n-6 and n-9 n-10, n-12, n-13, n-16 n-6(90%); n-9(10%) n-6 and n-9 n-9 n-9 n-6

n-10 n-9 n-9

al., 1980; Chang et al., 1982) or in the microsomal fractions (Ho et al., 1977; Siege1 et al., 1980; German and Kinsella, 1986a). In human platelets, 70% of the total activity is in the cytosol and 30% is found in the membrane fraction. The membrane-bound enzyme did not differ from the cytosolic one with respect to substrate positional specificity (Lagarde et al., 1984). Lipoxygenase catalyzes the insertion of oxygen into polyunsaturated fatty acids. The basic cis,cis-nonconjugated diene system is required for all substrate fatty acids. The normal product from the action of animal and plant lipoxygenases on such a substrate is a cis,trans-conjugated hydroperoxy acid (Papatheofanis and Lands, 1985). Most available information concerning the mechanism of catalysis has been obtained from experiments with soybean lipoxygenase-1 (Vliegenthart et al., 1979). At least three interconvertable forms of this enzyme occur, such as the ferrous form, which is silent in EPR spectroscopy and does not show particular features in the absorption spectrum. The yellow ferric form shows an EPR signal at g = 6 and a broad absorption in the near-UV region; the purple complex, the product from the interaction between the ferric enzyme and 13~,-hydroperoxylinoleicacid ( ~~L,-HPOD), shows an additional EPR signal at g = 4.3 and an absorption maximum at 580 nm. The ferrous lipoxygenase is converted to the ferric enzyme upon addition of

OXIDATION OF POLYUNSATURATED FATTY ACIDS

263

an equimolar amount of I~L,-HPOD.Stopped-flow kinetic measurements of this transition have revealed that the rate constant of this process is only about one-fifth of that of the dioxygenation of linoleic acid. In addition to the EPR and optical spectral changes, the formation of ferric lipoxygenase is accompanied by a quenching of the fluorescence maximum at 328 nm (Egmond el al., 1975a). Generally, the activation of enzyme can be initiated by either ferric lipoxygenase or ferrous lipoxygenase in the presence of oxygen. The activation of lipoxygenase requires the presence of a hydroperoxy fatty acid. The iron in soybean lipoxygenase is bound very tightly, since it cannot be removed without denaturation of the enzyme. There are some indications for the presence of methionine (Rapport er al., 1984)and tryptophan at or in the neighborhood of the catalytically active iron (Egmond et al., 1975a). A simplified scheme of the catalytic cycle of lipoxygenase is presented in Fig. 17 (Schewe er al., 1986). The lipoxygenase in the ferrous state is activated to the active ferric form by the presence of trace amounts of hydroperoxide. Hydrogen abstraction from the substrate fatty acid is initiated by this activated ferric form of the enzyme. The resulting fatty acid radical remains enzyme bound and the enzyme is reduced to a ferrous state. Oxygen is then stereospecifically introduced into the fatty acid substrate to form a hydroperoxy radical that removes an electron from ferrous iron, yielding the anion of the hydroperoxy fatty acid, the final product of the dioxygen reaction. By this inner electron transfer, the ferric state of the enzyme is regenerated. In this reaction scheme, the lipoxygenase reaction catalysis is initiated by hydrogen abstraction of fatty acid and not by activation of oxygen. This is supported by the fact that, even with the removal of oxygen from the reaction mixture, fatty acid dimers arise from the radical intermediates of free fatty acids generated via the anaerobic lipohydroperoxidase reaction. Various lipoxygenases differ in three substantial features: the site of primary abstraction, the direction of the double bond shift in the primary system, and radical leading to the l-hydroperoxy-2,4-rrans,cis-pentadiene the stereospecificity of both hydrogen abstraction and dioxygen insertion. There are also differences in the relative affinities between various polyenoic fatty acids and different lipoxygenases; hence different products may be generated (Tables I1 and 111)(Schewe et al., 1986). The specificity of lipoxygenase is a function of substrate fatty acid, pH, and other incubation conditions, e.g., the presence of calcium. For the formation of one conjugated diene radical during a lipoxygenase cycle, there are two possible positional isomers of oxygenation products (Fig. 18). The preference for the generation of one of two possible isomers of hydroperoxides by most lipoxygenases may be explained in

264

R. J. HSIEH AND J. E. KINSELLA

H

H LO-Fe(lll)

H+I

.LO-Fe(ll)-

w

1

H

H abstraction (pro-R or pro-S)

Rearrangement of the radical ( + 2 carbon atoms)

O2 insertion (ant rafaclally to H abstraction)

-6 H

inner electron transfer LO-Fe( II) LO-Fe(lll)

FIG. 17. The simplified reaction scheme of lipoxygenase-catalyzedlipid oxidation. LO, Lipoxygenase. [From Schewe et a/. (1986).1

two different ways: (1) better stabilization of one of the mesomeric structures of the fatty acid radical intermediate and (2) the orientation of the dioxygen introduced at the active site, so that only one of the possible mesomeric structures of fatty acid radical is transformed to the hydroperoxy fatty acid radical. Such an oxygen orientation is known for hemoproteins (Shaanan, 1982). According to the position of oxygenation, lipoxygenases may be categorized into two groups: (1) the position of hydrogen removal is determined by the distance from the methyl end of the fatty acid and oxygenation by a (+2) rearrangement, as catalyzed by lipoxygenases from soybean (Hamberg, 1971), reticulocytes (Kuhn et al., 1983),

OXIDATION OF POLYUNSATURATED FATTY ACIDS

265

TABLE 111 THE MAJOR TYPES OF CARBONYL COMPOUNDS GENERATED FROM VARIOUS ACYL HYDROPEROXIDES’

Fatty acid Oleate

Linoleate

Linolenate

Arachidonate

Hydroperoxide

Breakdown products

8-Hydroperoxide 9-Hydroperoxide 10-Hydroperoxide 1 I-Hydroperoxide 9-Hydroperoxide 10-Hydroperoxide 12-Hydroperoxide 13-Hydroperoxide 9-Hydroperoxide 10-Hydroperoxide 12-Hydroperoxide 13-Hydroperoxide 15-Hydroperoxide 1&Hydroperoxide 5-Hydroperoxide &Hydroperoxide 8-Hydroperoxide 9-Hydroperoxide I 1-Hydroperoxide 12-Hydroperoxide 14-Hydroperoxide 15-Hydroperoxide

2-Undecenal, decanal 2-Decenal, nonanal Nonanal, octane, I-octanol Octanal, heptane, I-heptanol 2.4-Decadiena1, 2-nonenal 2-Nonenal, 2-octene, 2-octen-1-01 2-Heptenal, hexanal Hexanal, pentane, 1-pentanol, pentanal 2,4,7-Decatrienal, 2.6-nonadienal 2.6-Nonadiena1, 2,S-octadiene, 2,5-octadien- 1-01 2,4-Heptadienal, or 2-hexenal 2-Hexenal, 2-pentene, 2-penten-1-01, 2-pentenal 2-Butena1, propanal Propanal, ethane, ethanol 2,4,7,10-Hexadecatetraenal,2,6,9-pentadecatrienal 2.6.9-Pentadecatnenal + 2,5,8-tetradecatrien-I-ol 2,4,7-TnedecatrienaI + 2,6-undecadien-l-ol 2,bDodecadienal + 2.5-undecadien-1-01 2,CDecadienal. 2-noneanl 2-Nonenal, 2-octene, 2-octen-1-01 2-Heptenal, hexenal Hexanal, pentane, I-pentanol, pentanal

“From Frankel (1983).

and bovine blood (Nugteren, 1975); or (2) the position of hydrogen removal is determined by the distance from the carboxyl end of the fatty acid and oxygenation by a (-2) rearrangement, such as occurs with the lipoxygenases from potato (Corey et al., 1980), corn (Hamberg, 1971), tomato (Matthew et al., 1977), wheat (Kuhn et al., 1983, and neutrophil (Borgeat et al., 1976). Dual positional specificity has been found in lipoxygenases from soybean (Hamberg, 1984), potato (Shimizu et al., 1984), wheat (Kuhn et al., 1985), human blood platelets (Hamberg, 1983), and reticulocytes (Bryant et al., 1982). For example, the oxygenation of arachidonic acid by lipoxygenase of reticulocyte produced 15-~,-and 12-~,hydroperoxy-eicosatetraenoic acid (HPETE) in a ratio of about 15 : 1. The formation of S H P E T E requires hydrogen removal at C-13 (n-8), that of 1ZHPETE at (2-10 (n-1 I), followed by a shift of the resulting radical in the direction of the methyl terminus of the substrate fatty acid as well as a chiral dioxygen insertion at n-6 and n-9 in the L, position, respec-

266

R. J. HSIEH AND J. E. KINSELLA

- H*

I

--\

/-\

R

Lipoxygenase

/

7R2

2

FIG. 18. The positional specificity of oxygen insertion in the lipoxygenase reaction. R,, Methyl end; R2,carboxyl end. [From Schewe et al. (1986).]

tively (Fig. 19) (Bryant et al., 1982).The positional specificity of reticulocyte lipoxygenase has been confirmed with several unsaturated fatty acid substrates. The high activity with n-6 and low activity with n-9 oxygenation was also observed with lipoxygenases from soybean (Hamberg, 1984), wheat (Kuhn et al., 1985), and human blood platelets (Hamberg, 1983). The lipoxygenase of potato tuber forms ~-D,-HPETE(n-16) from arachidonic acid and mainly 8-hydroperoxyeicosatrienoicacid (n-13) from bis-homoy-linolenic acid (20 : 3, cis,cis,cis-8,11,14) (Shimizu et al., 1984). The oxidation of linoleic acid (18 : 2 n-6) by reticulocyte lipoxygenase results in the abstraction of hydrogen atom from C-1 1 and oxygenation at both C-13 and C-9 in a ratio of 10 : 1 (Schewe et al., 1986). The formation of the 9-hydroperoxide of linoleic acid occurs to a minor extent with soybean lipoxygenase-1 (Vliegenthart and Veldink, 1982). Analyses of the enantiomeric composition of the products of soy and reticulocyte lipoxygenases from linoleic acid revealed that 13-hydroperoxide showed high stereospecificity (preference for 1 3 - ~ROOH), , whereas the 9-hydroperoxide fraction consists of a racemic mixture ( 9 - ~and , 9 - ~ , ) It . is reasonable to assume that, in the case of linoleic acid, the intermediate radical may dissociate from the enzyme to a limited extent and combine with oxygen in a nonspecific manner. The proportion of racemic linoleic acid

OXIDATION OF POLYUNSATURATED FATTY ACIDS 14

11

267

5

8

Arachidonic Acid Reticuiocyt Lipoxygenase

HOO 15-Hydroperoxide

12-Hydroperoxide

FIG. 19. The dual positional specificity of oxygen insertion of some lipoxygenases. [From Schewe ef a/. (1986).]

products varies, depending on the kind of lipoxygenase (type 1 or 2), pH, temperature, etc. Damage by aerobic storage or modification of lipoxygenase by organic mercurials resulted in partial loss of specificity (Spaapen et al., 1980a,b). Lipoxygenases are generally defined in terms of their ability to dioxygenate polyunsaturated fatty acids possessing a 1,4-cis,cis-pentadiene system. Among the naturally occurring fatty acids that meet this structural requirement, the following are most frequently used as lipoxygenase substrates: linoleic (9,12-di-cis-octadecadienoic),a-linolenic (9,12,15-allcis-octadecatrienoic), y-linolenic (6,9,12-all-cis-octadecatrienoic),arachidonic acid (5,8,11,14-all-cis-eicosatetraenoic),bis(homo)-y-linolenic (8,11,14-all-cis-eicosatnenoic),and 5,8,11-all-cis-eicosatrienoic,eicosapentaenoic, and docosahexaenoic acids. Fatty acids containing a trans double bond, such as linelaidic acid, are not oxygenated but some of them are competitive inhibitors of linoleate oxygenation by soybean lipoxygenase (Veldink et al., 1977). Thus, both cis and trans fatty acids are bound to the enzyme but the initial hydrogen abstraction is sterically hindered in the trans structure. Moreover, fatty acids with a conjugated diene system are strong competitive inhibitors of linoleate oxygenation. Several lipoxygenases have been described, and the major distinction

268

R. J. HSIEH AND J. E. KINSELLA

between isomers of this enzyme found in a variety of mammalian cell types is the positional specificity of the hydrogen abstraction and oxygen addition site on the substrate arachidonic acid. The IZlipoxygenase found in platelets, skin, and lung initiates the conversion of arachidonic acid to 12(L) hydroxy-eicosatetraenoic acid (12-HETE), via the corresponding hydroperoxy acid (12-HPETE) (Hamberg and Samuelsson, 1974).The lipoxygenases of various leukocytes have attracted an increasing interest and a number of enzymes have been described, including the 5-lipoxygenase from human neutrophils (Goetzl, 1980), 8-lipoxygenase from peritoneal macrophages (Rabinovich et al., 1981), 9-lipoxygenase from human neutrophils (Goetzl and Sun, 1979), 1I-lipoxygenase from human eosinophils (Goetzl, 1980), and 15-lipoxygenasefrom human hung (Hamberg et al., 1980). Arachidonic acid is the most studied substrate for animal lipoxygenases owing to its role as a main biological precursor of leukotrienes and other lipoxygenase products. In plants, a-linolenic acid is the precursor of jasmonic acid, a regulatory substance that inhibits growth and promotes senescence via a lipoxygenase pathway (Vliegenthart and Veldink, 1982). Many lipoxygenases attack not only unesterified polyenoic fatty acids but also their methyl esters. The rates of the dioxygenation of methyl linoleate were consistently 25% of that for free linoleic acid in the case of the lipoxygenases of reticulocytes, pea seeds, and wheat (Regdel et al., 1985). Soybean lipoxygenase-1 attacks methyl linoleate with low reactivity and low regio- and stereospecificity as compared to the reaction with free linoleic acid (Schewe et al., 1986). This dfierence may reflect the differences in solubility of the methyl ester rather than the particular properties of the enzymes. Methyl linoleate (Christopher et al., 1970), unfractionated soybean oil (Guss et al., 1968), mono- and dilinolein can be oxygenated by soybean lipoxygenase (Guss et al., 1968). The 15-lipoxygenasefrom reticulocytes shows the unique properties of attacking the polyunsaturated fatty acids esterified in the phospholipids of membranes to a sizeable extent even in the absence of phospholipase activity (Schewe et al., 1975). The unsaturated fatty acids in phospholipids are also attacked by other 15-lipoxygenases such as that of polymorphonuclear leukocytes (Jung et al., 1985). Nevertheless, reticulocyte lipoxygenase still prefers free polyunsaturated fatty acid as a substrate over phospholipid (Schewe et al., 1986). Linolenyl sulfate appeared to be a good substrate for soy lipoxygenase-1 whereas lipoxygenases-2 and -3 were totally inactive toward this substrate (Veldink et al., 1977). This suggested that the presence of a free carboxyl group was not a prerequisite for lipoxygenase substrate. Gill and skin tissues from fish contain a 12-lipoxygenase active toward

OXIDATION OF POLYUNSATURATED FATTY ACIDS

269

arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid (German and Kinsella, 1986a,b, 1985). The potential role of lipoxygenase to initiate and promote lipid oxidation in red meats and fish tissues is discussed below (Section IV). 4.

Cyclooxygenase

In addition to lipoxygenase, animal tissues contain cyclooxygenase which generates prostanoids via the stereospecific introduction of oxygen into arachidonic acid. Both of these enzymatic conversions, if perturbed, can cause uncontrolled lipid peroxidation, especially postmortem. In vivo, these oxygenations are also affected by metabolism of xenobiotics and may be perturbed in pathophysiological states. Cyclooxygenase catalyzes the conversion of arachidonic acid (20 : 4 n-6) to the cyclic endoperoxide prostaglandin PGG2 by a series of reactions in which two molecules of oxygen are inserted into the polyunsaturated fatty acid following activation of the fatty acid via homolytic hydrogen abstraction. Prostaglandin synthase contains cyclooxygenase and peroxidase and thus can potentially initiate lipid peroxidation via two separate catalytic activities: the highly substrate-specific oxygenation reaction and the relatively substrate-insensitive peroxidase cooxidation reaction. The enzyme has an absolute requirement for hydroperoxide as the activating cofactor and these same hydroperoxides, including the cyclooxygenase product PGH,, can at higher concentrations, irreversibly inhibit the enzyme. Thus, the enzyme is both regulated by and contributes to the very important balance between the formation and removal of lipid peroxides. This balance has been referred to as the peroxide tone. The reaction mechanism is of considerable importance both for the production of bioactive autocoids and potential initiation of lipid oxidation in vivo (Lands, 1985; Lands et al., 1982; Kanner et al., 1987). Both the cyclooxygenase and the peroxidase activity are located on the single heme group associated with the same protein complex. The cyclooxygenase free radical induced by hydroperoxides is identical to those reported for compound I of horseradish peroxidase. Since lipid hydroperoxides serve as essential activators of cyclooxygenase;at high concentrations hydrogen peroxide may activate the enzyme. This resembles the activity of metmyoglobin (MetMb) and methemoglobin (MetHb) as initiators of lipid peroxidation and it is assumed that heme-cyclooxygenase is activated via the same pathways, leading via peroxidation to a porphyrin cation radical, iron oxene (Kanner et al., 1987). Peroxidase cosubstrates, particularly phenolic agents, have been reported to increase cyclooxygenase activity in vitro by protection of the

270

R. J. HSIEH AND J. E. KINSELLA

enzyme from self-inactivation by the peroxy radicals or by the activated heme protein. (Smith and Lands, 1971). Phenolic compounds stimulate cyclooxygenase at low concentration and inhibit it at high concentration and different phenolic agents vary in their relative tendencies to activate or inhibit. This biphasic response indicates that low amounts of reducing agents act as protectors of the enzyme from self-inactivation whereas high concentrations are also competitive with the substrate for the activated catalyst. Phenolic compounds, which are radical trapping agents, stimulate cyclooxygenase activity under conditions where relatively high amounts of peroxides occur and inhibit cyclooxygenase activity when the amount of peroxide is relatively low. This behavior has been recognized in the varying effectiveness of certain nonsteroidal anti-inflammatory drugs, such as acid amidophenol, which are potent inhibitors at low peroxide levels, but very weak at high peroxide levels. (Smith and Lands, 1971; Lands et al., 1973, 1982; Lands, 1985). Thus, prostaglandin synthase is extremely sensitive to the endogenous level of lipid peroxides and to potential radical scavenging agents and inhibitors. The enzyme is potentially a major contributor to the proliferation of lipid peroxides and active oxidants in pathophysiological states. In this regard, high intakes of dietary polyunsaturated fatty acids, which may affect both cyclooxygenase and lipoxygenase activity, should be accompanied by appropriate amounts of antioxidants. There have not been any reports documenting the involvement of cyclooxygenase in initiating lipid oxidation in foods, though such reactions are possible. 111.

PRODUCTS OF LIPID OXIDATION

DECOMPOSITION OF ACYL HYDROPEROXIDES The acyl hydroperoxides formed by the various reactions described above are unstable and may be enzymatically modified to form various acyl derivatives, i.e., hydroxy, epoxy, and aldehydes, or they can spontaneously dismutate and undergo scission in reactions catalyzed by transition state metals to generate a wide range of volatile and nonvolatile derivatives (Lundberg, 1962). These compounds are very important components of the flavors and off-flavors of many foods of both plant and animal origin. The hydroperoxides of fatty acid can be reduced to hydroxy derivatives by glutathione peroxidase or nonenzymatically to yield a mixture of bitter trihydroxy derivatives (Fig. 20) via hydroxy epoxide intermediates (Bry-

OXIDATION OF POLYUNSATURATED FATTY ACIDS

27 1

HOO

12-Hydroperoxide of Arachidonic Acid

* 1

OH-

+

10,11,12-trihydroxy

8,l l,l2-trihydroxy derivative

FIG. 20. Formation of trihydroxy derivatives from the hydroperoxide of arachidonic acid.

ant and Bailey, 1979; German and Kinsella, 1986b). Moll et al. (1979) and Sessa (1979) attributed the bitterness in soybean flakes to the oxidation of phospholipids initiated by lipoxygenase and peroxidase activities. The components contributing to the bitterness were identified as 9,12,13-, 9,10,13-, 9,10,11-, and 11,12,13-trihydroxyand 9,12- and 10,13-dihydroxy derivatives of linoleic acid (Moll et al., 1979; Gardner, 1985). A mixture of 9,12,13-trihydroxy-lO-octadecenoicand 9,10,13-trihydroxy-l l-octadecenoic acids has a bitterness threshold of 0.6-0.9 mM, whereas 9-hydroxy-10,12-octadecadienoicand 13-hydroxy-9,ll-octadecadienoicacids have threshold values approximately 10-fold higher (Gardner, 1985). Similar hydroxylated fatty acids were responsible for the intense bitterness developed in aqueous suspensions of oat flour (Gardner, 1985). The hydroperoxides of unsaturated fatty acids and their secondary oxidation products are important precursors of volatile decomposition products. Hydroperoxides are readily decomposed thermally (Hiatt and

272

R. J. HSIEH AND J. E. KINSELLA

Irwin, 1968) or in the presence of metal ions (Ingold, 1962). The precursors and mechanisms of decomposition determine the types of volatile products formed and hence their impact on flavor quality. A major source of flavor and off-flavor generated from polyunsaturated fatty acids involves the homolytic cleavage of hydroperoxides and their derivatives (Frankel, 1983). Alkoxy radicals are produced from hydroperoxides by loss of hydroxyl radical. Alkoxy radicals can also be formed by interaction of peroxy radicals and homolytic cleavage of peroxides (Fig, 21). The alkoxy radicals from allylic hydroperoxides can undergo carbon-carbon homolytic cleavage on either side of the carbon bearing the oxygen to produce an alkyl radical on one side and a vinyl radical R'-CH=CH*) on the other side of the hydroperoxide group (Selke et al., 1978; Frankel, 1983). These free radicals may react with hydrogen or hydroxyl radicals to generate flavor compounds, such as hydrocarbons, alcohols, aldehydes, and ketones. The alkyl radicals can react with another hydrogen or hydroxyl radical, or molecular oxygen to from hydrocarbons, alcohols, or hydroperoxides, respectively (Fig. 21). The vinyl radical is very reac-

7 I

Ib I

' A!

RfH=CH+CHtCy-R

,

1

Alkoxyradlcal

cleavage R - C k C I t CHO 1-CH2-CH0

2 2-alkenal

alkyl radical

+

R1 -Ct$

He R 1 -CH3

hydrocarbon

OH-

R1-t$0~

alcohol

+

~

saturated aldehyde

RrCH=CH* radical OH*

R2-CH=CH2

R*H=CH-OH

I'

alkene

R2-

alkenol

CH2-CH0

aldehyde FIG. 21. A simplified scheme showing products formed from homolytic thermal cleavage of the hydroperoxide of an unsaturated fatty acid. [From Frankel (1983).]

OXIDATION OF POLYUNSATURATED FATTY ACIDS

273

tive and unstable and may also react with hydroxy radical, hydrogen radical, or molecular oxygen to generate aldehydes and olefins (Parson, 1973). 1.

Volatile Compounds from Acyl Hydroperoxides

Homolytic cleavage is the well-recognized pathway for the decomposition of the allylic hydroperoxides of oleate, linoleate, and linolenate (Frankel et al., 1981). The decomposition products from autoxidized and photosensitized oxidized methyl oleate, linoleate, and linolenate have been extensively studied and reviewed (Frankel, 1985a,b). In this section, the differences among the decomposition products derived from autoxidized and photosensitized oxidized methyl oleate, linoleate, and linolenate are discussed. Frankel et al., (1981) compared the volatile compounds from pure hydroperoxides of autoxidized methyl oleate (i.e., the 8-, 9-, lo-, and 1 l-hydroperoxides) with those from photosensitized oleate (9- and 10-hydroperoxides). Although the two starting hydroperoxide mixtures were different, both samples formed similar volatile products. However, there were exceptionally high amounts of 2-undecenal and methyl heptanoate, which originated from the 8-hydroperoxy oleate, present in the photosensitized oxidized oleate sample. This suggested that isomerization occurred and 8- and 1 I-hydroperoxides were formed (Chan et al., 1976; Frankel et a f . , 1981). The volatile compounds produced from the respective hydroperoxides of autoxidized and photosensitized linoleate are qualitatively similar, but vary significantly in their respective concentrations. For example, more volatile compounds such as 2, 4-decadienal and methyl octanoate (from the 9-hydroperoxide)and pentane, pentanal, and 1-pentanol (from the 13hydroperoxide) were formed from the autoxidized linoleate samples, while more volatile compounds such as methyl l0-oxo-8-decenoate and 1-octen-3-01(from 10-hydroperoxide) and 2-heptenal (from 12-hydroperoxide) were formed from the photosensitized oxidized linoleate samples (Frankel et al., 1981; Chan et a f . , 1976). 2-Heptenal, derived from the 12hydroperoxides in the photosensitized oxidation sample, is a distinguishing volatile compound. The l-octen-3-01 is a rearrangement product from 2-octen-1-01 probably derived from the 10-hydroperoxide(Frankel et af., 1981). This is an important product in autoxidized products of fats and foods containing linoleate (Selke et af., 1980). The autoxidation of linolenate hydroperoxides produces more decatrienal (from the 9-hydroperoxide), enthane (from the lQhydroperoxide), and methyl octanoate (from the 9-hydroperoxide) and less 2-butenal (from the 15-hydroperoxide) and methyl 10-oxo-8-decenoate(from the 10-hydroperoxide) than the photosensitized oxidation hydroperoxides (Frankel

TABLE IV EXAMPLES OF THE VARIOUS CARBONYL VOLATILE FLAVOR COMPOUNDS DERIVED VIA LIPID OXIDATION IN DIFFERENT BIOLOGICAL TISSUESa

Compounds Hexanal 4Heptenal 2,CHeptadienal 2-Hexenal 2,4,7-Decatrienal I-Octen-3-01 1,5-Octadien-3-ol 2,5-Octadien-l-ol 1.5-Octadien-3-0ne 2-Nonenal 2,bNonadienal

Origin

Concentrations (PPb)

Threshold (ppb)

18:2 18:3 18:3 18:3

0-1 1-10 1-10 1-10

1 10 17

n-3 18:3

1-10

150

1-10 10-100

10 10

n-6 n-3 n-3 n-3

n-6 n-3 n-3 n-3 n-6 n-3

“From Tress1 et al. (1981).

18:2 18:3 18:3 18:3 18:2 18:3

1-20 0.1-5

1-25 1-35

5

0.001

0.08 0.01

Sources Apple, grape, beans, tomato Oxidized fish, oxidized butter Oxidized fish, oxidized butter Apple, ripe banana, pear, plum Oxidized fish Mushroom, beans, seaweed Mushroom, seaweed Mushroom, seaweed Mushroom Cucumber, asparagus, wheat Cucumber, green banana, barley

OXIDATION O F POLYUNSATURATED FATTY ACIDS

275

et al., 1981). 2-Butenal is expected from the 15-hydroperoxy linolenate, and methyl 10-0x0-8-decenoate from 10-hydroperoxide (Frankel et al., 1981). Volatile compounds from the cyclization products derived from lo-, 12-, 13-, and 15-hydroperoxylinolenates were also reported (Chan et al., 1980; Frankel et al., 1981). In general, the composition of volatile compounds obtained from the fragmentation of hydroperoxides is controlled by the relatively competitive rates between carbon-carbon cleavages on both sides of the alkoxy group and by positional isomerization of the hydroperoxy group. The volatile products of autoxidized arachidonic acid are expected to be similar to those of linoleate because of the common n-6 structure, while those from autoxidized eicosapentaenoic and docosahexaenoic acids may be similar to those of linolenate because of the common n-3 structure. However, the autoxidation of arachidonic acid gives rise to 6 positional hydroperoxide isomers, whereas that of linoleate produces 2 hydroperoxides. The autoxidation of eicosapentaenoic acid and docosahexaenoic acid generates 8 and 10 positional hydroperoxide isomers, respectively, whereas that of linolenate produces only 4 hydroperoxides (Frankel, 1985a). For each hydroperoxide, there are two positions for homolytic cleavage to generate volatile compounds (Frankel, 1983). The patterns of volatile compounds generated from polyunsaturated fatty acids are much more complex than those obtained from linoleate and linolenate (Tables 111-V). TABLE V EXAMPLES OF THE VARIOUS CARBONYL VOLATILE FLAVOR COMPOUNDS DERIVED VIA LIPID OXIDATION IN FISH TISSUES~

Compounds

Origin

CHeptenal 2,CHeptadienal 2-Hexenal 2.4.7-Decatrienal I-Octen-3-01 I ,5-Octadien3-01 2,5-Octadien- 1-01 1.5-Octadien-3-one 2-Nonenal 2,6--NonadienaI

n-3 P U F A ~ n-3 PUFA n-3 PUFA n-3 PUFA n-6 PUFA n-3 PUFA n-3 PUFA n-3 PUFA n-6 PUFA n-3 PUFA

Concentrations (PPb)

Threshold (ppb)

1-10 1-10

1 10

1-10

17 150

1-10 10-100 10-100 1-10 0.1-5

0-25 0-35

10 10

0.001 0.08 0.01

Flavor note Creamy Rancid hazelnuts Green grass Oxidized fish oil Mushroom, melon-like Mushroom, seaweed Mushroom, seaweed Mushroom Cucumber-like Cucumber-like

“From Josephson e r a / . (1984a), Ke e t a / .(1975), McGill e t a / .(1977). and Swoboda and Peers (1977). b18:3 n-3, 2 0 5 n-3, and 22:6 n-3. ‘18:2 n-6 and 20:4 n-6.

276

R. J. HSIEH AND J. E. KINSELLA

2 . Secondary Oxidation Products

The secondary oxidation products obtained via the subsequent oxidation of the initial products of polyunsaturated fatty acids, such as dihydroperoxides, epoxy-hydroperoxides, hydroperoxy cyclic peroxide, and hydroperoxy bis-epidioxide, are all potential sources of volatile compounds via further degradation (Neff et al., 1982). These secondary products undergo fragmentation by mechanisms similar to those for the monohydroperoxides. Dihydroperoxide can serve as precursors of volatiles by carbon-carbon cleavage on either side of the alkoxy radicals (Fig. 22). For example, the 9,16-dihydroperoxideof linolenate can be thermally decomposed to give methyl 9-oxononanoate and propanal as the main products from the cleavage at the internal sides of the hydroperoxide groups (Frankel et al., 1981; Selke et al., 1978). Cleavage at the other sides of hydroperoxides give rise to methyl octanoate and ethane (Frankel et al., 1984) (Fig. 22). Allylic epoxy aldehydes, recently identified in oxidized butter fat and trilinolein, can be derived from epoxy-hydroperoxides(Fig. 23) (Gardner et al., 1978; Swoboda and Peers, 1978; Selke et al., 1980). Thermal decomposition of 12,13-epoxy-9-hydroperoxy-1O-octadecenoate produced 4,5-epoxy-2-decenal (Gardner et al., 1978; Selke et al., 1980). 16-Hydroperoxideof linolenic acid was further oxidized to 15,16-epoxy12-hydroperoxy-9,13-octadecadienoicacid, which via subsequent carbon chain cleavage between C-11 and C-12 yields 4,5-epoxy-2-heptenal (Peers et a/., 1984; Swoboda and Peers, 1978). Hydroperoxy cyclic peroxide are also potential precursors of volatile compounds (Frankel et al., 1981). The cleavage between the peroxy ring and the alkoxy group is the most important (Fig. 24). Cleavage of the peroxy ring and the carbon-carbon bond, p to the trans-olefinic bond is another major process. A less-favorable cleavage between the double Ethane

c-

Methyl octanoate * OH

OOH

Propanal

Methyl 9-oxononanoate

FIG. 22. The mechanism of breakdown of dihydroperoxide of linolenate. [From Frankel ei d.(1983).]

OXIDATION OF POLYUNSATURATED FATTY ACIDS

277

15,16-epoxy-l P-hydroperoxy9,l3-octadecadienolc acid

J 4,5-epoxy-2-heptenal

FIG. 23. An outline of the possible breakdown mechanism for the epoxy-hydroperoxide of linolenate. [From Peers cr a / . (1984).]

bond and the peroxy ring produces olefinic radicals that may react with hydroxyl radicals to form vinyl alcohols, which then may tautomerize to saturated aldehydes (Frankel et al., 1981). Another less-favorable cleavage occurs between the peroxy ring and the carbon-carbon bond next to the hydroperoxy group, after removal of the hydroperoxide by elimination of H,O,. The unique products from cyclic peroxide of linoleate are the 2-alkyl ketones (Frankel et al., 1982). Pentane, hexanal, and methyl 9-oxononanoateoriginate from 13-hydro-

F

Me 9-oxononanoate Me octanoate

C3-2)

3-H-

Hexanal Heptanal

c))3,-COOCH

Me 10-oxo-6-decenoate

FIG. 24. An outline of the possible breakdown mechanisms for the hydroperoxy cyclic peroxide of linoleate. [From Frankel ei o / . (1982).]

278

R. J. HSIEH AND J. E. KINSELLA

peroxy-l0,12-~yclicperoxide formed from 10-hydroperoxylinoleate (Fig. 24) (Mihelich, 1980; Frankel et al., 1981). Similarly, the 9-hydroperoxy10,12-cyclic peroxide from the 10-hydroperoxy linoleate can produce hexanal, methyl octanoate, and methyl 9-oxononanoate (Mihelich, 1980; Frankel et al., 1981). The diunsaturated hydroperoxy cyclic peroxides formed from autoxidized methyl linolenate can be degradea to form products such as furans, conjugated diunsaturated aldehydes, esters, and 2alkyl ketones (Frankel et al., 1983). Fragmentation of bis-epidioxide follows the same pattern as the corresponding monocyclic peroxides (Fig. 25). For example, methyl 9-oxononanoate, methyl octanoate, and propanal are the major thermal decomposition products of methyl 9-hydroperoxy-10,12,13,15-bis-epidioxytrans-16-octadecenoate (Frankel et al., 1983). Cleavage between the two peroxide rings result in furan ester formation. Thermal fragmentation of the hydroperoxy bicycloendoperoxides shows dominant cleavage between the hydroperoxide group and the allylic double bond. The cleavage across the endoperoxide ring is also very important in forming malonaldehyde (Pryor, 1976). 3. Acid-Catalyzed Decomposition

Volatile compounds can also be derived from acid-catalyzed selective heterolytic cleavage between the hydroperoxide group and the allylic double bond (Fig. 26). This process involves an acid-catalyzed carbonto-oxygen rearrangement of organic hydroperoxide. Application of this rearrangement to hydroperoxides of fatty acid predicts the generation of short-chain aldehyde. Kimoto and Gaddis (1969) reported that HCl-

Me octanoate

Me furan octanode

Me 9-oxononanoate

FIG. 25. An outline of the possible breakdown mechanism of bis-epidioxide formed from linolenate [From Frankel et a / . (1983).]

OXIDATION OF POLYUNSATURATED FATTY ACIDS

279

I*

I R -cH=CH 1

Hydroperoxide of fatty acid

I

5-

I

C5-

OH

CHO + OHC- CI$-R2 Aldehyde

FIG. 26. The simplified scheme showing acid-catalyzed breakdown of hydroperoxide of fatty acid. [From Frankel e t a / . (19831.1

treated Fuller’s earth selectively decomposed autoxidized trilinolein into volatile aldehydes such as hexanal and nonenal. Grosch et al. (1981) obtained hexanal from the 13-hydroperoxideof linoleic acid, and 2-nonenal and hexanal from the 9-hydroperoxide of linoleic acid by incubation with trichloroacetic acid in benzene. The 13-hydroperoxideof linoleic acid incubated with a Lewis acid, boron triflouride, in anhydrous ether is decomposed to hexanal and methyl 0x0- 10-dodecenoate, whereas 9-hydroperoxide of linoleic acid is decomposed to 2-nonenal and methyl 9-0x0nonanoate (Gardner and Plattner, 1984). Another thermolytic scheme, known as the Hock cleavage, has been recognized and generally accepted because it does not involve unstable vinylic radicals (Denny and Nickon, 1973). Hock cleavage (Fig. 27) in-

280

R. J. HSIEH AND J. E. KINSELLA ,Homolytic

$ 1

I

F$- C H= C

M H--CI+-R2

I

Rr CH =CH-CH-CH-R2

2

I L+

lb'

R.,-CH=CH-

CHO 2-AI kenal

+

+I

*C%-%

bH

R-CI$-CHO + OHC-CH-R2 1 2 Aldehyde

HO-CHr R2 Alcohol

+

C%-% Alkane

FIG. 27. The simplified scheme showing Hock cleavage of hydroperoxide of fatty acid. [From Frankel et a / . (1983).]

volves a mixed homolytic-heterolytic mechanism and produces the same aldehydes by hydrolysis (Frankel, 1983). By this mechanism, p-scission occurs homolytically on the alkyl side of the hydroperoxide to produce 2-alkenals, and heterolytically on the unsaturated side of the hydroperoxide to produce alkanals. The remaining free radical fragments can lead to hydrocarbons, alcohols, and esters. Heterolysis on the unsaturated side of the hydroperoxides may occur under neutral conditions because hydroperoxides are reported to have higher acid strengths than the corresponding alcohols (Frankel, 1983). 4 . Volatile Compounds in Foods

The formation of aldehydes, ketones, alcohols, and oxoacids (from linoleic and linolenic acids) following disruption of tissues is an important mechanism for flavor formation in many animal and plant tissues (Tressel et al., 1981;Josephson et al., 1987; Hsieh and Kinsella, 1989). Examples

OXIDATION OF POLYUNSATURATED FATTY ACIDS

28 1

of the array of six-, eight-, and nine-carbon carbonyls generated in various plant and animal tissues are summarized in Table IV. The unsaturated alcohols I-octen-3-01, 1,5-octadien-3-01,and 2,5-octadien-1-01 occur in many mushroom species (Wurzenberger and Grosch, 1986). Thus, linoleic acid is oxidized to its 10-hydroperoxide, which is then cleaved by a hydroperoxide lyase into 1-octen-3-01 and lO-ox0-8decenoic acid (Tressl et al., 1982; Wurzenberger and Grosch, 1984). In addition, Tressl et al. (1982) identified 1,5-octadien-3-01 and 2,5-octadien-1-01as components of volatiles obtained from linolenic acid in mushrooms. Octadienol has also been identified in red seaweed (Woolward et al., 1975), crustaceans (Whitefield et al., 1982), and emerald shiner (Josephson et al., 1984b). Lipoxygenases in tomato (Veldink et al., 1977) and soybean (Matoba et al., 1985) oxidize linoleic acid and linolenic acid to form both 9- and 13-hydroperoxides. The hydroperoxide lyase of both tissues acts specifically on 13-hydroperoxide of linoleic acid to produce hexanal. The sixcarbon aldehydes are common volatile compounds in tomato and soybean. Hexanal is the decomposition product of 13-hydroperoxylinoleic acid, whereas 2-hexenal is that of 13-hydroperoxylinolenicacid (Veldink et al., 1977; Galliard et al., 1977). Linoleic and linolenic acids can be oxidized by cucumber lipoxygenase to form both 13- and 9-hydroperoxides. The hydroperoxide lyase in cucumber can cleave both types of hydroperoxide isomers to form six- and nine-carbon aldehydes (Galliard et al., 1976). 2-Nonenal is formed from 9-hydroperoxylinoleic acid, whereas 9-hydroperoxylinolenic acid is the precursor of 2,6-nonadienal (Sekiya et al., 1977). Unsaturated nine-carbon aldehydes are the characteristic volatile flavor compounds of cucumber.

IV. OTHER EFFECTS OF LIPID OXIDATION

In Vivo EFFECTS OF PEROXIDATION Lipid oxidation has a range of detrimental effects on the quality attributes and the functional properties of foods (Simic and Karel, 1980), and lipid peroxidation in vivo has been implicated in the denaturation of proteins and inactivation of enzymes, the disruption of biological membranes, and in causing damage to genetic material (O’Brien, 1987; Chan, 1987; Esterbauer and Cheeseman 1987; Machlinn and Bendich, 1987). These effects may result form enzymatic or nonenzymatic oxidation of

282

R. J. HSIEH AND J. E. KINSELLA

polyunsaturated fatty acids into various reactive intermediates, free radicals, singlet oxygen, and hydroxyl radicals (Frankel, 1984; Esterbauer and Cheeseman, 1987; Sevanian, 1988). 1 . Effects of Lipid Oxidation on Proteins

Biochemical changes in proteins exposed to peroxidized lipids are similar to those induced by ionizing radiation, and include loss of enzyme activity (Cadenas et al., 1983; Benedetti et al., 1979; Tappel and Roubal, 1969); destruction of individual amino acids (Shimasaki et al., 1982); and polymerization, cross-linking, and scission (Logani and Davies, 1980; Zirlin and Karel, 1969). Free radicals generated by peroxidation of lipids have been reported to initiate free radical formation in proteins which may result in dimerization or polymerization. The polymerization process is damaging to enzyme activity and biomembranes in vivo (Logani and Davies, 1980) and causes loss in solubility and toughening of proteins (e.g., muscle) in food systems. Lipid peroxidation can cause destruction of amino acids (including arginine, serine, glutamic acid, methionine, tyrosine, tryptophan, phenylalanine, and threonine) (Roubal and Tappel, 1966) and cross-linking with proteins, e.g., N-acetylcysteine formed a thiol bond with linoleic hydroperoxide (Gardner et al., 1976). Reactions of histidine with methyl linoleate hydroperoxides generate imidazole lactic acid and imidazole acetic acid as major products (Yong and Karel, 1978). Interactions between peroxidized cardiolipin and albumin resulted in covalent bonding between the lipid and protein (Nielsen, 1978). Malondialdehyde, a product of fatty acid peroxidation, can cause cross-linkings between proteins (Esterbauer and Cheeseman. 1987). Free radicals can transfer from oxidized methyl linoleate to amino acids and proteins, and free radicals have been detected in oxidized tyrosine, arginine, histidine, tryptophan, and cysteine by electron spin resonance (Schaich and Karel, 1974). Oxidized glutathione and, to a limited extent, cystine also gave free radical signals. Free radicals were also detected in oxidized proteins. Glycine, arginine, histidine, and phenylalanine in bovine serum albumin can react with linoleic acid hydroperoxides and yield products with strong fluorescent properties (Shimasaki et al., 1982). Lysine is the most effective substrate for this reaction (Nielsen, 1981). These amino acid residues can form complexes with lipid hydroperoxides via their corresponding sulfhydryl or positively charged nitrogen moieties (Hidalgo and Kinsella, 1989). Radical transfer occurs when hydroperoxides and these complexes interact and hydroperoxide dismutates in the immediate vicinity of the amino acids (O’Brien, 1987). N-Acetylation of

OXIDATION OF POLYUNSATURATED FATTY ACIDS

283

amino acids and proteins greatly reduces the fluorescence of the products (Nielsen, 1981). The possibility that products of lipid oxidation may exert effects in vivo needs to be studied. The compound 4-hydroxy-2-nonenal, a decomposition product of linoleate peroxidation, can react with thiol groups of microsomal membrane proteins. This reaction decreases enzyme activity, protein synthesis, and causes loss of cell viability (Benedetti et a f . , 1979; Ferrali et al., 1980; Cadenas et a f . 1983). Hexanal and other saturated and unsaturated aldehydes could also be responsible for the destruction of cytochrome P -450 (White, 1981). Aldehydes such as transd-hydroxy2-octenal and trans4-hydroxy-2-pentena1, by their effects on protein thiol groups, have inhibitory effects on cell division in mammalian cell lines (Schauenstein, 1967). The 4-hydroxy-2-alkenal from the peroxidation of liver microsomal lipids inhibited protein synthesis and caused inflammation (Benedetti et al., 1981). The linoleic acid hydroperoxide-methemoglobin-catalyzed oxidation of glutathione, generated 40% sulfonic acid and 60% oxidized glutathione (Little and O’Brien, 1968). Inactivation of isocitrate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase by lipid peroxide is due to the oxidation of essential thiol groups in these enzymes (Parker and Allison, 1969). The formation of intramolecular disulfide bonds in glyceraldehyde-3-phosphate dehydrogenase by lipid oxidation caused extensive conformational changes and inactivation of the enzyme (Parker and Allison, 1969). The oxidation of sulfhydryl groups in papain by peroxides resulted in sulfenic acid formation and led to a loss of 70% of enzyme activity. Arsenite, thiols, and other nucleophils could reverse this inactivation (Lin et af., 1975). Oxidized lipids react with myosin in coho salmon to generate fluorescent products. These interactions result in oxidized myosin accompanied by denaturation, destruction, and quality changes in fish muscle lipids and proteins (Braddock and Dugan, 1973). Lysine, tyrosine, methionine, and arginine in fish myosin react with malondialdehydes (Buttkus, 1967). Insulin and gelatin reacted with autoxidized methyl linoleate, with loss in solubility reflecting their cross-linking with oxidized lipids (Andrews et a f . , 1965).

2 . Effects on Membranes Lipid peroxidation changes the physical structure of membranes and results in decreased rates of translational diffusion and rotational diffusion, suggesting changes in membrane fluidity (Dobretsov et af., 1976).

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R. J. HSIEH AND J. E. KINSELLA

The increase in membrane viscosity leads to an increase in the membrane rigidity. Lipid fluidity is important in membrane permeability, osmotic fragility, and the activity of certain membrane-bound enzymes and transport (O’Brien, 1987). Formation of conjugated dienes during lipid oxidation changes the double bond from a cis to a trans configuration, which may allow for tighter packing of the unsaturated chains. Formation of more rigid domains within the membrane bilayer decreases membrane fluidity (Coolbear and Keough, 1983). Fragmentation of fatty acid hydroperoxides, for example, by scission of alkoxy radicals to release volatile hydrocarbons, decreases the length of the unsaturated fatty acids and may change fluidity (Grzelinska et al., 1982). Hydroperoxides, alcohols, and aldehydes are considerably more polar than the fatty acids and their presence could result in an increase in membrane polarity and hydrophilicity. Furthermore, the appearance of these polar molecules could explain their movement to the membrane surface (Chapman and Wallach, 1968). Oxidation of the carbony1 groups formed could lead to the formation of negatively charged carboxylic acid groups and repulsion of these charges could contribute to disorder and increased fluidity (Logani and Davies, 1980). Polymerization of unsaturated fatty acids, covalent binding of unsaturated fatty acids to proteins, as well as protein polymerization would induce high rigidity within the membrane lipid layers (Barber and Thomas, 1978). Phosphatidylethanolamine and phosphatidylcholine are the principal lipids that are oxidized in biomembranes and thereby decrease membrane fluidity (O’Brien, 1987). Membrane lysis following lipid oxidation could be due to the formation of lysolecithin molecules in the membrane. Lysolecithin formation could explain the decreased cooperativity and enthalpy of the phase transition in membranes (Coolbear and Keough, 1983). Perturbation of membranes as a consequence of lipid peroxidation can cause uncoupling of oxidative phosphorylation in mitochondria (Vladimirov et al., 1978) and modification of ion permeability (Potapenko et al., 1972). In mitochondria, peroxidation caused membrane swelling, deterioration of electron transport, and organelle lysis (Narabayashi et al., 1982). Microsomal membranes undergoing peroxidation in vitro showed fragmentation, destruction of cytochrome P-450 (Hogberg et al., 1973), and loss of activities of glucose-6-phosphatase and UDP glucuronyl transferase (deGroot et al., 1985; Ferrali er al., 1980). The Ca2+- ATPase became inactivated because of oxidation of its essential sulfiydryl groups (Brownlee et al., 1977). Lipid peroxidation of lysosomes and erythrocytes caused lysis and enzyme release (Wills and Wilkinson, 1966; Brownlee et al., 1977). Various cytosolic enzymes containing essential -SH groups can also become inactivated during lipid peroxidation (Chio and Tapple, 1969). Lipid

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285

peroxidation can also exert comparable damaging effects in the nucleus (Baird, 1980), and 4-hydroxyalkenal inhibited protein synthesis in rabbit reticulocyte lysates Benedetti et al., 1981).

3. Effects on DNA Free radical metabolites are possible toxic intermediates or proximate carcinogens. Free radicals are electrophiles and can interact with the nucleophilic regions of macromolecules, such as proteins and DNA (Horton and Fairhurst, 1987). The interaction can result in a change in the physical structure of DNA, hinder the in vivo repair mechanism, and cause errors during DNA replication which may result in mutations and neoplasms. Cytotoxicity could also occur if DNA damage results in inhibition of RNA synthesis or DNA replication (O’Brien 1985). Weitberg (1987) recently reported that arachidonic acid induced sister chromatid exchange and exacerbated genetic damage observed with an oxygen radical-generating system. Vitamin E protected cells from oxygen radical-induced genotoxicity. Enzymes such as peroxidase, catalase, and superoxide dismutase remove different species of activated oxygen that promote lipid peroxidation (Fridovitch, 1976). Any pathological or degenerative conditions may decrease the concentrations of these protective enzymes, with consequent damage from the toxic effects of activated oxygen. Aliphatic aldehydes, decomposition products from lipid oxidation, may react with the amine groups of nucleic acid bases to form fluorescent Schiff bases (Summerfield and Tappel, 1983). Malondialdehyde (MDA) reacts with DNA to cause cross-linking which results in a loss of template activity and decreased hyperchromicity (Summerfield and Tappel, 1983). This has been demonstrated in nuclei isolated from liver and testes of rats fed 1,3-propanediol. It suggests that cross-linking involves interstrand guanine-cystosine bases in the interior of DNA rather than paired bases, which could explain why they may not be easily recognized by the normal repair system. The fluorescent nucleotide-aldehyde adducts were not enzymatically digestible. MDA also cross-linked DNA to histones (Summefield and Tappel, 1983). The mutagenicity of MDA may be due to DNA adduct formation, interstrand cross-linking, and polymerization (Basu el al., 1984). Other fluorescent compounds may also arise during lipid peroxidation. MDA may react with primary amines, and DNA may form fluorescent adducts with lipid peroxidation products other than MDA (Kikugawa and Ido, 1984; Fujimoto et al., 1984). MDA is not much less mutagenic than formaldehyde, glutaraldehyde, and glyoxal (Reiss and Tappel, 1973).

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Formaldehyde has been shown to be a carcinogen (Levin et ul., 1982). However, the mutagenicity and carcinogenicity of various aldehydes and dialdehydes are not well understood. The lipid peroxides such as cumene hydroperoxide and tert-butyl hydroperoxide are more mutagenic than the above aldehydes (Levin et al., 1982). The potential adverse effects of lipid oxidation and of products of lipid oxidation are relevant to the formation of these products in foods and the fatty acid composition of foods. The presence of reactive products in foods and the potential of dietary PUFA to undergo peroxidation in vivo underscore the need for more systematic research concerning the metabolic effects of these compounds and the associated antioxidant requirements (Machlin and Bendich, 1987). V. LIPID OXIDATION IN FISH

FACTORS AFFECTING OXIDATION I . Lipids and Fatty Acids

The demand for and consumption of fish and seafood products is increasing because of their nutritional benefits, i.e., high content of n-3 polyunsaturated fatty acid, low content of cholesterol, and high content of good quality protein (Stansby, 1982; Kinsella, 1987, 1988). The lipid content of fish is highly variable, both among species and within a given species (Kinsella, 1987). The lipid content in the edible flesh of individual fish may range from a minimum of 0.5% to a maximum of 25%. A value of 5% total lipid content has been suggested as a breakpoint between low-fat and medium-fat fish, with values over 15% being catalogorized as high-fat fish (Stansby, 1982). The lipids of fish occur primarily as triglycerides, with phospholipids amounting to around 0.5% of muscle (Kinsella et al., 1975). The major polyunsaturated fatty acids (PUFA) are eicosapentaenoic acid (EPA; C20 : 5 n-3) and docosahexaenoic acid (DHA; C22:6 n-3), (Kinsella, 1987). The presence of n-3 PUFA, EPA and DHA, distinguishes fish lipids (Table VI) from lipids of plant and other animal sources in terms of nutritional value and oxidative instability (Kinsella, 1987; Stansby 1982). Variations in the composition and content of PUFA are large. Several factors, e.g., geographic location, catch, season, sex, maturity, and feeding habit affect the composition of fish and thus their susceptibility to oxidative instability (Stansby, 1982; Kinsella, 1987). Overall, the highly PUFA render fish tissue extremely susceptible to autoxidation and rapid

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287

TABLE VI FAlTY ACID COMPOSITION OF TROUT GILL AND SKIN TISSUE

Gill Fatty acid

(wt%)

Skin (wt%)

14:O 16:O 16:I 18.0 18.1 18:2 n-6 18:3 n-3 18:4 22.1 20:4 n-6 20:4 n-3 205 n-3 2 2 5 n-6 225 n-3 22:6 n-3

2.95 16.74 7.59 5.14 24.64 4.27 7.22 1.13 4.75 2.20 0.43 5.59 0.63 1.03 15.70

3.91 14.21 8.67 3.16 27.44 6.07 10.30 1.91 4.98 2.85 0.81 3.99 0.63 1.21 9.34

deterioration, especially when not handled properly. More basic information is needed concerning initiation and propagation mechanisms in order to minimize oxidative deterioration of seafoods.

2. Factors Affecting Fish Lipid Oxidation The initiation of oxidation of n-3 PUFA may involve heat, light, metals, and enzymes (Frankel, 1980). Initiation takes place by abstraction of a reactive methylene hydrogen from PUFA, followed by 'introduction of one molecule of oxygen to form a hydroperoxide. The hydroperoxides formed can react with oxygen to form such secondary products as epoxyhydroperoxides, ketohydroperoxides, dihydroperoxides, cyclic peroxides, and bicyclic endoperoxides (Frankel, 1984). These secondary products decompose to form volatile breakdown products such as alkanal, alkenal, alkenone, alkadienal, and alkantrienal (Ke et al., 1975), or they can condense into dimers and polymers. Alternatively, lipid hydroperoxides and their breakdown products can interact with other biological molecules in fish, such as pigments, enzymes, proteins, membranes, and DNA. This results in discoloration, flavor odor and deterioration, loss of water-holding capacity, and texture change, etc. (Stansby, 1982; Tsukuda and Amano, 1968).

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The factors which influence lipid oxidation in fish include the fatty acid composition of lipids, their disposition, the presence or absence of activators and inhibitors (heme, metal ions, pH value, oxidative enzymes, tocopherol, carotenoids), and external factors such as storage temperature, time, light, oxygen pressure, water activity, and packaging conditions (Khayat and Schwall, 1983). The type of fatty acid present is a major factor in determining the oxidative stability of lipids. In general, the rate of autoxidation increases as the number of double bonds increases (Enser, 1974; Lundberg, 1962). The location of fatty acids within the glyceride molecule or phospholipid can affect the oxidation rate (Raghuveer and Hammond, 1967). Unsaturated acids located in position sn-2 of glycerides oxidize more rapidly than those in the 1 or 3 position. The disposition of lipids within the tissue also influences their oxidation rate. Lipids associated with the dark lateral muscle of fish tend to oxidize more readily than those in white muscle (Fischer and Deng, 1977; Ke et al., 1978; Mai et al., 1978). In frozen cod and haddock, the PUFA associated with the phospholipids oxidize faster than glycerolipids or free fatty acids (Hardy et al., 1979). The skin lipids in mackerel and oil sardine are more susceptible to oxidation than muscle lipid (Ke et al., 1977; Nair et al., 1976). The PUFA of lipids in the exposed surface of fish fillets and fish mince oxidize more rapidly than lipids within the tissue because of the relative concentration of oxygen (Bligh and Regier, 1976). The rate of PUFA oxidation varies with the oxygen pressure, i.e., at very low oxygen pressures the rate of oxidation is approximately proportional to the oxygen pressure, whereas at pressures higher than 100 mm Hg, there is no dependence on the oxygen pressure. At different oxygen concentrations, the termination process can be different and produce different oxidative products (Marcuse and Fredricksson, 1968). This is because the alkyltype radical is predominant at oxygen pressures of C 100 mm Hg, while at oxygen pressures > 100 mm Hg, the alkoxy radical is the majora species. Exclusive of oxygen, e.g., vacuum packaging, substantially reduced oxidative deterioration in frozen fish and fishery products (Lindsay, 1977; Josephson et al., 1985), while treatment with an oxygen absorber prevented the oxidation of n-3 polyunsaturated fatty acids in sardine oil during freezing storage (Suzuki et al., 1985). The temperature effect is complicated by the composition of the foods and other factors. Usually, storage temperatures as low as -30 to -40°C are recommended to extend the storage life of fish (Hardy and Smith, 1976; Ke et al., 1977). In fatty fish, oxidation takes place primarily in the depot fats which are composed of triglycerides. The rates of oxidation of PUFA and decomposition of hydroperoxides decrease with decreasing

OXIDATION OF POLYUNSATURATED FATTY ACIDS

289

temperatures usually by a factor of 2 to 3 for every 10°C decrease. Chain propagation and peroxide decomposition reactions are accelerated with increasing temperatures (Lundberg, 1962). This produces more free radicals that are available for the initiation and propagation of lipid oxidation. Different oxidative products can form at different temperature (Lundberg, 1962). At higher temperatures up to 120"C,where an induction period is not observed, the predominant products of lipid oxidation are carbonyl compounds, carbon dioxide, and lipid polymers (Lundberg, 1962). Conditions of cold storage are important, thus glazing or freezing fish reduces dehydration and protects fish from oxidation (Banks, 1952; Hardy, 1980). The loss of water may also facilitate the interaction between hydroperoxides and metal catalyst and increase the decomposition of hydroperoxides (Labuza, 1971).

3. Metal Catalysis of Fish Lipid Oxidation The kinetics of metal-catalyzed oxidation in fish tissues have been reviewed by Khayat and Schwa11 (1983). The transition metals, which include iron, copper, cobalt, and manganese, possess two or more valences states with suitable oxidation potentials that both decrease the induction period and increase the rate of lipid oxidation (Ke et al., 1977). The catalytic activity of metals which are oxidizable/reducibleby oneelectron transfer may proceed via three types of reactions (Walling, 1957): (I) Activation of hydroperoxides by reduction (a)

M"'+ROOH+

M("+')+ +OH-+RO*

(b) M("+')++ ROOH -+ M"' +ROO.+ H +

(11) Direct reaction of metal with oxygen

(111) Complex formation of metal compounds with oxygen and the subsequent formation of an HO- radical M2++ 0, + M2'0,* M2+0,+M2+(XH)+ M"X- +HO,+M,'

Lipid oxidation is readily induced in both lean and fatty fish by the ppm), and Zn2+(30 ppm) (Castell, addition of Fe2' (<5 ppm), Cu2+(4

290

R. J. HSIEH AND J. E. KINSELLA

1971; Ke and Ackman, 1976). The oxidation of skin lipids increases very rapidly with increased Fe2+and Cu2+at concentrations of <10 ppm (Ke and Ackman, 1976). Both heme (6-15 ppm) and nonheme iron (10 ppm) present in most fish tissues are powerful prooxidants in catalyzing fish lipid oxidation (Ke and Ackman, 1976). Catalysis by dry nonheme iron is pH sensitive, being more active at acidic pH values. However, pH had less effect on hemoprotein-catalyzed lipid oxidation (Liu and Watts, 190). Mizushima et al. (1977) found that Cu2+,Fe2+,Fe3+,and hemin accelerated lipid oxidation in Alaska pollack and mackerel oil. Iron (Fe2+),at 1 mg/kg, promoted the oxidation of mackerel muscle lipids and the Fez+ was three times more effective as a prooxidant for muscle lipids than Fe3+ (Ke and Ackman, 1976; Tichivangana and Momssey, 1982, 1984). Trace amounts of copper (1 mg/kg) induced the rancidity of both lean and fat fish muscle (MacLean and Castell, 1964; Tichivangana and Morrissey, 1984). The increased rate of lipid oxidation in cooked muscle reflects the release of nonheme iron during cooking. The addition of 2% ethylenediaminetetraacetic acid (EDTA) effectively chelated the nonheme iron and thus significantly reduced lipid oxidation (Igene et al., 1979). Yong and Karel(l978) reported that inorganic iron and copper were strong prooxidative catalysts in lipid oxidation, particularly in mackerel meat lipids. Various substances, such as pigments, heme compounds, organic acids, and amino acids catalyze lipid oxidation, particularly when in association with certain trace metals (Yu et al., 1974; El-Zeany et al., 1974; Castell and Bishop, 1969; Mizushima et al., 1977). Both heme and hemecontaining proteins are capable of initiating lipid autoxidation in model systems composed of phospholipid liposomes. Small heme compounds such as hematin have higher prooxidative potency than large heme compounds such as myoglobin and hemoglobin (Kaschnitz and Hatefi, 1975). This may reflect the higher diffusional mobility of hematin and/or better accessibility of its active site toward substrate lipids. The oxidation of lipids in beef, chicken, and pork muscles was positively correlated with myoglobin and total heme content, but not with nonheme content (Rhee and Ziprin, 1987). Fischer and Deng (1977) concluded that heme iron is the major catalyst in homogenates of the dark flesh of mullet. Lee and Toledo (1977) indicated that dark muscle was more susceptible to oxidation than white muscle, possibly because of the heme pigments and a greater concentration of unsaturated lipids in this tissue. The prooxidant catalytic activity of these extracts was dependent on their total heme protein content. The dark muscle of pelagic fish has a high myoglobin content which is very effective in catalyzing the oxidation of lipids (Young, 1974). Myoglobin has a higher catalytic effect than hemoglobin (Lillard, 1978),

OXIDATION OF POLYUNSATURATED FATTY ACIDS

29 1

hence the myoglobin-to-hemoglobin ratio as well as the total concentration of heme protein should be considered when determining the role of heme pigments as prooxidants (Lillard, 1978). In cooked meat, the myoglobin is converted to the active catalytic form, metmyoglobin, which accelerates development of oxidative rancidity in cooked fish or meats (Yamauchi, 1973). After prolonged cooking, the active ferric heme is destroyed and thus liberates iron that can cause nonheme catalysis of lipid oxidation (Igene et al., 1979; Rhee and Ziprin, 1987). Heating hemoproteins may also increase the exposure of the heme group to the lipid substrate (Tichivangana and Momssey, 1984). Metmyoglobin has the same prosthetic group as peroxidase and catalase, but its interaction with hydrogen peroxide occurs at a slow rate (Keilin and Hartree, 1950). The interaction of metmyglobin with hydrogen peroxide may produce a free radical (King and Winfield, 1963; Shinga and Imaizumi, 1975). The metmyoglobin radical may also be formed as a result of its reaction with organic hydroperoxide (Kalyanaraman et al., 1982). Metmyoglobin, activated by hydrogen peroxide, generates an active molecular species, perhaps a porphyrin cation radical, which causes rapid oxidation of membrane lipids in turkey muscle (Harel and Kanner, 1985; Kanner and Harel, 1985). Similar activated species has been reported following the interaction between methemoglobin and hydrogen peroxide (Kanner and Harel, 1985). The rate of hemoglobin catalysis varies with different lipid classes. In cod, phosphatidylethanolamine oxidizes rapidly in both the presence and absence of hemoglobin Phosphatidylcholine oxidizes only in the presence of hemoglobin, whereas neutral lipids exhibit a very low rate of oxidation even with added hemoglobin (Tichivangana and Morrissey, 1982, 1984). Recently, Kanner ( 1989 personal communication) observed that free iron was a major catalyst of peroxidation in turkey and chicken muscle. 4 . Enzyme-Involved Lipid Oxidation

The enzyme-catalyzed lipid oxidation system in the microsomal fraction of muscle tissue has been extensively studied (Lin and Hultin, 1976; Player and Hultin, 1977; McDonald et al., 1979). This enzyme system is unstable to heat, shows a preference for NADPH compared to NADH, and the reaction is inhibited by disruption of membranes with detergents. The rates of reaction are higher with Fez+than with Fe3+and the optimum pH of the reaction is around 6.5 (Love, 1983; Rhee et al., 1984). In contrast to fish, microsomes isolated from tissues of rats apparently require NADPH rather NADH for enzymatic lipid peroxidation (Svingen et al., 1978).

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R. J. HSIEH AND J. E. KINSELLA

The microsomal lipid oxidation have been demonstrated in various seafood muscle tissues, such as flounder (McDonald and H u h , 1987; Shewfelt and Hultin, 1983), red hake (McDonald et al., 1979), yellowtail (Murata and Sakaguchi, 1986), herring (Slabyi and Hultin, 1982), rainbow trout (Han and Liston, 1987), and mussels (Musgrave et al., 1987). The lipid oxidation initiated by microsomal enzymes is a free radical reaction (Hultin, 1986), while singlet oxygen and other reduced oxygen products may participate (McDonald and Hultin, 1987). The proposed reactions for the microsomal lipid oxidation in flounder muscle are summarized in Fig. 28 (McDonald and Hultin, 1987). The function of microsomal enzymes is to reduce Fe3+to Fe2+using electrons from NADH. The reduced iron reacts with oxygen to form an intermediate (Fe2+02,dioxygen ferrous ion complex, in equilibrium with Fe3+O-', i.e., perferryl ion). This released superoxide and Fe3' form a complex with ADP and the ADP-FE3+ complex keeps the iron ion soluble and modifies its redox potential. Upon reduction, the Fe2+ is released from the ADP complex. The enzyme-catalyzed reaction has a rate-limiting step prior to the formation of hydroperoxides: the abstraction of hydrogen from the substrate fatty acids, which is generally considered to be the rate-limiting step in nonenzymatic lipid oxidation (Simic and Karel, 1980). In contrast to chicken tissues, NADH was much more effective than NADPH in microsoma1 lipid oxidation in fish tissues (McDonald et al., 1979; Slabyj and H u h , 1982). The K,,, for NADH in the presence of 0.1 mM ADP was NADH

+ NAD

NAD

Y .~

Cyt. P450 reductate Oxidized Cyt. . p450 Reduced

01

ADP-Fe(lll)

I

W. P450 reductasel( Oxidized

Cystelne-Fe(ll)

Cyt. P-450 reductase Reduced

Cysteine-Fe(iii)

0;

I[

cvt. P450 reductate Ascorbate-Fe(II) Oxidized f 2 Cyt. P-450 reductate Ascorbate-Fe(lll) Reduced

0;

FIG. 28. The proposed reaction scheme of microsomal lipid oxidation in flounder muscle. [From Kanner et a/. (1987) and McDonald and Hultin (1987).]

OXIDATION OF POLYUNSATURATED FATTY ACIDS

293

approximately 1 pM,whereas it was 10 p M in the absence of ADP. This is significant because NADH is the common cofactor in the muscle tissue and the concentration of NADH present in the fresh flounder (Phillippy, 1984) and yellowtail (Murata and Sakaguchi, 1986) was higher than 30 p M . During frozen storage, there was a rapid decrease of NADH, though the concentration was still above the K , value. ADP and Fe3+are essential for lipid peroxidation in microsomes from mussels (Slabyj and Hultin, 1982; Musgrave et al., 1987). The ADP forms a complex with ferric ion to catalyze the initiation and help maintain iron in a soluble form (McDonald and Hultin, 1987). The optimum pH for microsomal enzymes in herring was between 6 and 7 (Slabyj and Hultin, 1982). The microsomal enzyme system provides an active mechanism for initiating lipid oxidation in fish and other muscle foods even during frozen storage (Apgar and Hultin, 1982). The extent and rate of lipid oxidation during frozen storage may not be significant enough to cause a change in quality. However, when frozen seafood is thawed, oxidative enzyme activity may increase and become an important factor in quality deterioration of thawed products. Microsomal oxidative activity can be retarded by using oxygen intermediate scavengers, such as catalase and superoxide dismutase, and free radical quenchers like propyl gallate, as discussed below. Peroxidase isolated from fish leukocytes is capable of initiating lipid oxidation in the presence of hydrogen peroxide and halides (Kanner and Kinsella, 1983a,b). Myeloperoxidase from trout leukocytes caused rapid hydroperoxidation of unsaturated fatty acids and bleaching of p-carotene in the presence of hydrogen peroxide and iodide or bromide (Kanner and Kinsella, 1983a,b). No destruction of p-carotene occurred when halogen ions, hydrogen peroxide, or the enzyme were omitted from the system. This reaction could involve any one of three mechanisms, viz, free radical addition, abstraction, or singlet oxygen oxidation. Free radicals of electron donor molecules are formed in the peroxidase reaction (Rutter and Hager, 1982; Kanner and Kinsella, 1983a). The radicals derived from this reaction could also interact with the electrons of the double bonds by addition or abstraction mechanisms. Both radical compounds could then interact with oxygen to form a hydroperoxide or react with other halide radicals. The activation energy for the addition of chlorine or bromine and especially iodine to a double bond is lower than the hydrogen abstraction step. Another possible mechanism could involve singlet oxygen generated by the interaction of hypochlorite (a by-product of the peroxidase reaction) with hydrogen peroxide and halide ions, especially bromide and chloride ions (Harrison and Shultz, 1976). Muscle foods, e.g., fish, are exposed to processing, cutting, chopping, deboning, etc. During these processes the blood components are spread

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R. J. HSIEH AND J. E. KINSELLA

over the surface of the product, and in an environment with plenty of oxygen this could facilitate the peroxidase reaction in neutrophils and monocytes. The concentration of iodide and chloride in fish tissue is also high enough to activate the peroxidase reaction (Kanner and Kinsella, 1983a,b). Thus, leukocytes may be one focus for the initiation of lipid peroxidation and thereby contribute to lipid deterioration and adversely affect the quality of fish and seafoods. Lipoxygenase occurs in the gill and skin (Table VII) tissues of fish (German and Kinsella, 1985, 1986a,b; Yamaguchi and Toyomizu, 1984; Hsieh et al., 1988b). This enzyme activity is localized in the microsomal fraction with a pH optimum around pH 7 (German and Kinsella, 1985, 1986a,b). The enzyme is insensitive to the cyclooxygenase inhibitors, indomethacin and aspirin, but is strongly inhibited by lipoxygenase inhibitors, stannous chloride (5 mM), esculetin (10 pM), and eicosatetraenoic acid (100 pIM) (German and Kinsella, 1986a.b; Hsieh and Kinsella, 1989). The gill lipoxygenase acts by a free radical mechanism and initiates the

TABLE VII THE RELATIVE ACTIVITY OF LIPOXYGENASE IN GILL AND SKIN TISSUES OF FISHES'

(Relative activity (%) Fish species

Gill

Skin

Fresh water drum White perch Emerald shiner White bass Gizzard shad Rock bass Brown bullhead Sheephead Rainbow trout Channel catfish Salmon Smallmouth bass Yellow perch Blue gill Pumpkin seed

160

17 8 40 ND~ 50 NDb 36 NDb I00 84 44 13 5 ND~ 20

156 154 151 140

136 133 I24 I00 88 79 43 40 31 26

"From Hsieh et al. (1988b). bND, Not determined.

OXIDATION OF POLYUNSATURATED FATTY ACIDS

295

peroxidation of polyunsaturated fatty acids, such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, to form specific 12- and 14-hydroperoxides (German and Kinsella, 1985). These hydroperoxides are further oxidized by an iron-catalyzed mechanism to generate hydroxy epoxides which are then hydrolyzed to trihydroxy fatty acid derivatives (German and Kinsella, 1986a; Pace-Asciak, 1984). The breakdown of these unstable hydroperoxides and secondary oxidation products (Table V) may contribute to the onset of lipid oxidation in fish tissues and produce reactive free radicals which subsequently initiate chain reactions causing further autoxidation. Trout gill lipoxygenase displays a broad pH optimum around pH 7 (German and Kinsella, 1986; Hsieh et d., 1988b). The enzyme is less sensitive to pH change in the alkaline range than in the acidic range. A decrease in pH from 7.5 to 6.0 resulted in the loss of 90% of the activity, while raising pH from 7.5 to 9.0 reduced the activity by only 40%. Similar optima pH were observed for lipoxygenases from human platelets (Wallach and Brown, 1971), rat lung tissue (Yokoyama et al., 1983), and skin tissue of the guinea pig (Ruzicka et al., 1983). Both eicosapentaenoic and docosahexaenoicacids, which are abundant in fish lipids, are facilely oxygenated by the lipoxygenase of trout gills, which exhibit no selectivity toward these over arachidonic acid. However, the enzyme showed much less reactivity (<20%) toward linoleic acid (Hsieh et al., 1988b). The K, of rainbow trout 12-lipoxygenase for arachidonic acid is 20 p.M as determined by oxygen consumption (German and Kinsella, 1986; Hsieh et d., 1988a). This value is similar to that of 1Zlipoxygenasefrom human platelets (Wallach and Brown, 1981)and skin tissue of the guinea pig (Ruzicka et al., 1983). The 12-lipoxygenase from human platelets shows similar affinities for substrate fatty acids, such as dihomo-y-linolenic acid, arachidonic acid, M) and 5,8,11,14,17-eicosapentaenoicacid, (K,, in the range of (Wallach and Brown, 1981). However, in the case of 1Zlipoxygenase from rat lung, eicosapentaenoic acid is used preferentially to arachidonic acid, while little reactivity is shown toward linoleic and linolenic acid (Yokoyama et al., 1983). Because of the potential role of lipoxygenase in causing off-flavors, practical methods for minimizing lipoxygenase activity are of interest. Gill lipoxygenase retained over 90% of its activity following heating between 10" and 30°C. The high activity of fish lipoxygenase at temperatures near freezing suggests that it may be important in initiating fish lipid oxidation under refrigeration conditions. At 40"C, 80% of activity was retained. However, an abrupt decline in activity occurred at 50°C and only minimal activity was observed at temperatures above 60°C (Hsieh et al.,

296

R. J. HSIEH AND J. E. KINSELLA

1988b). The thermal instability of gill lipoxygenase provides an approach for controlling the enzyme activity and fish quality and also to explain the improvement of fish shelf stability following heat treatments (ToledoFlores, 1987). The decrease in activity of gill lipoxygenase during storage was time dependent. The half-life of the enzyme (1.5 hr) at 4°C was shortened by the addition of substrate fatty acid, such as arachidonic acid, or the elevation of temperature to 25°C (German and Kinsella, 1986; Hsieh et al., 1988a). This loss of activity was not caused by the exhaustion of substrate fatty acids because addition of new substrate failed to restore the activity. The inactivation of lipoxygenases by oxygenated substrate also occurred within reticulocytes (Rapport et al., 1979). The instability of lipoxygenase may be caused by oxidation and the accumulation of excess amounts of enzyme-generated hydroperoxides (Egan et al., 1979). The stability of gill lipoxygenase is significantly improved by the addition of 1 mM glutathione to the preparation. Glutathione may be used by glutathione peroxidase in trout tissue (Bell et al., 1980) to reduce hydroperoxides to stable hydroxy analogs and reduce excess hydroperoxides. 2-Mercaptoethanol has a lower potency than glutathione in stabilizing lipoxygenase activity (Fig. 29). The effect of 2-mercaptoethanol may be ascribed to the maintenance of essential sulfhydryl groups in gill lipoxygenase in the reduced state or the liberation of glutathione to facilitate the glutathione peroxidase reaction and minimize self-inactivation (Hohe, 1982a,b). However, the addition of high concentrations (5 mM) of glutathione or 2-mercaptoethanol to the gill lipoxygenase preparation may reduce the available concentrations of hydroperoxides to such an extent that the lipoxygenase activity becomes limited (Fig. 29). The requirement for low levels of hydroperoxides (
OXIDATION OF POLYUNSATURATED FATTY ACIDS

0

1

1

I

I

1

2

3

4

297

i 5

Concentration ( mM )

FIG. 29. The effect of thiol reagents on the activities of gill lipoxygenase. Reactions were performed in a standard assay mixture containing(a) glutathione;(b) 2-mercaptoethanol;(c) iodoacetamide; or (d) p-chloromercuribenzoate. [From Hsieh ef al. (1988b).]

which reduced activity and adversely affected its affinity toward fatty acids, its substrate specificity, and kinetics (Grossman et al., 1984). pCMB inhibited lipoxygenases in platelets (Siege1 ef al., 1980) and skin tissue of guinea pig (Ruzicka et al., 1983). Modification of sulfhydryl groups of soybean lipoxygenase by alkylmercuric halides has also been assumed to block sulfhydryl groups in the active site of the enzyme (Spaapeen et al., 1980b). Oxidative enzymic activity may cause carotenoid bleaching deterioration in the skin of red fishes (Tsukuda and Amano, 1968; Stone and Kinsella, unpublished 1989). Lipoxygenase activity may provide an answer to the preferential oxidation of skin lipid to muscle lipids in frozen oil sardine (Nair et al., 1976), mackerel (Ke et al., 1977), and round fish (Yamaguchi et al., 1984). Lipoxygenase metabolites, such as hydroxy fatty acids, and leukotrienes from gill and skin tissues may provide biological signals for external stress and antimicrobial protection, and, in practical terms, they generate oxidative off-flavors in fish.

298

R. J. HSIEH AND J. E. KINSELLA

5. Generation of Volatile Compound in Fish

Various aliphatic aldehydes contribute strongly to the characteristic aroma of oxidized fish lipids (Tables IV and V). These carbonyl compounds impart flavors which have been frequently described as rancid, cod-liver-oil-like, and “painty.” Certain volatile carbonyls, arising from the autoxidation of polyunsaturated fatty acids, have been described as contributing typical fishlike aroma (Badings, 1973; Josephson et al., 1983; McGill et al., 1977). The relative concentrations greatly affect the perceived flavors of these compounds. 4-Heptenal appears to be characteristic of the cold-stored flavor of fish, especially frozen cod (McGill et al., 1977). Other volatiles, such as 2-pentenalY2-hexenal, 2,4-heptadienal, 2,CdecadienaL and 2,4,7-decatrienal tend to increase with the increase of time and temperatures of frozen storage. Production of these volatiles is directly related to the progress of oxidation of unsaturated fatty acids in fish (McGill et al., 1977). 2,CDecadienal and 2,4,7-decatrienal together are key components in the oxidized fish-oil “like” flavors (Meijboom and Stroink, 1972; Swoboda and Peers, 1977). The decrease in major polyenoic fatty acids of mackerel oil during autoxidation can be related to the formation of 2,4,7-decatrienal (Ke et al., 1975). The origins of volatile flavor compounds associated with fishlike aroma in fish flesh and fish oils have been the focus of much research. Fresh fish is characterized by a plantlike, green grassy aroma, reflecting the combination of carbonyls and alcohols derived from polyunsaturated fatty acids, conceivably by enzymatic mechanisms (Josephson et al., 1984b). Carbonyls and alcohols such as hexanal, 2-octenal, I-octen-3one, octen-3-01, 1,5-0ctadien-3-01, 2,5-ocadien-l-ol, 2-nonenal, and 2,6nonadienal are important components of fresh fish aroma and flavor and are present in lake whitefish, Northern pile, smelt, and shiner, rainbow trout, and bluegill (Josephson et al., 1984a). The characteristic aromas of fresh fishes vary among different species (Josephson et al., 1984a). Thus, 2-nonenal and 2,6-nonadienal are present in freshwater fishes (Josephson et al., 1984a), whereas I-octen-3-01, 1,5-0ctadien-3-01, and 25octadien1-01 occur in some species of fresh freshwater and also saltwater fishes (Josephson et al., 1983, 1984a). The presence of 2-nonenal, 2,6-nonadienal, 6-nonen-1-01, and 3,6-nonadien-l-ol in rainbow trout impart a cucumber-like aroma. The nine-carbon carbonyl compounds appear to be associated with species inhabiting cold waters. These species also appear to be more vulnerable toward handling damages than species that do not contain these compounds (Josephson et al., 1984a). The biochemical origins of these compounds are unknown.

OXIDATION OF POLYUNSATURATED FATTY ACIDS

299

In emerald shiner, the eight-carbon alcohols, 1-octen-3-01, 2-octenol, 1,5-octadien-3-01,and 2,5-octadien-1-01, are derived from enzymatic reactions (Josephson et al., 1984a), while carbonyls, 2,4-heptadienal, 33-octadien-Zone, 2,4-decadienal, may be nonenzymatically derived products of lipid oxidation (Mejiboom and Stroink, 1972; McGill et al., 1974, 1977; Swoboda and Peers, 1977). The 2-nonenal and 2,6-nonadienal may be formed either enzymatically (Galliard et al., 1976), or nonenzymatically (Lillard, 1978, Frankel, 1980). Hexanal, 2,4-heptadienal, and 2,4-decadienal are classical fishlike oxidative flavors derived via nonenzymatic mechanisms (Mejiboom and Stroink, 1972; McGill et al., 1974, 1977; Swoboda and Peers, 1977). Several enzymatic and nonenzymatic processes, such as autoxidation, photosensitized oxidation, lipoxygenase, peroxidase, and microsomal enzymes, act as initiators of lipid oxidation in fish tissues (Kanner and Kinsella, 1983a; Frankel, 1985a; Slabyj and Hultin, 1984; German and Kinsella, 1986a; Josephson et al., 1987). Lipoxygenase, present in gill and skin tissues of several species of fishes, catalyzes the oxidation of polyunsaturated fatty acids to produce hydroperoxides. The inhibition of lipoxygenase by BHA or esculetin inhibited the formation of specific hydroperoxides and also the resultant volatile products (Hsieh and Kinsella, 1989). This indicated that lipoxygenase activity is not only important as an initiator of lipid oxidation, but is also involved in the subsequent generation of oxidative volatile compounds (Fig. 30a,b). The products of lipoxygenase-initiated oxidation are specific. Arachidonic and eicosapentaenoic acids are oxidized by gill lipoxygenase to produce their respective 12-hydroperoxides(German and Kinsella, 1985). I n vivo, these hydroperoxides are reduced to their hydroxy analogs in the presence of glutathione peroxidase (German and Kinsella, 1986a,b). As lipid oxidation proceeds, especially postmortem, the hydroperoxides are further oxidized to epoxide, hydroxy, epoxide, trihydroxy fatty acids, etc. (Frankel, 1985a). In the presence of metal ions, these reactive oxygenated derivatives are cleaved by homolytic cleavage and p-scission to generate alkyl, alkoxy, or alkperoxy radicals, etc. (Frankel, 1985a,b; Hsieh and Kinsella, 1989). The decomposition products of enzyme-generated hydroperoxides of PUFA are quite specific. Thus, 12-hydroperoxyarachidonic acid decomposes to produce 3-nonenal, 1-octene, or 2-octene radicals (Hsieh and Kinsella, 1989). The 3-nonenal can isomerize into 2-nonenal and the 1octene radical may accept a hydrogen or hydroxyl radical to form l-octene or 1-octen-3-01. On the other hand, the 2-octene radical may receive a hydrogen or hydroxy radical to produce 2-octene or 2-octen-1-01 (Fig.

300

R. J. HSIEH AND J. E. KINSELLA

a 12-Hydroperoxide of arachidonic acid

CHO

3-Nonmal

m

1

or

Isomerization CHO

A 0 v b A A A J 2-Nonenal

I

+OH*NH*

2-Octen- 1-01

+

A &

l-Octen-3-ol

FIG. 30 (a), The proposed reaction scheme of lipoxygenase-catalyzed oxidation of arachidonic acid and the subsequentgeneration of oxidative flavor compounds. [From Hsieh and Kinsella (1989).]

30a). By similar mechanisms, the 12-hydroperoxy eicosapentaenoic acid can be decomposed to generate 2,6-nonadienal, 175-octadien-3-ol,and 2,5-octadiene-l-ol, respectively (Fig. 30b). The compounds 2-nonenal and 2,dnonadienal and 1-octen-3-01, 1.5-OCtadien-3-01, and 2.5-octadien-1-01 contribute to the fresh aroma of various species of freshwater and saltwater fishes (Josephson et al., 1983, 1984a,b). 4-Heptenal, 2,4-heptadienal, 2-pentena17 3-hexenal, 2,4-decadienal, and 2,4,7-decatrienal are the important compounds derived via oxidation which have been associated with fish off-flavors (McGill et al., 1977; Khayat and Schwall, 1983).

OXIDATION OF POLYUNSATURATED FATTY ACIDS

30 1

FIG. 30 (Continued) (b), The proposedreaction schemeof lipoxygenase-catalyzedoxidation of eicosapentaenoic acid and the subsequent generation of oxidative flavor compounds. [From Hsieh and Kinsella (19891.1

A key point in controlling lipid oxidation in muscle foods and fish is at the initiation stage of the process. Both enzymatic and nonenzymatic reactions may be responsible for initiation reactions in fish and seafood tissues (Kanner and Kinsella, 1983a,b; Slabyj and Hultin, 1984; German and Kinsella, 1986a,b; Josephson et al., 1987; Hsieh and Kinsella, 1989). Lipoxygenase and peroxidase are plausible sources of the initial peroxides, i.e., acyl hydroperoxides generated from PUFA. These are also sources of short-chain oxidative flavor compounds and free radicals, which may lead to extensive chain reactions and autoxidation. Proper postharvest handling methods, such as low-temperature stor-

302

R. J. HSIEH AND J. E. KINSELLA

age, avoidance of bruising or injury, possibly mild heat treatment (i.e., blanching), and the use of specific antioxidants may be employed to control or retard enzyme-initiated lipid oxidation and quality deterioration (Hsieh and Kinsella, 1989). VI. CONTROL OF LIPID OXIDATION

A. REACTANTS AND INHIBITORS 1 . Oxygen

Because oxidation of polyunsaturated fatty acids can proceed via a number of mechanisms, several strategies are possible for minimizing oxidation. Knowledge of the key initiating mechanism(s) facilitates control. Because oxygen is an essential reactant, the control of oxygen availability is a critical variable in minimizing oxidation of PUFA. The rate of oxidation of unsaturated fatty acids is affected by oxygen pressure, but only when the partial pressure of oxygen in the reaction system is less than 100 mm Hg (Pryor, 1973). The addition of oxygen to substrate is a diffusion-limited process (k = 109-1010M-'sec-I). The partial pressure of oxygen also influences termination reactions; when the partial pressure is 100 mmHg or greater, termination reactions involving only the peroxy radicals need to be considered. The level of available oxygen may be controlled by vacuum packaging (Lindsay, 1977; Josephson et al., 1985) by using oxygen absorbers (Suzuki et al., 1985) and oxygen scavengers, such as glucose oxidase and ascorbic acid oxidase (Scott, 1965). These measures reduce the rate and extent of lipid oxidation, especially when combined with antioxidants and low-temperature storage. 2. Active Oxygen Species

A general mechanism for protection of biological systems against the potential hazards of superoxide anion, hydrogen peroxide, and hydroxyl radical is to convert them to water by reduction (Fridovich, 1976). The control of superoxide anion is to prevent the possible direct initiation of lipid peroxidation by the perhydroxyl radical (H02*).In addition, superoxide dismutase accelerates the dismutation of superoxide anion to hydrogen peroxide, thereby preventing the Haber-Weiss reaction from producing hydroxyl radicals. Superoxide anion may be controlled by superoxide dismutase or reducing compounds such as ascorbic acid, thi-

OXIDATION OF POLYUNSATURATED FATTY ACIDS

303

ols, hydroquinones, and catechins, though these lack specificity and are not efficient (Korycka-Dahl and Richardson, 1980). Hydrogen peroxide is the precursor of hydroxyl radicals and also the activator of heme proteins. Catalase and glutathione peroxidase in animal tissues rapidly remove hydrogen peroxide. Glutathione peroxidase in the cytosol and mitochondria of animal cells is activated by hydrogen peroxide to an active compound which is then reduced to native state by glutathione (Flohe, 1982a). The hexose monophosphate shunt is the source of reducing equivalents used by the glutathione peroxidase system (Loven et al., 1983). Glutathione peroxidase also catalyzes the reduction of hydroperoxides, including those derived from unsaturated fatty acids (Flohe, 1982b). Singlet oxygen can be quenched by synthetic antioxidants, e.g., BHT (Foote, 1976). a-Tocopherol is an effective quencher of singlet oxygen, but its quenching effect is offset by its oxidation to unstable hydroperoxides (Clough et al., 1979). p-Carotene is also an effective quencher of singlet oxygen under certain conditions. Fridovich (1976)proposed that the first line of defense against hydroxyl radical-initiated lipid oxidation in biological systems is prevention of the formation of its precursors, hydrogen peroxide and superoxide anion, by catalase and superoxide dismutase. Hydroxyl radical scavengers, substances with a high reactivity toward hydroxyl radicals [the rate constant for this reaction being about lo9 M - ' sec-l (Kanner et al., 1987)l can be divided into two categories: those that easily cross cellular membranes, i.e., alcohol, dimethyl sulfoxide, thiourea, and benzoate, and those that cannot cross membranes, e.g., mannitol, sucrose, inulin (Dillard et al., 1982; Heikkilo et al., 1976). There is limited use of these in food systems. Natural antioxidants such a a-tocopherol or electron donors such as thiolcontaining compounds are good scavengers of hydroxyl radicals (Korycka-Dahl and Richardson, 1980). 3. Metals

Metals are important reactants involved in peroxidation. Iron is needed (Fenton reagent) in the Haber-Weiss reaction to generate hydroxyl radicals at a significant rate (Czapski and Ilan, 1978). It is also required to complex with oxygen to form the putative ferryl-iron complex in microsoma1 lipid oxidation (Hochstein et al., 1964). Also, ferric and ferrous iron are needed to catalyze the homolytic cleavage of hydroperoxides to free radicals for chain reactions (Frankel, 1983). Copper can replace iron as a catalyst in lipid peroxidation. In the presence of superoxide and hy-

304

R. J. HSIEH AND J. E. KINSELLA

drogen peroxide, copper acts as a Fenton reagent. However, copper can also scavenge electrons, and its ability to catalyze peroxidation can be altered by EDTA chelation (Samumi et al., 1981). Other transition metals that undergo univalent redox reactions, such as cobalt and manganese, can also participate in the initiation of lipid peroxidation (Aust and Svingen, 1982). Lipid hydroperoxides are relatively stable against uncatalyzed unimolecular decomposition (O'Brien, 1969). For example, the calculated half-life of H,Oz at 30°C is 10" years (Pryor, 1973). However, in the presence of transition metals, hydroperoxides are rapidly decomposed by a redox mechanism generated by free radicals (O'Brien and Little, 1969). The most stable oxidation states of iron in aqueous system are the ferric Fe" and ferrous Fez+(Spiro and Saltman, 1974). Free iron, especially ferric ion, is insoluble in water above pH 3; hence, they must be chelated with ligands, such as EDTA, ATP, or ascorbate, to form a soluble complex (Tien and Aust, 1982). The redox potential of iron is affected by the electronic states of these ligands. The type of chelation may influence the mechanism of initiation and the reaction intermediates formed (Marshall, 1985). It has been reported that ADP-chelated iron and EDTA-chelated iron may promote lipid oxidation in microsomes from chicken, beef, and fish tissues (Emster et al., 1982; Rhee et al., 1984; Hultin, 1986). Iron chelates with low redox potentials, such as EDTA, rapidly react with hydrogen peroxide and produce oxidative hydroxyl radicals (Walling et al., 1975). On the other hand, EDTA withdraws electrons from the ferrous-oxygen complex and reduces the stability of the perferryl complex. Thus, chelation of iron by EDTA may prevent the promotion of hydroperoxide-independent initiation of lipid oxidation. However, EDTA-chelated iron was potent in promoting the breakdown of lipid hydroperoxide via reductive activation of hydroperoxides by the ferrous ion (Aust and Svingen, 1982; Svingen et al., 1978). EDTA enhanced the generation of hydroxyl radical during the reduction of iron ions by ascorbate or cysteine in the presence of oxygen (Winterbourn, 1979; Kanner et d., 1986). This enhancement was inhibited by hydroxyl radical scavengers such as mannitol, formate, or histidine (Czapski et al., 1984; Kanner et al., 1986). The chelate complex of EDTA and iron ion can also promote the production of acyl radicals via the breakdown of lipid hydroperoxides (Aust and Svingen, 1982; Svingen et al.. 1978). This reaction can be controlled by free radical quenchers such as propyl gallate (McDonald and H u b , 1987) and BHT (Tien and Aust, 1982).

The inhibitory effect of EDTA was dependent on its concentration in relation to the concentration of the activated iron complex (Ernster et al.,

OXIDATION OF POLYUNSATURATED FATTY ACIDS

305

1982). At EDTA-to-iron ratios of less than one, maximal rates of peroxidation were obtained in fish; but at ratios greater than one, lipid oxidation was inhibited (Kanner et al., 1986). EDTA can deactivate iron ion by forming tightly bound ligands and preventing lipid oxidation (Waters, 1971). Thus, EDTA inhibits lipid oxidation in fish muscle by chelation of the iron ions released from hemoproteins by heat treatment (Tichivangana and Morrissey, 1984). Iron chelates with a low positive redox potential, such as ADP, are characterized by slow decomposition of hydrogen peroxide and low yields of hydroxyl radicals. Chelators like ADP, which may increase the electron density on the iron center and increase the stability of perferryl ion, are active promotors of hydroperoxide-independent initiation of lipid oxidation (Aust and Svingen, 1982; Ernster et al., 1982). The effects of ADP on lipid oxidation in fish muscle microsomes depend on the ratio of ADP to iron ion (Hultin et al., 1982). The optimum level of ADP is directly proportional to the concentrations of iron ion (Hultin et al., 1982). ATP provides a similar response to ADP, whereas that of AMP was 4 times less than ADP (Ernster et al., 1982; Hultin et al., 1982).

B. OXIDATIVE ENZYMES I . Microsomal Enzymes Singlet oxygen, hydroxyl radicals, and perferryl radicals are considered the most likely initiators of lipid oxidation in microsomal enzyme systems as described above. These reactive oxygen species then interact with unsaturated fatty acids to result in free radical-mediated peroxidation. Mannitol (100 mM), benzoate (100 mM), dimethyl sulfoxide (50 mM), and thiourea (20 mM) are free radical scavengers which can inhibit peroxidation of the lipids of microsomes prepared from red hake and flounder (Hultin et al.. 1982; McDonald and Hultin, 1987). This indicated that hydrogen peroxide, superoxide anion, and hydroxyl radical were involved in these oxidative reaction. NADH and the dinucleotide ADP are needed for the initiation of microsoma1 peroxidation (McDonald et al., 1979). An iron-oxygen perferryl ion complex with the nature of a free radical has been suggested (Ernster et al., 1982; Tien and Aust, 1982). The chelation of iron by ADP eliminated the inhibitory effects of catalase and mannitol (Tien et al., 1981). Iron chelates such as EDTA and sodium tripolyphosphate showed potent inhibitory effects on microsomal reactions by forming strong complexes with iron ion to prevent its participation in lipid oxidation (Hultin et al.,

306

R. J. HSIEH AND J. E. KINSELLA

1982; McDonald and Hultin, 1987). The inhibitory potencies of EDTA and ADP depend on their concentrations relative to iron ions (Kanner et al., 1986; McDonald and Hultin, 1987). Common antioxidants such as BHT, a-tocopherol, and propyl gallate also inhibited lipid oxidation by microsomes (McDonald and Hultin, 1987; Venekei, 1987).The a-tocopherol functions as an initiation preventor rather than a chain-breaking antioxidant (McDonald and Hultin, 1987). 2. Peroxidase

Peroxidases have been implicated in the quality deterioration of many foods, i.e., animal tissues, fruits, and vegetables (Muftugil, 1985; VamosVigyazo, 1981). The generation of oxygen free radicals, superoxide anion, hydrogen peroxide, hydroxyl radicals, and singlet oxygen has been suggested during the catalytic cycle of peroxidases in phagocytic cells (Cohen and Sinet, 1980; Fridovich and Porter, 1980; Gabig and Babior, 1981). Superoxide free radicals were reported during the oxidation of NADH and arachidonic acid by horseradish peroxidase (Halliwell, 1978a,b; Rosen and Klebanoff, 1979; Tauber and Babior, 1977). Hydrogen peroxide is essential for the initiation of lipid oxidation by peroxidase (Kanner and Kinsella, 1983b; Rosen and Klebanoff, 1977; Virion et al., 1981). During the reaction of myeloperoxidase with chloride, chlorine and hypochlorite may be generated (Harrison and Schultz, 1976; Klebanoff and Clark, 1978). The hypochlorite may interact with hydrogen peroxide and halide ions to produce singlet oxygen, which can initiate lipid oxidation (Harrison and Schultz, 1976; Harrison et al., 1978; Krinsky, 1981). Peroxidase activities can be controlled by catalase, superoxide dismutase, and quenchers of hydroxyl radicals, and singlet oxygen. The oxidation of unsaturated fatty acids catalyzed by lactoperoxidase is inhibited by free radicals scavengers, BHT, tryptophan, and methionine (Adeniji-Jones and Karnovsky, 1981; Kanner and Kinsella, 1983b). Ascorbate has been used to inhibit peroxidase activity and prevent the oxidation of anthocyanin pigments (Grommeck and Markakis, 1964).

3. Lipoxygenase Lipoxygenases can be inhibited by the acetylene analog of arachidonic acid, 5,8,11,1Ceicosatetraenoicacid (ETYA) (Kuhn et al., 1984). The oxygenation of the acetylenic fatty acid results in the competitive inhibition of lipoxygenase and the formation of 1 mol of methionine sulfoxide per mole enzyme (Gibson, 1977). 5,8,11-Eicosatriynoic acid (Hammarstrom,

OXIDATION OF POLYUNSATURATED FATTY ACIDS

307

1977) and 4,7,10,13-eicosatetraynoicacid (Sun et al., 1981) were also reported as suicidal substrates of lipoxygenase. Phenols and catechols inhibit lipoxygenase by virtue of their abilities to quench free radicals. 4-Nitrocatechol and some other 0-diphenolic compounds like protocatechualdehyde, 2,3-dihydroxyanthraquinone(histazine), catechol, 7,8-dihydroxy-4-coumarin (esculetin), and 3,4-dihydroxybenzonitrile inhibit active soybean lipoxygenase by interacting with its Fe3+ (Galpin et al., 1976). The inhibition of lipoxygenase by 4-nitrocatechol is noncompetitive (Spaapen et al., 1980a,b). Hydrogen peroxide is an effective irreversible inhibitor of soybean lipoxygenase-1 (Mitsuda ef d . , 1967; Aoshima e? d., 1977). It attacks the iron-containing center of lipoxygenase and generates a hydroxyl radical which causes structural alterations in the coordination sphere of iron (Egmond et al., 1975b). The tyrosine and histidine residues, essential for the catalytic activity of lipoxygenase, are modified by the hydroxyl radical (Aoshima et al., 1977; Mitsuda et al., 1967). Other antioxidant compounds, such as ferf-butyl hydroxyanisole (BHA), propyl gallate (PG), and nordihydroguaiaretic acid (NDGA), inhibit lipoxygenases from various sources (Agradi et al., 1981; van Wauwe and Goossens, 1983; Siddiqi and Tappel, 1975). Peroxy-enzyme radical complexes exist during the catalytic cycle of lipoxygenase with unsaturated fatty acids (Vligenthart and Veldink, 1982; Schewe et al., 1986). These release hydroperoxides which serve as sources of free radicals. At present it is not clear if lipoxygenase functions similarly to free radicals as an initiator of lipid oxidation. In this regard, specific inhibitory compounds could be useful in differentiating between lipoxygenase reactions and other free radical reactions. Compounds with free radical quenching ability should inhibit free radical oxidation, but may not be lipoxygenase inhibitors. Phenolic compounds, catechol, and pyrogallol, with oxidation potentials of 0.92,0.74, and 0.61 V, respectively, are traditionally considered as antioxidants which scavenge alkoxy and alkperoxy radicals (Scott, 1965; Sherwin, 1972). The relative effects of BHA, BHT (phenolics), caffeic acid, NDGA (catechol), and PG (pyrogallol) indicated that the inhibition of lipoxygenase by these antioxidants was not related to their oxidation potentials (Table VIII) (Hsieh et al., 1988a). BHA and BHT have similar oxidation potentials (0.65 and 0.68 V, respectively), but BHA was a much stronger inhibitor of lipoxygenase than BHT. NDGA, with a catechol structure, has a lower oxidation potential than BHA (Scott, 1965). Nonetheless, NDGA showed the same inhibition potencies as BHA. Caffeic acid with a catechol structure was a weaker lipoxygenase inhibitor than

TABLE VIII THE RELATIVE INHIBITORY EFFECTS O F VARIOUS PHENOLS, FLAVONOIDS, AND ANTIOXIDANTS ON FISH GILL 12-LIPOXYGENASE ACTIVITY

Test compounds Chrysin Apigenin Fistein Quercetin Myricetin Rutin Esculetin Esculin BHA BHT Catechind Epicatechind Naringenin'

PG NDGA

Radical quenchingb

3-c

5-c

3'-c

4'-c

5'-c

I C d W

H

OH

H OH

30

OH

H H

H

H

H

5

0.40

OH OH OH OH

OH

H H

0.25 0.40 2 7 0.02

0.68

OH OH OH Rutinose

H OH

OH OH

OH OH OH

OH

H

u)o 5

loo0

H OH

H

OH OH OH

H

H

H

OH

OH OH

H

H

H

3

I . 40 0.02 2.5

Oxidation potential' (volts)

0

0.94

-

0.92

-

-

0.76

0.72 0.76

0.68

0.28

-

-

0.65

-

-

"Reactions were perfomed in the standard radioactive and polarographic assays mixture containing I mg partially purified enzyme. The enzyme was preincubated with inhibitor at 25°C for 10 min, and then the substrate fatty acid was added for reaction. %ata derived from Baumann et al. (1988a.b). 0 denotes no quenching effect toward the free radical generated by human erythrocytes incubated with phenazine methosulfate, while 1 corresponds to 100% quenching effect. 'Data derived from Baumann er al. (1982); and Scott (1%5). %Van. 'Flavanone.

OXIDATION OF POLYUNSATURATED FATTY ACIDS

309

NDGA. Propyl gallate (with a pyrogallol structure) had a lower oxidation potential than esculetin (with a catechol structure), but they both showed similar inhibition potencies toward lipoxygenase (Hsieh and Kinsella, 1989). These findings suggest that the oxidation potential of an antioxidant does not predict its inhibitory potency on lipoxygenase. Comparable disparities in inhibitory potencies among BHA, BHT, and NDGA were reported for lipoxygenases in reticulocytes and mice skin (Nakadate et al., 1984; Schewe et al., 1986). Schewe et al. (1986) attributed the difference in inhibition of lipoxygenase to the structure and accessibility of antioxidants toward the catalytic site in the lipoxygenase where an enzyme radical complex is formed. C. ANTIOXIDANTS: FLAVONOIDS

1 . General Antioxidants Currently, there is a large number of antioxidants available. These include the most important of chemically produced FDA-approved phenolic antioxidants (Simic and Karel, 1980) and a large array of naturally occurring antioxidants, including tocopherol (see the chapter by Parker in this volume) and favonoids. On the basis of the autoxidation chain process, two mechanistically distinct classes of antioxidant can be used. The first operates by a radical chain-breaking mechanism by inactivating two important radicals species involved in the chain-propagatingstep, the alkylperoxy and alkyl radicals (Fig. 31). The other involves prevention of the introduction of chain-initiating radicals into the system. Because chain-breaking and preventive antioxidants interfere at different points in the autoxidation process, they mutually reinforce each other, resulting in synergism (Scott, 1965). Possible chain-ending reactions involve the interaction of the chain-breaking antioxidant with alkyl or alkperoxy radicals. The resulting antioxidant radicals may react with each other to terminate the chain reaction. Substituted phenolic compounds, e.g., butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT), are the most common anti-

ROO .+AH

A' +ROO' A'+A

-

e ROOH+A e ROOA AA

FIG. 31. The simplified radical chain-breaking mechanism. ROOH, Hydroperoxide of lipid; AH, antioxidant. [From Frankel (1985b).]

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oxidants. The type and location of the substituent groups greatly influence their efficacy, as discussed by Scott (1965). Thus, electron-releasing groups (e.g., methyl, methoxy) in the ortho and para positions markedly increase the antioxidant activity via donation of hydrogen, whereas substituents in the meta positions are much less effective. Electron-attracting groups (e.g., nitro, carboxy ,halogen) decrease activity, a-branched ortho alkyl groups considerably increase antioxidant activity but such groups in the para position decrease it (Scott, 1965). Tocopherols are chain-breaking antioxidants. They differ from one another only in the number and position of the methyl groups on the aromatic ring. a-Tocopherol, which is fully methylated, is the most prevalent naturally occurring antioxidant. The order of antioxidant activities of the tocopherols (a > p > y > 9)is the same as that of their biological activities in vivo (Burton and Ingold, 1986). a-Tocopherol interacts with peroxy radicals at a rate constant of 2.6 x lo6 M-' sec-', which is almost 100fold higher than that of BHT (2.4 x lo4 M-' sec-') (Burton and Ingold, 1986). Zama et al. (1979) reported that the rate of oxygen consumption in fish decreased as the concentration of added a-tocopherol was increased. Treatment of raw whole red sea bream (Pagrus major) and mackerel (Scomberjaponicus) with tocopherol prevented lipid oxidation. Deng et al. (1977) retarded the onset of oxidation in frozen mullet by adding ascorbic acid and tert-butyl hydroquinone in combination with vacuum packaging. Ascorbic acid may act as an antioxidant or prooxidant because of its ortho dihydroxyl group. In the dark flesh of mullet, ascorbic acid acted as an antioxidant at concentrations above 500 ppm and a prooxidant at concentrations below 500 ppm. The antioxidant to prooxidant shift was observed at 50 ppm in the white flesh (Deng et al., 1977). Ke et al. (1977) found that the order of effectiveness of antioxidants for inhibiting the oxidation in mackerel skin lipids was TBHQ > BHA > BHT at the concentration of 0.02%. TBHQ was the most powerful antioxidant for marine lipids and also retarded the formation of carbonyls from secondary oxidation reactions. BHA, PG, and BHT at 40 ppm were effective in preventing lipid oxidation in cooked fish samples (Shahidi and Rubin, 1987). Recently, considerable interest has developed in naturally occurring antioxidants. In this regard, it has been observed that various flavonoids may be effective inhibitors of lipid oxidation by a number of mechanisms. Though these currently are of minor importance compared to phenolic antioxidants and tocopherol, some important aspects of their properties are reviewed below.

OXIDATION OF POLYUNSATURATED FATTY ACIDS

31 1

2. Flavonoids Flavonoids occur in a variety of fruits, vegetables, leaves, and flowers. For centuries, plant preparations which contain flavonoids as the principal physiologically active constituents have been used by laymen and physicians in treating human diseases (Havsteen, 1983). These may function by altering the in vivo enzymatic oxygenation of PUFA. Flavonoids occur as aglycones, glycosides, and methylated derivatives (Kuhnau, 1970; Swain, 1976). The flavonoid aglycones all consist of a benzene ring (A) condensed with a six-member ring (C) which carries a phenyl ring as a substituent in the 2-position (B) (Fig. 32). The six-member ring condensed with the benzene ring is either a y-pyrone (flavonols and flavones) or its dihydro derivative (flavanols and flavanones). The position of the benzenoid substituent divides the flavonoid class into flavonoids (Zposition) and isoflavonoids (3-position).Flavonols differ from flavanones by a hydroxyl group in the 3-position and a C - 2 4 - 3 double bond. Anthocyanidins are closely related to the flavonoids. Their difference is in the C-ring, which is open in anthocyanidins. Flavonoids are often hydroxylated in positions C-3, 5 , 7, 3', 4', or 5 ' . When glycosides are formed, the glycosidic linkage is normally located in position 3 or 7 and the carbohydrate can be L-rhamnose, D-glucose, glucorhamnose, galactose, or arabinose (Kuhnau, 1970). Flavonoids structurally resemble

0

Flavone

Quercetin: 3,3',4',5,7-Rntahydroxyflavone Kaempferol: 3,4',5,7-Tetrahydroxyflavone Myrlcetln: 3,3',4',5,5',7-Hexahydroxyflavone

FIG. 32. The basic structure of the flavones quercetin, kaempferol, and myricetin. [From Herrrnann (1976).1

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nucleosides, isoalloxazine, and folic acid, and this similarity is the basis of many of the current hypotheses concerning their physiological actions (Havsteen, 1983). Flavonols are commonly found in vascular plants as aglycones and glycosides (Harborne, 1967; Wollenweber and Dietz, 1981). The glycosides are usually localized in hydrophilic regions such as vacuoles, while the free aglycones are localized in hydrophobic regions such as oil glands and waxy layers of plant cells (Wollenweber and Dietz, 1981). However, free aglycones are also distributed in the thylakoid membranes. Recently, the aglycones were found in cottonseed (Whittern et al., 1984) and in the sepals of several plants (Ishikawa et af., 1986), but their subcellular localization has not been examined. In plant cells, the concentration of flavonols is more than 1 mM (Furuya and Thomas, 1964), with Viestra et al. (1982) reporting that the concentration of flavonols in plant epidermal cells was 3-10 mM. The formation of flavone and flavonol glycosides depends on the action of light (Harborne, 1967; Mohr, 1969), and leaves usually contain higher concentrations of flavonols than other tissues of the same plants (Table IX) (Herrmann, 1976). Lettuce, endive, leek, kale, and brassica species contained about 2-50 and 10-60 mg/kg fresh weight of quercetin (3,3' ,4' ,5,7-pentahydroxyflavone)and kaempherol(3,4' ,5,7-tetrahydroxyflavone), respectively (Bilyk and Sapers, 1985; Wildanger and Herrmann, 1976). The flavonol concentration drops markedly from the outer to the inner leaves, reflecting less access to sunlight (Table IX). Myricetin (3,3',4',5,5',7-hexahydroxyflavone) was not detected in these samples (Bilyk and Sapers, 1985, Wildanger and Herrmann, 1976). Tea contains quercetin and kaempherol glycosides in concentrations exceeding 1% of its dry matter (Bokuchava and Skobeleva, 1969). Hops contain approximately 700 mg/kg and 550 mg/kg quercetin and kaempherol glycosides, respectively (Herrmann, 1976). Fruits such as apple and pear contain mainly glycoside of quercetin. Kaempferol is also frequently found in small quantities. The glycoside of myricetin is found in blackberry, 40-90 mg/kg fresh weight (Wildanger and Herrmann, 1976). Cultivated bilberries can contain up to 70 mg/kg of myricetin glycoside (Herrmann, 1976). Quercetin, Kaempherol, and myricetin (1-100 mg/lOO g dry matter) are the three most common flavonols occurring in many stone and berry fruits, such as plum, peach, apricot, cherry, blackberry, currant (Wildanger and Herrmann, 1976). In a considerable number of vegetables only small concentrations of flavone and flavonol glycosides have been detected, e.g., < I mg/kg fresh weight in carrots, radish, rutabaga, cauliflower, peas, cucumber, eggplant, and potato (Wildanger and Herrmann, 1976). On the other hand,

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TABLE IX CONCENTRATIONS OF QUERCETIN AND KAEMPFEROL IN DIFFERENT PLANT TISSUES

Sources

Quercetin (mgAcg fresh weight)

Kaempferol (mg/kg fresh weight)

Leaf lettuce Head lettuce Chives Leek Kale Ryegrass Broad bean Apple (skin) (meat) Pear (skin) Potato Tomato Pea

2-54 1-28 9 ND" 7-20 382 201 1 98 2 28 700 420 1500

0-2 0-2 54 20 13-30 ND" 1584 <1


"ND, Not determined.

the leaves of root vegetables, potatoes, and asparagus frequently contain concentrations in excess of 1 g/kg. Flavonoids also occur in other tissues of higher plants, such as wood, bark, stems, fruit, roots, flowers, pollen, and seeds (Pratt, 1976). The greatest natural source of flavans such as catechins and leucoanthocyanins is from wood and bark. Chalcones and aurones are mainly found in flower petals, and to a lesser extent in leaves and stems of some species but are not as widely distributed as other groups of flavonoid compounds. Wollenweber and Dietz (1981) demonstrated that the flavones and flavonols of leaf waxes and plant exudates occur in the free state, without glycosylation, and often in methylated form. Biflavonoids, which also lack glycosylation, are located in gymnosperm leaves at the outer wall or in the cuticle (Gadek et al., 1984). This contrasts with the more usual location of the corresponding flavonoid glycosides, which are found within the cell vacuoles of leaves or flowers. There is evidence that the pattern of glycosides may vary within the leaf, the epidermal cells, for example, having different glycosides from those in the mesophyll cells. This is true in the leaves of oat, barley, and rye. In the bean (Viciafaba), guard cells and upper epidermal cells contain major amounts of kaempferol glycosides (Weissenbock et al., 1984). Microscopic studies of anthocyanins in flowers show that, in most spe-

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cies, pigments are located in the upper epidermal cells, together with flavones. In a few species, such as the Boraginaceae, anthocyanins are present in mesophyll cells (Kay et al., 1981). The anthocyanin 3,5-diglucoside occurs in the cationic flavylium form in the intact petal of Mulva sylvestris (Harborne, 1986). 3. Antioxidant.Action

Flavonol aglycones in chloroplasts (Oettmeier and Heuple, 1972) may function as antioxidants, as do tocopherols in the thylakoid membranes, by scavenging radicals. Flavonol glycosides may also function as antioxidants in the hydrophilic regions, although they are not as effective as the aglycones for scavenging radicals (Takahama, 1983b). The antioxidative functions of flavonols appear to involve scavenging of superoxide anions (Takahama, 1985a, 1987), H,O, (Barz and Hoesel, 1979; Barz and Wiermann, 1981; Takahama, 1984a,b), singlet oxygen (Takahama et al., 1984; Sorata et al., 1982), and lipid peroxy radicals (Takahama, 1983b, 1985b; Whittern et al., 1984). The participation of hydroxyl radicals appears improbable because of the failure of inhibition of lipid oxidation by mannitol (Takahama, 1983a,b). They may function as UV-light filter protectors, based on their stability in UV light under aerobic conditions and their localization in the epidermis (McClure, 1975). Flavonols, such as quercetin and kaempherol, quench lipid free radicals and suppress lipid photoperoxidation (Takahama, 1982) and carotenoid photobleaching (Schenck et al., 1982). During the scavenging of active species of oxygen, the flavonols are oxidized (Takahama, 1983a). Kaempferol can be oxidized to 4-hydroxyphenylglyoxylicacid and phloroglucinolcarboxylic acid (Fig. 33) in yields of 19 and 21%, respectively (Nishinaga et ul., 1979; Takahama, 1987). Ascorbate, which occurs in high concentrations in some tissues such as spinach chloroplasts, can reduce the oxidized flavonols and enhance regeneration of these natural antioxidants (Takahama, 1984a,b; Takahama et al., 1984). The oxidized ascorbate can be reduced to ascorbate by glutathione (Foyer and Halliwell, 1976; Nakano and Asada, 1981). However ascorbate, due to its localization in the hydrophilic regions of biomembranes, cannot effectively suppress the radical reactions involved in the carotenoid photobleaching in the lipophilic regions (Takahama, 1984a,b). Flavones, such as fisetin, quercetin, and luteolin, form ligands with metal ions and reduce oxidation of lard (Hudson and Lewis, 1983; Mahgoub and Hudson, 1985). With catechin (owing to the absence of the carbony1 group), there is little complex formation (Hudson and Lewis, 1983).

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6 HO

"O@

COOH

+

OH Phloroglucinolcarboxylic Acid

co-coon 4-Hydroxyphenylglyoxylic Acid

FIG. 33. The possible mechanism showing the oxidation of kaempferol. [From Takahama ( 1987).I

The necessity of a 2,3 double bond for antioxidant activity is controversial (Hudson and Lewis, 1983). Glycosylation of 3-OH of quercetin to rutin either reduced the antioxidative activity (Letan, 1966a,b) or had no effect (Gardner, 1985; Hudson and Lewis, 1983). In foods, both quercetin and myrecetin, and their 3-glucosides, were effective antioxidants at concentrations ranging from 0.4 to 2.5 x lop5M in beef slices and carotene-lard solution. In nonenzymatic systems, their antioxidant effects are attributed to free radical acceptance and chelation of metal ions (Pratt and Watts, 1964). Aqueous and methanol extracts of glandless cottonseed prevent oxidation of linoleate in fresh beef homogenate catalyzed by metmyoglobin

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(Rhee et al., 1981; Ziprin et al., 1981). Rutin is one of the major quercetin glycosides in cottonseed (Whittern et al., 1984). Organic extracts of soybean protein products contain flavonoids (Dziedzic and Hudson, 1983; Hammerschmidt and Pratt, 1978; Naim et al., 1976) and these compounds inhibit lipid oxidation. Soy protein concentrates, defatted soy flour, soy protein isolate, and fresh and dried soybean hot water extracts exhibited appreciable antioxidant activity on lipids of beef slices, vegetable-beef soup, aqueous-lard, and aqueous-meat systems (Pratt, 1972; Sangor and Pratt, 1974). Pratt (1976) reported that phenolic acids have potent antioxidant activities. The 7,4’-dihydroxy- and 6-methoxyisoflavone was demonstrated in soybean products and exhibited potent antioxidative activity (Naim et al., 1976; Hammerschmidt and Pratt, 1978). Lipid extracts from the leaves of ryegrass, broad beans, and lucerne have marked antioxidative properties as stabilizers when added to lard. The antioxidative potencies of ryegrass, broad beans, and lucerne are caused by their phenolic compounds content, which amounts to 400, 2000, and 250 mg/kg dry matter, respectively (Hudson and Mahgoub, 1980). However, quercetin is four times less effective than the artificial antioxidants BHA, BHT, and propyl gallate in delaying the initiation of lard oxidation (Hudson and Mahgoub, 1980). Chalcones are more effective than the corresponding flavanones as antioxidants against lard oxidation. The 3,4’-dihydroxychalcones,such as butein and okanin, are particularly effective antioxidants in the concentration range of 0.025-0.1%. The effectiveness of the 3’,4‘-dihydroxy structure (catechol) in chalcones is dependent on the formation of extended forms of resonance-stabilized free radicals. Butelin is found in Acacia heartwood and as its 4’-glucoside in the petals of Coreopsis spp. (Geissman, 1941). Carnosic acid, which is present in rosemary oil, contains the catechol structure and is a highly active antioxidant (Bracco e? al., 1981). Generally, optimum antioxidant activity in flavonoids is associated with the following structural features: multiple phenolic hydroxyl groups, especially the 3’,4’-dihydroxy configuration; a carbonyl group at C-4; a free 3-OH group in preference to a 5-OH group, or the presence of both a 3- and 5-OH group (Hudson and Lewis, 1983; Dziedzic and Hudson, 1984). 4 . Effects on Lipoxygenase

Flavonoids, with free radical-quenching and metal-chelating capabilities, are potential inhibitors of enzyme-mediated lipid oxidation (Pratt ,

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1976; Baumann et al., 1980b; Havsteen, 1983). Certain flavonoids may specifically interfere with lipoxygenase reactions, and their inhibitory potencies may be related to particular structural features rather than their free radical-quenching abilities (Baumann et al., 1980a,b; Sekiya et al., 1982; Yoshimoto et al., 1983). Flavonoids, such as quercetin, are inhibitors of soybean lipoxygenase-dependent linoleate peroxidation (Takahama, 1985a,b; King and Klein, 1987). Kaempherol, caffeic acid, and chlorogenic acid are also effective inhibitors of soybean lipoxygenase (King and Klein, 1987). During the inhibition, quercetin apparently donates hydrogen (Takahama, 1983a,b). Carotenoid radicals, which are formed in chloroplasts, can also be reduced by quercetin (Takahama, 1982, 1984a,b). Specific requirements for the number and position of hydroxyl groups, as exemplified by the catechol (1,2-benzenediol) structure in the B rings of quercetin and fisetin, have been suggested for the inhibition of gill lipoxygenase-initiated lipid oxidation (Hsieh et d . , 1988a). Catechol, with an oxidation potential of 0.74 V, is easily oxidized and capable of scavenging superoxide and hydroxy radicals and inhibiting lipid oxidation (Hodnick et al., 1986; Pincemail et al., 1986). Replacement of the hydroxyl group in the catechol structure (B ring) with hydrogen (e.g., apigenin, with a phenol structure, and chrysin, with a benzene structure) resulted in a higher oxidation potential and higher radical quenching ability (Scott, 1965), but these had lower inhibitory potencies on gill lipoxygenase (Hsieh et al., 1988a). Myricetin, with a pyrogallol structure, has a lower free radical quenching ability, and was a less-potent lipoxygenase inhibitor than quercetin (Hsieh et al., 1988a). (Table VIII). Esculetin, with a catechol structure and oxidation potential of 0.76 V (Fieser, 1930), is a potent inhibitor of fish lipoxygenase (Table VIII). Glycosidation at one of the hydroxy groups of the catechol structure converts esculetin to esculin, which is a less-potent lipoxygenase inhibitor than esculetin (Hsieh et al., 1988a). However, the presence of catechol structure is not sufficient for flavonoids to be lipoxygenase inhibitors. Glycosidation of a hydroxy group, such as the 6-glycosidation of quercetin to rutin, changes neither the catechol structure nor the free radical quenching ability (Baumann et al., 1980b), but rutin is 20 times less potent than quercetin as a gill lipoxygenase inhibitor (Table VIII) (Hsieh et al., 1988). Alteration of the flavonoid structure from a flavone (such as quercetin) to a flavan (such as catechin and epicatechin), or from a flavone (such as apigenin) to a flavanone (such as narigenin) retains the catechol structure in the B ring and has similar free radical quenching abilities (Baumann et al., 1980b).Nonetheless, catechin and epicatechin are less potent than quercetin as 1Zlipoxygenase

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inhibitors (Table VIII). Naringenin is not as potent as apigenin in inhibiting lipoxygenase activity (Hsieh et al., 1988a). These findings indicate that small alternations in the flavonoid structure, which did not influence oxidation potential nor free radical quenching ability, markedly changed the inhibition potency of 12-lipoxygenase. Molecules with a catechol structure can form complexes with the active ferric form of soybean and reticulocyte lipoxygenases and thereby cause inhibition (Spaapen et al., 1980; Schewe et al, 1986). This implies that the small structural change such as glycosidation may influence the conformation of flavonoid and hinder its accessibility to the active site of lipoxygenase and lower its inhibitory potency. A dihydroxyl group on flavonoids was also required for inhibition of 5lipoxygenase (Yoshimoto et al., 1983) and 15-lipoxygenase (Baumann et al., 1980a; Yoshimoto et al., 1983). The position and number of hydroxyl groups in the coumarin skeleton are important for the inhibition of platelet 1Zlipoxygenase (Sekiya et al., 1982) and the 5-lipoxygenase in polymorphonuclear leukocytes (Kimura et al., 1985). Baicalein (5,6,7-trihydroxy-2-phenyl-4-hydro1-benzopyran-Cone) (Sekiya e? al., 1982) and esculetin (6,7-dihydroxy coumarin) (Sekiya e? al., 1982) are potent and selective inhibitors of platelet 12-lipoxygenase. Esculetin was a potent inhibitor of 5-lipoxygenase of human polymorphonuclear leukocytes (Hamberg, 1984) and of mastocytoma cells (Neichi et al., 1983). Daphnetin (7,8-dihydroxycoumarin) and fraxetin (6-methoxy7,&dihydroxycoumarin) also inhibit the formation of 5-lipoxygenase products. The monohydroxyl coumarins, umbelliferone (7-hydroxycoumarin) and scopoletin (bmethoxy-7-hydroxycournarin), and the coumarin glucosides, esculin and fraxin, also inhibited the formation of 5-HETE. However, 4-hydroxycoumarin and coumarin had lower inhibitory effects on 5-lipoxygenase and cyclooxygenase in the guinea pig (Kimura et al., 1985; Wauwe and Goossens, 1983). The results suggested that two adjacent phenolic hydroxyl groups at C-6 and C-7 or C-7 and C-8 in the coumarin skeleton are essential for inhibition of 5-lipoxygenase. The mechanism of the lipoxygenase inhibition by esculetin was of a noncompetitive type (Sekiya et al., 1982). Various hydroxyl positional isomers of flavonoids have been tested for their inhibitory effects on lipoxygenase. Cirsiliol (3’,4’,S’-trihydroxy-6, 7-dimethoxyflavone) and pedalitin (3‘4’,5’-trihydroxy-7-methoxyflavone) are inhibitors of 5-lipoxygenase of rat leukemia cells and 12-lipoxygenase of bovine platelet and porcine leukocytes (Kohno et al., 1985; Yoshimoto et al., 1983). Both IZlipoxygenase from human platelets and 5-lipoxygenase from human neutrophils were inhibited by phloretin, quercetin (3’,4‘,3,5,7-pentahydroxyflavone), morin (2’,4’,3,5,7-~entahydroxyfla-

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vone), and naringenin (4’,5,7-trihydroxyflavanone). 12-Lipoxygenase, but not 5-lipoxygenase, was inhibited by flavone, catechin (3’,4’,3,5,7pentahydroxyflavane), and rutin (3-glucoside of quercetin) (Corvazier and Maclouf, 1985). Morin, fisetin (3’,4’,3,7-tetrahydroxyflavone),kaempferol (4’,3,5,7-tetrahydroxyflavone),and esculetin inhibited epidermal 12lipoxygenase of mice, but catechin and epicatechin were inactive (Nakadate et al., 1984). Similar requirements of hydroxyl positional isomers of flavonoids were reported for the inhibition of 5-lipoxygenase from rat peritoneal macrophage and 12-lipoxygenasefrom human platelets (Welton et al., 1986). Some of the structural requirements essential for lipoxygenase inhibition have been suggested: glycosides are much less effective than the corresponding aglycones, the presence of the C - 2 4 - 3 double bond is essential, and a high number of hydroxyl groups enhances the inhibition. Quercetin (with a catechol ring) and myricetin (with a pyrogallol ring) inhibited platelet 12-lipoxygenase. Kaempferol (with a phenol ring) and morin (with a resorcinol ring) were less potent inhibitors of lipoxygenase, whereas flavone and flavanones were inactive (Landolfi et al., 1984; Michel et al., 1985). Quercetin is also an active inhibitor of soybean 15lipoxygenase, horse platelet IZlipoxygenase, and human platelet 124poxygenase (Welton et al., 1986). These authors question whether flavonoids with a catechol or pyrogallol ring can inhibit lipoxygenase, simply because of the free radical quenching properties. The inhibitory potencies of flavonoids are abolished or reduced if one of the hydroxyl groups is blocked by a sugar (quercetidrutin or esculetin/esculin) or if flavone is replaced by a flavanone (fustin/taxifolin) (Baumann et al., 1980a,b; Michel et al., 1985). Recently, flavonols have been reported to serve as antitumor agents (Havsteen, 1983). The involvement of reactive oxygen species in tumor promotion and the decrease of superoxide dismutase and catalase activities by tumor promoters (Solanki et al., 1981) have also been reported. The inhibition of tumor initiation by antioxidants other than quercetin has been reported (Ames, 1983). Flavonols suppress lipid metabolism by tumor promoters (Havsteen, 1983); however, the mechanism of suppression is not clear, though inhibition of lipoxygenase is possible. The possibility that flavonoids can be used to inhibit lipid oxidation in foods and lipid peroxidation in vivo warrants further study. These observations may also indicate that plant foods (fruits, vegetables, spices) may provide protection against certain pathophysiologies related to free radical reactions and oxidation reactions initiated by lipoxygenase and/or cyclooxgyenase. Conceivably, they may partly account for the beneficial therapeutic effects of plant foods. More research is needed to explore this possibility.

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VII. CONCLUSIONS

This review clearly indicates the ubiquitous nature and pervasiveness of lipid oxidation ex vivo in foods and in vivo,and demonstrates that oxidation generally has deleterious results in both systems. This dramatizes the need for more effective strategies for controlling lipid oxidation, both in food materials and in tissues in vivo. Existing practices using antioxidants, chelators, enzyme inactivation, and anoxic and low-temperature storage conditions have been discussed (Simic and Karel, 1980). However, because of increasing concern about the safety of some phenolic antioxidants and the apparent desirability of increasing the polyunsaturated fatty acid content (especially the n-3 eicosapentaenoic and docosahexaenoic acids), of foods, newer and more effective strategies are needed. Such strategies can be best designed by understanding the mechanisms of PUFA oxidation as reviewed above. Effective control of lipid peroxidation in vivo requires a balanced intake of antioxidants, e.g., tocopherol p-carotene, ascorbic acid, and essential components of enzymes that rigorously deactivate active oxygen species, e.g., selenium of glutathione peroxidase. The importance of vitamin E and related compounds is reviewed by Parker (this volume). Knowledge of the importance of the products of enzymatic oxidation of polyunsaturated fatty acids in biologically regulation, (i.e., prostanoids and leukotrienes generated by cyclooxygenase and lipoxygenase, respectively) and the many dangers of uncontrolled peroxidation has dramatized the need for a careful reassessment of dietary PUFA intake and of the most appropriate and adequate balance of dietary antioxidants. These questions are germane to the food science and nutritional aspects of food formulation and preservation.

ACKNOWLEDGMENT Part of this work was supported by the New York Sea Grant Program; and a US-Israel BARD grant.

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Agradi, E., Petroni, A., Socini, A., and Galli, C. 1981. In vitro effects of synthetic antioxidants and vitamin E on arachidonic acid metabolism and thromboxane formation in human platelets and on platelet aggregation. Prostaglandins 22, 255. Albrich, J. M., McCarthy, C. A., and Hurst, J. K. 1981. Biological reactivity of hypochlorous acid: Implications for microbiocidal mechanisms of leukocyte myeloperoxidase. Proc. Natl. Acad. Sci. (U.S.A.) 78, 210. Ames, B. N. 1983. Dietary carcinogens and anticarcinogens: Oxygen radicals and degenerative diseases. Science 221, 1256. Andrews, F., Bjorksten, J., and Trenk, F. B. 1965. The reaction of an autoxidized lipid with proteins. J. Am. Oil Chem. SOC.42, 779. Aoshima, H., Kajiwara, T.,Hatanaka, A., Nakatani, H., and Hiromi, K. 1977. Modification of lipoxygenase by hydrogen peroxide and photooxidation. Int. J. Pept. Protein Res. 10, 219.

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PROTEINS IN WHEY: CHEMICAL, PHYSICAL, AND FUNCTIONAL PROPERTIES J. E. KINSELLA AND D. M.WHITEHEAD Institute of Food Science Cornell University Ithacu, New York 14853

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Introduction Whey Products Structure of Whey Proteins A. p-Lactoglobulin B. a-Lactalbumin C. Bovine Serum Albumin D. Immunoglobulins E. Proteose-Peptones F. Lactofenin G. Lysozyme Whey Protein Preparation and Isolation Functional Properties of Whey Proteins A. Intrinsic Factors and Functional Properties B. Extrinsic Factors C. Variability in Whey Protein Preparations Hydration and Solubility Thermal Properties of Whey Proteins A. Effects of Heat on Conformation B. Factors Affecting Heat Denaturation Gelation A. Gel Structure B. Gelation of Whey Proteins C. Composition and Gelling Properties D. Whey Proteins as Gelling Ingredients Surface Activity of Whey Proteins A. Protein Adsorption B. Protein Structure and Film-Forming Properties C. Whey Protein Films D. Film-Forming Properties of Glycosylated p-Lactoglobulin

343 Copyright 8 1989 by Academic Press, Inc. AII rights of reproduction in any form reserved.

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J. E. KINSELLA AND D. M. WHITEHEAD

X. Foams

XI.

X11.

XIII. XIV. XV.

A. Protein Structure and Foam Stability B. Whey Protein Foams C. Factors Affecting the Foaming Properties of Whey Proteins Emulsions A. Emulsifying Properties of Whey Proteins B. Molecular Flexibility, Surface Hydrophobicity, and Emulsifying Properties C. Factors Affecting Emulsifying Properties Ligand Binding by Whey Proteins A. Lipid Binding B. Flavor Binding C. Ligand Binding by P-Lactoglobulin D. Interactions of Flavors with Whey Proteins Modification of Whey Proteins A. Enzymatic Modification B. Chemical Modification Nutritional Aspects of Whey Proteins Summary and Conclusions Research Needs References

1.

INTRODUCTION

The annual production of cheese whey is expected to exceed 23 x lo’ kg in the near future while current utilization of whey approximates 60% of this total. Whey represents a potentially significant source of functional protein ingredients for many traditional and novel food products. The unique structure and physicochemical properties of whey proteins govern their functional behavior in food systems and structure-function relationships must be elucidated and controlled in order to utilize this food resource fully. This review focuses on the structural properties of whey proteins and how these properties are influenced by environmental factors (e.g., pH, temperature, salts) in manifesting characteristic functional properties, such as gelation, foaming, and emulsifying activity, which are important in food applications (Fig. 1). Proteins are important structural components in many foods and they are also used as ingredients because of their nutritional value and physicochemical properties (Kinsella, 1976, 1982; Morr, 1984). The market for functional protein-rich ingredients is expanding and is currently supplied by various proteins (Table I). Proteins obtained from dairy products have traditionally been one of the main protein sources for humans. Currently, a selection of high-protein dairy powders, e.g., caseinates, coprecipitates, and whey powders are used, yet these display a highly variable range of

345

PROTEINS IN WHEY

PROTEIN STRUCTURE and CONFORMATION

OTHER MOLECULES PRESENT

tEMrEnlltunE

PR-INS

PR EDOMIN A N T INTRA- AND INTERMOLECULAR FORCES HYDROPHOBIC IONIC (HYDROGEN BONDINO AND ELECTROSTATIC) VAN DER WML'S INTERCTIONS DlSULFlDE BONDINO

FIG. I. Factors and forces involved in determining the functional properties of food proteins.

TABLE I APPROXIMATE AMOUNTS OF PROTEINS USED AS FUNCTIONAL INGREDIENTS IN FOOD MANUFACTURING IN THE UNITED STATES

Commodity source Milk Whey EfB Meat Fish Cereals Oilseeds Yeast

Estimated amounts used ( x lo6 kg) I36 168 6.8

14.0 145.3 I.4

Functional component protein Caseins, whey proteins Whey proteins Egg white proteins, lipoproteins Collagen, myosin Collagen, myosin Wheat and corn gluten Soybean concentrates, isolates, and flours Single-cell protein (SCP)

346

J. E. KINSELLA AND D. M. WHITEHEAD

functional properties because of differences in composition and processing treatments (Kinsella, 1984a). The anticipated market demand for functional ingredient proteins is expected to increase in the United States and worldwide as the food industry increases the formulation and fabrication of new foods from basic ingredients (Kinsella, 1985). To satisfy this demand, food processors require ingredient proteins with consistent functional properties that perform in food systems in a reliable manner and are compatible with automation. II. WHEY PRODUCTS

Production of fluid whey in the United States increased to 20 x lo’ kg (18 x lo9 kg sweet and 2 x lo9 kg acid whey) in 1985 and the volume is increasing annually (Clark, 1987). Approximately 60% of this total is processed principally for use in foods and animal feed, with dried whey powder being the predominant product (Table 11). Liquid whey, a byproduct of cheese manufacture, contains approximately 20% of the origi-

TABLE I1 DIRECT AND INDIRECT USES OF DRY WHEY POWDERS IN FOODS IN

(x

Dry sweet whey

Bakery Dairy Blends Dry mixes Confectionery Margarine Processed meats soups Chemicals Soft drinks Institutions Infant foods Other

1982 AND 1984

lo6 kg) Whey with lactose/salt

Whey protein concentrate

1982

1984

1982

1984

1982

I984

50.8 48.6 26.8 13.6 6.0 0.2 1.4 0.8 5.2 0.3

46.3 72.6 35.4 18.6 7.7 2.4 2.6 2.8 0.4 2.8 0.2

-

0.6 6.2 1.3 0.2 0.3 -

0.3 4.4 3.0

0.5 8.1 3.2 0. I 0.3

-

6.0

-

19.0

4.9 I .5 0.8 0.3

-

0.4

-

0.6

10.6 1.4

-

-

0.4 0.6

0.5

1.1

1.1 2.3

10.0

-

PROTEINS IN WHEY

347

nal protein of milk ranging from 4 to 7 g/liter of which 3.7,0.6,0.3, and 1.4 g represent the P-lactoglobulin (P-Lg), a-lactalbumin (a-La), serum albumin (BSA), and “proteose-peptone” fractions, respectively (Marshall, 1982). In addition, it contains other proteins such as lactoferrin, immunoglobulins, ceruloplasmin, and such milk enzymes as lysozyme, lipase, and xanthine oxidase, which are present in low concentrations. The major whey products, dry sweet whey (DSW), demineralized delactosed whey (DLMW), and whey protein concentrate (WPC), are used in several general food categories with dried whey predominantly being used in bakery, dairy, and dry mixedblends (Table 11). The recent data indicate increases in the use of dry whey and whey protein concentrate in certain food categories. Approximately 75% of total processed whey is used in the form of dry whole whey, i.e, whey condensed by evaporation and spray dried to a powder. About 2% of whole whey is demineralized by ion exchange or electrodialysis prior to spray drying and is suitable for use in infant formula and special nutritional products. About 14% of whey solids is used in the preparation of refined lactose and about 10% of total whey solids is subjected to ultrafiltration (UF) to produce WPC (Kosaric and Asher, 1985). A range of UF-derived, spray-dried WPC powders containing 35, 50, 60, and 80% protein; 46, 31, 21, and 3% lactose; and 8,6,4, and 3% ash have been produced (Kinsella, 1984a). Whey protein isolates (WPI), containing little else but protein, can be prepared by ion exchange and diafltration processes (Skudder, 1983; Zall, 1984). In addition, approximately 0.7 x lo6 kg of lactalbumin (heat-precipitated whey protein) is imported and used in the United States, mostly in pasta, bakery, soups, and cereal-based products. The use of whey in certain products is restricted by some intrinsic components, for example, in breadmaking, the maximum amount of whey powder that can be incorporated is limited by the ash and lactose content, which can impair hydration, retard yeast growth, cause excessive browning, and impart an undesirable “salty” taste. In addition, the presence of free thiol groups and proteose-peptone or other components may reduce loaf volume. This effect may be minimized by appropriate preheat treatments of whey (75”C/30 min) prior to drying (Webb, 1970). Whey proteins are very susceptible to denaturation. For example, during preparation of WPC, maximum care must be taken to minimize denaturation during preheating, evaporation, drying, and subsequent processing. The extent of denaturation is normally assessed by solubility loss at pH 4.5 (Morr, 1979a). Commercial whey protein concentrates may range in solubility from 24 to 93% and 65 to 100% at pH 6 and 7, respectively (Morr, 1979b). Maillard browning reactions can also result in gradual deterioration and loss of solubility.

348

J. E. KINSELLA AND D. M. WHITEHEAD

A wide array of whey proteins, whey powders, and whey powder blends are increasingly utilized in a broad range of products (Table 111). In most of these applications, whey powders have been sold as replacements for nonfat dry milk (NDM).Vendors tend to make many claims concerning the value of using whey powders in products; however, in many applications whey powder is being used simply as a minor leastexpensive “filler” ingredient, improving nutritional value by supplying essential amino acids and perhaps providing some quality attributes, e.g., enhanced crust browning of bread. The price advantage of whey powder over milk powder has contributed to its increasing use over the past decade, particularly in bakery applications. In most conventional uses, however, the high content of lactose and ash in whey powders limits their use at high application levels. Upgrading and further refining of whey powder to reduce lactose and ash will become more important in order to expand markets. For the longrun utilization and marketing, whey proteins must be concentrated and processed to retain essential functional properties. For whey proteins to

TABLE 111 EXAMPLES OF TYPES OF WHEY POWDERS AND BLENDS USED IN A VARIETY OF FOODS IN THE UNITED STATES

Type of ingredient” Dairy blend of WP + NDM + BM (16% protein) Fortifiedlmodified whey Whey + BM for flavor Modified whey Liquified whey Modified whey (20% protein, 15% ash) Whey + NDM Fractionated cheese whey Partially demineralized whey proteins Lactose hydrolyzed whey

Typical applications Baked goods Yeast-raised bakery products Breads; pancakes; biscuit mixes Potato-based products Beverages; processed foods Fortified snacks; flavored beverages; puddings; frostings; bakery applications Baked goods; beverages Whipped toppings; coffee whiteners; soft-serve ice creams; confectioneries; cereals; baked goods, margarines Baby foods; meat and sausage products Desserts; confectioneries; yogurts; beverages, baked goods; baby foods; low-sodium foods; whipped toppings; ice cream mixes

“WP, Whey protein; NDM, nonfat dry milk; BM, buttermilk powder.

349

PROTEINS IN WHEY

be fully utilized, it is necessary to understand the properties of the individual whey proteins in order to understand the structural features which contribute to their characteristic functional properties. 111.

STRUCTURE OF WHEY PROTEINS

The amino acid composition, sequences, and structural characteristics of the major whey proteins, p-Lg, a-La, and BSA, have been described previously (Whitney et al., 1976; Swaisgood, 1982; Eigel et al., 1984), and some notable characteristics are listed in Tables IV and V. Whey proteins are globular proteins with a limited number of disulfide bonds (Fox and Mulvihill, 1982; Swaisgood, 1982) which confer a certain degree of structural constraint and impart stability. Compared with the caseins, whey proteins are more heat-sensitive, less calcium-sensitive, and can engage in thiol-disulfide interchanges to form oligomeric structures (McKenzie, 1971; Fox and Mulvihill, 1982; Swaisgood, 1982).

TABLE IV PHYSICOCHEMICAL CHARACTERISTICS OF WHEY PROTEINS OF BOVINE MILKo

Protein component @-Lactoglobulin -Lactalbumin Serum albumin Immunoglobulin Proteose-peptones: Component 5 p -CN-SP (fl-105) Component 5 p -CN-SP (fl-107) Component 8-fast @ -CN-4P (fl-28) residual fragment @ -CN-IP (09-107) Lysozyme Lactofemn

Q

Molecular mass 18,600 14,200 66,000 15.0-96.0 X lo4

Approximate percentage in skim milk protein (dliter) 7-12 2-5 0.7- 1.3 1.9-3.3 2-6

Isoelectric

PH

Cystine groups

5.3 4.8 5.1 5.5-6.8

2 (1 -SH) 4 17 ( 1 -SH) 32

-

- -

1 I .500

3.7

0 -

13,000

4.5

0 -

4,100

3.0

0 -

18,000 76,500

0.13-0.32 0.02-0.35

5.2 9.5 NIA

0 3 19 -

“Data from Whitney ef a/. (1976); Eigel ef al. (1984); Reiter (1985); Kitchen (1985); Wang ef a / . (1984).

350

J. E. KINSELLA AND D. M. WHITEHEAD

TABLE V AMINO ACID COMPOSITIONS OF THE MAJOR PROTEINS OF WHEY FROM BOVINE MILK'

Acid ASP Asn Thr Ser Glu Gln Pro GlY Ala 112 c y s Val Met Ile Leu TYr Phe Trp LYS His Arg Total

p -Lactoglobulin

a -Lactalbumin

Bovine serum albumin

4

1

10

22 4 4 2 15 2 3

8 13 4 4 4 12 3 I

41 13 34 28 59 20 28 I5 46 35 36 4 14 61 19 27 2 59 17 23

I62

123

581

I1 5 8 7 16 9 8 3 14 5

9 12 7 7 8 5 2 6 3

10

6

8

Lysozyme

Lactofenin

13 10 8 8 10 4 9 11 5 6 6 2 10 5 7 7

15

34 43 17 78 14 58

I54

987

I 10 7

53

44 52 50 30 68

44 74 98 38 66 4 26 106

"Data from Swaisgood (1982); Brown (1977); Brew et al. (1968); Eitenmilter et nl. (1976); Wang et a/. (1984).

A. p-LACTOGLOBULIN p-Lactoglobulin (p-Lg), the major protein of whey, is the most extensively characterized and best described of all food proteins (McKenzie, 1971; Swaisgood, 1982; Morr, 1982; Eigel et al., 1984). The amino acid sequence of p-Lg is depicted in Fig. 2 (Creamer ef al., 1983). The protein exists as a dimer in solution because of electrostatic interactions between and Glu'" of one monomer with corresponding lysyl residues of another monomer (Creamer et al., 1983). The native conformation is sensitive to heat and pH; at temperatures below 25°C and pH values above 7.0, the protein forms octamers (Fig. 3) (Pessen et al., 1985). Native pLg possesses two disulfide bonds ( C y ~ ~ - C yand s ' ~Cy~'"-Cys"~), and a free thiol group (Cys'*') which is inaccessible to solvent at or below neu-

FIG. 2. Primary sequences of bovine p-lactoglobulin B. The residues marked with an asterisk vary from species to species with the genetic variant. The free sulfhydryl is shown as Cysl*'. [From Creamer el a/. (1983).]

-

82

I

I

I I

fi--@Irr?

I drnol. I rlor. wry occrlorotrd rapld In cold I

I I

I I I .

.

.

I

I

I I

I

3.58nm

I

rrvrrtlblr 1 Ironsformallon

---

\

2.0

I

3.0

3.7 I

4D

4.65 15.1

I

5.0 6.0 PH

I

I

'

I 7.5 1 7.0 8.0

I I I

---

I 9.7 9.0

FIG. 3. Schematic representation of changes in the structure of p-lactoglobulins as a function of pH. p. p-Lg. [Inset (molecular models)] (A) Dimer (B) Octamer, with square decahedral faces on top and bottom; 0 ,octamer bonds; x --- x , tetrad axis; circular lines indicate monomer equators and parallels perpendicular to the dimer axis; (C) Octamer, with square faces in front and back, tetrad axis perpendicular to plane of paper. [From Pessen el a / . (19851.1

352

J. E. KINSELLA AND D. M. WHITEHEAD

tral pH (Papiz et al., 1986). At pH 7.5, a reactive carboxyl group which displays many unique chemical properties becomes exposed and signals a conformational change in the protein molecule, as observed by changes in the optical rotatory dispersion (ORD) spectrum (Tanford et al., 1959). The free thiol group normally occluded in the protein dimer also displays increased reactivity at pH values above 6.5 and appears to facilitate thiol-disulfide interchange reactions which allow the formation of new structures upon heating. Above 65”C, p-Lg undergoes a timehemperature-dependent denaturation which is accompanied by extensive conformational transitions (molecular expansions) that expose highly reactive nucleophilic groups in hydrophobic regions (McKenzie, 1971; Kella and Kinsella, 1988). The secondary structure of p-Lg has been calculated from circular dichroism and infrared spectroscopy data and it contains approximately 15% a-helix, 51% p-sheet, 17% reverse turn, and 17% aperiodic structure (Table VI) (Creamer et al., 1983). The e-amino groups of Lys”’, Lys”*, and LysI4’form a diagonal band which interacts ionically with the sideand G1ulMin the adjacent monomer to chain carboxyl groups of impart additional stability to an a-helical segment (Creamer et al., 1983). Recently, the three-dimensional structure of p-Lg was determined by high-resolution X-ray crystallography (2.8 A) (Papiz et al., 1986). The pLg molecule has an unusual protein fold composed of two slabs of antiparallel p-sheet (Sawyer et al., 1985; Papiz et al., 1986). The core of the protein fold is an eight-stranded, cross-hatched p-barrel composed of apolar amino acid side chains lining the internal cavity. This hydrophobic core binds apolar ligands, e.g., p-Lg binds retinol (Fugate and Song, 1980) and there is a tryptophan residue associated with the binding (Papiz et al., 1986). This tryptophan at position 19 is highly conserved among different species of p-Lg (Pervaiz and Brew, 1985). The reported crystal structure of human plasma retinol-binding protein (RBP) (Newcomer et al., 1984) demonstrates a remarkable conformational homology to p-Lg, particularly with regard to the cross-hatched p-barrel region and location of an a-helical rod (Sawyer et al., 1985; Papiz et al., 1986; Monaco et al., 1987). Although p-Lg shows limited sequence homology to RBP, there are two regions which have four consecutive identical residues and both of these contain residues involved in disulfide bridging and retinol binding (Papiz et al., 1986). The conformational and chemical similarities between RBP and p-Lg have prompted speculation that the biological function of p-Lg is in vitamin A transfer from maternal milk to the neonate via specific receptors in the intestine (Papiz et al., 1986) and possibly facilitates vitamin A esterification (Ong, 1985).

353

PROTEINS IN WHEY

TABLE VI SECONDARY STRUCTURE ASSIGNMENTS FOR SEGMENTS OF BOVINE

p-

LACTOGLOBULIN B"

Gamier analysis

Most likely structural elements

Possible structural elements

Residues

Structure

Residues

p-Sheet

1-6

11-16

$-Sheet

7-10 11-16

-

20-33

$-Sheet

$-Sheet Reverse turn p-Sheet 17-20 25-28 29-34

34-37 38-45 46-5 1

Random coil @-Sheet Random coil

Residues 1-7

52-59 60-64 65-75 78-84 88-91 91-97 98-100 I0 I- 107 108-1 13 114-1 18 119-125 126- I28 129-1 43 144-149 150-1 55

Structure

-

-

39-45

-

-

a-Helix Reverse turn a-Helix p-Sheet Reverse turn $-Sheet Reverse turn p-Sheet Random coil Reverse turn p-Sheet Random coil a-Helix p-Sheet Random coil

Reverse turn 21-24 Reverse turn p-Sheet 35-38

Structure

Reverse turn

p-Sheet

-

49-52

-

61-64 65-76 80-85

54-58 Reverse turn a-Helix p-Sheet

-

-

101-107 112-1 15 117-123

p-Sheet Reverse turn P-Sheet

129-143 145-151

a-Helix p-Sheet

46-48 Reverse turn 53-60 p-Sheet

Random coil

92-96 97-100

p-Sheet Reverse turn

108-1 I 1

Random coil

a-Helix or

-

-

-

-

-

152-155 156-160 152-162

Reverse turn and a-Helix or random coil

-

"After Creamer el af. (1983).

(3-Lactoglobulin exists in at least five different genetic variants (Eigel et al., 1984). The two most common genetic variants, known as A and B, differ at positions 63 and 118, where an Asp and a Val in the A variant are substituted by a Gly and an Ala in the B variant (Braunitzer et al., 1973). Both the A and B forms undergo a variety of conformational

354

J. E. KINSELLA AND D. M. WHITEHEAD

changes and association states that have been diagrammatically presented in Fig. 3. Pessen and co-workers, (1985)investigated the slow, irreversible denaturation and rapid dimer e octamer equilibrium of p-Lg, both occurring when the protein is subjected to temperatures below 2°C. The 36,700-Da dimer is the kinetic unit persisting over the pH range 3 to 7;between pH 3.7 and 5.1, self-association of the p-Lg dimer to octamer occurs as the temperature is decreased to 2°C (Timasheff and Townend, 1964;Kumosinski and Timasheff, 1966). It is postulated that changes in molecular water surrounding the protein enhance hydrophobic interactions, thus promoting protein association. Above pH 6.5 the dimer dissociates because of excessive electrostatic repulsion (Brown and Farrell, 1978). The carboxyl group of AspMis believed to be involved in the dimer e octamer transition in p-Lg A since the transition does not proceed as readily in the B variant, where a glycine residue is substituted for the aspartyl residue (Kumosinski and Timasheff, 1966; Townend et al., 1969; Swaisgood, 1982). B. a-LACTALBUMIN a-Lactalbumin (a-La) accounts for 25% of whey protein. The ratio of a-La to p-Lg in bovine milk is approximately 1 : 3. a-Lactalbumin is a compact globular protein (14,OooDa) and possesses an excellent essential amino acid profile, being rich in lysine, leucine, threonine, tryptophan, and cystine (Table VI). It has four disulfide bonds linking residues 6 to 220,28 to 111, 61 to 77,and 73 to 91 (Fig. 4). The biological function of a-La is to modulate the substrate specificity of galactosyltransferase in the lactose synthetase complex, which is responsible for the synthesis of lactose in lactating mammary tissue (Hill and Brew, 1975). The association of a-La to the lactose synthetase complex catalyzes the addition of glucose to galactose in the Golgi apparatus (Jones, 1978). a-Lactalbumin avidly binds calcium, which may stabilize the molecule against irreversible thermal denaturation (Hiraoka and Sugai, 1984). The protein also contains a distinct binding site for a zinc ion (Murakami and Berliner, 1983). Studies of the mode of binding of these metal ions to aLa suggest that a balance between calcium and zinc concentrations in the Golgi lumen controls the protein conformation and the pattern of ion binding affects the catalytic properties ( Vmx) of the lactose synthetase complex (Musci and Berliner, 1985). Removal of bound Ca2+ by acid treatment reduces the transition temperature and facilitates the irreversible thermal denaturation and aggregation of the protein (Bernal and Jelen, 1984). Calcium binding may also be important in controlling the

PROTEINS IN WHEY

355

FIG. 4. Primary sequence of bovine a-lactalbumin, including the four disulfide bridges (labeled with Roman numerals). [From Vanaman et al. (1970).]

release of a-La from the Golgi membrane, an essential step in inducing lactation (Musci and Berliner, 1985). The high resolution (1.7 A) X-ray structure of a-La reveals a Ca2+binding fold which superficially resembles the EF-hand of other calciummodulated proteins (Stuart et al., 1986). a-La displays a clear homology

356

J.

E. KINSELLA AND D. M.

WHITEHEAD

in sequence and backbone conformation with hen egg-white lysozyme (L). The “parent” protein, c-type lysozyme, has a corresponding loop which binds three water molecules in place of the Ca“ ligand (Stuart et al., 1986). The significant structural homology shared between a-La and lysozyme is particularly interesting since both proteins apparently have a common ancestral origin but have evolved quite different functions (Shewale et al., 1984). Not only are the four disulfide bonds of a-La and lysozyme in similar locations but the discovery of a hydrophobic “box” region in a-La (Richardson and Brew, 1980; Sinha and Brew, 1981) that is homologous (both in types of amino acid residues involved and spatial orientation)to a similar hydrophobic region associated with the monosaccharide binding subsites D and E in lysozyme has prompted speculation on the biochemical significance of this region (Brew et al., 1968; Richardson and Brew, 1980). It has been proposed that a-La interacts with galactosyltransferase at the hydrophobic box region, essentially forming a stronger, hybrid monosaccharide binding site and creating the lactose synthetase complex (Powell and Brew, 1976; Shewale er al., 1984). In many previous studies on Ca” binding of a-La, it has been emphasized that removal of the bound Ca” results in a pronounced conformational rearrangement (Kronman et al., 1981; Murakami et al., 1982). However, a recent study of Ca2’ binding by a-La using proton nuclear magnetic resonance spectroscopy suggests that the global structures of the folded apo-form and holo-form of a-La are essentially the same (Kuwajima et al., 1986). Thermal unfolding of bovine a-La at pH 8.0 has been monitored by circular dichroism (CD) and proton nuclear magnetic resonance (‘H NMR) spectroscopy (Hiraoka and Sugai, 1984, 1985; Kuwajima et al., 1986). The apo-form of a-La follows a three-state mechanism of unfolding, involving a stable intermediate which retains native secondary structure but no characteristic tertiary structure, while the native form of the protein follows the two-state unfolding mechanism similar to native lysozyme (Tanford, 1970; Ikeguchi et al., 1986a,b). Calcium binding stabilizes apo-a-La since the equilibrium intermediate state is not observed by CD, ‘H NMR, or calorimetric techniques during thermal unfolding of the protein molecule (Hiraoka and Sugai, 1985; Ikeguchi et d., 1986a). Thermodynamic characteristics of the thermal unfolding between bovine apo- and holo-a-La have been compared to evaluate the role of bound Caz+in stabilizing the native protein. The heat capacity of the apoprotein is significantly lower than the Ca2+-boundprotein and the enthalpy of unfolding is greater for the apoprotein than for the holoprotein (Hiraoka and Sugai, 1984). Apo-a-La assumes a native-like structure at low temperatures

PROTEINS IN WHEY

357

(10°C) as suggested by the similarity in the CD spectrum to that of the native holoprotein, which has one Ca2+bound per protein molecule (Hiraoka and Sugai, 1985). These results would be expected if the intrinsic stability of the native form of the protein is attributable to calcium binding and the fact that lysozyme does not bind any calcium (Imoto et al., 1981).

C. BOVINE SERUM ALBUMIN The bovine serum albumin (BSA) component of whey is a large globular protein (66,OOODa) with a good essential amino acid profile (Table V). BSA has been extensively characterized and its chemical and physical properties are well known (Peters, 1975). The protein consists of a single polypeptide chain containing about 580 amino acid residues with 17 intrachain disulfide bonds and one free thiol group at residue 34. Although the precise three-dimensional crystal structure of BSA is not known, the distribution of disulfide bonds and the location of specific residues throughout the polypeptide chain suggest that the albumin molecule folds to form three structural domains and nine subdomains (Brown, 1977). The multidomain structure of BSA is responsible for the anomalous behavior of the protein under denaturing conditions (Pace, 1975). The domains of BSA are dissimilar in hydrophobicity, net charge, and ligand binding sites and each has a distinct function apart from the others (Peters and Reed, 1977). BSA avidly binds free fatty acids, other lipids, and flavors which can stabilize the molecule somewhat against thermal denaturation (Damodaran and Kinsella, 1980, 1981a). Isolated BSA is a versatile, highly functional protein (Waniska et al., 1981). There is some evidence that between 40" and 50"C, BSA partially unfolds, exposing apolar residues to the molecular surface, facilitating reversible protein-protein interactions. D. IMMUNOGLOBULINS Four of the five classes of immunoglobulins in serum, i.e., IgG, IgA, IgM, and IgE occur in bovine milk in small amounts (gramdliter), although the concentration in colostrum is high (Whitney et al., 1976; Eigel et al., 1984). IgG is the principal type in bovine milk and comprises about 80% of the total content of immunoglobulins. All classes of immunoglobulins exist as either polymers or monomers of a basic unit composed of four polypeptide chains linked covalently by disulfide bridges. The monomeric form consists of two identical light (approximately 20,000 Da) and two identical heavy (50,000 to 70,000 Da, depending on class) polypeptide

358

J. E. KINSELLA AND D. M. WHITEHEAD

chains. Each of the light and heavy chains contains constant (C-terminal half)and variable (N-terminal half) regions of amino acid residues responsible for various functions, e.g., membrane transport, and antigen binding (Gally, 1973). These proteins are very thermolabile and their functional properties in the whey protein fraction have not been determined. E. PROTEOSE-PEPTONES The proteose-peptone (PP) fraction of bovine milk has been characterized as a heterogeneous mixture of heat-stable, acid-soluble (pH 4.6) polypeptides precipitated by 12% trichloroacetic acid (TCA) (Whitney et al., 1976). The PP fraction is actually a heterogeneous group of phosphoglycoproteins formed following proteolysis of the N-terminal region in the sequence of p-casein by plasmin (Eigel, 1981). Andrews (1978)demonstrated that proteins previously called proteose-peptone components 5 and 8-fast are identical with p-casein residues 1-105, 1-107, and 1-28, respectively. The remaining fragments of p-casein (residues 2-105 and 29- 107)released during plasmin degradation correspond to PP component 8 (Eigel and Keenan, 1979;Eigel, 1981).It has been recommended that fragments resulting from proteolytic cleavage be named as derivatives of the parent polypeptide from which they originated (Eigel et al., 1984). Whey preparations contain highly variable amounts (from 2-20 giliter) of PP. These molecules are amphiphilic because of their charged phosphate groups and sequences of hydrophobic amino acid residues. The amount present may have an affect on the functional behavior of whey proteins, and more research is required to monitor the amounts and types present and their effects on specific functions, e.g., whipping and baking. There is some question as to whether this fraction is responsible for depression of egg white foams (Volpe and Zabik, 1975;Phillips et al., 1987). It is possible that the PP fraction may also contain other milk peptides or proteins, lipoproteins, and proteolipids that impart foam depressant activity.

F. LACTOFERRIN Lactoferrin is present in bovine milk at 0.02 to 0.35 g/liter, while there are considerably larger quantities (approximately 2-5 giliter) in human milk (Table V). Lactoferrin is a single glycoprotein with an approximate molecular mass of 80,000 possessing two attached carbohydrate groups (Reiter, 1985). Lactoferrin is an iron-binding protein, containing two ferric ion (Fe3') binding sites per protein (Aisen, 1980; MetzBoutique et al., 1984). Limited proteolysis, amino acid sequence, and metal-binding

PROTEINS IN WHEY

359

analyses have demonstrated that the polypeptide chain may be subdivided into two major domains, each of which has a nonidentical iron binding site, one glycan moiety, and a high degree of sequence homology to each other (Mazurier and Spik, 1980; MetzBoutique et af., 1984). Amino acid analysis reveals an unusually high half-cystine content (Table VI) (Wang et af., 1984). Lactoferrin is structurally and functionally homologous with two other nonheme, iron-binding proteins, serum transfertin and ovotransferrin (Aisen, 1980). These proteins reversibly bind two iron atoms per molecule with the synergistic binding of two bicarbonate or carbonate anions (Masson and Heremans, 1966). Because of their ability to complex free iron, all three apotransfemns exert a bacteriostatic effect in v i m (Oram and Reiter, 1968; Parry and Brown, 1974; Reiter, 1985). They may function as iron transport proteins, and lactoferrins may also possess a mitogenic function and stimulate the development of intestinal mucosa in the neonate.

G. LYSOZYME Bovine milk contains between 13 and 32 pg lysozyme/100 ml, depending on the stage of lactation, while human milk contains 10 mg/100 ml (Kitchen, 1985; Reiter, 1985). The enzyme is generally considered to be synthesized in the mammary gland (Reiter, 1985). Lysozyme isolated from bovine milk is apparently different both in molecular mass and amino acid composition than lysozymes from human milk or egg white sources (Tables IV and V ) (Eitenmiller et af., 1976; Kitchen, 1985). It has an isoelectric point of pH 9.5 and the pH of optimum enzyme activity is at 7.9, which is higher than that found for lysozymes from other sources. The enzyme is stable at low pH values (Eitenmiller et af., 1976). Reduction of the enzyme from bovine milk with mercaptoethanol followed by reoxidation resulted in a 3-fold increase in the specific activity. Reformation of disulfide bonds different from the original native structure, or an altered tertiary structure arising from new amino acid interactions are likely explanations for this effect (Friend et al., 1972). Lysozyme is considered to be an important component of the antibacterial system of milk, possibly affecting the general immune system as well (Reiter and Oram, 1982; Reiter, 1985). Its bactericidal and lytic action depends not only on the concentration of enzyme and the prevailing ionic strength but also on the nature of the anions; SCN- and HCO; promote bacterial cell wall lysis more effectively than C1- or F- (Goodman et af., 1981; Tortosa et al., 1981). There is a renewed interest in the bactericidal effect of lysozyme for applications in cheese-making, infant formula, and pharmaceutical products (Reiter, 1985).

360

J.

E. KINSELLA

AND D. M.WHITEHEAD

IV. WHEY PROTEIN PREPARATION AND ISOLATION

Knowledge of protein structure and the relationship to functional properties (combined with an awareness of the various factors affecting functional properties) and recognition of the critical functional requirements in different products are important in developing practical methods for preparing functional ingredients from whey. The methods employed in the isolation and fractionation of whey protein preparations should minimize denaturation and avoid some of the undesirable consequences discussed above. As a functional ingredient per se, whey powder contains too little protein for most uses requiring a protein-rich ingredient; hence a number of approaches have been used to recover or enrich the protein content of whey. The principle methods currently used for fractionation of whey are summarized in Table VII. The details of these methods have been reviewed (Marshall, 1982; Matthews, 1984; Pepper and Pain, 1987). In the conventional process, whey is centrifuged to recover fat, condensed by evaporation to increase solids content, then spray dried or used for lactose crystallization and, after desludging, the residual liquor is dried. This yields whey powder containing about 11-14% protein which may be variably denatured, particularly during the evaporatiodcondensation step. Several methods have been used for concentration of the proteins from whey. Lactalbumin, a denatured, protein-rich product, is made by heating whey liquor at pH 5-6, 85" to 100°C (steam injection) for 15-20 min, acidification to pH 4.5, recovering the precipitated protein by centrifuga-

TABLE VII SUMMARY OF CONVENTIONAL METHODS USED FOR PREPARATION OF VARIOUS WHEY PRODUCTS FROM RAW WHEY ~~

Method

Whey product"

Centrifugation Evaporation and crystallization Steam injection (90°C, 20 min) Electrodialysis Reverse osmosis Ultrafiltration Gel filtration Diafltration Ion-exchange chromatography

Fat Lactose Lactalbumin Whey powder (low ash) Whey powder (up to 24% protein) WPC (30-50% protein) WPC WPI (5690% protein) WPI

"WPC, Whey protein concentrate; WPI, whey protein isolate.

PROTEINS IN WHEY

36 1

tion, and either spray- or drum-drying to give a product with 85,4,1.5, and 3% protein, lactose, ash, and lipid, respectively (Marshall, 1982; Matthews, 1984). This product is low in lactose and ash and the proteins are a rich source of essential amino acids, e.g., lysine, methionine, cystine. Lactalbumin is relatively insoluble but has useful water sorption characteristics (Kinsella and Fox, 1986). Because of its nutritive value, it is frequently added to cereals, baked goods, confectionery foods, and snack foods. There may be some merit in examining the possibility of improving the solubility and functional attributes of lactalbumin by enzymatic and/ or chemical modification. A number of chromatographicmethods have been used to prepare concentrated whey proteins (Morr, 1979b; Marshall, 1982; Matthews, 1984). Gel filtration on Sephadex proved to be effective but impractical because of column plugging, effluent problems, etc. (Melachouris, 1984). Spherosil ion-exchange chromatography (microporous silica beads with high exchange capacity, good flow rates, and low compressibility) has been used (Skudder, 1983; dewit, 1984) to prepare a protein isolate (90% protein) and to prepare whey protein concentrate (66% protein) (Nichols and Morr, 1985). Both protein preparations have good solubility. However, the problem of concentrating the protein eluate by UF, the cost of materials, and volume of solvents required render this method impractical. Ionexchange chromatography is being used to prepare whey protein isolates (>90% protein) with excellent functional properties (Houldsworth, 1979; Marshall, 1982; Matthews, 1984). With the advances in membrane and separation technologies, ultrafiltration has emerged as the principle method for increasing the protein concentration of whey (Matthews, 1984; Modler, 1985). The whey is clarified at 50°C, subjected to UF (and perhaps diafiltration, depending on the final protein concentration desired) and then spray dried ( 175"-2OO0C inlet, 8O"-9O0C outlet). This method can provide preparations with protein concentrations of 30 to 90% and denaturation is minimal when heat treatments are carefully controlled. The use of reverse osmosis (RO) rather than evaporation to concentrate protein solutions reduces protein denaturation and combined RO-UF is a feasible method for preparing undenatured proteins with good functional properties (Pepper and Pain, 1987). This technology, with appropriate control, may be successfully used by whey processors in generating products with consistent functional properties that will compete more effectively with other functional protein ingredients. The differences in heat sensitivities of whey proteins can be exploited, e.g., the thermal separation of the major whey proteins a-La and p-Lg has been accomplished by choosing conditions for maximum precipitation of

362

J. E. KINSELLA AND D. M. WHITEHEAD

a-La. Thus heat treatment of whey at 65"C, in the pH range 4.1 to 4.3 for 10 to 20 min should selectively precipitate a-La while the supernatant is enriched in p-Lg and the aggregated a-La may then be resolubilized above pH 5.0 (Table VIII) (Pearce, 1983). This method may provide a practical approach for preparing concentrated a-La or p-Lg with low levels of contamination from lactose or ash. The addition of ferric chloride (4 mM) to whey at pH 3.0 precipitated all the proteins except p-Lg (Kuwata et al., 1985). The ferric chloride was removed from the resolubilized proteins by ion-exchange chromatography. The fraction containing p-Lg was recovered by heat denaturation (92*C, 15 min, pH 3.0) and isoelectric precipitation and was soluble at pH 6.8. When acid whey was treated with ferric chloride (7.5 mM, pH 4.3, 4"C),90% of the p-Lg was precipitated with BSA while 70% of the immunoglobulins and 95% of the a-La remained in solution and could be recovered following precipitation of iron at pH 8-9 at 4°C (Kuwata et al., 1985). Following neutralization of cottage cheese whey, the addition of Mg2+ or Zn2+salts (at 4 and 22 g/dl, respectively) resulted in the precipitation of 20 to 35% whey protein nitrogen at pH 6.7 and 10.5, respectively (Cerbulis and Farrell, 1986). Precipitation of whey protein nitrogen by zinc acetate is nearly complete and is highly dependent on pH, i.e., maximal precipitation occurred near neutral pH. However, by adding magnesium acetate and calcium hydroxide (4 g/dl) at pH 10.5, over 90% of the total whey nitrogen could be precipitated (Cerbulis and Farrell, 1986). A large-scale process for the separation of whey protein concentrates has recently been described in which the whey is cooled to 2°C at pH

TABLE VIII SELECTIVE AGGREGATION OF a-LACTALBUMIN FOLLOWING HEATING AT DIFFERENT

PHS AT

55°C FOR 10 mill" Turbidity

pH of heating at 55OC 3.0 3.5 4.0 4.5 5.0

a-Lactalbumin

P-Lactoglobulin

I mg/d

2 mdml

2 mg/ml

0.01 0.01 0.02

0.02 0.09 0.80

0.01 0.01 0.05 0.05

0.04

0.00

0.06 0.03

"Data after Pearce (1983).

1.10

4 mg/ml

0.01

0.04 0.08 0.09 0.06

363

PROTEINS IN WHEY

7.3 and the calcium content adjusted to 1.2 &kg (Maubois et a / . , 1987). Subsequently, the whey is rapidly heated to 50°C for 8 min to precipitate

the lipid fraction, which is then removed by microfiltration. In order to separate individual proteins, the pH of the whey is adjusted to 3.8 and heated to 55°C for 30 min, which causes aggregation of a-La, while p-Lg remains in the supernatant (Maubois et al., 1987). Rapid, reliable methods are needed to quantify the various protein components of whey and to differentiate between protein and nonprotein fractions. Proteins can be fractionated by gel chromatography, electrophoresis, ion-exchange chromatography, and, more recently, it has been shown that HPLC may provide a rapid method for separating and quantifying components (dewit, 1984; Brooks and Morr, 1984; Morr, 1984; Nichols and Morr, 1985). Methods for quantitative analysis of the extent of protein denaturation and its correlation to functional properties are required. Methods to assess denaturation may include physical measurements of aggregation, electrophoretic analysis, gel permeation chromatography, calorimetric analysis, and immunological assays (Aschaffenburg and Drewry, 1957; Wyeth, 1972; Ruegg ef al., 1977; Harper, 1984; deWit, 1984; Harper and Zadow, 1984). Because these methods are based on different physical or chemical properties of the protein, difficulties are encountered in ascertaining whether measurements of the extent of denaturation are comparable under differing experimental conditions.

TABLE IX COMPOSITION OF SOME WHEY PRODUCTS PREPARED BY DIFFERENT METHODS‘

Percentage of solids

Extent of denaturation

Methodh

Protein

Lactose

Ash

Fat

(%)

WPC (35%) WPC (70%) Lactalbumin Spherosil Ultrafiltered UF + diafiltered Ion exchange Whey powder Demineralized powder

35.0 76.0 86.0 66.0 37.0 83.0 90.0 12.0 13.0

40-60 8.0 3.5 2.0 51.0 5.0 3.3 74.0 81.0

2-20 3.0 1.5 20.0 7.0 2.8 2.0 9.0 0.8

2-4 8.0 3.6 8.0 3.0 7.0 1 .o

? ? 95 10 16 22 21 ? ?

1.o

1 .o

“Data after Marshall (1982); deWit (1984); Morr (1985); Nichols and Mom (1985). bWPC Whey protein concentrate; UF, ultrafiltrate.

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J. E. KINSELLA AND D. M. WHITEHEAD

Methods which involve the determination of protein by nitrogen content or the Lowry method may give falsely high values because of the presence of other polypeptides and PP components. Wide discrepanciesin values determined by the traditional methods were observed by Harper, (1984) and the data in Table IX illustrate the variability in the composition of whey products prepared by different methods (Marshall, 1982; dewit, 1984; Morr, 1985; Nichols and Morr, 1985). V. FUNCTIONAL PROPERTIES OF WHEY PROTEINS

The possession of a range of functional properties is as important as cost in selecting proteins for use in specific products. Functional properties of proteins are those physicochemical properties which govern the performance and behavior of proteins in food systems during their preparation, processing, storage, and consumption, i.e., properties affecting the final quality attributes of foods. Examples of the structural and functional roles of proteins include the caseins in cheese curd; the myofibrillar proteins which impart structure, water holding, juiciness, and texture to meat; gluten proteins in leavened bread; egg white proteins in whipped toppings, etc. (Kinsella, 1976, 1982, 1984b). The functional properties of food protein preparations reflect the manner in which the proteins interact with each other and other components in the system as determined by conditions of processing, storage, and perhaps by food preparation methods. Different proteins vary in their composition and properties and demonstrate different functional behavior, for example, caseins have good emulsifying properties but do not form gels whereas whey proteins have limited emulsifying properties but possess good gelling properties. Traditionally, the food industry has relied almost exclusively on abundant commodity proteins, e.g., milk proteins, gluten, gelatin. However, food processors are increasingly selecting protein ingredients for their specific functional properties and performance in particular food products (Kinsella, 1984b, 1985). Thus, as the functional requirements of protein ingredients are defined and the functional behavior of various proteins in food systems is described, other protein preparations may be developed to compete with the traditional proteins. Food manufacturers are increasingly designing new products and then seeking functional ingredients to meet the specifcations for that particular product rather than allowing the type of product to be limited by the properties of traditional protein preparations. In view of these developments, the producers of whey proteins now recognize that the requisite functional properties in the final preparation must meet a set of decisive criteria. It is no longer sufficient

PROTEINS IN WHEY

365

to locate a market after a by-product becomes available. Rather, the ingredient manufacturer must anticipate and recognize the specific needs of the food manufacturer and adopt and optimize manufacturing procedures for producing an array of ingredients with the requisite functional properties. Different food applications each require a different set of functional properties. For example, in a beverage, solubility, storage stability, flavor compatibility, controlled viscosity, and turbidity may be required in a range of different pH values. In reformed meat systems, a range of functional properties that change in a desirable manner with processing and cooking, e.g., in sausage-type products, emulsion stabilization, subsequent gelation, good adhesive properties, and water holding are important. In a dough system, the protein should not absorb excessive water, but should align with gluten fibrils while not disrupting the gluten network, nor weakening the viscoelastic properties, nor reducing loaf volume. Thus, each application requires specific functional attributes to obtain the desired performance in each system (Table X). In bakery applications, whey powders should readily hydrate and contain a minimum of PP and free thiol groups (which may exert a loaf depressant effect), not impair fermentation (low ash level), facilitate aeratiodleavening, and contribute flavor and color. In cake products, particularly in batter-based products, emulsifying activity and aeration capacity are necessary, and reasonable heat stability of the aerated matrix is also required. In regard to bakery uses, there is a tendency to promote whey powders as replacements for NDM; however, this may be incorrect because, in certain high-fat batters the presence of caseins, which impart emulsifying properties, is required for optimum performance (dewit, 1984). Interfacial film formation and foam stabilization during baking/ expansion are also criteria for functional ingredients in many air-leavened high-ratio cake products and meringues. Egg white proteins perform these functions optimally, whereas whey proteins lack suitable waterholding capacity and heat stability (devilbiss er al., 1974), despite claims that they are a suitable substitute for egg white. Much has been reported about the excellent functional properties of whey proteins based on laboratory research. However, in some cases, their functional properties have been overrated and overpromoted for applications in which these whey preparations are unsuitable. A. INTRINSIC FACTORS AND FUNCTIONAL PROPERTIES Functional properties reflect the intrinsic physicochemical properties of the proteins, i.e., amino acid composition and disposition of amino acid residues, conformation, molecular size, shape, “flexibility,” net charge,

TABLE X FUNCTIONAL PROPERTIES REQUIRED OF PROTEIN INGREDIENTS IN DIFFERENT FOOD PRODUCTS ~

~~

Functional properties

Frozen dessert

Confectionery

Bakery

Whipped toppings

Solubility Gelation Emulsifying Foaming Adhesion Flavor binding

+ + +

+ + + +

+

+

(+)

-

Processed meats

Coffee whiteners

-

-

+ +

+ -

?

?

367

PROTEINS IN WHEY

molecular hydrophobicity, substituent chemical groups (esterified phosphate, carbohydrate) and sulfhydryl groups (Table XII). Thus, knowledge of the relationship between intrinsic and extrinsic (temperature, pH, ion concentration, etc.) factors is critically important in elucidating and controlling functional behavior in different applications and in modifying proteins and/or processing conditions to optimize desirable functions (Table XI) (Kinsella, 1982, 1984a). The amino acid composition determines the folding behavior and reactivity of proteins. Proteins with a high content of polar and/or charged amino acids, which tend to be exposed to the aqueous phase, bind more water and are useful as emulsion stabilizers (Table XIII). Proteins with a high content of apolar amino acid residues (greater than 30% of total amino acids) display good surface activity (Kato and Nakai, 1980), but generally do not possess good gelling properties because of their predisposition toward extensive self-association and coagulation (McKenzie, 1971; Fox and Mulvihill, 1982; Swaisgood, 1982; Morr, 1985). The presence of cysteine and cystine residues greatly affects heat-induced polypeptide association via thiol-disulfide interchange reactions and subsequent precipitation, e.g., p-Lg (Sawyer, 1968). However, it is the disposition of certain amino acids along the polypeptide chain, rather than their total content, which is more critical in governing protein conformation and hence functional properties. The native structure of the globular proteins in whey represents a thermodynamic equilibrium, and protein conformation may fluctuate depend-

TABLE XI FACTORS INFLUENCING THE FUNCTIONAL BEHAVIOR OF PROTEINS IN FOOD ~

Intrinsic features Composition of protein Composition of protein Monomeric or oligomeric Protein blends Rigidityhlexibility Hydrophobicitylhydrohilicity Surface charge Bound flavor ligands

~~

Extrinsic factors Temperature PH O/R status Salts, ions Water Carbohydrates Lipids Gums Surfactants Tannins

Process; treatments; conditions Heating Acidification Counterions Ionic strength Reducing conditions Drying Storage conditions Modification Physical Chemical Enzymatic Genetic

368

J. E. KINSELLA AND D. M. WHITEHEAD

TABLE XI1 INTRINSIC FACTORS THAT AFFECT PROTEIN STRUCTUREFUNCTION RELATIONSHIPS IN FOOD SYSTEMS

Amino acid composition Amino acid sequence (disposition of amino acid side groups) Secondary and tertiary structure (conformational energy) Size, shape (topography) Net surface charge, effective hydrophobicity Intramolecular stabilizing forces (ionic and hydrophobic interactions) Quaternary structures Secondary interactions (intra- and interpeptide) Substituent groups (phosphoryl and carbohydrate groups) Bound and/or prosthetic groups (iron, calcium, lipids) Disulfidelsulfhydryl content

ing on environmental conditions (Creighton, 1985; Karplus and McCammon, 1986). The noncovalent forces involved in stabilizing native protein structure include hydrogen bonding, hydrophobic, van der Waals’, and electrostatic interactions, while covalent disulfide bonding is important in maintaining the structural integrity of extracellular proteins (Schulz and TABLE XI11 EXTRINSIC FACTORS WHICH CAN AFFECT COMPOSITION AND FUNCTIONAL BEHAVIOR OF WHEY PROTEIN PREPARATION

Preparation Milk composition Cheese manufacturing Whey handling Whey concentration Dried whey Heat treatments Fractionation methods Distribution and content of components Physical state of components

Variables” Stage of lactation, sanitation, storage time and conditions, somatic cell count, proteolysis by milk enzymes Sweet/acid whey; rennet type; calcium addition, fat separatiordwhey recovery Storage time, temperature, sanitation, clarification efficiency, residual enzymes Evaporation, ultrafiltration, dehydration conditions Storage, moisture content, chemicaVphysica1changes Time, temperature, pH, calcium content Heat precipitation, UF/RO, chromatographic method Lipid, ash, lactose, proteins Native/denatured, solubility, bound lipids

“UF, Ultrafiltration; RO, reverse osmosis.

PROTEINS IN WHEY

369

Schirmer, 1979). Alterations in environmental factors which impact on these forces can alter the conformation of proteins: salt may weaken ionic interactions (Schulz and Schirmer, 1979); cleavage of intramolecular S-S bonds by thiol groups can facilitate protein unfolding and protein-protein interactions, resulting in coagulation or gelation (Schmidt, 1981); acidic conditions can affect calcium binding (Bernal and Jelen, 1984); calcium binding may stabilize a particular conformation (Hiraoka and Sugai, 1984; Stuart et al., 1986); temperature treatments greatly affect protein conformation, as in the case of dissociation of p-Lg and exposure of the reactive thiol group of p-Lg upon heating (Watanabe and Klostermeyer, 1976). Whey proteins become denatured at temperatures above 65" to 70°C and may coagulate after heat treatment (Patocka et al., 1987). The nature, extent, and rate of denaturation can be influenced by a number of factors such as pH, ionic strength, protein concentration, time, and temperature of heating (Kilara and Sharkasi, 1986). The thermal denaturation of whey proteins is pH sensitive and the isoelectric pH of approximately 4.6 is used to recover heat-denatured whey proteins. The pH affects the rate of denaturation and coagulation by affecting the net charge of the proteins (Harwalkar, 1986). Thus, minimal coagulation occurs when whey proteins are heat-denatured above pH 6.5 (dewit, 1981) or below the critical pH range of 3.7 to 3.9 (Bernal and Jelen, 1985). The presence of calcium enhances protein aggregation following heating at particular pHs and this is attributed to neutralization of electrostatic repulsions (dewit, 1981;deWit and Klarenbeek, 1984; Bernal and Jelen, 1984; Patocka et al., 1987). Thus, the net effects of noncovalent forces and interactions play major roles in the functional behavior of whey proteins. For example, in gelation, a balance of attractive (hydrophobic and electrostatic interactions and hydrogen bonding) and repulsive (electrostatic) forces is necessary in discrete regions of interacting molecules for adequate network formation (Schmidt, 1981; Mulvihill and Kinsella, 1987, 1988). Surface hydrophobicity is important in flavor binding and effective hydrophobicity greatly affects film formation, which is required for foaming and emulsifying properties (Kinsella, 1981; Kato and Nakai, 1980; Nakai, 1983). B. EXTRINSIC FACTORS The functional properties manifested collectively by whey proteins are largely determined by a number of extrinsic factors: methods of preparation, isolation, drying, storage; extent of refining and purification, content and concentration of proteins, and environmental conditions; tempera-

370

J. E. KINSELLA AND D. M. WHITEHEAD

ture, pH, and solute concentration (Table XIII). For example, the method and conditions of drying can affect the extent of denaturation, particle size, rehydration, and dispersibility (Kinsella, 1984b). The efficiency of lipid removal may affect many properties related to surface phenomena (foaming, binding of flavor compounds, etc.) and sensory qualities of whey proteins.

C. VARIABILITY IN WHEY PROTEIN PREPARATIONS Variability in whey composition reflects the different sources, e.g., total solids may range from 5.8 to 7.0% in sweet and acid whey, and minerals like calcium and phosphate may range from 0.5 to 1.4% and 0.6 to 0.8% in different sweet and acid whey samples, respectively (Marshall, 1982). Generally, minerals are much higher in acid whey and differences in mineral content can markedly affect the functional performance of whey proteins; calcium can render proteins more susceptible to thermal precipitation, thereby affecting gelation, water sorption, viscosity, etc. (Melachouris, 1984). The composition of WPC can range from 30 to 95% protein, 1 to 80% lactose, 1 to 18% ash, and 1 to 9% fat (Marshall, 1982). Typical ranges of protein concentrations in commercial WPCs are 29 to 60% (Morr, 1979a). The protein content of cheese whey can vary in amounts of total solids in sweet (cheddar) and acid (cottage) whey by 11-15% and 10-15%, respectively. This can have a marked influence on functional behavior, especially of whey protein concentrates and isolates. The variability in composition is a challenge in producing whey with consistent functional properties. The protein distribution markedly affects the functional properties of a particular preparation; thus, greater amounts of p-Lg may increase sensitivity to heat precipitation and relative amounts of PP may adversely affect whipping and bakery uses (Jelen, 1973; Kinsella, 1984b; Phillips et al., 1987). In addition, whey fractions may contain sizable amounts of p-casein which dissociates from the micelle, particularly during cold storage of milk prior to cheese making, affecting the foaming and emulsifying properties of whey proteins. The concentration of whey proteins in milk and hence in whey varies with season and stage of lactation (Morr, 1982). Heat treatment of milk, e.g., pasteurization, ultrafiltration, and thermalization, may result in the enhanced association of p-Lg and K-casein, thus reducing the protein concentration in whey (Kilara and Sharkasi, 1986). The efficiency of cheese-making and whey separation influences the amount of protein in whey, which can range from 8 to 15% of total solids

PROTEINS IN WHEY

37 1

(Melachouris, 1984). Since proteins are the major functional components, this variability must be reduced in order to produce the consistent functional ingredients required by the food industry. Ultrafltration (UF) and diafiltration (DF) are techniques presently used to routinely manipulate the protein content of whey in a consistent manner (Matthews, 1984). High microbial counts (especially of psychotropic bacteria), somatic cells, and plasmin activity can all cause significant proteolysis in stored milk and thus affect whey composition. The content of PP is much higher in milk containing high numbers of somatic cells or plasmin activity (Schmidt et al., 1984). The presence of lipid materials, which can range from 0.4 to 1.0% of total solids (Melachouris, 1984), exerts a deleterious effect on most of the desirable functional properties, e.g., solubility and whipping of whey protein prepartions. Lipids also undergo extensive oxidation during storage, causing off-flavor development in most whey preparations. In addition, lipids cause problems in the fractionation and refining of whey proteins, especially by chromatographic and U F methods (Melachouris, 1984). Thus, removal of lipid by efficient centrifugation, clarification, or complexation techniques is important to ensure elimination of unpredictable and deleterious components. Polyphosphate treatment at pH 5.1 prior to clarification at pH 7.0 improves lipid removal (Grindstaff, 1977). The state of the component proteins in the preparation, i.e., native/ denatured, soluble/insoluble, is perhaps the major factor determining the functional behavior of whey proteins. Denaturation, mostly from thermal processes, is the principal factor limiting the application of whey proteins and represents the major challenge for the preparation of whey concentrates and isolates with consistent and reliable functional properties. The extent of protein denaturation significantly affects the utilization of whey proteins as functional ingredients (Melachouris, 1984). Denatured proteins have limited solubility, a primary prerequisite for most other functional uses. Thus, denaturing heat treatments have the greatest deleterious effect on functional properties of whey protein preparations. Even moderate heat treatments (45" to 60°C) may cause unfolding and partial denaturation of p-Lg, resulting in protein aggregation and loss of solubility. There is a close relationship between heat-induced alterations and denaturation temperatures of p-Lg and whey proteins. Generally, the rate of heat denaturation decreases with protein concentration, particularly for p-Lg (deWit and Klarenbeek, 1984). The extent of protein denaturation in whey samples ranges from 12 to >90% and depends on the heating procedures. Melachouris reported that 40,25,20, and 10% of various whey samples from the same plant contained 20-30, 10-20, 30-40, and

372

J. E. KINSELLA AND D. M. WHITEHEAD

40-50% denatured protein, respectively (Melachouris, 1984). Denatur-

ation must be minimized for whey proteins to compete successfully in the functional ingredients market. Advances in UF combined with rigorous control of spray-drying temperatures can overcome this problem in manufacturing functional proteins. Lactose tends to reduce whey protein aggregation during heat treatment, particularly in the isoelectric pH range (Hillier and Lyster, 1979). Sucrose inhibits whey protein thermal coagulation, although the disaccharide apparently promotes a conformational change in proteins (Garrett et al., 1988). Whey proteins are more stable in the neutral pH range (pH 6-7) and calcium facilitates heat-induced aggregation (dewit, 1981). Commercial whey preparations should consistently meet minimum standards in terms of protein content, extent of denaturation, and requisite functional properties. Manji and Kakuda (1987) recently reported the effective use of fast protein liquid chromatography (FPLC) in determining the extent of whey protein denaturation in variously heat-treated milk samples. Results from this method were compared with literature values obtained from differential scanning calorimetry (DSC), whey protein nitrogen index (WPNI), and Kjeldahl nitrogen (KN) analyses and generally showed that the WPNI method consistently yields lower percent denaturation values as compared to those obtained with either FPLC or KN. These investigators conclude that FPLC is an equivalent method to KN, and that the FPLC method may yield data showing the extent of denaturation of individual whey proteins (Manji and Kakuda, 1987). deWit and Klarenbeek (1984) demonstrated the effect of subtle differences in composition and degrees of denaturation of the functional performance of various whey protein preparations in different food systems. For example, in a meringue system, the presence of fat in the whey preparation was detrimental to foaming; in a madeira cake, the presence of a small amount of fat seemed to improve the performance of whey protein, indicating that perhaps emulsifying activity, in addition to aeration, is important in the madeira cake. This research indicated the sensitivity of different functional applications to variations in composition, and also revealed the potential for making whey preparations with different functional attributes which might be ideal for specific applications. Based on knowledge of functional criteria required in an ingredient, the conditions of preparation can be manipulated to optimize those functional properties. Data are needed to assess systematically the cumulative effects of heating during processing operations (e.g., pasteurization, evaporation, concentration, spray drying) on the extent of denaturation. Generally, conventional heat treatments are not severe enough to cause extensive

PROTEINS IN WHEY

373

denaturation, but modest heating around 55"-60"C can cause conformational changes in p-Lg which may subsequently slowly aggregate and lose solubility during storage. Whey proteins can undergo deterioration in functional performance during storage. The Maillard reaction results in discoloration, some polymerization, and loss of solubility. Lipid oxidation can result in cross-linking of the proteins and loss of functionality. The moisture content (water activity) must be controlled to minimize browning and appropriate packaging should be employed in storing whey proteins. For consistent quality control of the various factors affecting functional properties, WPC should be routinely analyzed for protein, nonprotein nitrogen (NPN), calcium, phosphorus, lipid content, and extent of protein denaturation. The processor can then be assured of consistent composition and quality. In addition to quality control, it is desirable to develop rapid tests for assessment of functional properties of each batch of WPC. This is critical for reliable functional performance of the product in food applications. In this regard, solubility, water adsorption, heat-induced gelation, and surface active properties such as foaming and emulsifying characteristics should become standard tests. In food applications, a range of functional properties may be required in ingredients during processing and preparation. These vary with the food in question and usually a mixture of proteins is required to provide the desired range of functional properties. Thus, water sorption, rapid solubilization, viscosity, emulsifying properties, gelation, and all functional properties involving protein-water interactions are frequently required in food products. Several aspects of the functional properties of whey proteins have been discussed and reviewed (Morr, 1979b, 1984; Harper ef al., 1980; deWit, 1984; Kinsella, 1984a; Melachouris, 1984; Mangino, 1984; Kilara, 1984; Modler, 1985). In general, whey proteins show good solubility and gelling properties plus adequate whipping properties in certain applications, but these are critically dependent on the content of undenatured protein, salts, and lactose. VI.

HYDRATION AND SOLUBILITY

Hydration, wetting, dispersibility, and dissolution are terms describing the interactions of proteins with water in different systems and are important criteria for assessing the suitability of whey protein preparations. Some of the physical characteristics (particle size, shape, state of agglomeration, porosity, etc.) and chemical properties (surface hydrophilicity, net charge, molecular hydrophobicity, adsorbed wetting agents, etc.)

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J . E. KINSELLA AND D. M. WHITEHEAD

which affect wettability and dispersibility have been discussed (Kinsella, 1984a). Rapid dispersibility and dissolution are required for most applications. Control of spray drying to yield particle sizes of 150-200 pm diameter, agglomeration, and/or use of food-approved wetting agents can facilitate wetting and dispersibility (Neff and Morris, 1968). The water sorption behavior of whey powders and proteins is important in optimizing storage conditions (Kinsella and Fox, 1986). The water-binding capacity of pure proteins can be estimated from their amino acid composition. However, protein conformation, surface polarity, ionic strength, ion species, pH, and temperature all affect the waterbinding capacity of proteins (Kinsella and Fox, 1986). In addition, the particle size of the protein in a food system, its porosity, molecular surface features, and interactions with other food components can affect the extent and rate of water binding and/or hydration. Such effects may supersede the basic compositional factors in determining water binding. Whey proteins show great variability in water binding, but generally have low water-holding capacity (Melachouris, 1984). Solubility is a prime requisite for a functional ingredient protein and is critically necessary for products such as beverages (Kinsella, 1976; Damodaran and Kinsella, 1980). Generally, whey proteins are the nitrogenous fraction remaining soluble in the supernatant at pH 4.6 after precipitation of casein. Thus, the loss of solubility at this pH is commonly used to assess the extent of protein denaturation (Guy et al., 1967). Operationally, solubility connotes the amount of protein that goes into solution at pH 6.5-7.0 at 25°C and is not sedimented by relatively low centrifugal forces. Usually, low concentrations (
PROTEINS IN WHEY

375

teins while avoiding denaturation. For example, in a beverage, solubility under a variety of pH, temperature, and ionic strength conditions may be required. The pH of the dispersing medium affects the net charge of proteins. Proteins with a net negative or positive charge tend to bind more water and are more soluble than if the net charge is minimal, i.e., at the isoelectric point, protein solubility is generally low, though whey proteins may remain hydrated and soluble at their isoelectric pH under certain conditions. Ionic strength affects protein solubility according to ion species and valency; salt concentrations up to 0.1 M enhance solubility but above 0.15 M can reduce it (Damodaran and Kinsella, 1981a). Ions in solution exert their influence by affecting the net charge of proteins, hydration, and electrostatic interactions. p-Lg displays the least solubility between pH 4 and 5 but in the presence of salts, the protein is soluble in this pH range. A high content of salts in whey and whey protein preparations limits their use in many foods, and electrodialysis and reverse osmosis have been employed to demineralize condensed whey preparations. Ion exchange is used less frequently (Houldsworth, 1979; Marshall, 1982). In acid whey, removal of salts by UF/DF may result in subsequent precipitation (acid coagulation) of p-Lg and some immunoglobulins in the pH range 4-5. Thus, precipitation of p-Lg in the isoelectric range at low ionic strength may be mistaken for denatured protein in determining the soluble protein content of whey samples. Heat treatments alter the conformation of whey proteins and often result in denaturation, loss in solubility, and aggregation. The solubility of p-Lg following heating for 15 min at 80°C in the presence of 0.01 M sodium chloride showed a typical bell-shaped solubility curved with a minimum close to the pZ of 5.28 (Townend and Gyuricsek, 1974). Increasing the sodium chloride concentration to 0.1 M caused a decrease in the amount of protein precipitated (increased solubility) by 50% and shifted the apparent isoelectric pH to 6.0. At 0.5 M sodium chloride, precipitation of the protein was inhibited completely. In contrast, calcium chloride at 0.01 M caused complete precipitation of 0-Lg at pH 5.5 and 0.7 M calcium chloride caused precipitation above pH 6. Heating in the presence of milk salts yielded a typical bell-shaped curve with a minimum between pH 5 and 7 (Townend and Gyuricsek, 1974). In contrast, Harwalkar and Kalab (1985b) reported that p-Lg unfolded but remained in solution upon heating at around 65°C and at low ionic strength (0.2 M). At higher ionic strengths and higher protein concentrations, denaturation upon heating was accompanied by aggregation in the form of either nonsyneresing gels or syneresing precipitates.

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J. E. KINSELLA AND D. M. WHITEHEAD

VII. THERMAL PROPERTIES OF WHEY PROTEINS

The effects of heat are greatly influenced by pH, ionic strength, the rate of heating, and, to a lesser extent, by protein and lactose concentrations. Upon mild heating (40"C), p-Lg undergoes a slight conformational change; further heating (50" to 60°C) causes unfolding and exposure of the thiol group (McKenzie, 1971; Kella and Kinsella, 1988). If p-Lg is allowed to cool, protein association and aggregation occur via disulfide bonding and entropic forces which are enhanced in the presence of calcium (Haque et al., 1987). This phenomenon may be encountered during lactose crystallization in whey liquors. Heat denaturation of whey proteins at very low pH (pH C3.0) imparts some unique phy sicochemical and functional properties (Modler and Emmons, 1977; Modler and Harwalkar, 1981). Modler and Emmons (1975, 1977) reported that heating at 90°C for 15 min at pH 2.5-3.0 yielded an isoelectric precipitate (Protolac, 60% protein) that was reasonably soluble, highly viscous, and capable of setting to form a soft coagulum. p-Lg has a stable conformation at pH 2 (Reddy et al., 1988), and Modler and Harwalker (1981) showed that concentration of whey by U F at pH 2.5-3.0 prior to or after heating altered some properties, i.e., viscosity and gelling of the isolated protein. A. EFFECTS OF HEAT ON CONFORMATION

Several investigators have studied the heat-induced unfolding of whey proteins (Larson and Rolleri, 1955; Ruegg et al., 1977; Elfagm and Wheelock, 1978; deWit, 1981). In this respect, differential scanning calorimetry (DSC) has provided much of the data on the thermodynamics of whey protein unfolding. Ruegg and co-workers (1977) showed that a-La and pLg (concentration range 3-9%, pH 7.0) had transition temperatures (T,) (maximum on DSC endotherm peak) at 65"and 73"C, respectively, during heating (from 20" to 110°C at 10°C per minute). The T, increased slightly with heating rate and the enthalpies of denaturation (AH)for a-La and pLg were 318 and 227 kJ/mol, respectively. Increasing the pH from 6.4 to 7.3 had negligible effects on AH, while the T, decreased from 79" to 74°C with the rate of denaturation of p-Lg increasing slightly. Harwalkar (1979) reported that increasing pH from 4.5 to 6.5 at zero ionic strength decreased AH for p-Lg from 0.85 to 0.60 J/g protein and the denaturation temperature from 83" to 77°C. The transition temperature also decreased with increasing ionic strength. Hegg (1980) demonstrated that the denaturation temperature of p-Lg increased from 78" to 83°C between pH 2 and 4 and then progressively decreased with increasing pH, being approx-

377

PROTEINS IN WHEY

imately 78°C at pH 6, 67°C at pH 7, and 60°C at pH 8. The enthalpy of denaturation (AH,) decreased with pH from 0.83 to 0.23 J/g at pH 6 and 8, respectively. The discrepancies in the published data may reflect differences in protein preparations, ionic strength, bound lipids, etc. (Hegg, 1980; deWit, 1984; Bernal and Jelen, 1985). Some thermal characteristics of the major whey protein components are summarized in Table XIV (deWit, 1984). Conflicting reports exist in the published literature concerning the kinetics of heat denaturation of p-Lg. While Gough and Jenness (1970) reported a mostly first-order process, Hillier and Lyster (1979) and Park and Lund (1984) described the thermal unfolding of p-Lg as following a second-order reaction rate; the denaturation temperature (83"-84"C) changed little with pH between pH 4.0 and 7.0, while AH increased from 0.95 to I. 11 J/g and the activation energy (E,) increased from 28.3 to 33.0 kJ/rnol. Harwalkar (1980) concluded that the heat denaturation of p-Lg occurs in two stages, both being first-order; the first proceeds relatively slowly and is reversible, while the second stage is rapid and irreversible. Recently, Harwalkar (1986) reported that p-Lg follows a pseudo first-order process in aqueous solution but displays a second-order process in whey as observed by O'Neill and Kinsella (1988). Protein unfolding can be caused by increasing the pH and this augments electrostatic repulsive forces between component polypeptides. At alkaline pH, the thiol group of p-Lg becomes exposed (Kella and Kinsella, 1988) and the thiol-disulfide interchange reaction is accelerated (dewit, 1984). Increasing ionic strength masks exposed charged groups, thus en-

TABLE XIV THERMAL DENATURATION TEMPERATURES ("C) AND ENTHALPIES OF WHEY P R O T E I N S ~ ' ~ ~

Td

a-Lactalbumin @-Lactoglobulin Bovine serum albumin Immunoglobulins Whey proteins

62 78 64 72 62-78

T,, 68 83 70 89 68-83

AH (kJ/mol) 253 31 I 803 500

-

"Data after deWit (1984). bTd,Initial denaturation temperature; T,,, temperature at maximum peak on thermogram peak temperature in DSC; AH is the enthalpy of denaturation.

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J. E. KINSELLA AND D. M. WHITEHEAD

hancing hydrophobic interactions. Calcium is very effective in this respect (Thompson et al., 1969; Bernal and Jelen, 1984). The temperature of denaturation of a-La is 65°C and the enthalpy of denaturation is 318 kJ/mol at pH 6.7 (deWit and Swinkels, 1980). a-La shows rapid and extensive renaturation, i.e., 80 to 90% reversibility at concentrations in the range of 3 to 9% (Ruegg et al., 1977). The a-La molecule is a relatively small protein (123 amino acid residues) with four disulfide bonds, which limits the number of conformations the protein can assume and may account for the resistance to denaturation and rate of renaturation. The enthalpies of denaturation of a-La in milk ultrafiltrate and phosphate buffer (pH 7) were 318 and 272 kJ/mol, respectively, indicating that lactose, calcium, and citrate may stabilize a-La. When a-La is heated for 10-30 min at lOO"C, only 40% of the protein molecules revert back to the native state; the remainder are irreversibly denatured (Schnack and Klostermeyer, 1980). On prolonged heating, various-sized oligomers of a-La are formed via rupture of disulfide bonds, which apparently permits thiol-disulfide exchanges between these molecules and new reactive groups that are simultaneouslyformed (Chaplinand Lyster, 1986). In contrast to p-Lg, a-La is unstable at pH values below 4.0 (deWit and Klarenbeek, 1984). This was supported by Kronman and co-workers (1981) when it was observed that, below pH 3.75, a-La is subject to heatinduced aggregation but this can be reversed by adjusting the pH above 5.2. Acid pH causes unfolding of a-La and facile protein-protein interactions. This may involve exposure of apolar residues and enhanced intermolecular aggregation. The effect of acid pH on heat stability may be related to its effect on calcium binding to the protein ( K , = 3 x 10-6 M -1) which stabilizes the molecule. Removal of calcium by acidification or chelation destabilizes a-La. Bernal and Jelen (1984) demonstrated that, following chelation of calcium, the T, and AH of a-La (holo-form) decreased from 61" to 41°C and 22 to 6 J/g, respectively, and eliminated renaturation of a-La (Table XV). It is conceivable that, on exposure of a-La to low pH (3.0), some bound calcium is removed from the molecule, rendering a-La more susceptible to irreversible heat denaturation. Thus, extensive dialysis of acidified whey (UF/DF) may destabilize a-La by removing calcium. This may contribute to the anomalous behavior of whey protein preparations, particularly under low-ionic-strength conditions. B. FACTORS AFFECTING HEAT DENATURATION During whey processing, proteins are subjected to many heat treatments which may be cumulatively deleterious. Calorimetry can be effectively used to monitor the extent of heat denaturation of whey proteins

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PROTEINS IN WHEY

(dewit, 1981, 1984). The method is sensitive to the progressive denaturation of whey proteins as evidenced by the progressive decrease in the principal endothermic peak with cumulative heating during whey processing (dewit, 1984). Harper and Zadow (1984) reported that a measurable portion of BSA and immunoglobulin molecules were denatured, as determined by rocket immunoelectrophoresis, following heating of the proteins at 72°C for 15 sec. Denaturation progressively increased for all whey proteins following heating at 72°C for 5 min. Extensive denaturation of whey proteins occurred after 5 min of heating, particularly as the temperature was increased from 70" to 85°C. UF concentration of whey enhanced the rate of thermal denaturation and the extent of denaturation increased with time of heating. Based on immunological and solubility (denaturation) data at pH 4.6, BSA and immunoglobulins (IgG fraction) were the most heat-sensitive of the whey proteins, followed by (3-Lg and a-La (Harper and Zadow, 1984). Although the thermal denaturation behavior of whey proteins reflects the collective responses of the component proteins, particularly those of p-Lg (deWit and Swinkels, 1980; deWit and Klarenbeek, 1984), the behavior is complicated by the effects of pH, ash (especially calcium), and the presence of chelating agents such as citrate and phosphate (de Rham and Chanton, 1985). Hermansson (1979) observed that, at pH 2.0 to 5.0, the thermal transition of whey proteins in water displayed a sharp peak with maximum transition temperatures increasing from 80" to 90°C up to pH 4.5 and then decreasing back toward 80°C as the pH was increased to 9.0. Significantly, above pH 6.0, the endotherms showed a gradual slope

TABLE XV THE EFFECTS OF CALCIUM REMOVAL ON SOME THERMODYNAMIC

PARAMETERS OF a-LACTALBUMIN""

Td

Extent of renaturation

rc)

AH

pH of medium

A

B

4.5-6.5

61

3.5

58

41 40

A

(JM

(%)

B

22

6

10.6

4

A 85

<so

B 0 0

"Data after Bernal and Jelen (1984). "a-Lactalbuminskim milk ultrafiltrate without (A) and with 0.1 M EDTA (B). Td, Denaturation temperature.

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J. E. KINSELLA AND D. M. WHITEHEAD

rather than a peak. Using turbidometry, it was reported that, above pH 3.5, the apparent transition temperature of very dilute solutions (0.09% in 0.2 M sodium chloride) of whey proteins increased from 58" to 75°C at pH 4.0 and 6.0, respectively. However, below pH 3.5 these whey proteins were stable at 90°C (Hermansson, 1979). At pH 3.0, 6.0, and 7.5, p-Lg had peak denaturation temperatures of 82", 78", and 71°C and AH of 16.6, 15.9, and 11.2 J/g, respectively, indicating that increasing pH rendered pLg susceptible to thermal denaturation (dewit, 1981; deWit and Klarenbeek, 1984). This behavior is consistent with previously inaccessible hydrophobic patches becoming exposed to the aqueous exterior and resulting in a decrease in AH,,, i.e., as the molecule unfolds, increasing surface hydrophobicity causes a disruption in the hydrogen bonding network between polar groups on the protein and water molecules which surround the protein (Pace, 1983). The addition of calcium after heating results in greater flocculation than if present during heating (de Wit, 1981). However, very high heat treatments (160°C) reduce the sensitivity to calcium, reflecting greater polymerization of the proteins and perhaps occlusion of the charged anionic groups (deWit and Klarenbeek, 1981). Normal whey contains 2-3 mM calcium, yet above pH 6.5, the proteins are not heat precipitated because the calcium is apparently chelated. However, UF/DF of whey may increase calcium sensitivity and decrease solubility. De Rham and Chanton (1985) carried out a detailed study on the effects of ions in the heat-induced insolubilization of whey proteins of normal and demineralized whey (DW). The proteins in DW remained soluble following heating (90°C, 15 min) at pH 6.5 and above. Heating at pH 4.5 and 5.5 decreased solubility by 33 and 50%, respectively. Removal of lactose and nonprotein materials slightly increased thermal sensitivity below pH 6.0. When DW was heated at pH 7.2, a marked increase (2-fold) in solubility at pH 4.5 to 6.5 was observed, with 100% solubility at pH values greater than 5.5. The addition of calcium to DW before testing reduced solubility, and less calcium (2 mM) was needed at pH 6.0 than at pH 7.0. Chelation of calcium with citrate eliminated this effect. Sweet cheese whey contains approximately 8 mM Ca2+,3 mM MgZ+,15 mM Na+, 36 mM K', 8 mM citrate, 12 mM phosphate, and 32 mM C1- (de Rham and Chanton, 1985). The pH of whey during UF significantly affects the residual ash content; at lower pH values a reduction in the residual calcium content is obtained. Citrate is rapidly reduced by UF, irrespective of the pH. Generally, the heat stability of whey proteins above pH 6.0 is improved after U F at acidic pH (4.5-53, reflecting lower calcium levels in those products. The addition of citrate to chelate calcium improves solubility (above pH 6.0) following heating. Replacement of calcium with

PROTEINS IN WHEY

38 1

sodium (or potassium) by ion exchange also improves the heat stability and solubility of whey proteins (dewit, 1981; Johns and Ennis, 1981; de Rharn and Chanton, 1985). Aggregation and precipitation of p-Lg are greatly facilitated by pH and presence of calcium ions (Elfagm and Wheelock, 1978). Calcium possibly interacts with the negatively charged carboxyl groups of the protein, reducing the net charge to zero and causing isoelectric precipitation (Zittle and DellaMonica, 1956; Zittle et al., 1957). These data indicate that whey proteins, particularly when concentrated by UF, are more susceptible to thermal denaturation and that disaccharides such as lactose and sucrose may retard thermally induced denaturation of whey proteins (dewit, 1981; Bernal and Jelen, 1985; Garrett et al., 1988). Concentration of proteins prior to heat processing may be conducive to excessive denaturation, particularly of the IgG and BSA components. Other factors can affect the rate of heat denaturation, e.g., lactose concentration reduces susceptibility to heat denaturation and low concentrations of lipids enhance the thermal stability of p-Lg. Binding of 1-2 molecules of sodium dodecyl sulfate (SDS) to p-Lg increased its denaturation temperature from 82" to 90°C (Hegg, 1980). Lipid binding by BSA stabilizes the molecule but, at high concentrations of lipid, the protein is destabilized (Hegg, 1980). Thus, lactose, citric acid, pH, ionic environment, and calcium concentration can be manipulated to minimize the heat-induced insolubilization of whey proteins. VIII. GELATION

Whey proteins can form gels that range in properties from viscous fluid soft, smooth pastes or curds to stiff, rubbery gels. These gels vary in hardness, cohesiveness, stickiness, color, and mouthfeel (Hillier and Cheeseman, 1979; Schmidt et al., 1979; Johns, 1979; Dunkerley and Hayes, 1981; Hillier et af., 1980; Johns and Ennis, 1981; Schmidt, 1981; Kornhorst and Mangino, 1985; Zirbel, 1987). WPC gels also vary in visual appearance from firm elastic transparent gels to opalescent curdlike coagula. At low protein concentrations and low ionic strength, weak gels with gray or translucent appearances are obtained. In many cases, evaluations of whey gels are based on subjective assessment; thus, standardization of methodology and terminology is necessary. Gelation occurs when there are a limited number of specific protein-protein interactions following heating, whereas coagula or curds form when extensive random interactions occur. Gelation, i.e., the property of forming a structural network which maintains shape, has mechani-

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J. E. KINSELLA AND D. M. WHITEHEAD

cal strength, viscoelasticity, and retains entrapped water with minimum syneresis, is an important functional attribute of functional proteins in many food applications. Protein solutions (1-10 g/dl), when heated above a critical temperature, undergo conformational changes and on cooling may set to viscous, soft, opaque coagula or clear viscoelastic gels depending on the type of protein, concentration, heating rate, and environmental conditions, especially pH and calcium (Mulvihill and Kinsella, 1988). Thermally induced gelation may be viewed as a two-stage sequential process; and first phase involves heat-induced conformational changes in a protein which may involve unfolding of some polypeptide segments followed by subsequent protein-protein interactions resulting in a progressive build up of a network structure when protein-protein interaction is limited, i.e., approximately two interaction sites per molecule (Bernal and Jelen, 1985). Thermal activation of protein molecules to expose interactive sites may involve only very minor changes in overall molecular conformation. Where the protein concentration is adequate and the heating rate slow, protein-protein interactions leading to a three-dimensional network capable of entraining water are favored. If aggregation is more rapid than unfolding, a precipitate may form (Ferry, 1948). For the formation of an appropriate three-dimensional network, a balance between attractive and repulsive forces is necessary. In the formation of a gel from whey proteins, limited unfolding exposes hydrophobic segments on proteins which tend to associate on recooling, facilitating bonding between contiguous molecular groups. Networks thus formed vary considerably in structure depending on the number and relative reactivity of interactive sites, and the degree of electrostatic repulsion between the solute molecules. These are markedly affected by pH and ionic strength. The particular changes that occur in the secondary structure of proteins and the manner in which the network builds from individual molecules affect gel texture. Small conformational changes may facilitate protein-protein association to form long beads, or molecules may completely unfold to form a random network. For example, in the case of glycinin, the globular molecules associate to form chains with cross-linking or branching. In other cases, polymerization of small aggregates or of dimers and trimers into thick filaments for the formation of ropelike arrangements can occur (Clark et d.,1981). In BSA gels, irregular chains or filaments of protein molecules, approximately 1-2 protein molecules thick, form junction points or branch points. These filaments form a network with a density reflecting the degree of branching and cross-linking. Clark and co-workers (1981) concluded that, for BSA with a filament thickness of 6 to 12 pm (approximately double that of the undenatured molecule), thickening was caused by unfolding of the native molecule. p-

PROTEINS IN WHEY

383

Lg gels contain large spherical protein aggregates linked together to form a continuous branching network at both high and low ionic strength (Harwalkar and Kalab, 1985b). Some proteins, e.g., lysozyme form networks composed of straight, rodlike filaments, whereas other protein molecules seem to be assemblies of small, irregular protein clumps with a beadlike repeating structure (Clark et al., 1981). A. GEL STRUCTURE Proteins vary considerably in the amount of native tertiary and secondary structure which is lost prior to aggregation and gel formation. Little work has been done to quantify the extent of unfolding prior to gelation. In the case of BSA gels, there is limited unfolding and the molecules string together to form irregular chains 2 to 3 times as thick as the native molecule (Clark et al., 1981). Limited conformational changes occur during heating and gelation. Clark et al. (1981) reported an increase in p-sheet and unordered structure with a concomitant decrease in the a-helical content of BSA, which is common in fibrous protein systems, following heating. Timasheff et al. (1967) noted that aggregation of p-Lg was accompanied by an increase in P-sheet and unordered structure. Generally, clear gels reflect the formation of uniform networks of fine filaments and there is greater linear aggregation with frequent cross-linking and/or branching processes. As network density becomes less regular, gels increase in turbidity. In the case of BSA gels, gel clarity was decreased by adding salts or decreasing pH toward the isoelectric point, i.e., minimization of net charge (Clark et al., 1981). In the case of coagula, phase separation occurs and the protein network is composed of aggregates of protein separated by regions of water. Noncovalent protein-protein interactions occur in the formation of both reversible and irreversible gels (Table XVI). The number and type of noncovalent interactions (hydrophobic and van der Waals’ interactions, hydrogen bonding, and ionic interactions between charged amino acid side chains) vary with the protein, pH, heat treatment, and ions present. Cross-linking is essential for gel formation, which together with the solvent provides the fluidity, mechanical strength, elasticity, and flow behavior of gels (Schmidt, 1981). The degree of cross-linking can be variable, and thus provides a mechanism by which the strength of gels may be manipulated. Understanding the nature of the interactions provides the means to control the extent of cross-linking, permitting manipulation of gel properties. Some ion cross-linking may be important in calciuminduced gels (Schmidt, 1981; Mulvihill and Kinsella, 1987). In the formation of irreversibly set gels, covalent disulfide cross-linking may occur

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J. E. KINSELLA AND D. M. WHITEHEAD

TABLE XVI POSSIBLE FORCES INVOLVED IN THE FORMATION AND STABILIZATION OF PROTEIN FILMS AND GELS

Type Hydrophobic van der Waals Hydrogen bonding

Energy (kJ/mol) 4.212.6 8.433.6

Interactive distance (A)

Groups involved

3-5

Apolar

2-3

NH-OC

Effects Strengthening and initiating contacts Strengthens associations

0 Ionic Cation bridging Covalent

42-84 25.250.4 336378

1I

2-3 2-3

C-0:- +NH, Calcium

Strengthens cross-links Cross-linking

1-2

RSH:S-S

Bridging, ordering, strengthening

(Huggins et al., 1951). Electrostatic interactions (the net result of attractive and repulsive forces) are critical in determining gel formation and gel properties. Thus, the gelation of p-Lg at pH 7.0 or above is enhanced by calcium chloride (3-6 mM) or sodium chloride (0.2-0.4 mM). Gel strength is also affected by salts but their effects on gel properties vary with anion type. This may reflect differential binding by the anions, exerting variable effects on electrostatic interactions (Mulvihill and Kinsella, 1987). B. GELATION OF WHEY PROTEINS Whey proteins have excellent gelling characteristics, particularly above pH 7.0. Undenatured, soluble whey proteins prepared under mild processing conditions form irreversible gels at the appropriate pH, ionic strength, and protein concentration. Heating p-Lg above pH 6.5 causes formation of gels that become clearer as pH is increased (dewit, 1984). Coagula are formed on heating p-Lg at pH values below 6.5 and concentrations greater than 7 g/dl, reflecting extensive enhanced attractive interactions. The higher gelation tendency in the region above pH 8 may reflect some disulfide cross-linking and matrix formation via thiol-disulfide interchange. Whey proteins can form clear gels or opaque gels. Clear gels tend to be more elastic and hold water more effectively. Apparently, they possess finer filaments, smaller mesh size, more even spacing of intermo-

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385

lecular linkages, and are generally formed at higher pH values (Schmidt, 1981). Although much information exists concerning factors affecting gel formation and properties of whey protein gels (Dunkerley and Hayes, 1981; Schmidt, 1981; Mulvihill and Kinsella, 1987), there are limited data on the kinetics of gel formation and gelling properties of the individual proteins. Dynamic shear measurements at small total strain amplitudes during heatinduced gelation of whey protein concentrates were made and the changes in the storage modulus (G’) and the loss modulus (G”)were calculated by Beveridge et al. (1984). As the gel network formed, the samples became more elastic. WPC (7 g/dl protein solution) showed a slow increase in G’ (2 x lo3 dynekm’) on heating (85°C for 10 min) compared to egg white in which G’ was 4 x lo3 dyne/cm2 after 30 min. However, on cooling, the G’increased to 20 x lo3 dynelcm’, i.e., the elastic properties increased. It was concluded that aggregates were formed during the initial 3-10 min of heating and that these associated to form a weak viscous network on continued heating (Beveridge et al., 1984). Disulfide bonds may be involved at this stage. However, on cooling, an extensive elastic network is formed due to extensive hydrophobic, ionic interactions and hydrogen bonding. The increased elasticity reflected the increased number of cross-links formed during cooling. Reheating the gels reduced the elasticity somewhat but it significantly increased on cooling (Beveridge et al., 1984). Little is known of the effect of additional materials, such as lipids, on the gel-forming characteristics of whey proteins. Sternberg et al. (1976) reported a decreased tendency of whey protein solutions to gel after addition of milk lipids. On heating in the presence of calcium, Yamauchi et al. (1980) observed gelation of a coconut oillwater emulsion of whey protein. Gelation did not occur when calcium was omitted. Jost and co-workers (1986) investigated the amount of lipid which could be incorporated into whey protein gels without compromising desired gel textural qualities. Generally, the firmness of heat-induced gels increased with increasing emulsion fineness and was affected by changes in the protein and oil content. The pH range within which oillwater emulsions stabilized by whey protein could get extended from pH 3.5 to 8.0 (Jost et al., 1986). C. COMPOSITION AND GELLING PROPERTIES

The properties of whey protein gels are affected by protein concentration, time and temperature of heating, pH, ionic strength, etc. (Schmidt, 1981). The appearance of whey protein gels varies from translucent and

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J. E. KINSELLA AND D. M. WHITEHEAD

elastic to curdlike brittle aggregates. Weak translucent gels are obtained at lower protein concentrations (5%) and low heating temperatures (55" to 70°C). Stronger and more aggregated opaque gels which form at higher protein concentrations and higher heating temperatures (90°C) reflect a more extensive network. Translucent gels are usually obtained when protein is heated at low ionic strength (Schmidt, 1981). Gel strength increases with the concentration and purity of the protein. Prior denaturation of the protein reduces gelation and gel strength (Langley et al., 1986). Commercial samples of WPC (10 g/dl protein solution) vary tremendously in gel formation and gel properties when studied under identical conditions. The time required for gelation may range from 1 to 17 min at lOO"C, and some samples did not form a gel even after 30 min heating. This may reflect extent of prior denaturation, composition of the whey, pH, and ion concentration (Schmidt, 1981; Langley et al., 1986). Dynamic rheological techniques (Mitchell, 1980) permit the viscoelastic properties of individual whey proteins to be assessed separately. BSA was characterized as having good, p-Lg intermediate, and a-La poor thermal gelling properties under conditions of pH 6.6, 1% NaCl, and heating range from 30" to 95°C (heating rate l"C/min). The BSA gels were purely elastic, while the p-Lg gels had viscous elements (Paulsson et al., 1986). In protein mixtures, the influence of various whey proteins at equal concentrations on heat-induced gelation decreased in the order BSA, p-Lg, a-La and the gelation behavior was influenced mostly by the protein in highest concentration (Paulsson et al.. 1986). The presence of lactose and ash may impair gelation by interfering with network formation, but generally increasing the protein concentration, relative to nonprotein components, improves gelation. Thus, gels formed following dialysis of WPC are more translucent, stronger, more cohesive, less springy, and more chewy, than gels from the nondialyzed WPC. The pH has a marked effect on gelling properties of whey proteins, depending on the particular preparation, and this effect may be related to calcium content. Gels formed at low pH (pH <6) are more coagulated and less elastic than gels formed at pH 7 to 9 (Hillier et al., 1980; Schmidt, 1981). The addition of 0.1 M phosphate enhances the rate of gelation at higher pH values, possibly due to chelation of calcium (Schmidt, 1981). Exposure of WPC to pH 9 followed by readjustment to pH 7 prior to heating facilitates the subsequent gelation of WPC. This may reflect some unfolding and/or rupture of disulfide bonds under alkaline conditions. The pH of the solution during heating markedly affects gelation reactions; thus increasing the pH from 7 to 11 reduces gel strength, particularly above pH 9. The relative strengths of gels made by heating whey protein solutions at 100°C for 15 min at pH 7, 9, 10, and 11 progressively de-

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387

creased from 100, 57, 23, 0 (N/m*), respectively (Schmidt et al., 1979; Schmidt, 1981). Exposure to alkaline pH conditions accelerates heat-induced gelation and reduces the temperature treatment required for gelation. Hillier and Cheeseman (1979) and Hillier et al. (1980) confirmed the effects of pH on the gelling time. As the pH of the solution was increased above 6.5, gelling time of deionized WPC (10 g/dl) increased, particularly at pH 9 and above. Below pH 6.4 and especially around pH 5 , the proteins precipitated on heating. Thus pH, by affecting protein conformation and net charge, markedly affects the rate and extent of gelation, i.e., where electrostatic repulsion is excessive a limited network is formed. The effects of pH in turn are influenced by the presence of ions. Calcium may reduce net repulsion above the isoelectric pH and facilitate hydrophobic interactions. Calcium may also serve in cross-linking adjacent anionic molecules, thus facilitating network formation (Mulvihill and Kinsella, 1988). Because the degree of electrostatic interactions between proteins is critical in forming a three-dimensional matrix with desirable water-holding capacity, the heat-set and thermal denaturation characteristics of two acidic proteins, BSA and WPI, containing added basic protein components, e.g., clupeine (pZ = 12), hydrolyzed clupeine, or modified p-Lg (pZ = 9.9, at low ionic strength, were examined by Poole et al. (1987a). An optimum concentration ratio of 3 : 0.3% (w/v, BSA : clupeine) yielded a gel at pH 8 comparable to that formed with BSA alone at pH 5 . However, using the same concentrations of hydrolyzed clupeine, a much weaker gel was formed, demonstrating the importance of size of the basic protein in determining whether cross-linking or aggregation occur. Modified p-Lg with a net positive charge also enhanced gelation, but the minimum total protein concentration required for gelation was higher (4 : 0.4% w/v, BSA : p-Lg), reflecting weaker interactions with BSA possibly because of a lower pZ than clupeine. Clupeine promoter the gelation of WPI but only at a critical ratio above 5 : 0.4% (w/v, WPI : clupeine). However, the gel had a coarser texture and poorer water-holding capacity than the corresponding BSA gels. It was concluded that basic protein may promote gelation by cross-linking (partially) denatured acidic proteins via electrostatic attractive forces. DSC and spectral analyses demonstrated that basic proteins tend to destabilize acidic proteins at a pH value greater than the pZ, thus facilitating protein unfolding (Poole et al., 1987a). The gel structure of whey proteins is affected by salt concentration (Table XVII). The action of sodium chloride can be explained in terms of a charge-shielding effect of counterions which surrounds like-charged macromolecules in solution, causing repulsion (Dickinson and Stainsby,

388

J. E. KINSELLA AND D. M. WHITEHEAD

TABLE XVII EFFECT OF SALTS AND THIOL GROUPS ON THE STRENGTH OF WHEY PROTEIN GELS*

Treament

Penetration (dcm)

Hardness (kg)

Whey protein control (WPC) WPC + NaCl (0.2 M) WPC + CaCI,(IOmM) WPC + cysteine (25 mM)

533 996 1785 1980

0.56 0.82 0.89 0.69

“Data after Schmidt et nl. (1979).

1982). Thus, in the presence of sodium chloride (0.2 M), a coarse gel of large aggregates is formed compared to finer gels developed in the absence of salt (Schmidt et al., 1979). The addition of calcium (5-20 mM) tends to restore the strength of whey protein gels formed at pH values of 7 and above (Schmidt et al., 1979; Mulvihill and Kinsella, 1988). This cation reduces electrostatic repulsion and permits greater protein-protein interactions and network formation. The effect of calcium addition varies with each whey preparation, reflecting variability in composition, e.g., citrate, phosphate, calcium content. Dialyzed WPC shows greater response to calcium addition; addition of 10 mM calcium to dialyzed WPC improved gelation and gel strength (Schmidt, 1981). The rheological properties of gels made from p-Lg are affected by addition of calcium (Table XVIII), suggesting that, in the absence of added salts, electrostatic repulsive forces, which are enhanced at high pH, predominate to prevent gelation but dissipate on increasing the ionic strength (Mulvihill and Kinsella, 1987, 1988). Maximum gel strength occurs at 10 mM CaCl, in contrast to 200 mM NaCl, reflecting the greater effectiveness of calcium compared to sodium in increasing rheological parameters related to gel strength (Mulvihill and Kinsella, 1987). In the case of gels made at pH 6, replacement of 30,67, and 100% of the calcium in UF/WPC with sodium progressively increased the hardness, springiness, and chewiness of 10% WPC gels (Johns and Ennis, 1981). Additionally, gels became more translucent with calcium replacement, and the time for gelation increased from 44 to 166 min on heating at 70°C at pH 6.7 (Johns and Ennis, 1981). Calcium may interfere with gelation by causing excessive coagulation following heating rather than enabling the protein network to form properly.

389

PROTEINS IN WHEY

TABLE XVIII EFFECTS OF CALCIUM CHLORIDE CONCENTRATION ON THE RHEOLOGICAL PROPERTIESOF p-Lg GELS~.’

[CaC121(mM)

Stress (kN/m*)

5

0.5 7.0 2.5 1.5

10

I5 20

AMD (kN/m’) 2.0

16.5 11.5

8.6

“Data after Mulvihill and Kinsella (1988). ”Gels measured at 20% compression. AMD, Apparent modulus of deformability.

Thiol groups at reasonably low concentrations facilitate gel formation by enhancing the rate of unfolding of proteins since reduction of strained disulfide bonds permits greater conformational freedom and perhaps greater interactions between unfolded molecules (network formation). However, excessive disulfide reduction may impair network formation (Schmidt, 1981). Disulfide bonds are important in stabilizing the structure of gels since, on addition of thiol reagents, they can be disrupted (dewit, 1981). The addition of cysteine (up to 10 mM) prior to heat treatment enhances gelling properties, whereas, at higher concentrations, gelation is impaired (McKenzie, 197 1). The content of apolar amino acids in proteins influences the changes which occur on heating because a high molar percentage of apolar side groups can overcome the repulsive electrostatic forces arising when pH values are outside the isoelectric region (Circle et al., 1964; Shimada and Matsushita, 1981). Thus, small proteins with high molar percentages of apolar amino acids (>33%), such as p-Lg and a-La, are concentration independent in gelling systems (Shimada and Matsushita, 1980). However, the disposition of apolar amino acid side chains along the polypeptide chain may be more important to the development of intermolecular interactions than the total content of these residues. D. WHEY PROTEINS AS GELLING INGREDIENTS WPC has been promoted as a replacement for egg white proteins in gelling applications (Melachouris, 1984). At protein concentrations of 10 g/dl, WPC forms gels when heated above 85°C for 15 to 30 min. Egg white

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E. KINSELLA AND D. M.

WHITEHEAD

proteins gel at 60°C and many applications of egg white in food processing are based on this feature. Thus, when replacing egg white with whey protein, the food may have to be heated above 80°C for adequate gel formation and this may be incompatible with other functional attributes of the particular product. This is particularly true of cakes; for example, in angel food cake the extra heat may cause oven-collapse. The addition of polyphosphates to whey protein treated with pepsin improved its gelling and whipping properties, which were comparable to those of egg white (Nakai and Li-Chan, 1985), but this preparation was found to be unsuitable as an egg white substitute in angel food cakes because of the instability of whey protein foams. The differences in thermal characteristics of egg white and whey must be recognized before advocating WPC as a suitable replacement. Thus, the temperatures required to induce gelation and the effects of the various components on the temperature of gelation are extremely important. The gelling behavior of proteins is important in processed and reformed meat products for setting into a matrix but also because the binding (water, fat, flavors) properties are related to gelling behavior (Acton, 1972). In this respect, whey proteins have acceptable binding properties but, for optimum performance, their thermal properties should be compatible with those of the myofibrillar proteins. Thus, when functional proteins are added to a food system to impart gelling properties, it is necessary to know the required processing temperatures to obtain that functional attribute. For example, myosin gels at 6O"C, whereas whey protein concentrate may require higher temperatures, e.g., 80"C, to obtain an equivalent extent of gelation. This may or may not be compatible with the food product in question. Therefore, in seeking to substitute WPC for other possible gelling proteins and products, the conditions required for optimal gelation and development of requisite gel properties must be clearly defined and be compatible with the processing and preparation of the product to which it maybe added. Thus, in certain products, e.g., meats, WPC may not form a gel with the firmness required for slicing, whereas in other products, depending on concentration, heating temperatures, etc., the gel strength may be excessive. These concerns underline the necessity of testing whey proteins in actual food systems under commercially compatible conditions before claiming specific functional attributes. In comparing gelling properties of whey protein preparations, it is important to standardize conditions of protein preparation, isolation, concentration, pH, temperature, time, and ionic strength. Standardized tests of all the physical properties of whey protein gels should be outlined to enable data from different laboratories to be compared validly. Using standardized testing, Melachouris (1984) demonstrated marked variability

PROTEINS IN WHEY

39 1

in gelling properties and gel strength of WPC lots containing 50% protein. Variability in gelation behavior of whey preparations reflects differences in extent of protein unfolding during drying, composition and ion concentration, e.g., of calcium and lipids, all of which affect gelling rates and gel properties. The extent of denaturation of WPC produced by U F may range from 10 to 50%, but is usually within the range 20 tO 30%. Measures to minimize denaturation by controlling heat and/or the use of additives to minimize protein denaturation should be explored (Melachouris, 1984). IX. SURFACE ACTIVITY OF WHEY PROTEINS

A. PROTEIN ADSORPTION The surface active properties of proteins are important in a number of functional applications, e.g., foams and emulsions (Graham and Phillips, 1976a,b; Kinsella, 1981). Many proteins are surface active, that is, they adsorb at surfaces or interfaces at a rate which is initially diffusion controlled (Graham and Phillips, 1976a; MacRitchie, 1978; Phillips, 1981). Adsorption is thermodynamically favored because of the concomitant loss of some conformational and hydration energy by the protein molecules. The initial protein molecules arriving at an interface unfold to a varying extent (depending on their inherent molecular properties) and spread to cover a maximum area of the surface, forming an extended film (Graham and Phillips, 1979; Phillips, 1981). Additional molecules may adsorb provided they can overcome the interfacial pressure barrier and the “energy” barrier (electric potential) created by the initially adsorbed molecules. Once adsorbed at an interface, protein molecules undergo spreading and rearrangement in order to achieve a state of lowest free energy. Thus, extensive rearrangement of polypeptide chains and interactions between polypeptides occur at the interface over time. Rearrangement involves the hydrophobic components of polypeptides orienting toward the nonaqueous phase while polar moieties become exposed to the aqueous phase (Phillips, 1981). The extent of unfolding and reorientation depends on the nature of the protein, molecular flexibility, and protein concentration at the interface. Adsorption gradually causes a decrease in interfacial tension which is proportional to the number of groups penetrating the interface. From the changes in the surface tension, surface pressure, rate of adsorption of the protein to the surface, area cleared per mole of protein, and work of compression during penetration of the protein into the interface, the approximate number of amino acid residues penetrating the interface can be estimated (MacRitchie, 1978; Waniska and Kinsella,

392

J. E. KINSELLA AND D. M. WHITEHEAD

1985). High surface tension or low surface pressure indicates a high surface free energy. As protein molecules adsorb at an interface, the increase in surface pressure reflects a lower surface free energy (Joly, 1972). Generally, the first layer of protein at an interface contributes the most to surface pressure development. Thus, protein preparations with small surface active components, e.g., PPs in whey preparations, rapidly decrease surface pressure, but because of their small molecular size, their presence may not be conducive to the formation of a continuous cohesive film. Furthermore, these same components facilitate foam formation but may actually impair foam stability. Rapid adsorption is important for entrapping air or encapsulating an oil droplet. As protein concentration increases, thicker films are formed (3-5 mg/m2)and tighter packing gives a more condensed compressed film with 3-8 mdm2 protein (Graham and Phillips, 1976a). Noncovalent interactions operate between adjacent molecules at the interface to form a cohesive film (Graham and Phillips, 1979, 1980). Under equilibrium conditions, the surface activity of a protein is influenced by its rate of diffusion, which is in turn affected by molecular properties (e.g., size, composition, conformation), temperature, pH, ion species, and concentration. In this regard, small, randomly structured, i.e., flexible, amphipathic proteins (e.g., p-casein, PP) exhibit excellent interfacial properties in terms of surface pressure development. However, in food systems, equilibrium conditions may not prevail and generally there is a large energy input during mixindwhipping to form an interfacial film. Furthermore, continuous cohesive films that possess mechanical strength and appropriate viscoelastic properties are required in most food systems (Kinsella, 1981; Halling, 1981). Low-molecular-weight, randomly structured molecules do not form strong films, whereas larger protein molecules, which are moderately stable and retain a certain critical amount of tertiary structure at an interface, are generally better surface active agents because of greater intermolecular interactions (Graham and Phillips, 1979; Song and Damodaran, 1987). The rate of surface pressure increase reflects the nature of the protein and its molecular “flexibility” (Graham and Phillips, 1979). As increasing amounts of protein are adsorbed, the extent of molecular rearrangement decreases and the film changes from an expanded to a compressed film. Globular proteins generally form more condensed interfacial films than extended proteins such as the caseins (Graham and Phillips, 1976a, 1979). Presumably, this is because globular proteins such as BSA and p-Lg retain a greater amount of residual secondary and perhaps tertiary structure when adsorbed at the interface, facilitating greater polypeptide entanglements and interactions to yield a condensed film. In an expanded film,

PROTEINS IN WHEY

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the extent of unravelling and loss of tertiary structure may be significantly greater for globular proteins than for extended proteins (Graham and Phillips, 1976a; MacRitchie, 1978). Globular proteins such as p-Lg, BSA, and a-La form condensed fdms with relatively high surface viscosity. The viscoelastic properties of BSA and p-Lg films reflect a continuous twodimensional network, formed via extensive protein-protein interactions (Kim and Kinsella, 1985; Waniska and Kinsella, 1985). B. PROTEIN STRUCTURE AND FILM-FORMING PROPERTIES Different proteins behave differently at interfaces and protein adsorption at an interface is highly dependent on the nature of the protein and predominating forces, i.e., molecular size and shape, and conformational, ionic, and hydrophobic characteristics. It is essential to understand how each of these parameters affects the dynamics of the adsorption and spreading processes. In order to elucidate the contribution of molecular conformation, different proteins with varying structural characteristics have been extensively studied (Graham and Phillips, 1979, 1980; Damodaran, 1989). p-Casein adsorbs and spreads rapidly at the interface to occupy the maximum area per molecule compared to BSA or lysozyme (Benjamins et al., 1975; Phillips, 1981). BSA rapidly diffuses to the interface where some of the molecular domains reorient, but the bulk of the tertiary structure is retained and anchors in the interface (Graham and Phillips, 1979). Ovalbumin, a compact and nonhydrophobic protein molecule, takes 0.36 sec to form a film of surface concentration 1.8 mg/m2 from a 0.03% solution (Bull, 1972). It was concluded from these results that differences in the surface behavior could be attributed to differences in conformational characteristics. However, differences in amino acid composition and disposition along polypeptide chains also exist and should not be discounted in contributing to the surface behavior of these proteins. One approach to assess the influence of protein conformation on interfacial properties has been to study the adsorption and molecular behavior at fluid interfaces of structural intermediates of BSA, i.e., protein molecules which have been partially unfolded and permitted to refold in variable amounts of time (Song and Damodaran, 1987). The kinetic data from rates of surface pressure development for six structural intermediates of BSA, ranging from 0 to 50% a-helix, suggest that an optimum ratio of ordered to disordered structure is apparently essential to facilitate maximum changes in surface behavior (Song and Damodaran, 1987). Because of the importance of charge in influencing the behavior of proteins in solution, i.e., during adsorption, unfolding and protein-protein

394

J. E. KINSELLA AND D. M. WHITEHEAD

interactions at the interface, the pH and ionic strength have dramatic affects on the film-forming properties of proteins. A high net charge retards adsorption and protein-protein interactions in the film, causing the formation of expanded films with limited cohesiveness. Maximum adsorption rates occur near the isoelectric point if the protein is soluble, and strong viscous condensed films with maximum protein-protein interactions are obtained (Halling, 1981; Kinsella, 1981). Salt can enhance the rate of adsorption by minimizing electrostatic repulsion between protein molecules and facilitate molecular packing in the film (Cumper, 1953). Adsorption is increased by temperature as long as the protein remains soluble (Halling, 1981). The effects of aging on the surface rheology of interfacial films of several proteins (e.g., BSA, p-Lg, ribulose 1,5-bisphosphate carboxylase, Kand p-caseins) have been reported and discussed (Benjamins er al., 1975; Kim and Kinsella, 1985; Waniska and Kinsella, 1985; Barbeau and Kinsella, 1986). Rates of surface pressure development, a function of protein concentration and conformation at an aidwater interface, surface viscosity, and film hysteresis increased as the protein films aged. The pH of the solutions also affected these parameters, i.e., maximum values were attained at or near the isoelectric points of the proteins. The data indicate that there is a limit to the number of polypeptide segments which can occupy the interface (MacRitchie, 1978) and significant protein-protein interactions occur between segments both above and below the plane of the interface (Graham and Phillips, 1979, 1980). The' physical properties of the film once formed are the principal determinants of foam stability. Stability depends on the extent of protein-protein interactions, film thickness, mechanical strength, viscoelasticity, and on various external factors such as temperature and pH that alter the nature and magnitude of protein-protein interactions. Thicker films are usually more stable because of better rheological properties and mechanical strength. Thus, films of amphipathic bulky proteins which retain tertiary structure are usually thicker and more stable, (e.g., BSA versus pcasein) and this is reflected in more stable foams and emulsions. Such films have better viscoelastic properties and can adapt to shocks, compression, and distortion without rupture. For example, BSA forms viscous films while caseins show limited viscosity because of weak protein-protein interactions in the film (Graham and Phillips, 1979; Phillips, 1981).

The viscoelastic behavior of protein films has been explained in terms of protein-protein interactions; however, the nature of such interactions remains largely unknown. Several investigators have proposed a correlation between surface hydrophobicity and the surface activity of proteins

PROTEINS IN WHEY

395

(Scriven and Sternling, 1960; Kato and Nakai, 1980; Townsend and Nakai, 1983; Nakai, 1983; Voutsinas et al., 1983). It is conceivable that at or near the isoelectric pH of a protein, hydrophobic interactions are facilitated, thus enhancing hydrogen bonding and electrostatic interactions between polypeptides in the film. This premise has been supported by experiments on the effects of salts enhancing the foam stability of a protein (Barbeau and Kinsella, 1986). The surface activity (rate of surface pressure development) of p-Lg was maximum around pH 4.9, which is slightly below the isoelectric point of 5.25 (Waniska and Kinsella, 1985). The highest rates of adsorption and rearrangement are observed when proteins possess limited net charge (Graham and Phillips, 1979), reflecting minimum electrostatic repulsion between polypeptides at the interface (MacRitchie, 1978). Surface viscosity, surface elasticity, and dilatational moduli are measurements of the cohesiveness, rigidity, compressibility, and extensibility of films (Graham and Phillips, 1976a,b). All are relevant to foam stabilization and indicate the yield strength, elasticity, and flexibility of the film and determine its capacity to withstand the normal shocks and stresses encountered in foams. Films with a yield point and viscosity can dissipate sheardshocks and the elasticity helps restore the film. Excessive viscosity results in brittle films (MacRitchie, 1978).

C. WHEY PROTEIN FILMS Whey proteins possess good surface active properties. Jackson and Pallansch (1961) reported that the relative rates of adsorption of whey proteins occurring at an interface were in the order BSA > a-La >> pLg. Partial unfolding of p-Lg improved its rate of adsorption. Native pLg, a-La, and BSA showed rather similar aidwater interfacial adsorption behavior. Heating of a-La and/or p-Lg markedly accelerated the rate of surface pressure development, particularly during the initial 3- to 5-min adsorption period (Jackson and Pallansch, 1961). After 10 min, the surface pressures at surface concentrations of 2 m g h ’ were 12 dynekm for native and 16 dynekm for heat-treated p-Lg. The surface pressure for native a-La was approximately 16 dynekm and 21 dyne/cm for heattreated a-La, while the value for heated-treated BSA was approximately 15 dynekm. The initial development of surface pressure was closely related to the diffusion coefficient during the first minutes of measurement (Jackson and Pallansch, 1961). Tornberg reported that p-Lg adsorbed more rapidly than BSA and observed that the ability of whey proteins to form an interfacial film was enhanced by 0.2 M salt. The rate of diffusion and adsorption of whey

396

J.

E. KINSELLA

AND D. M. WHITEHEAD

protein was greatly decreased below concentrations of 1 mg/dl (Tornberg 1978a,b). Shimizu and co-workers (1981) reported that whey proteins form films containing 2-3 mg tightly polymerized protein per mz of film surface. The amount of protein in the interfacial film was 2.0, 7.6, 3.0, and 2.7 mg/mz at pH 3, 5, 7, and 9, respectively. Analyses of the film material indicated that a-La, BSA, and p-Lg were preferentially adsorbed at pH 3, 5, and 7-9, respectively. Waniska and Kinsella (1985) demonstrated that the rapid adsorption of p-Lg was accelerated at pH 5.3, close to the isoelectric point, although true steady-state “equilibrium” was not attained until after 360 min. The maximum surface pressure reflected both the adsorption of the protein and the subsequent rearrangement of the adsorbed protein molecules in the film. Maximum surface viscosity occurred in the pH range 5-6 and decreased by 40% at pH 7.0. These values correspond to the pH at which foams of p-Lg show maximum strength. In the case of BSA, surface pressure development, surface viscosity, surface yield stress, and film elasticity all showed maximum values in the pH range 5-6. Significantly, maximum foam stability was observed in the same pH region, indicating a relationship between film and foaming properties (Kim and Kinsella, 1985).

D. FILM-FORMING PROPERTIES OF GLYCOSYLATED P-LACTOGLOBULIN The balance of forces operating between molecules at the interface is subject to protein-protein and protein-solvent interactions, and disruption of these interactive forces by altering protein structure and conformation may either enhance or diminish protein film-forming properties. Protein hydrophilicity,as well as protein hydrophobicity, net charge, and osmotic and steric effects, contribute to protein film formation since the hydrophilic segments of proteins resist penetration of the interfacial film as this would require dehydration of the tightly bound water groups, a thermodynamically unfavorable process (Cumper and Alexander, 1951 ; Phillips, 1977; Waniska and Kinsella, 1987). The cumulative effects of glycosylation on P-Lg were an enhanced molecular weight, reduced net charge, increased relative viscosity, and a significant reduction in the amount of a-helical content. These parameters directly reflected changes in the size, shape, and conformation of the derivatized protein molecules. Because of apparent enhanced hydrophilic interactions between the modified proteins in water, a reduction in ionic and hydrophobic interactions resulted in destabilization of the native

PROTEINS IN WHEY

397

structure of p-Lg (Waniska and Kinsella, 1987; Kinsella and Whitehead, 1988).

The added glycosyl moieties on p-Lg enhanced hydrogen bonding between neighboring protein molecules and solvent and alteration of net charge on the molecule caused changes in the nature and magnitude of intermolecular forces between protein molecules in the film, i.e., reduction in sensitivity of the protein to pH because of diminished elctrostatic forces (Kuntz and Kauzmann, 1974; Waniska and Kinsella, 1987). Changes in the hydrodynamic volume of modified proteins apparently contributed to the reduced rate of diffusion to the interface, thereby slowing the rate of surface adsorption. Loss of conformational energy upon glycosylation (loss of some secondary structure) resulted in a decreased gain in free energy of the modified proteins upon adsorption at the interface. The combination of these factors reduced the extent of protein interactions, i.e., hydrogen bonding and electrostatic interactions, which resulted in weakened films in foams and emulsions (Kinsella and Whitehead, 1988). X. FOAMS

A typical foam is composed of millions of bubbles each encapsulated by a protein film and separated by thin water-filled canals (lamella). Foam bubbles tend to assume polyhedral shapes and water is held in the space between adjacent bubbles by capillarity, by binding to exposed polar residues of the protein, and to some extent by negative pressures in the Plateau border regions. Maintenance of the lamella is essential for foam stability because contact between adjacent bubbles results in disproportionation, coalescence, and collapse of the foam. Separation of adjacent bubbles is aided by electrostatic repulsion between the adjacent films, by steric hindrance (which retards contact), and by retention of the lamellar water column. This is affected by its viscosity (more viscous fluids are held more effectively), and to some degree by the negative pressure at Plateau borders (Halling, 1981; Kinsella, 1981). Foam instability is enhanced by fluid drainage from the lamella due to gravity and rupture of the film resulting from shocks, etc. Drainage can be slowed either by increasing viscosity or by using film materials with polar water-binding groups which possess high surface viscosity. Such features as high yield values and surface viscoelasticity reflect strong cohesion between the protein molecules in the film and also reflect the properties of the component proteins, i.e., their surface activity, molecular flexibility, and ability

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J. E. KINSELLA AND D. M. WHITEHEAD

to interact with neighboring molecules. Proteins that form cohesive films with high surface viscoelasticity generally form more stable foams (Graham and Phillips, 1976a; Halling, 1981). The foaming capacity of proteins is related to the rate of decrease in surface tension and rate of film formation, while foam stability very much depends on the nature and strength of the film, reflecting the extent of protein-protein interactions within the film matrix per se (Table XIX). Thus, while flexible proteins like p-casein can reduce surface tension very rapidly and facilitate a large volume increase in a short time, the foam is relatively unstable because of low resistance to shear, reflecting limited interactions. Graham and Phillips (1976a) and Kim and Kinsella ( 1985) have elucidated relationships between film-forming behavior and foaming properties of BSA and p-casein. p-Casein is very surface active and rapidly forms foams, but because of limited protein-protein interactions, the foams collapse easily. In contrast, globular proteins such as BSA, which retain considerable tertiary structure at the interface, form stable foams because of more extensive intermolecular network formation. A. PROTEIN STRUCTURE AND FOAM STABILITY The capacity of proteins to form foams is important but the ability to form viscous foams that are stable is required in food applications. Structural features which favor rapid foam formation, i.e., low molecular weight, amphipathic flexible molecules, may not be conducive to the formation of stable foams. Upon formation of a protein-encapsulated bubble, the component proteins should interact extensively via hydrogen bonding, and electrostatic and hydrophobic interactions (and perhaps disulfide bonds) to form a strong viscoelastic, continuous, cohesive, polyTABLE XIX SOME FACTORS AFFECTING STABILITY OF PROTEIN-BASED FOAMS

Enhance stability

Reduce stability

Increased viscosity of aqueous phase Protein concentration and film thickness Film mechanical strength and yield stress Surface viscoelasticity Gibbs-Marangoni effect Film net charge Heterogeneous proteins with residual tertiary structure

Drainage (gravitational) Disproportionation Mechanical shockdvibrations Capillary pressureldrainage Permeable film Surface active lipids Temperature Overwhipping

PROTEINS IN WHEY

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meric, impervious film that retains the air and provides a strong structural matrix for the foam. Several forces act to destabilize a foam but drainage of the lamellar water is a major cause leading to foam collapse. This can be minimized by maintaining capillary hydrostatic pressure, increasing viscosity, and preventing approach and contact of the films of adjacent bubbles; an event resulting in eventual coalescence and rupture of the bubbles. During drainage, van der Waals’ attractive forces between adjacent films increase as the distance apart decreases. These are counteracted by repulsive forces acting between protein groups, i.e., electrostatic forces, steric hindrance, volume restriction, and osmotic effects between protein moieties. However, over time, bubbles coalesce by disproportionation, films become thin and rupture, and fluid is lost via drainage, resulting in eventual collapse of foams (MacRitchie, 1978; Halling, 1981; Phillips, 1981). The rates of these consecutive events are greatly affected by the properties and composition of the protein films. A heterogeneous population of proteins with a range of properties, for example, the proteins of egg white, form more stable films. Soluble proteins which can orient and interact to form thicker, more viscous films generally create the best foams. It has been reported that acidic-basic protein mixtures generally display improved foaming properties as a result of enhanced electrostatic interactions between protein molecules at the bubble surface (Poole et af., 19876). However, the differences in pls of the acidic and basic proteins must be sufficiently large so that at intermediate pHs, interaction is strong enough to yield good foaming properties. The ability of basic proteins, e.g., lysozyme and clupeine with PI>9.0, to enhance the foaming power of acidic proteins (e.g., p-Lg BSA) is attributed to not only net charge effects but also to the molecular size and conformation of the complex (Poole et af., 1987b).The addition of salts improves foaming by modifying net charge (masking of net repulsive forces), enhancing adsorption, and perhaps preventing excessive denaturation during foam formation (Halling, 1981; Kinsella, 1981). For optimal foam formation a protein should rapidly diffuse to the newly created interface and possess sufficient segmental molecular flexibility to spread at the interface, reduce interfacial tension, facilitate surface expansion, and encapsulate the nascent air bubble with a protein membrane. The protein, in adjusting to new thermodynamic conditions, undergoes some conformational changes: the hydrophobic segments or loops occupy the apolar air phase, while the polar and/or charged groups occupy the aqueous phase, and the major bulky tertiary structure occupies the interface (Graham and Phillips, 1976a; Phillips, 198I). Ideally, segments of the protein in the aqueous phase should be charged to repel the adjacent film and entrap water, thereby retarding drainage. Simultane-

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ously, there should be extensive protein-protein interactions (ionic, hydrogen bonding, van der Waals’ and hydrophobic interactions) to form a continuous, three-dimensional cohesive film that is impermeable to air and possesses mechanical strength, viscoelasticity, and extensibility in order to stabilize the film against shocks, gravity, and rupture. The component protein molecules in the film (or in the aqueous solution) should possess sufficient mobility to occupy thin or weakened segments of the film. Input of energy by whipping is required to expand and crate new interface and perhaps aid in spreading the protein at the interface.

B. WHEY PROTEIN FOAMS Whey proteins are reasonably good foamindwhipping proteins but commercial WPC preparations vary immensely in whipping properties because of variability in extent of denaturation of the proteins, high ash content, the presence of lipids and possibly PPs (devilbiss et al., 1974; Richert et al., 1974; Richert, 1979; Phillips et al., 1987). The term “foaming properties” can be misleading, as researchers have reported good foams from whey preparations when the reported overruns may range from 300 to 1500%. Egg white is the premier ingredient in protein-stabilized foams; under normal conditions it forms foams with a 10-fold increase in volume on whipping for 5 min and the foam is stable for 30 min. Most importantly, egg white foam can withstand the addition of other ingredients, e.g., sucrose, and it sets to a rigid, permanent structure on heating. Unfortunately, while whey protein concentrates form good foams, they are not very tolerant of other components and in applications involving heating, the functional performance of whey protein foams is poor. DeVilbiss et al. (1974) studied the overrun stabilities of whey protein foams from 11 different WPC samples containing from 29 to 88% protein and observed remarkable differences in foaming and drainage that appeared to be independent of protein concentration, denaturation, and pH of the respective dispersion. A WPC solution (20% total solids) formed an angel food cake foam similar to that of egg white (devilbiss et al., 1974). However, when the batters were baked at 191”C, the WPC foam collapsed after 15 min of heating. By increasing WPC to 25% total solids, cake volume was retained; however, the texture was crumbly and the cake was filled with large holes, which reflects an unstable foam. In an angel cake, the foam must be capable of incorporating the other ingredients without detriment to the foaming properties and baking. In contrast to egg white, which attained maximum stability after 5 min, WPC required 10 min of whipping time to attain maximum stability. The presence

PROTEINS IN WHEY

40 1

of fat in excess of 0.5 to 0.9% significantly depressed foaming performance. The angel food cake test provides a practical method for evaluating WPC in an actual application, and such tests may become an integral method of evaluating model foams (Harper, 1984). In the application of WPC in foam batters, the addition of 44% sucrose to WPC increased their apparent viscosities from 8 to 34 and 9 to 79 centipoise, respectively, for 14 and 20% WPC total solids (devilbiss et al., 1974). The addition of sucrose also enhanced foam density from 0.16 to 0.2 g/ml, but the foam was too light to hold the other flour ingredients and the batter collapsed on mixing. Increasing the solids concentration reduced the rate of drainage of lamellar water, i.e., at 5 and 15% total solids, the times required for 3 ml to drain were 6 and 15 min, respectively. At around 15% total solids, egg white foam shows superior water retention properties. The addition of sucrose to WPC dispersions containing 20% total solids shows superior water retention compared with egg white, suggesting that drainage is not a major problem with WPC foams containing added sucrose (devilbiss et al., 1974). On whipping of WPC or egg white, there is a progressive increase in the amount of protein denaturation and an enhanced stability of the foam. Heating WPC (10 g/ dl protein) at 55°C for 30,60, and 90 min at pH 4.7 increased the insoluble proteins from 10 to 15 to 2096, respectively. There was a progressive increase in the stability of foams made from WPC containing 10 to 18% denatured protein (deVilbiss et al., 1974). Richert et al. (1974) studied the effects of heat, pH, calcium, redox potential, and sodium lauryl sulfate on the foaming properties of WPC. Heating WPC dispersions above 70°C for 30 min caused protein aggregation, particularly in the pH range 4 to 5 and this impaired foaming behavior. WPC dispersions heated above 70°C required longer whipping times and produced diminished foams with varying stability; overruns were reduced from approximately 1200 to 700%, the foams were very viscous, dense, and resembled whipped cream, while drainage was markedly reduced. Heating, pH, redox potential, and calcium level all had inconsistent effects on whipping time, overrun, drainage, and viscosity of whey protein foams. Higher temperatures of heating and increased reducing conditions reduced the foam volume. Compared to unheated controls, heating at 50", 60", or 65°C for 30 min increased the whipping time from 9 to 13 min, enhanced the overrun, and increased the stability of WPC foams. Heating WPC dispersions from 65" to 70°C improved foaming, whereas higher temperatures impaired foaming properties (Table XX). Maximum apparent stability was observed at approximately pH 7 and 70°C. This has been explained as a balance between the disaggregation effect of pH and the tendency toward aggregation at higher temperatures.

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TABLE XX EFFECT OF HEAT TREATMENT ON FOAMING PROPERTIES OF WHEY PROTEIN CONCENTRATEa

Heating (“C/30 min)

Unheated 65 70 75 80 85

Denatured protein (%)

Overrun

10 20 15 23 38

800 1500 1500 1350 1150 800

60

(%I

Stability (min) 4 14

-

-

“Data after Richert ef al. (1974).

As heating temperature was increased from 65” to 85”C, the denatured protein content increased from 40 to 80%, while the maximum overrun decreased from 1500 to 800%. Increasing the pH from 5 to 7 improved foam stability, and this effect was enhanced at higher temperatures, indicating that “partial” unfolding of whey proteins enhances foaming properties (Richert er al., 1974). Spray-dried WPC powders prepared from cheddar cheese whey and casein whey, containing 49 to 63 and 76 to 80% protein, respectively, were evaluated for whipping and foaming properties (Haggett, 1976). Dispersions of 10% protein preparations, adjusted to pH 6.0 or 8.5, were whipped using a typical electric mixer for 6 min. Generally, higher foam volumes, i.e., 1000 versus 1450% overrun, were observed at pH 6.0 and 8.5, respectively (Haggett, 1976). The presence of sucrose in the mix significantly depressed foaming. Heat treatments up to 55°C for 5 to 10 min enhanced whipping, particularly at pH 6.0, and also improved stability. Whey pasteurization enhanced the foaming properties of the treated proteins. Heating WPC containing antifoaming lipids may cause the formation of lipoprotein complexes which precipitate out of the foaming mix and facilitate foam formation (Haggett, 1976). C. FACTORS AFFECTING THE FOAMING PROPERTIES OF WHEY PROTEINS The whipping properties of WPC are influenced by the source, method of manufacture, pasteurization, extent of clarification (removal of precipitated materials and lipids formed during pasteurization), methods used

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for concentrating the protein (ion exchange, diafiltration, gel chromatography), and conditions of drying. The amount of fat, calcium, and other components present in WPC preparations markedly affected foaming properties (Cooney, 1974; deVilbiss et al., 1974; Richert et al., 1974; Haggett, 1976; Richert, 1979; Phillips et al., 1987). The foaming properties of WPC generally increased with increasing solids content, with an optimum observed around 10 g/dl protein. This has been attributed to the viscosity effect and increased protein concentration. Solids content ranging from 2.5 to 35% and protein from 1.5 to 17.5% yielded overruns from 300 to 1400% without any apparent consistency and foam stability was highly variable. Generally, foam stability increased at higher solids though exceptions have been observed (devilbiss et al., 1974; Richert et al., 1974; Richert, 1979). The effects of pH are significant, but wide variations have been noted depending on the composition of the whey foam, the salt concentration, and the presence of glycomacropeptides in renneted wheys. Maximum foam volumes in the pH range 5-6 and maximum stabilities have been reported around the isoelectric pH, presumably because of enhanced protein-protein interactions in the film (Cooney, 1974). Richert (1979) has noted the difficulty in concluding anything about the affects of pH on foaming other than it is an important variable which must be controlled. Whey protein concentrates (73% protein) that displayed 90% solubility and were prepared from renneted casein by ultrafiltration and spray drying had excellent foaming properties (Morr, 1985). Overrun was much better at pH 4.5 than at pH 9, while stability was better at pH 9. Ions, by affecting protein conformation and solubility, influence film formation and hence foaming properties. Some investigators have reported that calcium chloride decreased the overrun and firmness of WPC foams, whereas others reported these properties as improved. Calcium is more effective than equimolar concentrations of sodium in decreasing foam stability and this has been attributed to a decrease in the thickness of the electrical double layer, thereby facilitating coalescence of proteincoated air bubbles (Cooney, 1974). Foam stability decreased linearly with the square root of ionic strength and maximum overrun occurred at 0.05 M NaCl. The effects varied with ion species and concentration; the progressive replacement of calcium with sodium reduced foaming capacity but apparently had little effect on foam stability (Johns and Ennis, 1981). The addition of sucrose or soluble starch to WPC solutions before whipping decreased overrun (Haggett, 1976). The effects of sucrose on protein solutions are attributed to the increase in viscosity of the solution, but it is also conceivable that sucrose, by increasing the stability of the protein, minimized unfolding at the interface, thereby decreasing foam-

404

J. E. KINSELLA AND D. M. WHITEHEAD

ing. Phillips clearly showed that sucrose improved the heat stability of pLg and whey protein foams (Phillips, 1988). The presence of small amounts of fat in whey protein preparations causes foam instability, particularly if the lipids are composed of monoglycerides and polar lipids. These may cause desorption and also weaken the protein film. Centrifugation of WPC solutions to reduce lipid content resulted in a marked increase in foam overrun (Cooney, 1974). The addition of phospholipids to WPC solutions increased overrun but decreased stability (Cooney, 1974). Heating WPC solutions reduced the negative impact of the lipid materials, conceivably by enhancing binding to the proteins. The addition of basic proteins (e.g., clupeine) to acidic proteins (e.g., BSA, WPI, WPC) dramatically enhanced the lipid tolerance in protein foaming systems (Poole et al., 1986). However, the foaming power is highly dependent on both the type of acidic protein and the type of lipid incorporated; BSA was more effective than either WPI or WPC. Lecithin, which can disrupt the film more effectively than other types of lipids, can only be tolerated at low levels compared with corn oil and butterfat (Poole et al., 1986). The addition of clupeine markedly improved the foaming performance of whey protein and egg albumen contaminated by lipids, while also withstanding the addition of sugar, and such mixtures could produce novel aerated foods (Poole et al., 1986). Generally, mild heat treatment induces the partial unfolding of whey proteins, thereby reducing whipping times, increasing overrun, and enhancing the stability of WPC foams. However, several investigators have reported contrary results (devilbiss et al., 1974; Richert et al., 1974; Richert, 1979). The efficacy of heating is influenced by the pH of the protein solution; mild heating (below 70°C) is preferred at acidic pH, e.g., pH 5.0, whereas higher temperatures (above 80°C) are preferred around neutral pH (Richert et al., 1974). Whey protein preparations contain varying quantities of PPs which are surface active and, at concentrations of 1-2%, they can be potent defoaming agents (Volpe and Zabik, 1975). However, it has been shown that the pp was not the foam depressant in whey preparations but a high-molecular weight (> lO0,OOO) lipoprotein component may be responsible (Phillips, 1988). Because of the confusing state of the literature, it is recommended that foaming properties be evaluated for each particularly designed food system. Generally, the literature indicates that whey protein foams do not possess the same properties as egg white foams and whey proteins cannot presently be used interchangeably without prior modification. There is a need for standardized methods and criteria in assessing the whipping properties of whey proteins. Furthermore, data relating whipping behavior in model systems to actual food systems is questionable. The approaches of Harper (1984) and deWit (1984) in using appropriate

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food systems warrant evaluation. A method has recently been described for the evaluation of foams which offers both practicality and standardization (Phillips et al., 1987).

XI.

EMULSIONS

Emulsions are heterogeneous systems consisting of one or more phases dispersed in a continuous phase. Stabilization of an emulsion system is achieved by amphiphilic surface active agents possessing an affinity for both phases. The major function of emulsifiers is to reduce the interfacial energy and facilitate dispersion of the discontinuous phase. In proteinstabilized emulsions, the role of the protein is to form an interfacial membrane rapidly around the oil droplet to prevent coalescence, flocculation, creaming, and oiling-off. Different criteria are required in a fluid compared to a viscous emulsion: in salad dressing, the rheological and viscoelastic properties of the film formed are critical, while in sausage-type products, thermal stability of the film and the ability to set to age1 on cooling are important properties of the component proteins (Kinsella, 1984b). Protein must be soluble to be an effective emulsifier (Halling, 1981; Kinsella, 1982). The ability of a protein molecule to unfold at the interface, exposing hydrophilic and hydrophobic residues toward their preferred aqueous and nonaqueous environments on either side of the Gibbs’ surface is critical (Dickinson, 1986). The capacity of a protein to produce an emulsion of large interfacial area correlates strongly with its ability to lower the interfacial tension at the oil/water interface. Factors which influence the conformation of protein, i.e., those that facilitate the unfolding of protein at the oil/water interface subsequent to adsorption, may improve the emulsifying properties of whey protein (Hayes et al., 1979; Shimizu et al., 1981). A. EMULSIFYING PROPERTIES OF WHEY PROTEINS

The adsorption of whey proteins onto the surface of a fat globule is selective and influenced by pH, presence of salts, protein concentration, and temperature (Yamauchi et al., 1980). The creaming stability of whey protein-coconut oil dispersions was minimal at pH 5 , while viscosity and adsorption of protein were maximum at pH 5 , suggesting that emulsion stability is dependent on the electrostatic nature of the proteins (Yamauchi et al., 1980). However, the effective hydrophobicity, i.e., the abundance of apolar groups exposed at the molecular surface, may also be an important factor in protein adsorption because, initially, hydrophobic interactions are predominant between the protein and oil.

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J. E. KINSELLA AND D. M. WHITEHEAD

Whey proteins can be effective emulsifying agents (Graham and Phillips, 1976b; Tornberg and Lundh, 1978; Morr, 1981; Kinsella, 1983) and display average emulsifying properties (dewit, 1981 ;Reimerdes and Lorenzen, 1983; dewit, 1984). The emulsifying properties of whey proteins may be improved by partial unfolding, especially during the process of emulsion formation, e.g., homogenization in food systems (Tornberg and Hermansson, 1977). Slack et af. (1986) assessed the emulsifying properties of oil/water emulsions stabilized with either p-Lg-enriched or a-La-enriched WPC samples. Similarities in the data for emulsifying capacities and stabilities between WPC and p-Lg-enriched samples suggest that the origin and processing of whey have little effect on their emulsifying ability. The results also indicate that p-Lg-enriched samples are adequate emulsifying agents while emulsions made with a-La-enriched samples display average emulsion capacity but poor stability (Slack et al., 1986). The emulsifying activities (EA) of reduced, urea-denatured, and acylated BSA over the pH range 2-10 were determined in order to assess the importance of protein structure and charge on emulsifying properties (Waniska et al., 1981). Reduction of disulfide bonds permitted the BSA to unfold into a more expanded conformation. This resulted in a decreased EA compared with the native protein, suggesting that native BSA, with a greater degree of tertiary structure, formed a stronger and more cohesive film. Furthermore, the EA of reduced BSA was more sensitive to pH changes compared with native BSA, indicating greater response to electrostatic repulsions, particularly at the higher pH values. Complete disruption of the tertiary and secondary structure of BSA by urea eliminated its EA (Waniska et al., 1981). Shimizu et al. (1981) observed that emulsions of coconut oil stabilized with whey proteins contained three times more protein associated with the interfacial material at pH 5 than at pH 7. At pH 9, p-Lg was selectively adsorbed and was the predominant protein in the isolated interfacial material. At pH 3 the amount of a-La associated with the membrane had progressively increased. Apparently, pH-dependent conformational changes affected adsorption and emulsifying properties, i.e., alkalineinduced molecular expansion of p-Lg causes the protein to become adsorbed more readily, while in the acidic pH range a-La may lose the stabilizing effect of bound calcium, permitting facile adsorption and spreading. B. MOLECULAR FLEXIBILITY, SURFACE HYDROPHOBICITY, AND EMULSIFYING PROPERTIES Ample evidence exists which indicates that the ease of protein unfolding and the accessibility of hydrophobic residues at an interface are

PROTEINS IN WHEY

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closely related (Kato and Nakai, 1980; Kato et al., 1981; Morr, 1981; Voutsinas et al., 1983). The number and disposition of apolar groups contribute to the effective hydrophobicity of proteins by promoting a greater affinity of the protein for the oil phase, which facilitates reorientation of polypeptide segments. Shimizu et al. (1985) investigated the relationship between protein hydrophobicity and emulsifying properties of p-Lg at different pH values. Although the surface hydrophobicity of p-Lg changed on lowering the pH, no significant difference in the secondary structure of the protein between pH 3 and 7 was observed by circular dichroism (CD) spectral analysis. Results from sedimentation velocity analysis, surface tension measurements, and urea and guanidine hydrochloride denaturation experiments strongly indicated that p-Lg has a relatively rigid conformation at pH 3 and resists surface denaturation (Shimizu et al., 1985). This structural feature may explain the reduced protein adsorption and low emulsifying activity observed at pH 3. Changes in the emulsifying properties of several food proteins, e.g., pLg and BSA, were monitored during heat denaturation and the results were correlated with changes in surface hydrophobicity, which were measured with the fluorescent probe cis-parinaric acid (Kato et d.,1983). The emulsifying activity and emulsion stability of all proteins examined displayed a linear correlation with surface hydrophobicity, although protein structure, measured by CD spectroscopy, was altered significantly during heat denaturation, particularly for BSA and p-Lg (Kato et al., 1983). The emulsifying activity of p-Lg and BSA decreased, along with a reduction in the surface hydrophobicity, in proportion with the amount of heat-induced denaturation. This may be explained by the fact that both native BSA and p-Lg are hydrophobic proteins and the significant structural changes observed at the thermal transition points triggered conformational changes which reduced the accessibility of apolar residues to hydrophobic probes. Kato et ul. (1985) have asserted that the functional properties of proteins, particularly surface active properties, cannot be attributed to surface hydrophobicity alone. For example, a-La shows average emulsifying and foaming properties but has a low surface hydrophobicity (Kato et al., 1985). A possible relationship between protein flexibility and surface behavior has been pointed out by several investigators (Shimizu et al., 1981; Townsend and Nakai, 1983; Kato et al., 1985, 1986). The presumption that flexible protein molecules are more susceptible to surface denaturation (and thus to molecular rearrangements) at interfaces than rigid protein molecules has been demonstrated by proteolysis of adsorbed proteins (Kato et d.,1985, 1986). BSA and p-Lg are susceptible to protease digestion and this correlates with a high emulsifying activity index compared with lysozyme and ovalbumin, which were resistant to protease

408

J. E. KINSELLA AND D. M. WHITEHEAD

digestion and displayed inferior emulsifying properties (Kato et al., 1985). Since protein unfolding is known to increase protease susceptibility (Privalov, 1979), it is probable that ovalbumin and lysozyme retain more folded conformations at the interface because of their more compact conformations since they form less cohesive films. Thus, molecular flexibility along with surface hydrophobicity may be considered important structural factors governing the surface properties of proteins. C. FACTORS AFFECTING EMULSIFYING PROPERTIES Compositional factors such as lipid, ash, and sulfhydryl content also contribute to the emulsifying properties of whey protein concentrates. Peltonen-Shalaby and Mangino (1986) have proposed using the sulfhydryl content of WPC samples to predict the performance of various WPC samples in aerated emulsion systems. Whey proteins had good emulsifying properties in fluid emulsions but were less effective than isolated p-Lg, which was inferior to casein, in ease of emulsion formation (Pearce and Kinsella, 1978). Heat-denatured WPC in solutions of sodium phosphate displayed good emulsifying properties at pH 6.4 and maximum stability at pH 7.0 (Mutilangi and Kilara, 1985). The emulsifying capacity of whey proteins is greatly affected by the extent of denaturation and loss of solubility and this may account for some of the variations observed in the emulsifying properties of different whey preparations. The emulsifying systems used in many studies which rely on a high energy input may partly overcome this problem by enhanced physical spreading of the protein at the interface during homogenization. The various factors which affect the stability of emulsions made with milk proteins have recently been reviewed by Leman and Kinsella ( 1989). XII. LIGAND BINDING BY WHEY PROTEINS A.

LIPID BINDING

The interaction between ionic surfactants such as sodium dodecyl sulfate and whey proteins is pH dependent (Brown, 1984). Apparently, the initial interaction (in the pH range 5.5 to 7.0) is electrostatic, followed by hydrophobic interactions between the lipid hydrocarbon chain and the protein (Brown, 1984). This is consistent with the fact that, at pH 5 , a greater amount of whey protein remains with emulsion droplets that have been subjected to low centrifugal forces than at pH 7, where only one-

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third of the protein remains associated with the emulsion (Yamauchi et al., 1980). Binding of ionic surfactants to p-Lg at high molar ratios of surfactant to protein (S/P = 1600) increased the -helical content to twice that observed when the protein is in buffer alone (Mattice et al., 1976) and perhaps this stabilizes the protein against thermal denaturation (Hegg, 1980). Both p-Lg and BSA can bind a variety of apolar molecules, especially fatty acids and amphipathic lipids (McMeekin et al., 1949). Binding of the detergent n-octyl benzene-p-sulfonate (OBS) to p-Lg is concentration dependent: at low OBS concentrations, the p-Lg dimer bound two to three molecules of detergent (intrinsic dissociation constant of 1.59 x lop5M) and binding was attributed to hydrophobic interactions between the protein and the apolar tail region of the OBS molecule (Hill and Briggs, 1956). At higher detergent concentrations, a stoichiometry of 1 mol of detergenvmol of protein monomer for native, S-carboxymethylated-, alkylated-, and carboxypeptidase A-hydrolyzed p-Lg was observed and the binding interaction involved the charged head group of the OBS molecule with basic amino acid residues of p-Lg (Seibles, 1969). Recently, studies using gel filtration chromatography demonstrated that p-Lg can bind p-nitrophenyl phosphate @-NPP), an analog of retinol, with a stoichiometry of 1 mol ligand per 18,360-Da monomer (Farrell et al., 1987). Fluorescence intensity of the protein was quenched upon binding p-NPP, and the CD spectra (260 to 300 nm region) implicated tryptophan and phenylalanine residues in the binding of the ligand (Farrell et al., 1987). These results coupled with the K D values obtained from gel filtration clearly indicated the formation of a complex between p-Lg and p-NPP. The K Dfor the p-Lg-p-NPP complex is independent of pH in the range 4.0 to 7.5. In addition, a complex of p-Lg and dodecyl sulfate bound retinol in an manner analogous to the native protein (Fugate and Song, 1980). Robillard and Wishnia (1972) estimated that the apolar binding site of p-Lg may be limited in size to single ring aromatics. The tryptophan and phenylalanine residues which are purportedly involved in binding of p-NPP (or retinol) have been located at or near the p-barrel structure recently elucidated by crystallographic studies (Sawyer et al., 1985 ;Papiz et al., 1986; Monaco et al., 1987). This structure has been identified as an ideal structural motif for the transport of small apolar molecules such as retinol (Sawyer, 1987). Retinol forms the tightest binding complex with p-Lg (Table XXI) and the strong structural homology between retinolbinding protein (RBP) and p-Lg points to a possible evolutionary and functional relatedness (Sawyer et al., 1985; Papiz et al.. 1986). However, further experimental evidence is required in order to establish the biological role of retinol binding and transport for p-Lg (Sawyer, 1987).

410

J. E. KINSELLA AND D. M. WHITEHEAD

TABLE XXI COMPARISON OF THE DISSOCIATION CONSTANTS ( K D )OBTAINED FOR BINDING OF NONPOLAR COMPOUNDS BY P-LACTOGLOBULIN A

Ligand

KD ( P ‘ w

Reference

Toluene Pyridoxal phosphate p-Nitrophenyl phosphate Retinol

2200 320 31 0.020

Robillard and Wishnia (1972) Farrell ef al. (1987) Farrell el a / . (1987) Fugate and Song (1980)

B. FLAVOR BINDING Because overall flavor, as perceived by the consumer, is important in determining the acceptability of many foods, interactions of flavors with proteins is of practical interest. A problem rarely mentioned is the offflavor associated with whey proteins. The components responsible for off-flavor development have not been quantitatively identified, but may include some volatile fatty acids, carbonyls from oxidized lipids (e.g., phospholipids, lactic acid/esters), and amino acid products. These conceivably are bound to whey proteins such as BSA and p-Lg, both of which have a high affinity for apolar ligands (O’Neill and Kinsella, 1987a). The binding of flavors and off-flavors to food components, especially proteins, is a challenging problem as food technologists attempt to fabricate and flavor new foods using protein ingredients. For example, the flavor characteristic of meats consists of at least 400 flavors and, to manufacture meat analogs with the appropriate flavor, correct concentrations, binding characteristics (i.e., relative binding affinities, release rates, and partitioning of individual flavor components between food components), and, particularly, partitioning between the complex food medium and the appropriate oral chemoreceptors need to be determined. The consequences of flavor binding to the food industry are actually twofold because, in addition to off-flavor development, loss of desirable flavor in the formulated food may also occur (Mills and Solms, 1984). Furthermore, though functionally acceptable, many protein preparations possess undesirable flavors, and release of these reversibly bound off-flavor compounds hinders their more widespread use in foods (Kinsella and Damodaran, 1981). The ability to either mask undesirable flavor(s) or simulate the desired food flavor is significantly influenced by the flavor-binding capacity of the chosen protein. The structure of some proteins may enhance or dimin-

PROTEINS IN WHEY

41 1

ish their capacity to adsorb added flavors and variable amounts must be added to obtain the desired flavor impact. Binding of flavor compounds by protein results in the suppression of their primary flavoring impact. Therefore, techniques for controlling adsorption of specific flavors must be sought and used in fabricated foods. The excessive binding of flavors by proteins, the uneven retention of flavors during processing treatments and storage, and/or the preferential release (or retention) of some components of a flavor blend during mastication, are problems confronting the manufacturer of fabricated foods. Because the perceived flavor is ultimately most important in determining food acceptability, the phenomenon of flavor binding and release is extremely significant. Information concerning the interaction of selected flavor compounds has previously been reviewed (Beyeler and Solms, 1974; Franzen and Kinsella, 1974; Damodaran and Kinsella, 1980; Kinsella and Damodaran, 1981; Kinsella, 1989). For the fabrication of food, knowledge of the affinity of food components for flavor compounds and the differences in binding characteristics of different food components are critically important. Quantitative data are needed to determine the binding of flavors to proteins in order to compare the binding affinities of different proteins, and to determine if there are differences between the extent of binding of components in a flavor blend. Furthermore, the rate and extent of release of flavor compounds from proteins during ingestion and mastication are important. However, little information is available in this highly relevant area. The binding of saturated aldehydes and methyl ketones (e.g., heptanol and nonanone) to unprocessed whey protein has been studied, with emphasis on the effects of lactose, salt, and residual fat, in order to understand off-flavor development in commercial products (Mills and Solms, 1984).The lactose (up to 82 g/lOO g protein) and salt (0.5 M ) contents of all samples tested had little effect on the binding of heptanol; however, the effect was more marked with nonanone. Very little difference in the binding of heptanal was observed when the fat content of the whey powder was reduced from 4.82 gAO0 g protein to 0.91 g/lOO g protein, whereas the binding of nonanone decreased by 50% with the same decrease in fat content (Mills and Solms, 1984). The effect of pH and temperature on the binding of flavor compounds is of particular interest since many different conditions of pH and temperature are employed during the industrial isolation of whey proteins (Marshall, 1982). At pH 6.89 and 25"C, the binding of heptanal was greater than the binding of nonanone, while at pH 4.66, the opposite effect was observed (Mills and Solms, 1984). As temperature was increased from 25" to 50°C.the amount of heptanal irreversibly bound to whey protein

412

J . E. KINSELLA AND D. M. WHITEHEAD

increased to the extent that, at the highest total heptanal concentration, only 10% of the heptanal bound at 50°C was released, while binding of nonanone was completely reversible at both pH values and temperatures (Mills and Solms, 1984). Thus, the binding of some classes of compounds to whey protein may be achieved by careful choice of pH, temperature, and processing conditions to minimize the level of bound off-flavor compounds. The predictable effects of various flavors bound by proteins are also complicated by the highly variable, intrinsic properties of the protein components in a food system, i.e., conformation, state of the protein (native/denatured), surface area and topography, and presence of other factors, such as lipids and additional nonspecific interactions between the flavor compounds and proteins (Franzen and Kinsella, 1974; Damodaran and Kinsella, 1980). In order to optimize flavoring of food proteins and to develop practical methods for the removal of off-flavors, the mechanisms and thermodynamics of flavor binding need to be understood. Studies to elucidate the characteristics of flavor-protein interactions have been attempted by employing the classical approach for protein-ligand associations (Damodaran and Kinsella, 1980). For a protein (P)having a number of equal and independent binding sites, the interaction between the ligand, i.e., flavor molecule (L),and the protein may be represented by the equation. P

+ nL = PL,

Based on this model, the interaction between flavor molecules and protein can be represented thermodynamically by the Scatchard equation: V/[L] = nK - VK where f? is the number of moles ligand bound/mole protein, [L] is free ligand concentration, n is total number of binding sites, and K is the intrinsic binding constant. A plot of f?/[L]versus f?gives a straight line with a slope of - K and an intercept equal to nK. This equation assumes no protein-protein interactions at higher concentrations and may only apply to single polypeptide chains, e.g., BSA or p-Lg, such that, at a given free ligand concentration, the molal ratio of binding is the same irrespective of protein concentration (Kinsella and Damodaran, 1981). When analyzing binding, it is important to measure accurately the amount of ligand that is actually associated with the protein. Using a liquid-liquid partition equilibrium method, the kinetics and thermodynamics of flavor binding to BSA and p-Lg have been investigated. The protein in solution was

PROTEINS IN WHEY

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equilibrated with the flavor ligand and the amount of flavor bound to BSA or p-Lg was determined by gas chromatography (Damodaran and Kinsella, 1980; O'Neill and Kinsella, 1987a). Differences in the slopes of the binding curves of 2-heptanone and 2nonanone indicated differences in the affinity of these components for the binding sites in BSA. The initial number of binding sites (approximately 6 to 7) was similar for both compounds. Increasing the chain length of the ligand by two methylene groups increased the binding energy sixfold, indicating that hydrophobic interactions were dominant in the binding of these apolar ligands to BSA. The curvilinear relationships of the binding isotherms were positive, possibly reflecting unfolding of the protein molecule at higher ligand concentrations and resulting in the exposure of nonspecific binding sites which are unavailable in the native protein. Such structural changes were monitored from changes in the UV absorption and fluorescence emission spectra of the protein-ligand complex. Thus binding of these ligands at low concentrations stabilized the BSA molecule initially, but above this concentration the molecule began to unfold (Damodaran and Kinsella, 1980). The presence of certain types of salts affected flavor binding. Ligand binding affinity progressively increased with anion concentration (SO2; > C1- > Br-), in the order of the lyotropic series of anions. This is the order in which these anions stabilize protein structure via enhancement of hydrophobic interactions (Damodaran and Kinsella, 1981a).

C. LIGAND BINDING BY P-LACTOGLOBULIN p-Lg and RBP display a strong sequence homology with respect to several amino acid locations that have been demonstrated to be critical for the maintenance of structure and retinol binding (Pervaiz and Brew, 1985). X-Ray crystallographic analyses reveal striking conformational similarities between p-Lg and RBP, most notably in a cross-hatched, eight-stranded 9-barrel (the core of which is lined with apolar side chains) (Newcomer et al., 1984; Papiz et al., 1986). This particular structural feature has been identified in several extracellular transport molecules and is ideally suited for binding small apolar molecules (Sawyer, 1987). Two molecules of retinol are bound per dimer of p-Lg with a dissociation constant of 2 x lo-" M. The binding is primarily hydrophobic in nature because it is pH independent, and is unaffected by the presence of urea (8 M ) or SDS (Fugate and Song, 1980). The TrpI9 residue (located at the bottom of the p-barrel) apparently is the binding site for the p-ionone moiety of retinol in RBP and is highly conserved in p-Lgs from several species sequenced to date (Newcomer et al., 1984; Pervaiz and Brew,

414

J. E. KINSELLA AND D. M. WHITEHEAD

1985). Although the binding of retinol by p-Lg could reflect a general affinity for small apolar molecules, the relationship between p-Lg and RBP infers that binding is a significant and specific phenomenon that suggests a specific biological function for p-Lg (Pervaiz and Brew, 1985). p-Lg is remarkably stable at pH values below 3.0 (Kella and Kinsella, 1988), an attribute that enables the protein molecule to remain intact under the prevailing acidic conditions of the stomach (Reddy et al., 1988). Thus, it is possible that a biological role of p-Lg may be vitamin A transport in the bovine neonate via p-Lg-specific receptors in the small intestine (Papiz et al., 1986). p-Lg readily binds carbonyls, e.g., alkanones and methyl ketones (O’Neill, 1986), and the double reciprocal plots of 2-heptanone, 2-octanone, and 2-nonanone binding to native p-Lg B indicate that there is one binding site per p-Lg monomer. The free energies of association are compared with those of BSA (Table XXII) (Damodaran and Kinsella, 1981b; O’Neill and Kinsella, 1987a). The binding affinity of alkanones for p-Lg increases with chain length and for each additional methylene group there is a corresponding change in free energy of -3.36 kJ/mol. The effect of chain length on the free energy of association suggests that the protein-ligand interaction is primarily hydrophobic in nature. The binding constants for the interactions of alkanones with p-Lg are higher than those obtained for either BSA or soy protein (Damodaran and Kinsella 1980, 1981b; O’Neill and Kinsella, 1987b). A binding constant of 930 M-’for p-Lg was obtained for heptanone, which is approximately 2.5 times that of soy protein (O’Neill and Kinsella, 1987b). Urea at increasing concentrations caused unfolding of p-Lg and pro-

TABLE XXII THERMODYNAMIC(AG, kl/mol) AND BINDING CONSTANTS ( K , M-’) FOR BOVINE SERUM ALBUMIN AND P-LACTOGLOBULIN”

Ligand 2-Heptanone Protein‘

BSA P-Lg

2-Octanone’

2-Nonanone

K

AG

K

AG

K

A

270 150

- 13.8 - 12.5

(810) 480

(- 16.6)

1800 2440

- 18.5 - 19.4

- 15.4

“Data from O’Neill and Kinsella (1987a) and Damodaran and Kinsella (1980). ”Constants in parentheses are calculated values. ‘Protein concentration was 0.974% at 25°C in 1 M NaCI.

PROTEINS IN WHEY

415

gressively decreased the binding affinity of alkanones, although the number of binding sites remained unchanged (O’Neill and Kinsella, 1987a). Heat treatment of p-Lg at 75°C caused an increase in the number of binding sites for alkanones but the binding affinities of the protein decreased because of heat-induced conformational changes (O’Neill and Kinsella, 1988). Similar results were observed when the free carboxyl groups of pLg were esterified, possibly reflecting a tendency of the modified protein to form hydrophobically associated aggregates (O’Neill and Kinsella, 1988). Thus, the nature and extent of the interactions between flavors and proteins are altered by heat treatment, and proteins may bind more or less of a given flavor compound, depending on the intensity of heat treatment. Because heat-denatured p-Lg forms aggregates with an increased number of binding sites, the strength of the binding is reduced; hence, substitution of heat-treated p-Lg for native p-Lg in a whey-based flavored food might result in increased binding of that flavor than allowed for in the original flavor formulation, resulting in a stronger or unbalanced perceived flavor. D. INTERACTIONS OF FLAVORS WITH WHEY PROTEINS a-La also binds aldehydes and methyl ketones (Franzen and Kinsella, 1974; Jasinski and Kilara, 1985), although the binding capacity is lower than that of p-Lg. Interestingly, whey protein preparations (88% protein) exhibit a very high flavor-binding capacity that apparently exceeds the sum of the binding capacities of the component proteins (Jasinski and Kilara, 1985). This may reflect unfolding and/or denaturation of the proteins, which increases the number of nonspecific binding sites (Franzen and Kinsella, 1974; O’Neill and Kinsella, 1988), and also the presence of lactose, which avidly binds flavors (Nickerson er al., 1976). In considering the binding of flavors to proteins, the association constant is an index of the tendency of that compound to partition to the protein and bind. For sensory perception of the flavor, it must be released from the protein and bind to the chemoreceptor complex; if the binding association is very high, the release rate is low and there is minimal availability of flavor for perception. The rate of association which reflects the diffusion rate constant may be on the order of 10” to 10”. If the association constants are around lo8, then the equilibrium of the on/off reaction rates means that a sizable concentration of the flavor is in the unbound form and available for detection. Thus, the association constant may be a useful index of the efficacy with which flavors are released from food components. It is relatively easy to formulate a blend of flavors in free solution to

416

J. E. KINSELLA AND D. M. WHITEHEAD

provide a desirable sensory impact. However, if a similar concentration is blended in the presence of proteins or other components which bind specific flavors to differing extents, the perceived flavor may not be the same as the original blend. This is a major challenge in formulating food flavors for fabricated and processed foods. Because the difference between desirable and undesirable flavor impact is often one of concentration, knowledge of the flavor binding behavior of food components is critical in determining the acceptability of foods. XIII. MODIFICATION OF WHEY PROTEINS

The importance of functional properties for the proper utilization of whey proteins underscores the need to develop methods allowing manipulation of certain properties in order to suit specific applications. Modification of proteins to alter native chemical and physical properties has been reviewed (Kinsella, 1976; Feeney and Whitaker, 1977; Kinsella and Shetty, 1979; Kinsella, 1982; Richardson and Kester, 1984; Richardson, 1985; Kinsella and Whitehead, 1987). Whey protein modification can be accomplished by enzymatic, chemical, physical, or genetic methods (Feeney and Whitaker, 1977; Fujimaki et af., 1977; Kinsella and Shetty, 1979; Fox et al., 1982; Richardson and Kester, 1984; Richardson, 1985; Kinsella and Whitehead, 1988). A. ENZYMATIC MODIFICATION Protein modification via enzymes generally involves limited proteolysis to yield a mixture of polypeptides. The use of enzymatic modification methods has the advantage of milder reaction conditions and the potential for stereochemical specificity (Adler-Nissen, 1986; Dickinson and Stainsby, 1987). Apparently, there is an optimum degree of hydrolysis beyond which any improvements gained in functional behavior are lost (McNairey, 1984) and there are different degrees of hydrolysis for certain properties, e.g., emulsification and foaming, but the reason for this is unclear. Interpretation of the physicochemical data of partial hydrolysates is complicated by the general lack of information concerning distributions of fragment sizes and peptide composition (Dickinson and Stainsby, 1987).

Enzymes may be used to form intermolecular cross-links, or attach or remove specific functional groups to the protein. Treatment with proteases reduces molecular size and may enhance the hydrophobic/hydrophilic balance, either by generating extra terminal amino and carboxyl

PROTEINS IN WHEY

417

groups or by attaching hydrophobic substituent groups, such as L-leucine N-alkyl esters via the plastein reaction (Yamashito et al., 1979; Arai et al., 1986) to enhance the surface active properties of proteins. Partial proteolysis may facilitate unfolding of polypeptides and thereby enhance certain functional properties, e.g. solubility and also increase the heterogeneity of the protein species (peptide molecular weight distribution), which may enhance foaming properties. Deamidation has been reported to improve many functional properties, particularly solubility, of proteins (Matsudomi et al., 1982). Enzymatic deamidation of glutaminyl and asparaginyl residues in proteins offers advantages over mild acid treatment because the latter treatment results in denaturation of protein and also cleavage of peptide bonds (Kato et al., 1987). Limited proteolysis has been used to improve the foaming properties of whey protein preparations (Kuehler and Stine, 1974; Horiuchi et al., 1978), but because of inadequate protein-protein interactions in the protein films, proteolysis reduces foam stability. Partial proteolysis of whey protein concentrates with trypsin greatly improved thermal stability and improved emulsifying properties (Hidalgo and Gamper, 1977). During proteolysis, there is a tendency to form bitter-tasting peptides (Kilara, 1985), although this is less of a problem with whey proteins than with caseins (Richardson and Kester, 1984). However, enzymes have been utilized in plastein reactions to reduce bitter peptides produced by limited proteolysis (Noguchi et al., 1975; Eriksen and Fagerson, 1976). Overall, proteolytic treatment may provide a practical approach for increasing the use of whey proteins in beverages because of improvement in solubility and stability in acid pH and to heating. (Dickinson and Stainsby, 1987). Enzymes have been used to add functional groups to proteins, e.g., attaching of phosphoryl groups using protein kinase (Bingham, 1976). In addition, transglutaminase has been used to form new intermolecular cross-links, thereby modifying the viscosity, surface activity, and gelling properties of whey proteins (Nio et al., 1986a,b). Tanimoto and Kinsella (1987) demonstrated that cross-linking of p-Lg using transglutaminase increased viscosity and greatly improved the heat stability of the crosslinked protein compared with native p-Lg. B. CHEMICAL MODIFICATION A limited amount of work has been conducted on the chemical modification of whey proteins. A number of the functional side chain groups in a protein, particularly the €-amino group of lysyl residues, can be modified to create protein products with remarkably different chemical and

418

J. E. KINSELLA AND D. M. WHlTEHEAD

physical characteristics. For example, whey proteins with a number of reactive polar ester or amino groups can be chemically modified to improve their surface activity (Feeney and Whitaker, 1977; Richardson, 1985; Arai et al., 1986). Methods to modi€y whey proteins chemically include acylation, phosphorylation, amidation, esterification, reductive alkylation, and thiolation (Richardson and Kester, 1984). 1 . Acylation

Acylation of protein r-amino groups with acid anhydrides is a common method of protein modification. Acetylation eliminates a positive group which reduces the electrostatic attraction between charged groups and reduces the tendency toward gelation (Richardson and Kester, 1984). Succinylation enhances hydration and solubility, improves thermal stability and surface active properties, and generally results in unfolding of the protein molecule as extent of modification increases (Waniska et al., 1981; Shetty and Kinsella, 1982). This is reflected by a marked increase in intrinsic viscosity, an indicator of molecular shape and size, because of the increase in electrostatic repulsive forces, i.e., increase in net negative charge (Shetty and Kinsella, 1982). Presumably, the higher net negative charge of succinylated proteins (at a given pH) should impart enhanced charge repulsive forces between protein encapsulated air or oil droplets at interfaces, thus favoring more stable foams or emulsions (Kinsella, 1976, 1982). Succinylated BSA (>90% lysyl residues modified) exhibited improved emulsifying activity, above pH 5 , compared to native BSA (Waniska et al., 1981), reflecting a more flexible conformation that facilitates diffusion to the interface and rearrangement of polypeptide segments within the interfacial film (Phillips, 1981). However, acetylation of BSA had little effect on protein solubility and resulted in a reduced emulsifying activity between pH 4 and 7 (Waniska et al., 1981). Studies on foams generated from succinylated p-Lg suggest that ionic forces exert considerable influence on molecular interactions in the surface film (L. Phillips, 1988). This is shown by a progressive increase in surface pressure at 25% succinylation of the protein with a concomitant reduction in foam stability of about 3-fold. Although more protein was initially available at the interface as a result of relaxing of protein conformation, critical protein-protein interactions, i.e. ,ionic, hydrophobic, and possibly steric, were diminished to an extent that inhibited stable film formation. Succinylation has been used to enhance the functional behavior of heatdenatured whey proteins. Succinylation of heat-coagulated WPC improved several properties, e.g., solubility and emulsifying power, but did

PROTEINS IN WHEY

419

not improve whippability. The modified proteins absorbed 12-fold and 2fold the molecular weight in water and fat, respectively, and displayed excellent emulsifying properties (Thompson and Reyes, 1980). This product was an effective replacement for sodium caseinate in coffee whiteners, egg yolk in salad dressings, and NDM in meat patties, ice cream, and instant pudding (Thompson and Reyes, 1980; Thompson et al., 1982). 2 . Phosphorylation

Phosphate groups can be covalently attached to free €-amino groups on proteins with phosphorus oxychloride to increase the net negative charge (Woo et al., 1982; Woo and Richardson, 1983; Matheis and Whitaker, 1984). Chemical phosphorylation of proteins may also involve derivatization of the hydroxyl group of serine, the imidazole nitrogen of histidine, and other free amino groups. Furthermore, it may cross-link proteins to varying degrees via phosphate bridges or isopeptide linkages (Woo et al., 1982). Phosphorylation generally enhances several physical properties of proteins, such as solubility (by increasing net hydration of protein chemical groups), electrostatic repulsive forces, water absorption, water-holding capacity, and viscosity, all of which can be exploited for use in commercial food products. The lysyl residues of p-Lg were phosphorylated with phosphorus oxychloride, incorporating approximately 13. mol phosphate per mole of protein and resulted in loss of the native protein conformation (Woo et al., 1982). Phosphorylated p-Lg readily gelled without heating, via calcium cross-linking, to yield a fluid, yogurt-like gel (Woo and Richardson, 1983). Modified p-Lg also displayed markedly improved emulsion stability (creaming properties), presumably because of enhanced electrostatic repulsive forces between emulsion droplets.

3. Amidation and Esterification Generally, proteins possess a net negative charge around pH 7 and this charge can be effectively reduced by derivatization of the carboxyl group of the aspartic and glutamic acid. residues. Amidation is accomplished via a carbodiimide-mediatedcondensation of ammonium ions with carboxylic groups forming asparagine and glutamine, respectively. Esterification is achieved using an acidified alcohol medium (Mattarella et al., 1983). Amidation of 78% and esterification of 83% of-the carboxyl groups of pLg increased its isoelectric pH to 10 and 9.8, respectively. Intermediate levels of esterification yielded proteins with a range of isoelectric points (Mattarella et al., 1983; Mattarella and Richardson, 1983; Halpin and

420

J. E. KINSELLA AND D. M. WHITEHEAD

Richardson, 1985). Modification increased the random configuration of pLg. These positively charged derivatives may form complexes with negatively charged protein components, in the neutral pH range, because of strong electrostatic interactions. This may improve emulsifying and foaming properties since the increased association of modified whey proteins with other proteins during film formation should increase the viscosity of films, resulting in enhanced foam and emulsion stability (Poole et al., 1987b). Richardson and Kester (1984) reported that ethyl-esterified p-Lg absorbed to the interface four times more rapidly than the nonmodified protein. Further research on the properties of amidated/esterified whey proteins is warranted. 4.

Thiolation

The presence of thiol and disulfide groups in proteins markedly affects their functional behavior and provides an excellent approach for modification via disulfide reduction, thiol oxidation, and thiol-disulfide interchange reactions (Cup0 and Pace, 1983; Kella et al., 1986). Alteration of molecular conformation, protein flexibility, and intramolecular crosslinking to enhance solubility, adjust intermolecular interactions, and improve viscosity and network formation necessary in gelation is possible. Free thiol (-SH) groups are involved in many important reactions of proteins required in gelation, thermostability, viscosity, and complexation with other proteins. Reduction of p-Lg with mercaptoethanol or dithiothreitol enhanced its aggregation tendencies and increased its flavor-binding capacity (O’Neill and Kinsella, 1988). Thiolation of proteins is generally accomplished using N-acetylhomocysteinethiolactone (N-AHTL) and S-acetylmercaptosuccinic anhydride (S-AMSA) (Feeney and Whitaker, 1977; Richardson and Kester, 1984). Thiolation with S-AMSA involves alkylation of the amino groups so that the -SH function is protected in the form of an acetylthiol group. The acetyl group is subequently removed by nucleophilic displacement with hydroxylamine (Richardson and Kester, 1984). The reaction with N-AHTL under alkaline conditions involves an imidazole-catalyzedalkylation of the amino groups of the protein followed by opening of the thiolactone ring with exposure of a new -SH group (Richardson and Kester, 1984). 0-Lg has been successfully thiolated using both of these reagents, which acylated the e-amino groups and reduced the isoelectric pH of derivatized p-Lg compared to the native protein. Intermolecular disulfide cross-linking occurred on oxidation of thiolated p-Lg and resulted in highmolecular-weight polymers that apparently possessed enhanced resistance to heat, increased viscosity, improved foaming power, and gelation

42 I

PROTEINS IN WHEY

properties (Richardson and Kester, 1984). In t h e presence of calcium ions, these polymers formed strong, transparent, heat-stable gels. 5. Reductive Alkylution

Methylation or ethylation of amino groups can be accomplished via a reaction using formaldehyde and a strong reducing agent, such as sodium borohydride or cyanoborohydride. Reductive methylation causes minor changes in the conformation of proteins. This is a useful way of labeling proteins with radioactive derivatives, such as [ ''C]formaldehyde. In this way, any changes occurring during processing may be easily monitored in order to study factors affecting protein-protein interactions, surface accumulation of p-Lg, etc. (Richardson and Kester, 1984). 6.

Glycosylution

Is it possible to alter the size, conformation, and physicochemical character of proteins with high-molecular-weightderivatives, e.g., single sugar groups, using relatively mild conditions (Waniska and Kinsella, 1984b). Covalent attachment of carbohydrate residues of varying sizes, e.g., malTABLE X X l l l DIFFERENCES I N COMPOSITION BETWEEN HUMAN A N D BOVINE MILK"

Human milk

Total proteins Caseins Whey proteins a-Lactdlbumin P-Lactoglobulin Lactoferrin Serum albumin Lysozyme kA IgG IgM

Others Lactose Minerals

Bovine milk

gldl

76

ddl

0.89 0.25

100

0.64

65

-

-

0.17 0.05

17 6 6

3.30 2,60 0.70 0.12 0.12 Trace 0.03 Trace

II

0.003

8

0.003

0.25

0.05 0 . 10

0.003 0.002 0.07 7.4 0.2

"Data after Hambraeus (1984).

35 17

96

I 00 79 21 2.5

9.0 I .0 3.0

0.06

0.15

4.8 0.8

4.5

422

J. E. KINSELLA AND D. M. WHITEHEAD

tosyl and p-cyclodextrinyl, to the €-aminogroups of p-Lg altered molecular size, conformation, and several physicochemical properties, including surface hydrophobicity (Waniska, 1982; Kinsella and Whitehead, 1988). Many surface active properties, including the foaming characteristics, of p-Lg were improved as a result of alterations in the molecular nature of the protein induced by the introduction of sugar moieties, i.e., net charge, hydrophobicity, and hydrophilicity (Waniska and Kinsella, 1984a, 1987; Kinsella and Whitehead, 1987). Synthetic glycoproteins of p-Lg have been reported to display high solubility at low ionic strength and improved heat stability compared to the native protein (Kitabatake et al., 1985). XIV. NUTRITIONAL ASPECTS OF WHEY PROTEINS

Infant formula provides a significant potential market for refined whey proteins. Ideally, in preparing commercial formulations the end-product must simulate as close as possible the composition of human milk, particularly with regard to protein content. The disparities in the caseidwhey protein ratios and lactose content between human and bovine milks also require adjustment. In human milk, casein (2.5 g/liter) accounts for only 20% of the total nitrogen. Whey proteins represent 70% of human milk protein and consist mostly of a-La, lactoferrin, lysozyme, y-globulins, and serum albumin (Table XXIII) (Hambreus, 1984). Trace amounts of p-Lg may be present. Lactoferrin is an iron-binding protein which can be involved in supplying iron to the child by facilitating intestinal absorption, in addition to having an apparent bacteriostatic effect and possibly stimulating development of the gastric mucosa (Reiter, 1985). Lysozyme is relatively abundant in human milk and also exerts an antimicrobial function in the intestinal tract (Wharton, 1980; Hambreus, 1984). Lysozyme, like lactoferrin, is relatively resistant to proteolytic digestion. These proteins are present in trace levels in bovine milk. Human milk also contains significant amounts of immunoglobulins (IgG fraction) compared to bovine milk. In the manufacture of infant formula, bovine milk is fortified with additional whey to correspond with the ratio of casein to whey protein (20 :80) present in human milk as closely as possible. The whey is demineralized by ion exchange, electrodialysis, and/or UF/DF to adjust the concentration of minerals, lactose, and NPN materials in the diluent whey (Marshall, 1982; Morr, 1982; Jost e? al., 1987). However, protein compositional differences still exist because bovine milk contains p-Lg, which accounts for approximately 60% of total whey protein whereas human

PROTEINS IN WHEY

423

milk contains higher levels of a-La, lactoferrin, lysozyme, and immunoglobulins and lacks P-Lg. It has been suggested that addition of bovine colostrum may “boost” immunoglobulin concentrations in infant and baby foods. There is concern about limited pepsin digestibility of p-Lg and the allergenic reaction frequently elicited by bovine p-Lg when present in sufficient quantities; consequently, there is interest in reducing and/or eliminating it from infant formula. Thus, methods such as gel filtration, selective precipitation, and enzymatic hydrolysis have been pursued to reduce or remove p-Lg from whey. The addition of ferric chloride (7 mM) to whey at pH 4.5 selectively precipitates p-Lg (Kuwata et al., 1985). However, this method also results in removal of the immunoglobulinsfrom the precipitate. If the whey is adjusted to pH 3 in the presence of ferric chloride (4 mM), then all proteins except p-Lg remain in solution. Excess iron is then removed by ion exchange or U F to yield a whey protein preparation devoid of p-Lg. Selective heat precipitation by pH adjustment is also an alternative because, between pH 2.6 and 3.0, most whey proteins, except p-Lg, are thermally coagulated. The selective thermal precipitation technique of Pearce (1983) for separation of p-Lg from other whey proteins may be useful in this regard. During enzymatic hydrolysis, cleavage of the polypeptide chains destroys existing epitopes on native protein, thereby rendering the protein nonantigenic. Treatment of whey proteins with proteases reduces the antigenicity of both p-Lg and a-La by three orders of magnitude (Pahud et al., 1985; Jost et al., 1987). For specialized dietary uses, there is a growing need for hydrolyzed proteins with a balanced essential amino acid composition. In cases where injury or disease prevents digestion and/or absorption of ingested protein, intravenous feeding of complete whey protein hydrolysates may be of critical nutritional value (Manson, 1980). XV.

SUMMARY AND CONCLUSIONS

There is abundant information concerning the functional behavior of whey proteins in model systems. The data on functional properties reported by different researchers, however, reveal wide discrepancies in values. For example, in the case of comparable whey preparations, apparent solubilities may range from 10 to 100%; strength of gels from 0.3 to >10 N , foam overruns from 250 to 1500%, and foam stabilities from 0.5 to 30 min. Many of the data are of limited value in assessing the true

424

J. E. KINSELLA AND D. M. WHITEHEAD

functional characteristics of different preparations, treatments, or processing effects. Reports to date are useful in indicating the relative behavior of different proteins; however, the data do not always predict the performance of such proteins in actual food systems. This reflects the fact that in foods, extensive interactions with other components may occur, resulting in modified behavior of the proteins. Harper, (1984) has advocated the testing of these various preparations in simulated food systems which should validly relate the behavior to performance in commercial systems. Emphasis on standardization of specific protocols, with regard to order of addition in ingredients, temperature, pH control, and amount of energy input during mixing, homogenization, emulsification, etc. deserves serious consideration. While this approach is justifiable in terms of providing valuable data to commercial users, it does not minimize the importance of examining these proteins in model systems where the physicochemical basis of each functional attribute can be described in molecular terms (Kinsella, 1987). Such information is necessary to expedite appropriate methods of processing in order to control compositional variability, extent of denatauration, and possible protein modification. In addition, rapid, reliable tests for routine quality assurance that can provide practical information concerning functional applications would be of great value. Whey protein preparations vary immensely in functional behavior and are presently relegated to limited use as functional ingredients in the food industry. This need not be the case since conventional and new technologies permit rigorous control of production protocols, e.g., careful control of heat treatments can result in the production of whey protein preparations with consistent, reliable functional properties (dewit, 1981, 1984; Harper, 1984; Morr,1985). As the market for functional proteins continues to expand, the whey industry must seek the means to refine whey protein products; determine useful functional properties; develop standardized manufacturing protocols; demonstrate the effectiveness of whey as a functional ingredient; promote, and then market, whey on the basis of performance at competitive cost. RESEARCH NEEDS Because of the enormous resources of whey available worldwide, there is ample justification in continuing research for its improvement in effective use for the food industry. This is underscored by the urgent need to develop standard methodology for evaluating the functional properties of whey protein concentrates and for correlating these properties with specific commercial uses of the protein, which may encourage effective utili-

PROTEINS IN WHEY

425

zation of whey proteins. Standard methods should emphasize the use of model systems which closely approximate end-use applications. Examples of research needs include Examination of the functional properties of whey proteins in relation to their composition, as affected by lipids, salts, and proteose-peptone content. Development of methods applicable to commercial practice for eliminating lipids from whey protein concentrates. Evaluation of the functional properties of whey protein fractions and proteins isolated by different methods. Elucidation of relationships between protein structure and specific functional properties, with emphasis on the effects of heat treatments. Investigation of processes for production and modification of whey proteins designed for specific functional applications. Development of practical methods for concentration (including, freeze concentration and drying) and isolation of highly functional products. Examination of the usefulness of whey proteins for blending with and/or complementing other food proteins. Determination of the effects of modification by physical, enzymatic, chemical, or genetic methods on functional properties. Determination of physicochemical attributes required in proteins for optimum performance in specific functions. ACKNOWLEDGMENTS This review was supported in part by grants from the National Dairy Promotion Board and the Wisconsin Milk Marketing Board.

REFERENCES Acton, J. C. 1972. Effect of heat processing on extractability of salt-soluble protein, tissue binding strength and cooking loss in poultry meat loaves. J. Food Sci. 37, 244. Adler-Nissen, J. 1986. Hydrolysis of food proteins for food uses. In “Enzymic Hydrolysis of Food Proteins” (J. Adler-Nissen. ed.), p. 100. Elsevier, Amsterdam. Aisen, P. 1980. The transfemns. In “Iron in Biochemistry and Medicine” (A. Jacobs and M. Worwood, eds.), Vol. 2, p. 87. Academic Press, New York. Andrews, A. T. 1978. The composition, structure and origin of proteose-peptone component 5 of bovine milk. Eur. J . Biochem. 90, 59. Arai, S., Watanabe, M., and Hirao, N. 1986. Modificaiton to change physical and functional

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properties of food proteins. I n “Protein Tailoring for Food and Medical Uses” (R. E. Feeney and J. R. Whitaker, eds.), p. 75. Dekker, New York. Aschaffenburg, R.,and Drewry, J. 1957. Improved method for the preparation of crystalline P-lactoglobulin and a-lactalbumin from cow’s milk. Biochem. J. 65, 273. Barbeau, W. E., and Kinsella, J. E. 1986. Physical behavior and functional properties: relationship between surface rheology and foam stability of ribulose 1.5-biphosphate car. 169. boxylase. Colloids S u ~ 17, Benjamins, J., De Feijter, J . A., Evans, M. T. A., Graham, D. E., and Phillips, M. C. 1975. Dynamic and static properties of proteins adsorbed at the aidwater interface. Trans. Faraday SOC.59,218. Bernal, V., and Jelen, P. 1984. Effect of calcium binding on thermal denaturation of bovine a-lactalbumin. J. Dairy Sci. 67, 2723. Bernal, V., and Jelen, P. 1985. Thermal stability of whey proteins-a calorimetric study. J. Dairy Sci. 68, 2847. Beveridge, T., Jones, L., and Tung, M. A. 1984. Progel and gel formation and reversibility of gelation of whey, soybean, and albumin protein gels. J. Agric. Food Chem. 32, 307. Beyeler, M., and Solms, J. 1974. Interaction of flavor model compounds with soy protein and bovine serum albumin. Lebenms.-Wiss. Technol. 7, 217. Bingham, E. 1976. Modification of cash by phosphatases and protein kinases. J. Agric. Food Chem. 24, 1094. Braunitzer, G., Chen, R., Shrant, B., and Stangl, A. 1973. Complete covalent structure of bovine P-lactoglobulin B. Hoppe-Seyler’s Z. Physiol. Chem. 354, 867. Brew, K., Vanaman, T. C., and Hill, R. L. 1968. The complete covalent structure of bovine a-lactalbumin. Proc. Natl. Acad. Sci. U.S.A. 59, 491. Brooks, J. R., and Morr, C. V. 1984. Phosphorus and phytate content of soybean protein components. J. Agric. Food Chem. 32, 672. Brown, E. M. 1984. Interactions of P-lactoglobulin and a-lactalbumin with lipids. J . Dairy Sci. 67, 713. Brown, E. M., and Farrell, H.M.,Jr. 1978. Interaction of p-lactoglobulin and cytochrome C: Complex formation and iron reduction. Arch. Biochem. Biophys. 185, 156. Brown, J. R. 1977. Structure and evolution of serum albumin. Proc. FEBS Meet. SO, 1. Bull, H. B. 1972. Adsorbed surface films of egg albumin. J. Colloid Interface. Sci. 41, 305. Cerbulis, J., and Farrell, H. M., Jr. 1986. Extraction of whey proteins with magnesium and zinc salts. J. Dairy Sci. 69, 2604. Chaplin, L. C., and Lyster, R. J. 1986. Irreversible heat denaturation of bovine a-lactalbumin. J. Dairy Res. 53, 249. Circle, S. I., Meyer, E. W., and Whitney R. W. 1964. Rheology of soy protein dispersions, effect of heat and other factors on gelation. Cereal Chem. 41, 157. Clark, A. H., Saunderson, D. H. P., and Suggett, A. 1981. Infrared and laser raman spectroscopic studies of thermally-induced globular protein gels. Inf. J. Pept. Protein Res. 17, 353. Clark, W. S., Jr. 1987. Status of whey and whey products in the U S today. Bull. IDF (212), 6. Cooney, C. M. 1974. A study of foam formation by whey proteins. Diss. Abstr. I n f . 36/03; 1123-B. Creamer, L. K., Parry, D. A. D., and Malcolm, G. N. 1983. Secondary structure of bovine P-lactoglobulin B. Arch. Biochem. Biophys. 227,98. Creighton, T. E. 1985. The problem of how and why proteins adopt folded conformations. J . Phys. Chem. 89, 2452. Cumper, C. W. 1953. The staibilization of foams by proteins. Trans. Faraday Soc. 49, 1360.

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INDEX A Absorption calcium from CCM, 147 dietary requirements and, 118-1 19 effects of complexation by ligands yielding soluble complexes, 146-147 fiber and phytate, 134-142 lactose, see Lactose effect oxalic acid, 142 protein and phosphate, 143-144 supplements, 144-147 mechanisms, 114-1 17 net absorption, calculation, 121-122 net amount necessary to balance urinary losses, 111 regulation, 114-1 17 folates age effects, 68-69 altered gastrointestinal function and, 67-68

conjugases and, 55-56 deconjugation of dietary polyglutamyl folate, 54-55 dietary components and, 71-72 ethanol ingestion effects, 69-70 folate-binding protein in jejunal brush border membrane, 54 hepatic, 54 K,,, transport values, 53 pH dependence, 53-54 pH effects, 67 tocopherols effects of gastric or intestinal destruction, 169-170 efficiency, 168-171 via lymphatic system, 168-169

mechanism, 168-17 1 Acid-catalyzed decomposition, acyl hydroperoxides, 278-280 Acylation, whey protein modification by, 418-419

Acyl hydroperoxides carbonyl compounds from, table, 265 decomposition, 270-273 products from homolytic thermal cleavage, 272 volatile compounds from, 273-275 Adenosine diphosphate chelation, 304-305 control of microsomal enzyme-associated lipid oxidation, 306 Adipose tissues tocopherol content, 178-179 tocopherol mobilization and turnover, 188 a-tocopherol release, 188-189 Adrenocortical cells, a-tocopherol binding sites, 199 Adsorption, whey proteins, 391-393 Affinity chromatography, polyglutamyl folates, 38 Age effects calcium absorption, 117-1 18 folate bioavailability, 68-69 surface rheology of whey protein interfacial films, 394 vitamin E status, 19&191 Aldehydes binding to unprocessed whey proteins, 41 1

a-lactalbumin binding, 415 Amidation, whey protein modification by, 419420

Amino acid composition effect on functional properties of whey proteins, 367

439

440 Amino acid composition (continued) whey proteins from bovine milk, table, 350 Amino acids apolar, effect on whey protein gels, 389 primary sequences of P-lactoglobulin B, 35 1 Antacids, antifolate effects, 71 Antifolates dietary, 78-80 ethanol, 69-71 therapeutic drugs, 70-71 Antioxidants ascorbic acid, 310 butylated hydroxyanisole, 309-3 10 butylated hydroxytoluene, 309-3 10 classes, 309 flavonoids, 311-314 inhibitory effects on fish gill 12-lipoxygenase, table, 308 naturally occurring, 310 tocopherols, 310 Arachidonate autoxidation, mechanism, 247 carbonyl compounds generated from, table, 265 di-, tri-, and tetrahdyroxy derivatives, formation from hydroperoxide precursors, 248 Arachidonic acid autoxidation, 247-248 hydroperoxide, trihydroxy derivative formation, 271 lipoxygenase-catalyzed oxidation and generation of oxidative flavor compounds, 300 Ascorbate, effects on folate retention, 49-50 Ascorbic acid inhibition of fish lipid oxidation, 310 oxidative losses to reduced folates, 15 a-tocopherol interactions, 21 1-212 Aspirin, antifolate effects, 71 Autoxidation arachidonic acid, 247-248 docosahexaenoate, 250 docosahexaenoic acid, 248-250 eicosapentaenoic acid, 248-250 linoleic acid, 242-243 linolenic acid, 243-246

INDEX

lipids, 239-249 oleate, 241 oleic acid, 241-242

B Baicalein, lipoxygenase inhibition, 318 Bilayers, phospholipid, a-tocopherol effects, 215-216 Bile, a-tocopherol metabolites in, 199-201 Bioassays, for folate bioavailability, 58-61 Bioavailability calcium definition, 119-120 diet effects on urinary excretion, 120 effects of fiber and phytate, 134-142 oxalic acid, 142 protein and phosphates, 143-144 supplements, 144-147 lactose effect, 124-125 in animals, 125-126 in elderly subjects, 134 in human infants, 132-134 in humans, 128-132 measurement, 120-121 animal models, 124 balance methods, 121-122 isotopic tracer techniques, 122-123 folates absorption, 53-57 aging effects, 68-69 assessment methods bioassays with animals and humans, 58-61 isotopic methods, 61-64 deconjugation, 54-55 dietary antifolates, 78-80 dietary components and, 71-80 endogenous, in various foods, table, 72 ethanol effects, 69-71 folate-binding protein role in jejunal brush border membranes, 54 gastrointestinal functions and, 67-68 hepatic uptake and secretion, 54 intestinal conjugases, 55-56 in vivo turnover kinetics, 57-58 K,,,transport values, 53 in legumes, 74

441

INDEX in milk, 75-76 nutrient interactions, 79-80 in orange juice, 74-75 pH dependence, 53-54 in vegetables, 75 in yeast, 73-74 monoglutamyl folates, inherent availability, 65-66 polyglutamyl folates, inherent availability, 64-65 tocopherols diet composition effects, 171-173 fiber effects, 171-173 pectin effects, 171-172 wheat bran effects, 172-173 Bovine serum albumin flavor binding, 412-413 structure, 357 thermodynamic and binding constants, table, 414 Brain tissue, a-tocopherol turnover, 187 Brans cereal, effect on calcium absorption, 138139

wheat, effect on calcium absorption, 137-138 tocopherol bioavailability, 172- 173 But ylated hydroxyanisole as antioxidant, 309-310 inhibition of lipoxygenase, 307 Butylated hydroxytoluene, inhibition of lipoxygenase, 307

C Caffeic acid, inhibition of soybean lipoxygenase, 317 Calcium absorption. see Calcium absorption balance, see Calcium balance binding, a-lactalbumin, 354-356 bioavailability, see Calcium bioavailability concentrations in intra- and extracellular fluids, 112-1 14 content, various foods, table, 106-107 cytosolic free concentrations, 112 effects on p-lactoglobulin gels, 388

thermal denaturation behavior of whey proteins, 379-381 whey protein foams, 401 homeostasis, 112-1 14 intakes within groups, 105-106 long term, 112 table of, 106 plasma concentrations, 112-1 14 Recommended Dietary Allowance, 105106, 108-112

Calcium absorption from CCM, 147 during development, 115-1 16 dietary requirements and, 118-119 effects of cereals, 136-139 complexation by ligands yielding soluble complexes, 146-147 fruit and vegetable fiber, 139-141 protein and phosphates, 143-144 purified fibers, 141-142 supplements, 144- 147 mechanisms, 114-117 net absorption, calculation, 121-122 net amount to balance urinary losses, 111

regulation, 114-1 17 Calcium balance definition, 108, 121 excretion and, 108 in males, 1 I I measurement of calcium bioavailability with, 121-122 measurements, I08 in perimenopausal women, 110-1 I 1 Calcium bioavailability definition, 119-120 diet effects on urinary excretion, 120 effects of fiber and phytate, 134-142 oxalic acid, 142 protein and phosphates, 143-144 supplements, 144-147 lactose effect, 124-125 in animals, 125-126 in elderly subjects, 134 in human infants, 132-134 in humans, 128-132 measurement, 120-121

442

INDEX

Calcium bioavailability (conrinued) animal models, 124 balance methods, 121-122 isotopic labeling of foods, 123-124 isotopic tracer techniques, 122-123 Calcium salts, water solubility and calcium content, table, 145 Carbohydrates, effect on calcium absorption, 130-131 Carbonyl compounds from acyl hydroperoxides, table, 265 volatile flavor compounds from lipid oxidation of biological tissue, table, 274 of fish tissues, table, 275 p-Carotene, quenching of singlet oxygen, 303 Catalase inhibition of oxidative losses to reduced folates, IS removal of hydrogen peroxide with, 303 Catechols inhibition of lipoxygenase, 307 structure, role in lipoxygenase inhibition, 317-3 18 CCM salt, calcium absorption from, 147 Cellobiose, effect on calcium absorption, 127 Cellulose, effect on calcium absorption, 141 tocopherol bioavailability, 173 Cereals bran, effect on calcium absorption, 138139 fiber, effect on calcium bioavailability, 136-139 Chard, total folate content, table, 40 Chelating agents, effects on thermal denaturation behavior of whey proteins, 379-381 Chemical modification, of whey proteins, 417-422 Chlorogenic acid, inhibition of soybean lipoxygenase, 317 Chromanoxyl radical, a-tocopherol ascorbic acid quenching, 21 1 formation, 208 regeneration by, 21 1-212

Chylomicrons, role in tocopherol transport, 183- 184 Citrus pectin, effect on calcium absorption, 141 Competitive binding methods, for folate determination, 22-26 alternative approach to, 25-26 differences between procedures, 23 plasma and red cell folate measurement, 24 problems and limitations, 24-25 selectivity of assay, 23 Concentration-dependent self-association phenomenon, folates, 6 Conjugases, enzyme efficiency in folate analysis, 18-19 Cooking, folate stability during, 47-51 Copper fish lipid oxidation, 289-290 peroxidation, alteration with EDTA chelation, 304-305 Cyclooxygenase, lipid oxidation, 269-270 Cystine groups, whey proteins, table, 349 Cytochrome P-450,lipid oxidation, 256-259

D DEAE-cellulose, for monoglutamyl folate separations, 27 Decomposition, acyl hydroperoxides, 270273 acid-catalyzed, 278-280 secondary oxidation products, 276-278 volatile compounds from, 273-275 Deconjugation, enzymatic, of folates, 17-20 autdytic deconjugation with endogenous conjugases, 17-1 8 efficiency of, 18-19 pteroylpolyglutamate hydrolases in, 17 Degradation , oxidative, folates energies of activation, table, 50 free radical mechanism, 45 resistance to, 45 tetrahydrofolic acid, mechanism, 42-43 thermal, 5-methyl-tetrahydrofolate,49 Denaturation, whey proteins assessment methods, 363

443

INDEX effect on gelation and gel strength, 386 enthalpies, table, 377 fast protein liquid chromatography, 372 heat effects on emulsifying properties, 407 factors affecting, 378-379 susceptibility of whey products to, 347 utilization and, 371-373 Deoxyribonuleic acid, lipid oxidation effects, 285-286 Depletion-repletion bioassay, for folate bioavailability, 59 Development, lactose effect in elderly subjects, 134 in human infants, 132-134 Dietary fiber, see Fiber Dietary folates, see Folates, bioavailability Diethylaminoethyl-cellulose,for monoglutamy1 folate separations, 27 Diets, low-fat, low-cholesterol, effect on vitamin E status, 190 Differential scanning calorimetry thermodynamics of whey protein unfolding, 376-377 whey protein denaturation, 372 Diglutamate, determination in conjugase assay by reversed-phase HFLC. 31 7,8-Dihydrofdate, preparation enzymatic reduction, 9 folk acid-sodium dithionite reaction, 8 Dihydroperoxides linolenate, breakdown mechanism, 276 from polyunsaturated fatty acid oxidation, 276-277 Dipalmitoylphosphatidylcholineliposomes, a-tocopherol transfer to rat liver mitochondria, 195 Diphenylhydantoin, antifolate effects, 71 Dissociation constants, binding of nonpolar compounds by P-lactoglobulin, table, 410 Diurnal variation, in plasma a-tocopherol concentration, 176 Docosahexaenoate, autoxidation, mechanism, 250 Docosahexaenoic acid, autoxidation, 248250 Dowex 2, for monoglutamyl folate separations, 26

E Eicosapentaenoate, autoxidation, mechanism, 249 Eicosapentaenoic acid autoxidation, 248-250 lipoxygenase-catalyzed oxidation and generation of oxidative flavor compounds, 301 5,8, I I , 14-Eicosatetraenoic acid, inhibition of lipoxygenases, 306-307 Electrochemical techniques, for detection of eluted folates, 30-33 Electrostatic interactions, effect on whey protein gelation, 387 Emulsions characterization, 405 whey proteins factors affecting, 408 properties, 405-406 protein conformation and, 406-408 surface hydrophobicity and, 406-408 Energy of activation, degradation of folates in aqueous solution, table, 50 Enthalpy of denaturation, whey proteins, 377-378 Enzymatic deconjugation, folates, 17-20 autolytic deconjugation with endogenous conjugases, 17-18 efficiency of, 18-19 pteroylpolyglutamate hydrolases in, 17 Enzymatic modification, whey proteins, 416-417 Enzyme-catalyzed lipid oxidation cyclooxygenase, 269-270 cytochrome P-450, 256-259 fish lipids, 291-297 lipoxygenase, 261-269 peroxidase, 259-261 Enzymes, microsomal, see Microsomal enzymes Epoxy-hydroperoxides linolenate, breakdown mechanism, 277 from polyunsaturated fatty acid oxidation, 276-277 Erythrocyte ghosts a-tocopherol concentrations, 212 a-tocopherol transfer from liposomes to, 195-196

444

INDEX

Erythrocyte hemolysis a-tocopherol acetate effects, 219 for vitamin E status, 189 Erythrocytes membranes a-tocopherol binding, 198-199 a-tocopherol turnover, 187 tocopherol association with, 177 tocopherol-deficient and tocopherol-sufficient, a-tocopherol transfer, 185 Esculetin, lipoxygenase inhibition, 317, 318 Esterification, whey protein modification, 4 19-420

Ethanol, ingestion effects on folate bioavailability, 69-71 Ethylenediaminetetraacetic acid chelation, 304-305 control of microsomal-enzyme-associated lipid oxidation, 305-306 Excretion, urinary calcium balance measurements and, 108 diet effects, 120 a-tocopherol metabolites, 202-204 Extraction, folates, 14-17 aqueous, effect on losses during cooking, 49

free folate during, 17 heat effects during, 16 inhibition of oxidative losses, 15 nonthermal procedures, 16 pH effects during, 15-16

F Fast protein liquid chromatography, whey protein denaturation, 372 Fats effect on whey protein foaming properties, 404 tocopherols in, 159-164 Ferric iron chelation, 304 fish lipid oxidation, 289-290 Ferrous iron chelation. 304 fish lipid oxidation, 289-290 Ferrous sulfate, effects on folate retention, 49-50

Fiber, effects on calcium bioavailability, 134-135 cereals, 136-139 fruits and vegetables, 139-141 mechanism, 135-136 pH dependence of calcium binding by fiber, 136 purified fibers, 141-142 folate bioavailability, 76-78 tocopherol bioavailability, 171-173 Fibroblasts, uptake of tocopherols, 180-182 Films, whey proteins, 395-3% adsorption. 391-393 effects of aging, 394 ionic strength effects, 394 pH effects, 394 protein conformation and, 393-395 stability, 394 viscoelastic behavior, 394 Finnish oils, tocopherols and tocotrienols in, 162 Fish lipids oxidation enzyme-catalyzed, 291-297 factors affecting, 287-289 metal catalysis of, 289-291 volatile compound generation, 298-302 polyunsaturated fatty acids in, 286 Fish oils effect on tocopherol bioavailability, 173 tocopherol content, 164 Flavonoids antioxidant action, 314-316 distribution, 312 effects on lipoxygenase, 316-319 formation, 312 in higher plants, 313-314 hydroxyl positional isomers, effects on lipoxygenase inhibition, 318-319 inhibitory effects on fish gill 12-lipoxygenase, table, 308 optimum antioxidant activity, 3 16 structural alterations, effects on lipoxygenase inhibition, 3 17-318 structural requirements for lipoxygenase inhibition, 319 structure, 311-312 Flavonols, as antitumor agents, 319 Flavor binding, whey proteins, 410-413 thermodynamic representation, 412

INDEX

Fluorescence, detection of eluted folates with, 28-29 Foams characterization, 397-398 stability, protein structure and, 398-400 whey proteins, 400--402 foaming properties, 402-404 Folate-binding protein, in jejunal brush border membranes, 54 Folates antifolates dietary, 78-80 ethanol, 69-71 therapeutic drugs, 70-71 bioavailability absorption, 53-57 aging effects, 68-69 assessment methods bioassays with animals and human, 58-6 1 isotopic methods, 61-64 deconjugation, 54-55 dietary components and, 71-80 dietary fiber effects, 76-78 endogenous, in various foods, table, 72 ethanol effects, 69-71 folate-binding protein role in jejunal brush border membranes, 54 gastrointestinal functions and, 67-68 hepatic uptake and secretion, 54 inherent, 64-66 intestinal conjugases, 55-56 in vivo turnover kinetics, 57-58 K,,, transport values, 53 in legumes, 74 in milk, 75-76 nutrient interactions, 79-80 in orange juice, 74-75 pH dependence, 53-54 in vegetables, 75 in yeast, 73-74 chain length, determination with HPLC methods, 34-38 chemical properties concentration-dependent self-association phenomenon, 6 inherent, 4-6 ionic, 4-5 pH effects on behavior, 5-6 structural, 4

445

chemical structures, 2-3 C-9-N-I0 bond, 34-38 determination analysis of polyglutamyl forms, 33-38 HPLC methods, 26-38 ligand binding methods, 22-26 microbiological methods, 21-22 separation and analysis in monoglutamy1 form, 26-33 deuterated, synthesis, in vivo application, and mass spectral analysis, 12 distribution in various foods, table, 41 enzymatic deconjugation, 17-20 extraction, 14-17 free folate during, 17 heat effects, 16 inhibition of oxidative losses, 15 nonthermal procedures, 16 pH effects during, 15-16 H,folate, see 7,8-Dihydrofolate H,folate, see 5,6,7,8-Tetrahydrofolate intrinsic stability, 40-47 in vivo turnover kinetics, 57-58 isotopically labeled and stable, preparation, 12-13 one-carbon-substituted, formation, 9-10 oxidation potentials, electrochemical detection in reversed-phase HPCL systems, 30-32 polyglutamyl, see Polyglutamyl folates radioisotopically labeled, preparation, 11-12 reactions of nitrite ions with, 44-45 Recommended Dietary Allowance, 52 Recommended Dietary Intake, 52 retention, ascorbate/ferrous sulfate effects, 49-50 in spinach and Swiss chard, table, 40 stability during cooking or thermal processing, 47-5 I effects of aqueous extraction, 49 in milk products, 51 retention after cooking or thermal processing, table, 48 Folic acid determination in conjugase assay by reversed-phase HPLC, 3 1 energy of activation for degradation in aqueous solution, table, 50

446 Folk acid (continued) reaction with sodium dithionite reaction for (6-ambo)- 5,6,7,8-tetrahydrofolate preparation, 8 retention in fortified food, 52 Formylation, formyl-tetrahydrofolates,10

INDEX

H

Haber-Weiss reaction, 237-238 Heart, microsomes, peroxidation, a-tocopherol effects, 213 Heat effects, see also Temperature effects; 5-Formyl-tetrah ydrofolate Thermal processing 6-ambo form folates during extraction, 16 availability, 9 ligand binding by p-lactoglobulin, 415 preparation, 10 oxidation, 78 a-tocopherol content of vegetable oils, 10-Formyl-tetrahydrofolate,oxidation, 46, 164-165 78 whey proteins Formyl-tetrahydrofolates,formylation beconcentrate foaming properties, table, 402 forelafter pteridine reduction, 10 Fortification of foods, folic acid retention, conformation, 376-378 52 foams, 401 Frozen storage, effect on folate in human partial unfolding, 404 milk, 51 solubility, 375 Fructose, effect on calcium absorption, Heme, fish lipid oxidation, 290 127 Hemolysis, erythrocyte Fruit fiber, effect on calcium bioavailability, a-tocopherol acetate effects, 219 139-141 vitamin E status with, 189 High-performance liquid chromatography, folates chain length determination, 34-38 G C-9-N-10 bond, 34-38 detection of eluted folates Gas chromatography-mass spectrometry, electrochemical techniques, 30-33 folate bioavailability measurement, fluorescence, 28-29 61-62 microbiological assays, 29-30 Gelation, whey proteins, 381-385 factors affecting gel composition and UV absorbance, 28 ion-exchange, monoglutamyl folates, 27properties, 385-389 forces in gel formation and stabilization, 28 reverse-phase table, 384 gel structure, 383-384 monoglutamyl folates, 27-28 Gel filtration chromatography polyglutamyl folates as azo dye derivaP-lactoglobulin binding of p-nitrophenyl tives of p-aminobenzoyl(po1y)gluphosphate, 409 tamates, 35 separation of polyglutamyl folates, 33 pteroyltriglutamate, diglutamate, and whey protein preparation, 361 folk acid in conjugase assay, 31 Gelling applications, whey proteins in, 389reduced folates, 30 39I urinary folates, with UV absorbance, Glucose, effect on calcium absorption, 127 29 Glutathione, reduction of a-tocopherol inHock cleavage, 279-380 termediate oxidation products, 212 Homolytic cleavage, hydroperoxides of Glutathione peroxidase, removal of hydropolyunsaturated fatty acids, 272gen peroxide with, 303 275 Glycosylation, whey proteins, 421-422 Hydration, whey proteins, 373-374

447

INDEX

Hydrocarbon gases, measurement for vitamin E status, 191 Hydrogenation, effect on tocopherol content of vegetable oils, 163 Hydrogen peroxide control, 303 formation, 237 inhibition of soybean lipoxygenase, 307 Hydroperoxy bis-epidioxide, from polyunsaturated fatty acid oxidation, 276277 Hydroperoxy cyclic peroxides linolenate, breakdown mechanism, 277 from polyunsaturated fatty acid oxidation, 276-277 Hydrophobicity effects on emulsifying properties of whey proteins, 406-408 on functional properties of whey proteins. 369 Hydroxyl radical formation, 236 scavengers, control of lipid oxidation with, 303 4 a-Hydroxy-5-methyl-tetrahydrofolate, structure, 46

I Immunoglobulins, structure, 357-358 Infant formula, whey proteins and, 422423 Intakes calcium within groups, 105-106 long term, 112 table of, 106 folates, recommended, 52 tocopherols, 166-168 vitamin D, calcium absorption and, 115I16 Interstitial retinol-binding protein, role in tocopherol binding and transport, 194 Intestines calcium absorption during development, 117-1 18 dietary requirements and, 118-1 19

effects of fiber and phytate, 134-142 oxalic acid, 142 lactose effect, see Lactose effect regulation, 114-1 17 folate absorption, 53-57 age effects, 68-69 altered gastronintestinal function and, 67-68 conjugases and, 55-56 deconjugation of dietary polyglutamyl folate, 54-55 dietary components and, 71-72 ethanol ingestion effects, 69-70 folate-binding protein in jejunal brush border membrane, 54 K, transport values, 53 pH dependence, 53-54 pH effects, 67 pteroylpolyglutamate hydrolases, 55-56 tocopherol absorption destruction effects, 169- 170 efficiency, 168-171 via lymphatic system, 168-169 mechanism, 168-1 7 1 Ion-exchange chromatography monoglutamyl folates, 26 polyglutamyl folates, 33-34 whey protein preparation, 361 Ionic strength, effects on foaming properties of whey proteins, 403 heat treatment of whey proteins, 376-378 Ion permeability, membranes, a-tocopherol effects, 218 Isoelectric pH, of whey proteins, table, 349 Isotopic labeling calcium bioavailability foods, 123-124 tracer techniques, 122-124 folate bioavailability, 61-64 folates with deuterium and other stable isotopes, 12-13

J Jejunal brush border membranes, folatebinding protein in, 54

448

INDEX

.

K Kaempferol inhibition of soybean lipoxygenase, 3 I7 oxidation, mechanism, 3 15 structure, 31 1 Kidney calcium handling, protein and phosphate effects, 143-144 urinary excretion a-tocopherol, 202-204 y-tocopherol, 205-206 &-tocopherol,205-206 Kjeldahl nitrogen analyses, whey protein denaturation, 372

L a-Lactalbumin, structure, 354-357 Lactobacillus casei assay for folate in foods, 21-22 biological synthesis of long-chain folates, 11-12 Lactobacillus casei reductase, reduction of oxidized folates, 9 Lactofenin, bovine milk, structure, 358359 P-Lactoglobulin binding of nonpolar compounds, dissociation constants, table, 410 film-formingproperties, 396-397 flavor binding, 412-413 genetic variants, 353-354 glycosylated, film-forming properties, 396-397 ligand binding, 413-415 secondary structure, 352-353 structure, 350-354 thermodynamic and binding constants, table, 414 Lactose effect, 124-125 in animals, 125-126 effect of carbohydrates, 130-13 1 other sugars, 127 in elderly subjects, 134 in human infants, 132-134 in humans, 128-132

Legumes, folate bioavailability, 74 Leukocytes, tocopherol association with, 177 Ligand binding folate determination methods, 22-26 alternative approach to, 25-26 differences between procedures, 23 plasma and red cell folate measurement, 24 problems and limitation, 24-25 selectivity of assay, 23 by whey proteins flavor binding, 410-413 by P-lactoglobulin, 413-415 lipid binding, 408-409 thermodynamic and binding constants, table, 414 Linoleate carbonyl compounds generated from, table, 265 oxidation autoxidation mechanism, 242 free radical and photosensitized, distributions of isomeric hydroperoxides, table, 254 by singlet oxygen, 253 Linoleic acid autoxidation, 242-243 a-tocopherol sources and, 167-168 Linolenate autoxidation and formation of hydroperoxy cyclic peroxide, 244 bicycloendoperoxide formation from I3hydroperoxide precursor, 245 bis-epidioxide, breakdown mechanism, 278 carbonyl compounds generated from, table, 265 dihydroperoxide breakdown mechanism, 276 formation from hydroperoxide precursors, 246 epoxy-hydroperoxide, breakdown mechanism, 277 free radical and photosensitized oxidations, distributions of isomeric hydroperoxides, table, 254 hydroperoxy cyclic peroxide, breakdown mechanism, 277 Linolenic acid, autoxidation, 243-246

449

INDEX

Lipids binding by whey proteins, 408-409 effect on whey protein foaming properties, 404 fish, see Fish lipids oxidation, see Oxidation Lipid transfer protein, tocopherol transfer between lipoproteins and, 175 Lipoprotein lipase, role in a-tocopherol transfer, 183-184 Lipoproteins high-density, mediation of tissue a-tocopherol uptake, 183 low-density, mediation of tissue a-tocopherol uptake, 180-182 plasma, tocopherol in distribution, 174 diurnal/seasonal variations, 176 exchange between classes, 174-175 incorporation, tracing with d- a-tocopherol acetate, 176-177 lipid transfer protein role, 175 postprandial changes, 176 very-low-density, mediation of tissue atocopherol uptake, 183 Liposomes dipalmitoylphosphatidylcholine, a-tocopherol transfer to rat liver mitochondria, 195 phosphatidylcholine, a-tocopherol transfer to rat liver microsomes, 194 Lipoxygenases activity in fish gill and skin tissues, table, 294 control of oxidative activities, 306-309 effects of flavonoids, 316-319 fish lipid oxidation, 294-297 gill, effect of thiol groups, 2%-297 lipid oxidation, 261-269 metabolites, 297 oxygen insertion, positional specificity, 266-267 from plant and animal tissues, positional specificity, table, 262 Liquid-phase methods, for pol yglutamyl folate synthesis, 7 Liver folate uptake and secretion, 54 microsomal enzyme-catalyzed lipid oxidation, 256-259

microsomes a-tocopherol concentrations, 2 12 a-tocopherol transfer factors, 194-195 mitochondria. a-tocopherol concentrations, 212 role in a-tocopherol clearance from blood, 184 tissue tocopherol content, 178-179 a-tocopherol turnover, 187 a-tocopherol metabolism, 199-201 y-tocopherol metabolism, 205-206 8-tocopherol metabolism, 205-206 Lung microsomes, a-tocopherol effects on peroxidation, 213 tissue, a-tocopherol turnover, 187 Lymphatic system, tocopherol absorption via, 168-169 Lysine, effect on calcium absorption, 127 Lysozyme, bovine milk, structure, 359

M Maillard reaction, whey proteins, 373 Malondialdehyde, mutagenicity, 285-286 Mannitol, effect on calcium absorption, 127 Margarines, tocopherol content, 163 Mass spectrometry deuterated folates, 12 folate bioavailability measurement, 61-62 Membranes lipid oxidation effects, 283-285 permeability to ions/small molecules, atocopherol effects, 218 2-Mercaptoethanol, inhibition of oxidative losses to reduced folates, 15 Metal-oxygen complexes, initiation of polyunsaturated fatty acid oxidation. 237-238 Methotrexate, effects on folate absorption and metabolism, 70 Methylation, whey proteins, 421 Methyl ketones binding to unprocessed whey proteins, 41 I a-lactalbumin binding, 415 5-Met hyl-tetrahydrofolate 6-ambo form, availability, 9

450

INDEX

5-Methyl-tetrahydrofolate (continued) chemical synthesis of 6-ambo or 6 s form, 10 energy of activation for degradation in aqueous solution, table, 50 oxidative degradation, 45-47 thermal degradation, 49 Metmyoglobin, fish lipid oxidation, 291 Microbiological assays, folate determination, 21-22 eluted folates, 29-30 and ion-exchange chromatography, 33 modifications, 22 Microsomal enzymes, lipid oxidation catalysis, 256-259 control, 305-306 fish lipids, 291-297 Microsomes heart, peroxidation, a-tocopherol effects, 213 liver a-tocopherol binding, 196-197 a-tocopherol concentrations, 2 12 a-tocopherol transfer factors, 194-195 lung, peroxidation. a-tocopherol effects, 213 Milk composition (bovine, human), 421-423 frozen storage, effect on folate content (human), 5 1 pasteurization effect on folate binding, 76 ultrahigh-temperature methods, effects on folate activity, 51 tocopherol composition (human), 179 Milk products folate bioavailability, 75-76 folate stability in, 51 Mitochondria, liver, a-tocopherol concentrations, 212 Mobilization, tocopherols, 186-189 Molecular mass, whey proteins, table, 349 Monoglutamyl folates bioavailability comparative, 65-66 table, 66 chiral stability, 13 deuterated, synthesis, 11-12 HPLC methods of separation and analysis, 26-33

racemization of glutamate a-carbon product, 13 stability in aqueous solution, 45 Monolayers, phospholipid, a-tocopherol effects, 215 Myricetin lipoxygenase inhibition, 317 structure, 31 1

N Nitrite ions, reaction with folates, 44-45 p-Nitrophenyl phosphate, P-lactoglobulin binding, 409 Nordihydroguaiaretic acid, inhibition of lipoxygenase, 307 Nuclear magnetic resonance, deuterated folates, 12-13

0 Oils, tocopherols in, 159-164 Oleate carbonyl compounds generated from, table, 265 oxidation autoxidation, 241 free radical and photosensitized, distributions of isomeric hydroperoxides, table, 254 by singlet oxygen, 252 Oleic acid, autoxidation, 241-242 Orange juice, folate bioavailability, 74-75 0 steoporosis calcium supplements and, 144-145 risk factors, 104 Oxalic acid, effect on calcium absorption, 142- 143 Oxidation antioxidants ascorbic acid, 15, 310 butylated hydroxyanisole, 309-3 10 butylated hydroxytoluene, 309-3 10 classes, 309 flavonoids, 31 1-314 inhibitory effects on fish gill 12-lipoxygenase, table, 308

45 1

INDEX naturally occurring, 310 tocopherols, 310 fish lipids enzyme-catalyzed, 291-297 factors affecting, 287-289 metal catalysis of, 289-291 volatile compound generation, 298-302 folate resistance to, 45 folates, energies of activation, table, 50 10-formyl-tetrahydrofolate,46 4 a-hydroxy-5-methyl-tetrahydrofolate, 46 5-methyl-tetrahydrofolate,4 5 4 7 polyunsaturated fatty acids acid-catalyzed decomposition, 278280 autoxidation, 239-249 control antioxidants, 309-310 flavonoids, 311-319 lipoxygenase, 306-309 microsomal enzymes, 305-306 peroxidase, 306 decomposition of acyl hydroperoxides, 270-273 enzyme-initiated, 256-270 oxygen activation, 234-238 peroxidation in vivo effects on DNA, 285-286 on membranes, 283-285 on proteins, 282-283 photosensitized singlet oxygen, 249256 reactants and inhibitors active oxygen species, 302-303 metals, 303-405 oxygen, 302 secondary products, 276-278 volatile compounds from acyl hydroperoxides, 273-275 volatile compounds in food, 280-281 tetrahydrofolic acid, mechanism, 42-43 a-tocopherol antioxidant functions in membranes, 207-211 Oxidation potentials, folates, electrochemical detection in reversed-phase HPCL systems, 30-32 Oxygen levels, control, 302 Oxygen scavengers, for lipid oxidation control, 302

P Palm oil tocopherol losses due to processing, 160 tocotrienols in, 160 Pasteurization, milk effect on folate binding, 76 by ultrahigh-temperature methods, effects on folate activity, 51 Pectin citrus, effect on calcium absorption, 141I42 effects on tocopherol bioavailability, 171I72 Perhydroxyl radical, formation, 237 Permeability, membranes to ions/small molecules, a-tocopherol effects, 218 Peroxidase control of oxidative activities, 306 fish lipid oxidation, 293 lipid oxidation, 259-261 Peroxidation lung and heart microsomes, a-tocopherol effects, 213 membrane, inhibition by a-tocopherol, 210 membrane lipids, a-tocopherol role, 213 PH isoelectric, whey proteins, table, 349 P-lactoglobulin changes as function of, 35 I pH dependence calcium binding by fiber, 136 folate absorption, 53-54 pH effects on folates behavior, 5-6 intestinal absorption, 67 stability during extraction, 15-16 on whey proteins binding of flavor compounds to, 41 1412 film-forming properties, 394 foams, 401, 403 functional properties, 369 gelling properties, 386-387 gelling time, 387 heat treatment, 376-378 solubility, 375 thermal denaturation behavior, 379-381

452

INDEX

Phenols inhibition of lipoxygenase, 307 inhibitory effects on fish gill 12-lipoxygenase, table, 308 Phenoxyl radical, a-tocopherol, 208-210 Phosphates, effect on calcium absorption, 143-144 Phosphatidylcholine liposomes, a-tocopherol transfer to rat liver mitochondria, 194 Phospholipase A,, platelet, a-tocopherol effects, 219-220 Phospholipid bilayers, a-tocopherol effects, 215-2 I6 Phospholipid monolayers, a-tocopherol effects, 215 Phosphorylation, whey protein modification by, 419 Photooxidation, polyunsaturated fatty acids, 249-251 products, 251-256 Phytate, effect on calcium absorption, 134142 Plasma calcium concentrations, 112-1 14 folate concentrations, 60 lipoproteins, tocopherol distribution, 174- I77 Platelets membrane fluidity, a-tocopherol effects, 2 17-2 I8 phospholipase Az activity, a-tocopherol effects, 219-220 tocopherol association with, 177 a-tocopherol concentrations, 212 a-tocopherol content, vitamin E status and, 189-190 Polyglutamyl folates affinity chromatography, 38 bioavailability , 64-65 chain length determination with HPLC methods, 34-38 chiral stability, 13 C-9-N-I0 bond, 34-38 deuteration, 11-12 dietary, deconjugation, 54-55 enzymatic deconjugation, 18 HPLC methods, 34-38 ion-exchange separations, 33-34

long-chain reduced (6S), biological synthesis, 11-12 separation by gel filtration chromatography, 33 synthesis, 6-8 liquid-phase methods, 7 pteroic acid preparation, 7-8 solid-phase methods, 6-7 Polyunsaturated fatty acids in fish lipids, 286 oxidation control antioxidants, 309-310 flavonoids, 31 1-314 antioxidant action, 314-316 effects on lipoxygenase, 316-317 lipoxygenase, 306-309 microsomal enzymes, 305-306 peroxidase, 306 oxidation mechanisms enzyme-initiated, 256-270 free radical oxidation, 239-249 oxygen activation, 234-238 photosensitized singlet oxygen, 249256 oxidation of fish lipids enzyme-catalzyed, 291-297 factors affecting, 287-289 metal catalysis of, 289-291 volatile compound generation, 298-302 oxidation products acid-catalyzed decomposition, 278-280 decomposition of acyl hydroperoxides, 270-273 secondary products, 276-278 volatile compounds from acyl hydroperoxides, 273-275 volatile compounds in food, 280-281 oxidation reactants and inhibitors active oxygen species, 302-303 metals, 303-305 oxygen, 302 peroxidation in vivo effects on DNA, 285-286 on membranes, 283-285 on proteins, 282-283 Postprandial changes, in tocopherol content of lipoprotein fractions, 176 Primethamine, effects on folate absorption and metabolism, 70

453

INDEX

Propyl gallate, inhibition of lipid oxidation by microsomes, 306 lipoxygenases, 307 Proteins conformation, effect on functional properties of whey proteins, 367-369 dietary, effect on calcium absorption, 143-144 lipid oxidation effects, 282-283 Proteose-peptones, bovine milk. structure, 358 Pteroic acid, for polyglutamyl folate synthesis, 7-8 F'teroylpolyglutamate hydrolases in folate deconjugation, 17 identification and properties, 55-56 Pteroyltriglutamate, determination in conjugase assay by reversed-phase HPLC, 31 Pyrogallol, inhibition of lipoxygenase, 307

Q Quenching, by a-tocopherol oxygen- or carbon-centered fatty acyl radicals or active oxygen, 208-2 I I singlet oxygen, 210, 303 Quercetin inhibition of soybean lipoxygenase-dependent linoleate peroxidation, 3 17 structure. 3 I I

Redox potential, effects on whey protein foams, 401 Reduction methods chemical. for (6-ambo)-5,6,7,8-tetrahydrofolate, 8 electrochemical. for (6-ambo)-5,6,7,8tetrahydrofolate, 9 enzymatic, for (6S)-5,6,7,8-tetrahydrofolate, 9 Reductive alkylation, whey proteins, 421 Refrigeration. effect on folate in human milk, 51 Repair, membrane proteins by a-tocopherol, 210-211 Retention folates after cooking or thermal processing, table, 48 ascorbate/ferrous sulfate effects, 4950

folic acid in fortified food, 52 Retinal outer rod segments, a-tocopherol concentrations, 212 Retinal pigment epithelium, tocopherols in, I80 Reverse osmosis, whey protein preparation with, 361 Reverse osmosis-ultrafiltration, whey protein preparation with, 361 Risk factors, osteoporosis, 104

S

R Radioimmunoassays, for folate determination, 26 Radiolabeling calcium bioavailability, 123-124 folate bioavailability, 61-64 folates, 11-12 Recommended Dietary Allowance calcium, 105, 108-1 12 folate, 52 tocopherols, 167 Recommended Dietary Intake folates, 52 tocopherols, 166

Salfasalazine. antifolate effects, 71 Salicylazosulfapyridine, antifolate effects, 70-7 1 Salts calcium, solubility, table, 145 effect on whey protein gel structure, 387388 Seasonal variation, in plasma a-tocopherol concentration, 176 Self-association phenomenon, concentration-dependent, of folates, 6 Singlet oxygen formation, 235 oxidation inhibition, 255-256

454

INDEX

Singlet oxygen (continued) linoleate, 253 oleate, 252 photosensitized lipid oxidation, 249-256 quenching by a-tocopherol, 210, 303 Sodium dithionite, reaction with folic acid, 8 Sodium lauryl sulfate, effects on whey protein foams, 401 Sodium tripolyphosphate, control of microsomal-enzyme-associated lipid oxidation, 305-306 Solid-phase methods, for polyglutamyl folate synthesis, 6-7 Solubility calcium, effect on bioavailability, 145147 calcium salts, table, 145 whey proteins, 374-375 Soybean oil, steam refining, tocopherol losses due to, 160, 162 Spinach, total folate content, table, 40 Stability films, protein structure and, 394-395 foams overrun, 400-401 protein structure and, 398400 folates during cooking or thermal processing, 47-5 1 effects of aqueous extraction, 49 intrinsic, 4 0 4 7 in milk products, 5 I monoglutamyl folates in aqueous solution at room temperature, 45 Steam refining, effect on tocopherol content of soybean oil, 160, 162 Storage effect on fish lipid oxidation, 289 effects on whey proteins, 373 oils, effect on tocopherol content, 163164 Streptococcus faecium, for biological syn-

thesis of long-chain folates, 11-12 Sucrose, effect on calcium absorption, 127 whey protein foaming properties, 401, 403-404 Sugars, effect on calcium absorption, 127

Superoxide anion control, 302-303 formation, 236 Superoxide dismutase, control of superoxide anion with, 302 Supplements, calcium, effects on calcium bioavailability, 144-147 Swiss chard, total folate content, table, 40

T TEAE-cellulose, for monoglutamyl folate separations, 26 Temperature effects, see also Heat effects on binding of flavor compounds to whey proteins, 41 1-412 on fish lipid oxidation, 288

5,6,7,8-Tetrahydrofolate energy of activation for degradation in aqueous solution, table, 50 oxidative degradation by air, 44 preparation 6-ambo form chemical reduction of 7.8-Hzfolate, 8 electrochemical method, 9 folk acid-sodium dithionite method, 8 hydrogenation of folic acid in glacial acetic acid, 8 6 s form enzymatic reduction with dihydrofolate reductases, 9 labeling at C-6 with tritium or deuterium, 1 1 Tetrahydrofolic acid, oxidative degradation mechanism, 42-43 Therapeutic drugs, antifolate effects, 70-71 Thermal processing, see also Heat effects folate stability during, 47-51 Thermal separation, whey proteins, 361-362 Thiolation, whey protein modification by, 420-42 I Thiol groups, effect on gill lipoxygenase, 296-297 whey protein gels, 388-389 a-Tocopherol antioxidant chain-breaking, 3 10 function in membranes, 207-21 I

INDEX

binding to membranes, 196-199 bioavailability bran effects, 172-173 fish oil effects, 173 concentration in membranes, 212-214 in vegetable oils, table, 161 content of vegetable oils, heat effects, 164-165 deuterated, incorporation into lipoproteins, 176-177 distribution in plasma lipoproteins, 174 diurnal/seasonal changes in plasma concentrations, 176 exchange between lipoprotein classes, 174-175 exchange rate across bilayers, 2 14 form in vegetable oils, 159 inhibition of lipid oxidation by microsomes, 306 membrane peroxidation, 210 intakes, 166-167 intestinal absorption, 168-170 linoleic acid sources and, 167-168 measurement for vitamin E status, 189 in membranes, physical effects acyl chain order of phospholipid bilayers, 216 hemolysis inhibition in human erythrocytes, 219 permeability to ions or small molecules, 2 18-219 in phospholipid vesicles, 217 platelet membrane fluidity, 217-218 platelet phospholipase A, activity, 219220 stabilization, 214-215 thermotropic behavior, 215-2 16 metabolism biliary metabolites, 199-202 polar metabolites, 200-202 quinone derivative, 199-201 urinary metabolites, 202-204 in milk (human), 179 mobility in membranes, 212-214 in ocular tissues, 180 phenoxyl radical formation, 208-201 postprandial changes in lipoprotein fractions, 176

455

protection and repair of membrane proteins, 210-21 I quenching of reactive oxygen species, 210 of singlet oxygen, 210, 303 radical quenching capability. 208-201 regeneration from a-tocopheroxyl radical, 21 1-212 removal from tissue, role of plasma lipoproteins, 185 RRR and SRR forms, 187-188 structure, I59 tissue levels, 178-179 tissue mobilization and turnover, 186-189 y-tocopherol interactions, 206207 transfer between tocopherol-deficient and tocopherol-sufficient erythrocytes, 185 transfer factors between dipalmitoylphosphatidylcholine liposomes and rat liver microsomes, 195 from liposomes to human erythrocyte ghosts, 195-196 from phosphatidylcholine liposomes to rat liver mitochondria, 194 6-tocopherol effects, 195 a-Tocopherol-binding protein, 191-194 a-Tocopherol hydroquinone, metabolism, 201 a-Tocopherol quinone, formation and distribution, 200-201 f3-Tocopherol antioxidant function in membranes, 207 in vegetable oils, 159-160 y-Tocopherol antioxidant function in membranes, 207 bioavailability, wheat bran effects, 173 concentration in vegetable oils, table, 161 content of soybean oil, heat effects, 165 effect on a-tocopherol transfer, 195 form of, in vegetable oils, 159 intakes, 166 intestinal absorption, 170-171 lipoprotein lipase-mediated transfer to fibroblasts. 183 metabolism, 205-206 in milk (human), 179 structure, 159 tissue levels, 179 a-tocopherol interactions, 206-207

456

INDEX

8-Tocopherol antioxidant function in membranes, 207 concentration in vegetable oils, table, 161 effects on a-tocopherol transfer, 195 metabolism, 195 structure, 159 tissue levels, 179 Tocopherols in animal fats, 164 antioxidant function in membranes, 20721 I binding factors, 191-194 binding to membranes, 196-199 bioavailability diet composition effects, 171-173 fiber effects, 171-173 pectin effects, 171-172 wheat bran effects, 172-173 dietary intake, 166-168 in fats and oils, 159-164 in Finnish oils, 162 in fish and fish oils, 164 food processing/preparation effects, 164166

intestinal absorption effects of gastric or intestinal destruction, 169-170 efficiency, 168-171 via lymphatic system, 168-169 mechanism, 168-171 losses due to processing, 160 extraction method, 162 hydrogenation effects, 163 steam refining, 160, 162 storage effects, 163-164 metabolism regulation, 206-207 a-tocopherol, 199-205 y-tocopherol, 205-206 8-tocopherol, 205-206 mobilization and turnover in tissues, 186189 physical effects in membranes, 214-220 plasma concentration, diurnal and seasonal variations, 176 in plasma lipoproteins, 174-177 exchanges between, 1 7 4 I75 isotopically labeled, incorporation over time, 176-177

postprandial changes, 176 recommended daily intake, 166 removal from tissues, 185 sources, 167 status assessment in humans, 189-191 in tissues human, 178-179 ocular, 280 transfer factors, 194-196 transport in blood, 174177 uptake by tissues, 180-186 biphasic patterns of disappearance and accumulation, I 8 4 185 chylomicron role, 183-184 donation from formed elements of blood, 185-186 high-density lipoprotein-mediated transfer, 183 lipoprotein lipase-mediated transfer. 183- I84 receptor-mediated low-density lipoprotein internalization, 180-182 tissue low-density lipoprotein clearance and, 182 very-low-density lipoprotein-mediated transfer, 183 vitamers in vegetable oils, table, 161 Tocopheronic acid, structure, 203 Tocopheronolactone, structure, 203 Tocopheronolactone glucuronide, structure, 203 Tocotrienols in Finnish oils, 162 in vegetable oils, 160 Trace metals, effect on folate bioavailability, 79-80 Transfer factors, a-tocopherol, 194-196 Transition metals initiation of lipid oxidation, 238 oxidation of fish lipids, 289-291 Transport, tocopherols in blood, 174-177 Transport values, K,,,, folates, 53 Triethylaminoethyl-cellulose, for monoglutamyl folate separations, 26 Trimethoprim, effects on folate absorption and metabolism, 70 Turnover folates, in vivo kinetics, 57-58 tocopherols, 186-189

INDEX

457

U

W

Ultrafiltration, whey protein preparation with, 361 Ultra-high temperature pasteurization of milk, effect on folate activity, 51 Ultraviolet absorbance, detection of eluted folates with, 28 Urine calcium balance measurements and, 108 diet effects, 120 a-tocopherol metabolites, 202-204 y-tocopherol metabolites, 205-206 8-tocopherol metabolites, 205-206

Wheat bran, effects on calcium absorption, 137-1 38 tocopherol bioavailability, 172-173 Whey products demineralized delactosed whey, 347 dry sweet whey, 347 powders as replacement for nonfat dry milk, 348 types used in food, table, 348 upgrading and refining, 348-349 uses, table, 346 susceptibility to denaturation, 347 use of, 347 whey protein concentrate, 347 Whey protein nitrogen index, whey protein denaturation, 372 Whey proteins adsorption, 391-393 emulsifying properties characterization, 405-406 factors affecting, 408 protein conformation and, 406-408 surface hydrophobicity and, 406-408 films, 395-3% effects of aging, 394 pH and ionic strength effects, 394 protein structure and, 393-395 stability, 394 viscoelastic behavior, 394-395 flavor binding, 410-413 thermodynamic representation, 412 and flavors, interactions, 415-416 foaming properties, 402-404 foams, 400-402 functional properties, 364-365 effects of variability in protein preparations, 370-373 extrinsic factors affecting, 369-370 factors affecting, table, 367 intrinsic factors affecting, 365-369 requirements in food products, table, 366 gelation, 381-385 factors affecting gel composition and properties, 385-389 forces in gel formation and stabilization, table, 384

v Vegetable oils, tocopherols losses due to extraction method, 162 hydrogenation, 163 processing, 160 steam refining, 160, 162 storage, 163-164 vitamers in, table, 161 Vegetables fiber, effect on calcium bioavailability, 139-141 folate bioavailability, 75 Viscoelasticity, whey protein films, 394 Vitamin B,, deficiency, effects on folate bioavailability, 79 Vitamin D, and calcium intake, effects on calcium absorption, 115-1 16 Vitamin ¶thyroid hormone system, regulation of plasma calcium concentrations, 113-1 14 Vitamin E status, assessment in humans, 189-191 Volatile compounds from acyl hydroperoxides, 273-275 flavor compounds from lipid oxidation in biological tissues. table, 274 in fish tissues, table, 275 in food, 280-281 generation in fish, 298-302

458 Whey proteins (continued) gel structure, 383-384 as gelling ingredients, 389-391 hydration, 373-374 infant formula, 422-423 lipid binding, 408-409 modification chemical, 417-418 acylation, 418-419 amidation and esterification, 419-420 glycosylation, 421-422 phosphorylation. 419 reductive alkylation, 421 thiolation, 420-421 enzymatic, 4 16-417 nutritional aspects, 422-423 preparation and isolation chromatographic methods, 361 combined reverse osmosis-ultrafiltration, 361 compositions of derived proteins, table, 363 conventional methods, 360-361 denaturation assessment methods, 363 reverse osmosis, 361 thermal separation, 361-362 ultrafiltration, 361 research needs, 424-425 solubility, 374-375 structure amino acid compositions, table, 350 bovine serum albumin, 357 emulsifying properties and, 406-408

INDEX

film-forming properties and, 393-395 foam stability and, 398-400 immunoglobulins, 357-358 a-lactalbumin, 354-357 lactofemn, 358-359 P-lactoglobulin, 350-354 lysozyme, 359 physicochemical characteristics, table, 349 proteose-peptones, 358 surface activity, 391-397 thermal properties, 376 heat denaturation, 378-379 heat denaturation temperatures and enthalpies, table, 377 heat effects on conformation, 376-378 White blood cells, tocopherol association with, 177 World Health Organization, recommended dietary allowances for calcium, table. 109

Y Yeast, folate bioavailability, 73-74

Z Zinc-folate interactions, effects on folate bioavailability , 79-80