Development and processing of vegetable oils for human nutrition

Development and Processing of Vegetable Oils for Human Nutrition

Copyright © 1995 AOCS Press

Development and Processing of Vegetable Oils for Human Nutrition

Editors Roman Przybylski Bruce E. McDonald Department of Foods and Nutrition University of Manitoba Winnipeg, Canada

Champaign, Illinois

Copyright © 1995 AOCS Press

AOCS Mission Statement To be a forum for the exchange of ideas, information, and experience among those with a professional interest in the science and technology of fats, oils, and related substances in ways that promote personal excellence and provide high standards of quality. AOCS Books and Special Publications Committee E. Perkins, chairperson, University of Illinois, Urbana, Illinois T. Foglia, USDA—ERRC, Philadelphia, Pennsylvania M. Mossoba, Food and Drug Administration, Washington, D.C. Y.-S. Huang, Ross Laboratories, Columbus, Ohio L. Johnson, Iowa State University, Ames, Iowa J. Lynn, Lever Brothers, Edgewater, New Jersey G. Maerker, Oreland, Pennsylvania G. Nelson, Western Regional Research Center, San Francisco, California F. Orthoefer, Riceland Foods Inc., Stuttgart, Arkansas J. Rattray, University of Guelph, Guelph, Ontario A. Sinclair, Deakin University, Geelong, Victoria, Australia G. Szajer, Akzo Chemicals, Dobbs Ferry, New York L. Witting, State College, Pennsylvania B. Szuhaj, Central Soya, Ft. Wayne, Indiana Copyright © 1995 by AOCS Press. All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without written permission of the publisher. The paper used in this book is acid-free and falls within the guidelines established to ensure permanence and durability.

Library of Congress Cataloging-in-Publication Data Development and processing of vegetable oils for human nutrition/ editors, Roman Przybylski and Bruce E. McDonald. p. cm. Includes bibliographical references and index. ISBN 0-935315-66-7 (alk. paper) 1. Oils and fats, Edible. 2. Vegetable oils. 3. Nutrition. I. Przybylski, Roman. II. McDonald, B.E. (Bruce Eugene), 1933– . TX407.034D49 1995 664′.3—dc20

Printed in the United States of America with vegetable oil-based inks. 00 99 98 97 96 95 5 4 3 2 1

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95-33314 CIP

Preface The recommendation that consumers reduce total fat to 30 percent and saturated fat to 10 percent of their total energy intake has had a tremendous effect on the food industry, particularly the fats and oils industry. Other major developments that have affected the edible fats and oils industry include the findings that (i) monounsaturated fatly acids are as effective as polyunsaturated fatty acids in lowering blood cholesterol; (ii) hydrogenated fats, or more precisely the trans fatty acids found in hydrogenated fats, may have an undesirable physiological effect; and (iii) n-3 fatty acids are important dietary constituents in health and disease. Several new oilseed varieties have already been developed and many others are under development in response to these findings. The development of novel oilseed varieties has produced a scramble among regulatory agencies to develop guidelines governing the licensing and release of these new crops. An added problem for governmental agencies, particularly in light of new agreements covering the international movement of food products, is the need to develop and standardize food labeling regulations. These developments were major factors in the decision to organize a conference on the development and processing of vegetable oils for human nutrition. The Canadian Section of the AOCS was invited to organize the conference in conjunction with its Annual Meeting on October 2–4, 1994. The Conference was a success thanks to the efforts of the Organizing Committee and its chairman James Daun, the support of sponsors and donors, and the distinguished group of speakers. Current nutrition issues and the contributions of processing, genetic engineering, and plant breeding were reviewed, as well as the role of government agencies in the development of novel oilseed crops. This monograph covers all of these issues, beginning with an up-to-date coverage of nutritional issues, followed by a discussion of current developments in processing vegetable oils for human consumption and the modification of traditional oilseed sources by genetic manipulation. The monograph concludes with a synopsis of the regulatory requirements in Canada, the United States, and Europe for the registration of novel oilseed crops and the nutrition labeling of these new oils. As the editors, we would like to thank the speakers for their cooperation in providing us with manuscripts. We are especially grateful to Angela Dupuis for willingly and patiently transcribing the manuscripts to a common format and

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her very significant efforts toward the success of this publication. We are grateful for the unique satisfaction that comes with having contributed to the knowledge on this subject. Roman Przybylski and Bruce E. McDonald Department of Foods and Nutrition University of Manitoba Winnipeg, Canada

Copyright © 1995 AOCS Press

Contents Preface Chapter 1

Food Fats and Fatty Acids in Human Nutrition Joyce L. Beare-Rogers

Chapter 2

Nutrition and Metabolism of Linoleic and Linolenic Acids in Humans E.A. Emken

Chapter 3

Trans Fatty Acids in Canadian Breast Milk and Diet W.M.N. Ratnayake and Z.Y. Chen

Chapter 4

Food Industry Requirements for Fats and Oils: Functional Properties T.K. Mag

Chapter 5

Hydrogenation: A Useful Piece in Solving the Nutrition Puzzle Robert C. Hastert and Robert F. Ariaansz

Chapter 6

Interesterification: Current Status and Future Prospects Suresh Ramamurthi and Alan R. McCurdy

Chapter 7

Sources of Oilseeds with Specific Fatty Acid Profiles W.A. Keller

Chapter 8

Production of Oilseeds with Modified Fatty Acid Composition Rachael Scarth

Chapter 9

Classification of Oils with Modified Fatty Acid Compositions as Novel Foods Frank W. Welsh

Chapter 10 Food Labeling in Canada Ian Campbell Chapter 11 Safety Evaluation and Clearance Procedures for New Varieties of Oilseeds in the United States and Canada Donna Mitten, Keith Redenbaugh, and Julianne Lindemann

Copyright © 1995 AOCS Press

Chapter 1

Food Fats and Fatty Acids in Human Nutrition Joyce Beare-Rogers 41 Okanagan Drive, Nepean, Ontario, K2H 7E9, Canada

This paper will deal principally with the fatty acids in food fats.

Total Dietary Fat A first consideration should be the amount of fat or fatty acids in the diet. It has long been appreciated that a caged experimental animal given a high-fat diet eventually becomes obese. An excellent demonstration in humans showed the interaction of the level of fat, provided covertly in an ad libitum diet, and level of physical activity (1). At the lowest level of fat to maintain energy balance, physical activity produced a negative energy balance. The intermediate level of dietary fat caused a positive shift in balance with a pronounced difference between sedentary and active individuals. At the highest level, 60 en%, both groups of individuals had a positive energy energy balance, but the energy storage was greater in inactive individuals. Particularly within the range of usual fat intake, there is a tradeoff with physical activity where the effect of fat is offset by the utilization of energy. Energy Storage Another aspect of fat ingestion is that appetite regulation fails to respond to fat in the same way that it does to carbohydrate (2). Individuals tend to be insensitive to the level of fat in a meal and are consequently apt to overeat. Excess carbohydrate is stored only to a limited extent and is then converted to fat. The cost of metabolic conversion is relatively high for carbohydrate and protein, but fatty acids are easily added to stored energy. Therefore, for sedentary individuals the recommendation for an upper range of fat intake has been 30% of energy.

Fatty Acids for Infants Fat and saturated fatty acids supply the energy consumed in cellular growth at certain stages of life, particularly infancy. Most human milk provides fat in which the total proportion of saturated fatty acids shorter than 18 carbon atoms is approximately equal to the monounsaturated fatty acids, principally oleic (3–5). Since the fat in human milk is 45–55% of the total dietary energy, the saturated component provides about 18% of the energy, considerably more than the ceiling of 10% that

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is frequently recommended. Questions about the fatty acid composition of infant formula have usually revolved around the essential fatty acids and the role that docosahexaenoic acid plays in neural membranes. Here it is important that the n-6 fatty acids be considered along with the n-3 fatty acids. Of course, the maternal diet is the main source of fatty acids for the fetus. Koletzko reported that the trans fatly acids in infant blood were inversely correlated with the long-chain n-3 and n-6 fatty acids (6). This apparent interference with the conversion of essential fatty acids was therefore thought to involve the desaturases, but placental receptors also may be sites of influence.

Lipoproteins The greatest debate about dietary fatty acids revolves around their effects on blood lipoproteins, that is, the concentration of high-risk, low-density lipoproteins (LDL), the ratio of LDL to HDL (not just total cholesterol, but the distribution of the particles in which it is carried), and the concentration of Lp(a) that limits plasmin production and promotes clotting and vascular smooth muscle proliferation. The saturated fatty acids, although frequently considered together, do have different degrees of influence on the concentration of LDL. Laurie and myristic acids, which are usually found in the same oils, are more hypercholesterolemic than palmitic acid (7,8). Palmitic acid is more hypercholesterolemic than stearic acid (9), which is considered neutral in terms of modifying cholesterol levels. However, stearic acid may not be neutral in thrombotic tendency or in its effect on arrhythmia. Whether a vegetable oil is considered hypo- or hypercholesterolemic depends upon the reference oil. Thus, palm oil is hypocholestcrolemic with respect to coconut oil but hypercholesterolemic with respect to corn oil (10).

Prediction by Equations The early equations of Keys et al. and Hegsted et al. emphasized that a change in plasma cholesterol was proportional to twice the amount of energy supplied by saturated fatty acids minus the amount of energy supplied by polyunsaturated fatty acids plus a small factor for dietary cholesterol (11, 12). In all later regression lines, the greatest adverse effect on plasma cholesterol levels was also associated with the intake of saturated fatty acids. The attempts to reduce the consumption of saturates have sometimes led to extreme proposals, the idea apparently being that since high amounts are bad, intermediate amounts must be barely tolerable and low amounts must be best. The quest for extremely low dietary levels of saturated fatty acids seems futile because if there are insufficient dietary saturated fatty acids to occupy the 1-position of membrane phospholipids, they have to be synthesized by the body. The effect of linoleic acid in reducing plasma cholesterol is thought to be nonlinear, plateauing at about 5% of energy, and having a range of 3–10% according to

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an individual’s responsiveness (13). Below the so-called threshold, an increase of 3% of energy as linoleic acid decreased plasma cholesterol by 35 mg/dL. A similar change in plasma cholesterol above the threshold required 13% of energy as linoleic acid. In this range, there is relative insensitivity to changes in dietary fatty acids, although the lowest concentrations of blood cholesterol occurred with high intakes of linoleic acid (14). The fact that individuals have different thresholds helps to explain some of the disparity in experimental results.

High Intakes of Oleic Acid The reported equivalence of oleic acid and linoleic acid in reducing LDL-cholesterol may have been related to high thresholds for dietary linoleic acid (15). Linoleic acid appeared to reduce HDL-cholesterol, but the high intake of linoleic acid in this study would be difficult to attain or maintain with ordinary foods. In another study without a group fed a high level of linoleic acid, Grundy et al. showed that a diet high in oleic acid was preferable to a low-fat diet (high carbohydrate) in sustaining HDL-cholesterol while decreasing LDL-cholesterol (16), Test fats consisting of butterfat, beef fat, cocoa butter, and olive oil produced no differences in HDL-cholesterol (17). Low-density lipoprotein cholesterol was highest with butterfat and significantly lower with cocoa butter, indicating that the position of the fatty acids on the acylglycerols was important. It appears that in at least some situations, saturated fatty acids in the 2-position are the most hypercholesterolemic.

α-Linolenic Acid and Postinfarct Patients A comparison was made between the usual postinfarct prudent diet and a Mediterranean diet that used a margarine made from canola oil (18). Improved mortality after a first myocardial infarction was attributed to the increased intake of α-linolenic acid. Although this observation is encouraging for canola oil, it must be remembered that many dietary features differed between the two dietary groups, and that more definitive work is required.

Trans Fatty Acids and Lipoproteins The impact of dietary fats containing trans monounsaturated fatty acids, as determined in Trappist monks, has stood the test of time (19). In the presence of dietary cholesterol, the trans fatty acids were associated with serum cholesterol levels that were higher than those obtained with oleic acid and slightly lower than those obtained with a mixture of lauric and myristic acids. The much quoted paper of Mensink and Katan (20), was the first to show that trans monounsaturated acids increased LDL-cholesterol and decreased HDL-cholesterol, worsening the LDL/HDL ratio. Although this 3-week study was criticized for the high level of trans fatty acids (11% of dietary energy) and the means of

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production by chemical isomerization rather than by commercial hydrogenation, these initial findings have been confirmed. The positional trans isomers used in the study were similar to those found in partially hydrogenated soybean oil; the positional cis isomers had one type, the 8-octadecenoic acid, that was higher than ordinarily found (21), but no significance has been attached to it. Another study from the same laboratory (22), had a lower level of trans fatty acids, 7.7% of dietary energy instead of 11%. The results of the two separate studies suggested a doseresponse to trans fatty acids. The finest precision yet seen in the determination of lipoprotein levels appeared in Judd et al. (23). The levels of trans fatty acids tested for 3 and 6 weeks were 3 and 6% of dietary energy. Unfortunately, only the data from the longer period were published; data from 3 weeks would have facilitated comparison with the results obtained in the study of Mensink and Katan. Again, the trans fatty acids were associated with increased LDL-cholesterol and decreased HDL-cholesterol when compared with oleic acid. It must be remembered that the original purpose of these experiments was to determine how trans fatty acids should be regarded, given that saturated fatty acids were already designated as hypercholesterolemic. Also, products promoted as being low in saturated fatty acids were sometimes high in trans fatty acids. At issue is whether saturated fatty acids should be replaced by trans fatty acids. More sensibly, both should be reduced in the total diet. The average intake (50th percentile) of any substance gives no indication of the risk to vulnerable individuals. Information on at least the 90th percentile of intake and the associated food patterns is required. Investigations of trans fatty acids should therefore provide estimates of possible human exposure along with intake guidelines for essential fatty acids, particularly for pregnant and lactating women. The assessment of intake of trans fatty acids loses accuracy when a part of the diet is self-selected. For many foods, the fatty acid composition is not accurately known, and the possible combinations are considerable. The most reliable data on fatty acid consumption are obtained from the analysis of all foods given to the participants of a study. Values for the coefficients of variation of total cholesterol for example, calculated from papers dealing with dietary trans fatty acids (20,22–27), are given in Table 1.1. Flynn et al. tested margarine versus butter with two eggs/day in an otherwise self-selected diet. Judd et al., Lichtenstein et al., Mensink and Katan, and Zock and Katan provided all foods to the test subjects. Wood et al. supplied test fats that were one-half of the total fat in diets that were rotated every 6 weeks. The precision of the experiment of Judd et al. (23) stands out, partly because of the control in subject selection and in the analytical procedures. The selection of subjects for this study raised questions about the general applicability of the results. The subjects had normal levels of all blood lipids, were free of any disease, and maintained their usual exercise program without gaining weight, in spite of a mean energy consumption of 3227 kcal/day for the men and 2025 kcal/day for the women. These healthy athletic subjects did not reflect the larger community, and

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TABLE 1.1 Precision in Human Studies on Trans Unsaturated Fatty Acids Study Flynn et al. Judd et al. Lichenstein et al. Mensink and Katan Wood et al. Zock and Katan

Foods provided

Foods selfselected +

+ + + + +

Coefficient of variationa % 19.3 4.7 10.7 15.3 14.6 14.7

a

For total cholesterol. Sources: Mensink and Katan (21), Zock and Katan (22), Judd et al. (23), Flynn et al. (24), Lichenstein el al. (25), and Wood et al. (26,27).

might be expected to represent a group with a well-regulated cholesterol metabolism. They constituted such a finely tuned bioassay that as little as 3% of energy from trans fatty acids produced a statistically significant result. Another effect of trans fatty acids on lipoproteins pertains to the risk factor of Lp(a) involved in thrombogenesis. Investigators in Australia and The Netherlands found Lp(a) to be elevated in persons consuming trans fatty acids (28,29). In North America, where this effect has not been observed, the situation will have to be clarified with the most sensitive methods available. Trans Fatty Acids in Epidemiological Studies In an analysis of tissue fatty acids of individuals who died from ischemic heart disease, the adipose tissue fat had increased trans fatty acids and decreased shorter chain fatly acids (30, 31). It was concluded that the victims had consumed more hydrogenated fat and less ruminant fat than the controls. Also, in patients undergoing coronary angiography, the level of trans fatty acids was 1.38% versus 1.11% of fatty acids in controls (32). Such results were said to be consistent with the hypothesis that dietary trans fatty acids are a risk factor. Recent studies (33, 34), however, raise questions with this hypothesis, although issue also has been taken with the results and conclusions of these studies (35). More controversial estimates of exposure to trans fatty acids came from semiquantitative, food-frequency questionnaires. Answers to questions about “how often over the previous year” a given portion of a specified food had been consumed became the source of data. Clinical studies in which dietary variables are known and controlled exhibit a scientific rigor that is unfortunately lacking in the responses to semiquantitative questionnaires. In adult men (mean age 62 yr; range 43–85 yr) assessed by a food-frequency questionnaire, total fat was given as 60 g/day and the trans fatty acid intake as 2.1 g at the 10th percentile and 4.9 g at the 90th percentile (36). These low values are inconsistent with other data. Nevertheless, the energy-adjusted intakes of trans fatty acids were reported to be positively correlated with LDL-cholestcrol and inversely correlated with HDL-cholesterol.

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In the Nurses’ Health Study (37), data on consumption came from the same type of questionnaire, with average values used in the assessment for such foods as margarine, cookies, biscuits, cake, and white bread. The intake of trans fatty acids was reported to have varied from 2,4–5.7 g/day or 1.3–3.2% of energy, and to be correlated with the risk of cardiovascular disease. Another paper claiming an association between the intake of trans fatty acids and the risk of cardiovascular disease involved questionnaires administered 8 weeks after patients had been discharged from hospital after a first myocardial infarction (38). The patients were matched with residents of the same town. The average consumption of trans fatty acids was 1.5% of energy for men and 1.7% of energy for women. These levels were about one-half of that calculated for the average trans fatty acids in the American diet (39). The relative risk of myocardial infarction for each quintile of energy-adjusted intake of trans fatty acid was 1.0, 0.89, 0.52, 0.93, and 2.28, respectively; that is, only the last value exceeded the first. The third quintile appeared to be the best. Overall, the epidemiological studies emphasize the need for additional research on the physiological effects of trans fatty acids and that, in the interim, prudence be exercised in the consumption of these fatty acids.

Idealized Dietary Fat Biotechnologists have challenged nutritionists to provide them with the fatty acid profile of the ideal vegetable oil. What is important is the lipid content of the total diet. For one oil to have an impact, it would have to be an appreciable contributor to the dietary fat. This does happen with some types of food patterns, but in most mixed diets there is some trade-off between foods high and low in a particular fatty acid. It is the ultimate blend that counts. For the total fatty acids in an adult diet, the saturated fatty acids (mostly palmitic) could be 10–25%, linoleic acid could be 10–20%, α-linolenic could be about 2%, and the rest could be oleic acid. The only virtue of a very low level of saturated fatty acids in a vegetable oil would be to dilute those from other sources. Since food preparation involves fats used in different ways, there might be an ideal salad oil, an ideal spread, an ideal cooking fat, and so on. To propose a fatty acid composition for an ideal vegetable oil, one would need information about the other foods to be consumed. It is the total dietary fatty acids that are important in nutrition. References 1. Stubbs, R.J., and A.M. Prentice, Am. J. Clin. Nutr. 62: 330–337 (1995). 2. Flatt, J.P. in Obesity, edited by P. Bjorntorp and B.N. Brodoff, J.P. Lippincott Co.,1992, pp. 100–116. 3. Sanders, T.A.B., F.R.Ellis, and J.W.T. Dickerson, Am. J. Clin. Nutr. 31: 805 (1978). 4. Carlson, S.E., P.G. Rhodes, and M.G. Ferguson, Am. J. Clin. Nutr. 44: 798 (1986). 5. Chen, Z.-Y., G. Pelletier, R. Hollywood, and W.M.N. Ratnayake, Lipids, in press. 6. Koletzko, B., Ada Paed. 81: 302 (1992).

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 36. 33.

34. 35. 37. 38. 39.

McGandy, R.B., D.M. Hegsted, and L.M. Meyers, Am. J. Clin. Nutr. 23: 1288 (1970). Sundram, K., K.C. Hayes, and O.H. Siru. Am. J. Clin. Nutr. 59: 841 (1994). Bonanome, A., and S.M. Grundy, N. Eng. J.Med. 318: 1244 (1988). Kris-Etherton, P.M., J. Derr, D.C. Mitchell, V.A. Mustad, M.E. Russel, E.T. McDonell, D. Salabsky, and T.A. Pearson, Metabolism 42: 121 (1993). Keys, A., Anderson, J.T., and F. Grande, Lancet 2: 959 (1957). Hegsted, D.M., R.M. McGandy, M.L. Myers, and F.J. Stare, Am. J. Clin. Nutr. 17: 281 (1965). Hayes, K.C. and P. Kosla, Fed. Am. Soc. Exp. Biol. J. 6: 2600 (1992). Hegsted, D.M., L.M. Ausman, J.A. Johnson, and G.E. Dallal, Am. J. Clin. Nutr. 57: 875 (1993). Mattson, F.H., and S. Grundy,.J. Lipid Res. 26: 194 (1985). Grundy, S.M., L. Florentin, D. Nix, and M.F. Whelan, Am. J. Clin. Nutr. 47: 965 (1988). Denke, M.A., and S.M. Grundy, Am. J. Clin. Nutr. 54: 1036 (1991). De Lorgeril, M., S. Renaud, N. Mamelle, P. Salen, J.-L. Martin, I. Monjaud, J. Guidollet, P. Touboul, and J. Dclaye, Lancet 343: 1454 (1994). Vergroesen, A.J., and J.J. Gottenbos, in The Role of Fats in Human Nutrition, edited by AJ. Vergroesch, Academic Press, London, 1975, pp. 1–32. Mensink, R.P., and M.B. Katan, N. Eng. J. Med. 323: 429 (1990). Mensink, R.P., and M.B. Katan, N. Eng. J. Med. 324: 339 (1991). Zock, P.L., and M.B. Katan, J. Lipid Res. 33: 399 (1992). Judd, J.T., B.A. Clevidence, R.A. Muesing, J. Wittes, M.E. Sunkin, and J.J. Podczasy, Am. J. Clin. Nutr. 59: 861 (1994). Flynn, M.A., G.B. Nolph, G.Y. Sun, M. Navidi, and G. Krause, J. Am. Coll. Nutr. 10: 93 (1991). Lichtenstein, A.H., L.M. Ausman, W. Carrasco, J.L. Jenner, J.M. Ordovas, and E.J. Schaefer, Arter. Throm. 13: 154 (1993). Wood, R., K. Kubena, B. O’Brien, S. Tseng, and G. Martin, J. Lipid Res. 34: 1 (1993). Wood, R., K. Kubena, S. Tseng, and G. Martin, J. Nutr. Biochem. 4: 286 (1993). Nestel, P.J., M. Noakes, G.B. Belling, R. McArthur, P. Clifton, E. Janus, and M. Abbey, J. Lipid Res. 33: 1029 (1992). Mensink, R.P., P.L. Zock, M.B. Katan, and G. Hornstra, J Lipid Res. 33: 1493 (1992). Thomas, L.H., and R.G. Scott, J. Epid. Comm. Health 35: 251 (198 I). Thomas, L.H., J.A. Winter, and R.G. Scott, J. Epid. Comm. Health 37: 22 (1983). Siguel, E.N., and R.H. Lerman, Am. J. Card. 71: 916 (1993). Troisi, R., W.C. Willet, and S.T. Weiss, Am. J. Clin. Nutr. 56: 1019 (1992). Aro, A., F.M. Kardinaal, I. Salminen, J.D. Kark, R.A. Riemersma, M. DelgardoRodriquez, J. Gomez-Aracena, J.K. Huttunen, L.Kohlmeier, B.C. Martin-Moreno, V.P. Mazaev, J. Ringstad, M. Thamm, P. van’t Veer, and F.J. Kok, Lancet 345: 273 (1995). Roberts, T.L., D.A. Wood, R.A. Riemersma, P.J. Gallagher, and F.C. Lampe, Lancet 545: 278 (1995). Letters to Editor, Lancet 345: 1107 (1995). Willet, W.C, M.J. Stampfer, J.E. Mason, G.A. Colditz, F.E. Speizer, B.A. Rosner, L.A. Sampson, and C.H. Hennekens, Lancet 341: 581 (1993). Ascherio, A., C.H. Hennekens, J.E. Buring, C. Master, M.J. Stampfer, and W.C. Willet, Circulation 89: 94 (1994). Hunter, J.E., and T.H. Applewhite, Am. J. Clin. Nutr. 54: 363 (1991).

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Chapter 2

Nutrition and Metabolism of Linoleic and Linolenic Acids in Humans E.A. Emken USDA1, ARS, NCAUR, 1815 N. University Street, Peoria, illinois, 61604, USA.

Introduction The importance of the early observations reported in 1956 by Sinclair (1), that Eskimos had little or no cholesterol deposits in their coronary arteries and a low incidence of coronary heart disease because of the n-3 fatty acids in their diet, was largely ignored by public health and medical organizations. In fact, Sinclair’s theory was termed imaginative by Key’s, who was a leading authority on heart disease and diet (2). A dramatic change in the health and medical community’s perception of the nutritional importance of n-3 fatty acids occurred when Bang et al. reported in 1971 that the high intake of n-3 fatty acids from fish was a key factor in the low mortality rate from coronary heart disease observed in Greenland Eskimo populations (3). Since those early times, there has been a growing accumulation of evidence that indicate n-3 long-chain fatty acids (LCFA) are associated with various antiatherogenic properties and a number of other health benefits, although this issue is still controversial (4–6).

Biological Properties It is now appreciated that n-3 and n-6 fatty acids have very different physiological effects. One reason is the difference in the physiological properties of the eicosanoids produced by the lipoxygenase and cyleooxygenase pathways from 20:5n-3, 20:3n-6, and 20:4n-6. In most cases, in vitro studies have shown that the physiological effects of the 1- and 3-series of prostaglandins formed from 20:3n-6 and 20:5n-3 are opposite the effects of the 2-series of prostaglandins formed from 20:4n-6. These results have led to the hypothesis that a balance between the various eicosanoids and their n-6 and n-3 precursors is necessary to regulate many physiological functions. These observations for the n-3 and n-6 LCFA have raised several questions concerning the nutritional importance of linolenic acid present in plant sources. A 1 Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. Portions of this paper have been published in the Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.

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basic question is whether the conversion of linolenic acid (18:3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) in humans has quantitative importance. Of practical concern is whether linolenic acid from plant sources is a viable alternative to dietary sources containing preformed n-3 LCFA.

Requirements Evidence from animal studies indicates that competition between fatty acids from the n-3 and n-6 families influences the incorporation of these fatty acids into tissue lipids and mediates their biological effects (4–6). These results raised the question of whether the actual amounts of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) or the 18:2n-6/18:3n-3 ratio in the diet has more nutritional importance. It is difficult to determine exactly what the best 18:2n-6/l8:3n-3 ratio for the human diet is. Examples of some of the 18:2n-6/18:3n-3 ratios recommended are 6:1–10:1 (7), 5:1 (8), 4:1–6:1 (9). An interesting recent study reported that rats fed diets with a 4:1–5:1 ratio of 18:2n-6 to 18:3n-3 were smarter, healthier, and tougher than rats fed diets with an n-6 to n-3 ratio of 3:1 or 6:1 (10). The estimates given in Table 2.1 for a hypothetical diet provide some guidance for the actual amounts of dietary 18:2n-6 and 18:3n-3 required to meet essential fatty acid recommendations (11–16).

Metabolism and Effect of Diet Experiments with animal models have provided most of the information on the effect of varying the balance between 18:2n-6 and 18:3n-3 (4–6,17–18). Studies with radioisotope–labeled substrates have been particularly useful for investigating TABLE 2.1 Estimated Recommendations for Essential Fatty Acids Translated for 2400 Kcal–Based Diet Containing 90 g (34% Energy) of Total Fat Fatty acid Linoleic acid Adult/infant Pregnant mother Lactating mother Adult Linolenic acid Adult Infant Adult 20:5n-3 plus 22:6n-3 Adult Adult

Total calories (%)

(g)

Total fat (%)

2–3 4.5 6.0 2.4

5–8 12 16 6.4

6–9 13 18 7.1

11 11 11 12

1.0 2–3 0.3

2.7 — 0.8

3.0 — 0.9

13 11 14

0.13 0.27

0.3–0.4 0.7

0.4 0.8

15,16 13

Source: Dietary Fats and Oils in Human Nutrition (11). Bourre et al. (12,14), Simopoulos (12), and Bjerve et al. (15,16).

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Reference

the oxidation and conversion of 18:2n-6 to 20:4n-6 and 18:3n-3 to 20:5n-3 and 22:6n3 (4–6,19–21). By contrast, experiments in humans using isotope-labeled n-6 and n-3 fatty acids are limited. Results for the conversion of 18:2n-6 in vitro have been reported for human liver microsomes (22–23) and human leukocytes (24). In vivo data have been published for one study with deuterium-labeled 20:3n-6 (25), two studies with C-labeled l8:2n-6 (26–27) and two studies with deuterated 18:2n-6 (28–29). We have recently reported results that directly compare the metabolism of deuterium-labeled linolenic acid and linoleic acid in young adult male subjects that had been previously fed diets containing two different levels of linoleic acid (30). The results were used to address the question of whether an increase in dietary 18:2n-6 intake influences incorporation and desaturation of 18:3n-3 and l8:2n-6. The experimental design consisted of feeding four subjects a triacylglycerol (TAG) mixture containing both deuterated 18:2n-6 (3.0–3.5 g) and 18:3n-3 (3.0–3.5 g). Three additional subjects were fed a deuterated TAG mixture that contained 2.2 g of deuterated 18:3n-3 as the only polyunsaturated fatty acid. In addition to labeled linoleic acid and linolenic acid, the mixtures of deuterated fats contained 2 or 3 of the following deuterated fatty acids: 16:0, 18:0, or 18:1. The deuterated TAG mixtures were fed after the subjects had fasted for 12 hr. Blood samples were collected over a 48-hr period. Methyl esters of the plasma lipids were analyzed by gas chromatograph-mass spectrometry methods (31). The subjects were fed control diets for 12 days prior to being fed the deuterated TAG mixtures. The composition of the control diets provided 35–36% of calories from fat, 43–44% from carbohydrates, and 21% from protein. The saturated fat (SAT) diet contained 15.1 g 18:2n-6 and 1.9 g 18:3n-3 (n-6/n-3 ratio = 8; P/S = 0.35) and the polyunsaturated fat (PUFA) diet contained 29.8 g of l8:2n-6 and 1.0 g 18:3n-3 (n-6/n-3 ratio = 30; P/S = 0.85). The amounts of 18:2n-6 in the diets were chosen to bracket the 21 g of l8:2n-6 estimated for a typical U.S. diet (n-6/n-3 ratio = 11; P/S = 0.59) [32]. 14

Incorporation of Fatty Acids into Body Lipids Results for the chylomicron triglyceride samples showed that the deuterated 18:2n-6 to 18:3n-3 ratio in the chylomicron TAG samples was slightly lower (ca. 8%) than for the 18:2n-6 to 18:3n-3 ratio in the mixture fed. This difference indicates that 18:3n-3 may be absorbed slightly more efficiently than 18:2n-6, but the difference was not significant. Differences between subjects were relatively small for the concentrations of deuterated l8:2n-6 (range 17.1-19.4 µg/mL) and l8:3n-3 (range 18.5–23.8 µg/mL) in the chylomicron TAG samples. These results indicate that the fatty acid composition of the prefed diets had no significant effect on absorption of 18:2n-6 and 18:3n-3. Examples of time course curves for incorporation of deuterated l8:2n-6 and 18:3n-3 into plasma phosphatidylcholine (PC) are plotted in Figure 2.1.

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Fig. 2.1. Examples of time course curves for incorporation of deuterated 18:2n-6 and 18:3n-3

into plasma phosphatidylcholine samples from male subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. Source: Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.

Qualitatively, these curves illustrate that phosphatidylcholine acyltransferase is more selective for 18:2n-6 than 18:3n-3. The mean values for the integrated areas of the time course curves for plasma PC samples from the subjects from the PUFA diet group were 363 ± 52 µg/mL 18:2n-6 and 58.5 ± 42 µg/mL 18:3n-3. Mean plasma PC values for subjects from the SAT diet group were 600 ± 6.5 µg/mL 18:2n-6 and 66.0 ± 21.7 µg/mL 18:3n-3. These results indicated that the higher 18:2n-6 content of the PUFA diet reduced the amount of deuterated 18:2n-6 incorporated (P < 0.02) but not the amount of 18:3n-3 incorporated into plasma PC. This difference

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in the ratio of deuterated 18:2n-6 to 18:3n-3 concentrations in plasma PC indicated that phosphatidylcholine acyltransferase is six to nine times more selective for 18:2n-6 than 18:3n-3. The mean concentrations for deuterated 18:2n-6 and 18:3n-3 in plasma TAG are compared in Figure 2.2 to concentration data for the deuterated 16:0, 18:0, and 18:1 fatty acids that were also part of the mixtures of deuterated TAG fed to these subjects. The general pattern for the deuterated fatty acids incorporated arc similar for subjects fed the SAT and PUFA diets. However, the concentration for the 18-carbon fatty acids were consistently lower for the subjects fed the PUFA diet. Concentration data for total plasma lipids for each subject are compared in Figure 2.3. The total lipid data show an overall preferential (ca. threefold) incorporation of 18:2n-6 relative to l8:3n-3. The higher 18:2n-6 content of the PUFA diet reduced the incorporation of deuterated 18:2n-6 and 18:3n-3 by about 40%. The combined TAG, PC, and total lipid data suggest the possibility that the higher 18:2n-6 content of the PUFA diet increased fatty acid oxidation by about 30%, which is reasonably consistent with the 9% increase in fat oxidation (based on use of O water methods) when a diet with a P/S ratio of 1.65 was fed in place of a P/S 0.24 ratio diet (33). The plasma TAG and total lipid data in Figures 2.2 and 2.3 provide evidence that incorporation of deuterated 18:2n-6 and 18:3n-3 in the major lipid classes were 18

Fig. 2.2. Concentration (µg/mL plasma) of deuterated fatty acids in plasma triacylglycerol samples from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. Bars indicate high and low values. For the unsaturated fatty acids, the SAT vs. PUFA diet data are significantly different (P < 0.05). Source: Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.

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Fig. 2.3. Concentration (µg/mL plasma) of deuterated 18:2n-6 and 18:3n-3 in plasma total lipid

samples from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. P < 0.05 for the 18:3n-3 SAT versus PUFA diet means. P < 0.17 for the 18:2n-6 SAT vs. PUFA diet means. Source: Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.

significantly depressed by increased dietary 18:2n-6 intake. Why do dietary 18:2n6 levels influence both the amount of the deuterated 18:2n-6 and 18:3n-3 incorporated into plasma lipids and the amount converted to n-6 and n-3 LCFA metabolites? A possibility consistent with fatty acid oxidation data is that a larger portion of the deuterated l8:2n-6 and 18:3n-3 was diverted into the ß-oxidation pathway when dietary 18:2n-6 levels were increased (33). Higher oxidation percentages would result in a general reduction of the concentration of deuterated fatty acids in plasma lipids which, in turn, would reduce the amount of 18:2n-6 and l8:3n-3 available for conversion to LCFA metabolites. An explanation for why 18:2n-6 increases fatty acid oxidation is that dietary 18:2n-6 reduces acyltransferase activity by reducing the synthesis of the mRNA, that codes for synthesis of the acyltransferase enzymes (20). Reduction in acyltransferase activity could allow a larger portion of the fatty acid pool to be diverted into the ß-oxidation pathway. A general reduction in the incorporation of non–n-6 and n-3 deuterium-labeled fatty acids (16:0, 18:0, and 18:1) that were fed to these subjects at the same time was also observed. This observation is consistent with the possibility of a general nonselective increase in fatty acid oxidation or storage in tissues when 18:2n-6 intake is increased. Desaturation-Elongation of 18:2n-6 and 18:3n-3 Concentration data for the individual n-3 and n-6 LCFA metabolites of 18:3n-3 and 18:2n-6 are shown in Figure 2.4. The concentration of all the individual n-3

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Fig. 2.4. Concentration (µg/mL) of individual deuterated n-3 and n-6 long-chain fatty acid metabolites in plasma total lipids from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. Bars indicate high and low values. Source: Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.

and n-6 LCFA metabolites were consistently lower for subjects that were prefed the high 18:2n-6 (PUFA) diet. Variability between subjects for the concentration of individual n-3 and n-6 LCFA metabolites was fairly large. However, when sums for the various n-3 and n-6 metabolites were compared (Figure 2.5), the variability between subjects was much smaller. The variability between the concentration data for the individual n-3 and n-6 LCFA metabolites indicates a considerable subject-dependent difference in the rate of conversion of the deuterated 18:2n-3 and 18:3n-3 to the major metabolites (20:4n-6, 20:5n-3, and 22:6n-3). Concentration data for sums of the n-3 and n-6 LCFA metabolites from individual subjects along with the means for subjects fed the SAT and PUFA diets are compared in Figure 2.5. These results demonstrate that conversion of 18:3n-3 to n-3 LCFA metabolites was considerably higher (ca. 3.7 times) than conversion of 18:2n-6 (P < 0.001) and that dietary 18:2n-6 significantly reduced (P < 0.01 for 18:3n3 and P < 0.09 for 18:2n-6) total conversion (ca. 68%) of both 18:2n-6 and 18:3n-3. The concentration data shown in Figure 2.5 can be converted to percent conversion data by dividing the n-3 LCFA metabolite data by the total for 18:3n-3 plus n-3 LCFA metabolites, Percent conversion data for 18:2n-6 can be calculated in a similar manner. The results are shown in Figure 2.6. The average percent conversion was about 40% lower for l8:3n-3 and 56% lower for l8:2n-6 when the subjects were fed the diet enriched in 18:2n-6. Expression of the µg/mL data as percent data distorts the conversion data because of the large difference between the amount of 18:3n-3 and 18:2n-6 incorporated into plasma lipids due to the high selectivity for 18:2n-6 discussed earlier.

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Fig. 2.5. Concentration (µg/mL) of the sums for n-3 and n-6 long-chain fatty acid metabolites

in plasma total lipids from subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. For SAT versus PUFA, 18:2n-6 means (P < 0.09) and 18:3n-3 means (P < 0.01) are significantly different. Note: deuterated 18:2n-6 was not included in the mixture of deuterated fats fed to subjects 5, 6, and 7. Source: Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.

Fig. 2.6. Percent of n-3 and n-6 long-chain fatty acid metabolites in plasma total lipids from

subjects fed diets containing 15 g (SAT diet) and 30 g (PUFA diet) linoleic acid. For SAT versus PUFA, 18:3n-3 means (P < 0.07) are significantly different. Means for 18:2n-6 are not significantly different. Source: Proceedings for the Scientific Conference on ω-3 Fatty Acids in Nutrition, Vascular Biology and Medicine, April 17–19, 1994, Houston, Texas, American Heart Association.

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The sum of the concentrations for the deuterated n-6 LCFA metabolites was much lower than the sum of the concentrations for the deuterated n-3 fatty acid metabolites (Figure 2.5). Comparison of these data clearly show that desaturation-elongation of deuterated l8:3n-3 was greater than for deuterated 18:2n-6. Deuterated 20:5n-3 (34.3 µg/mL) and 22;6n-3 (29.8 µg/mL) represent 6.0% and 3.8% of the total amount of labeled 18:3n-3 in total plasma lipids, respectively. In contrast, deuterated 20:4n-6 (7.2 µg/mL) represents 0.5% of the labeled 18:2n-6 in total plasma lipids. Average total percent conversion of deuterated 18:3n-3 for all subjects (15.3%) was higher than that of deuterated l8:2n-6 (1.6%). This low percent conversion of deuterated 18:2n-6 is consistent with both in vivo and in vitro data from other human studies (22–29). The difference in the amounts of deuterated 18:2n-6 and l8:3n-3 converted to long-chain polyunsaturated fatty acid metabolites is not easily explained. A higher amount of n-6 LCFA metabolites would be expected, since the concentration of deuterated 18:2n-6 in plasma total lipids (1260 µg/mL) is about three times higher (P < 0.001) than the concentration of deuterated 18:3n-3 (450 µg/mL). If one accepts that ∆-6 desaturase is the rate-limiting step in the conversion pathway and if the rate constant is similar for both 18:2n-6 and 18:3n-3 (5,19), then the concentrations of deuterated n-6 and n-3 LCFA should be proportional to the concentrations of 18:2n-6 and 18:3n-3 in plasma lipids. A difference in the selectivity of ∆-6 desaturase and/or the rate constant for 6-desaturation for 18:2n-6 and 18:3n-3 is a plausible explanation for the difference in conversion observed in this study. These in vivo data for deuterated n-6 and n-3 LCFA metabolites indicate that ∆-6 desaturase is about four times more selective for 18:3n-3 than l8:2n-6. This selectivity is somewhat higher than the difference in ∆-6 desaturation for 18:2n-6 and 18:3n-3 of 1.5–3.0 times reported for in vitro studies with rat liver microsomes (20,34,35). Effect of Dietary Linoleic Acid The influence of the rather large difference in dietary linoleic acid levels in the SAT and PUFA diets are illustrated by the concentrations of deuterated 18:2n-6 and 18:3n3 and their deuterated n-3 and n-6 LCFA metabolites in plasma total lipids (Figures 2.4 and 2.5). The concentrations of the deuterated fatty acids were clearly lower for the subjects fed the PUFA diet. These results indicate that the metabolism of both the 18:3n3 and 18:2n-6 was altered when subjects were fed diets containing different levels of 18:2n-6 (15.1 g vs. 29.8 g). This effect of dietary 18:2n-6 is consistent with animal data showing that 18:2n-6 competes with itself and with 18:3n-3 (4,5,18,20). The approximate twofold difference in dietary 18:2n-6 content lowered deuterated 18:2n6 and 18:3n-3 concentrations in plasma total lipids by 37–39% and deuterated n-6 and n-3 LCFA metabolite concentrations by 65–70%. The ratio of deuterated 18:2n-6 to 18:3n-3 and deuterated n-6 to n-3 LCFA metabolites were not influenced by the 18:2n-6 content of the diets. These results suggest that the absolute amounts of dietary 18:2n-6 and 18:3n-3 have a greater influence than the 18:2n-6/18:3n-3 ratio.

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Nutritional Implications The amounts of n-3 and n-6 LCFA synthesized per day from 18:3n-3 and 18:2n-6 in a typical U.S. diet can be estimated from the deuterated LCFA data. Based on a total plasma volume of about 3000 mL (39 mL/kg body wt) and the concentration of deuterated n-3 LCFA metabolites (Figure 2.5), the total amount of deuterated n-3 LCFA metabolites in plasma lipids was 351 mg or 127 mg/g of deuterated 18:3n-3 fed (SAT diet) and 126 mg or 43 mg/g deuterated 18:3n-3 fed (PUFA diet). By extrapolation from the metabolite weight data, the 2 g of 18:3n-3 in a typical U.S. diet is estimated to provide 186 mg/day of n-3 LCFA. Based on a similar calculation, 537 mg/day of n-6 LCFA is estimated to be synthesized from the 21 g of dietary l8:2n-6 in typical U.S. diets, Estimates based on plasma concentration data indicate that dietary 18:3n-3 provides about 50% of the n-3 LCFA daily requirement for adults. The estimates based on the total weight of deuterated LCFA metabolites are believed to be the most reliable, although they underestimate conversion of both l8:3n-3 and 18:2n6 because the plasma data do not include the amounts of deuterated LCFA metabolites that were incorporated into tissue lipids. Alternatively, the amount of long-chain n-3 and n-6 fatty acids synthesized from dietary 18:3n-3 and 18:2n-6 can be calculated from the percent conversion data in Figure 2.6. The percent conversion calculated for a typical U.S. diet is about 15% for 18:3n-3 and about 1.8% for 18:2n-6. Thus, about 300 mg of n-3 LCFA metabolites/day is estimated to be synthesized from 2 g of l8:3n-3 in a typical U.S. diet and 378 mg of n-6 LCFA metabolites/day is estimated to be synthesized from 21 g of 18;2n-6. Based on the percent conversion data, the 18:3n-3 in a typical U.S. diet is estimated to provide 75–85% of the 350–400 mg of longchain n-3 fatty acids/day that has been estimated to be required by adults (15,16). From these and other data used to estimate the requirements for essential fatty acids in humans, it is clear that most U.S. and Canadian diets contain a large surplus of 18:2n-6, but diets do not contain a surplus of n-3 fatty acids. If the 18:3n3 provided by soybean and canola oils are not included in dietary estimates, both the U.S. and Canadian diets would be deficient in 18:3n-3. Therefore, the concern is that the development of the new low (2–3%) 18:3n-3 soybean and canola oils may have a negative nutritional and health impact if they were to replace the conventional soybean and canola oils that contain 7–10% 18:3n-3. References 1. 2. 3. 4. 5.

Sinclair, H.M., Lancet 1: 381 (1956). Keys, A., J.T. Anderson, and F. Grande, Lancet 1: 66 (1957). Bang, H.O., J. Dyerberg, and A.B. Nielsen, Lancet 1: 1143 (1971). Nestel, P.J., Ann. Rev. Nutr. 10: 149 (1990). Ackman, R.G., and S.C. Cunnane, Adv. Appl. Lipid Res. 1: 161 (1992).

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6. Malasanos, T.H., and P.W. Stacpoole, Diab. Care 14: 1160 (1991). 7. Lasserre, M., F. Mendy, D. Spielmann, and B. Jacotot, Lipids 20: 227 (1985). 8. Crawford, M.A., Polyunsaturated Fatty Acids and Eicosanoids, edited by W.E.M. Lands. The American Oil Chemists’ Society, Champaign, Illinois, 1987, pp. 270–295. 9. Galli, C, and A.P. Simopoulos (eds.) General Recommendations on Dietary Fats for Human Consumption, Dietary ω-3 and ω-6 Fatty Acids: Biological Effects and Nutritional Essentiality. NATO Series A, Life Sciences, Plenum Press, New York, 1989, pp. 403–04. 10. Yehuda, S., and R.L. Carasso, Proc. Nat. Acad. Sci. 90: 10345 (1993). 11. Dietary Fats and Oils in Human Nutrition. A Joint FAO–WHO Report, Food and Agricultural Organization of the United Nations, Rome, 1977, pp. 23–30. 12. Bourre, J.M., M. Piciotti, O. Dumont, G. Pascal, and G. Durand, Lipids 25: 465 (1990). 13. Simopoulos, A.P., J. Nutr. 119: 521 (1989). 14. Bourre, J.M., O. Dumont, G. Pascal, and G. Durand, J. Nutr. 123: 1313 (1993). 15. Bjerve, K.S., I.L. Mostad, and L. Thoresen, Am. J. Clin. Nutr. 45: 66 (1987). 16. Bjerve, K.S., S. Fischer, F. Wammer, and T. Egeland, Am. J. Clin. Nutr. 49: 290 (1989). 17. Hagve, T.–A., and B. Christophersen, Biochim. Biophys. Acta 796: 205 (1984). 18. Vamecq, J., L. Vallee, P. Lechene de la Porte, M. Fontaine, D. de Craemer, C. van den Branden, H. Lafont, R. Grataroli, and G. Nalbone, Biochim. Biophys. Acta 1170: 151 (1993). 19. Yamazaki, K., M. Fujikawa, T. Hamazaki, S. Yano, and T. Shono, Biochim. Biophys. Acta 1123: 18 (1992). 20. Sprecher, H., in Dietary ω–3 and ω–6 Fatty Acids: Biological Effects and Nutritional Essentiality, edited by C. Galli and A.P. Simopoulos, NATO Series A, Life Sciences, Plenum Press, New York, 1989, pp. 69–79. 21. Brenner, R.R., in The Role of Fats in Human Nutrition, 2nd edn., edited by A.J. Vergroesen and M. Crawford, Academic Press Inc., London, 1989, pp. 45–79. 22. de Gomez Dumm, I.N.T., and R.R. Brenner, Lipids 10: 315 (1975). 23. Poisson, J.-P., R.-P. Dupuy, P. Sarda, B. Descomps, M. Narce, D. Rieu, and A.C. de Paulet, Biochim. Biophys. Acta 1167: 109 (1993). 24. Cunnane, S.C., P.W.N. Keeling, R.P.N. Thompson, and M.A. Crawford, Brit. .J. Nutr. 51: 209 (1984). 25. El-Boustani, S., J.E. Causse, B. Descomps, L. Monnier, F. Mendy, and A. Crastes de Paulet, Metabolism 38: 315 (1989). 26. Nichaman, M.Z., R.E. Olson, and C.C. Sweeley, Am. J. Clin. Nutr. 20: 1070 (1967). 27. Ormsby, J.W., J.D. Schnazt, and R.H. Williams, Meta. Clin. Exptl. 12: 812 (1963). 28. Emken, E.A., W.K. Rohwedder, R.O. Adlof, H. Rakoff, and R.M. Gullcy, Lipids 22: 495 (1987). 29. Emken, E.A., R.O. Adlof, D.L. Hachey, D. Garza, M.R. Thomas, and L. Brown-Booth, J. LipidRes. 30: 395 (1989). 30. Emken, E.A., R.O. Adlof, and R.M. Gulley, Biochim. Biophys. Acta 1213: 277 (1994). 31. Rohwedder, W.K., S.M. Duval, D.J. Wolf, and E.A. Emken, Lipids 25: 401 (1990).

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32. Nichaman, M.Z., Nutrition Monitoring in the United States: An Update Report on Nutrition Monitoring, DHHS Publication 89–1255, Life Science Research Office, Hyattsville, Maryland, (1989). 33. Peter, J.H., and D.A. Schoeller, Metabolism 37: 145 (1988). 34. Hrelia, S., M. Celadon, C.A. Rossi, P.L. Biagi, and A. Bordoni, Biochem. lnt. 22: 659 (1990). 35. Brenner, R.R., R.O. Peluffo, A.M. Nervi, and M.E. De Tomas, Biochim. Biophys. Acta 176: 420 (1965).

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Chapter 3

Trans Fatty Acids in Canadian Breast Milk and Diet W.M.N. Ratnayakea and Z.Y. Chenb a Nutrition Research Division, Food Directorate, Health Protection Branch, Health Canada, Ottawa, Ontario, K1A 0L2, Canada; and bDepartment of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.

Introduction Many commercial dietary fats available to the consumers in industrialized countries are prepared by the process of partial hydrogenation. This process converts liquid oils to solid fills that have the elasticity and texture desired for many food preparations. Negative aspects of this practice are the substantial reduction in the proportion of essential fatty acids in the dietary fats with a concomitant formation of trans and cis isomers of oleic and linoleic acids. The content of trans fatty acids (TFA) in dietary fat varies. The average daily intake of TFA for the U.S. population has been estimated to be at least 8 g or 3.7% of total energy (1,2). A recent estimate of TFA intake for the Canadian population is not available, nevertheless many of the food items in the Canadian retail market contain significant amounts of TFA. For example, Canadian margarines may contain up to 50% TFA (3). Cookies, biscuits, donuts, deep-fried foods, and many other common snacks made from partially hydrogenated vegetable oils also contain substantial amounts of TFA (4). It is well established that the fatty acids in breast milk reflect those of the maternal diet (5–14). The presence of TFA in human milk is a concern, because of their possible negative nutritional and physiological effects on the recipient infant. Human infants absorb and metabolize trans isomers and incorporate them into plasma and tissue lipids (15). Negative effects of TFA, such as perturbations of essential fatty acid and prostaglandin metabolism (16), and formation of unusual long-chain polyunsaturated fatty acids were observed in rodents (17–19). In human infants, TFA seem to impair the biosynthesis of n-6 and n-3 long-chain polyunsaturated fatty acids (LCP) and the individual’s growth (20). During late fetal and early postnatal growth, considerable amounts of n-6 and n-3 LCP are accreted in neural and other tissues (21). Phospholipids of the central nervous system and of retinal photoreceptor cells are particularly rich in arachidonic (20:4n-6, AA) and docosahexaenoic (22:6n-3, DHA) acids (22). Studies with infant animals have indicated that a deficiency of DHA in the brain and retina may impair development of visual acuity, and possibly also discrimination of learning (23–26). Although the presence of TFA in human milk has been recognized for a long time, the literature data are not complete and generally may not be accurate, due to the difficulty of analyzing TFA. Recent reviews on human milk fatty acids have not

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mentioned the content of TFA and other unusual isomeric fatty acids (27–30). There is also a lack of information about these fatty acids in human breast milk from Canadians. Therefore, using a combined procedure of silver nitrate-thin layer chromatography (AgNO3-TLC) and gas-liquid chromatography (GLC), we analyzed the fatty acids of mature breast milk of 198 women across Canada. The TFA data were then utilized to estimate the trans-octadecenoic (t-18:1) content in the Canadian diet. These estimates were calculated using an equation based on a relationship between t18:1 in milk and dietary fat (11). Part of this study has been published elsewhere (31).

Materials and Methods The human milk samples used in this study were from the 1992 collection of Health Protection Branch’s ongoing monitoring program of chlorinated hydrocarbon contaminants in the breast milk of Canadian women (unpublished work of W.H. Newsome, Health Protection Branch, Ottawa). Samples of mature milk (3–4 weeks of parturition) were collected from lactating women from across Canada (20–25 samples per province), except from Prince Edward Island and the two territories. Donors were requested to express about 3–4 mL of their milk manually during each feeding, starting from the very first feeding to the last feeding of the day. The sample from each donor thus represented the accumulated milk collection per day. A total of approximately 25–50 mL from each mother was collected in brown bottles with polytetrafluoroethylene-lined screw caps. Mothers were requested to refrigerate the milk samples between collections. The day following the 24-hr collection, the samples were shipped in dry ice to Ottawa and stored at -24°C until analysis. Fat from a 5 g milk sample was extracted using 25 volumes of CHCl3-MeOH (2:1, v/v) containing 0.02% butylated hydroxy toluene as an antioxidant and triheptadecanoin (1 mg/mL) as an internal standard to quantitate total milk fat by GLC. The extracted fat was methylated with BF3-MeOH and analyzed by GLC using an SP-2560 flexible fused silica capillary column (100 m × 0.25 mm i.d., 20 µm film thickness). Column temperature was programmed from 150 to 180°C at a rate of 0.5°C/min, and then to 210°C at a rate of 3°C/min. A typical GLC trace of a human milk fatty acid methyl ester (FAME) profile is shown in Figure 3.1. Single step, direct GLC analysis cannot accurately determine the total t-18:1 due to overlap of high delta 18:1 trans isomers (12t-16t) with c-18:1 isomer peaks (32). In the human milk of this study, 20.8% (range 9–30%) of the total t-18:1 isomers overlapped with c-18:1 isomers. Therefore, the total t-18:1 and c-18:1 levels in the milk samples were determined using AgNO3-TLC in conjunction with capillary GLC. Silver nitrate thin-layer chromatography was performed as described previously (33). The t-18:1 band was isolated and analyzed by GLC. The proportion of t-isomers that overlapped with the c-18:1 isomer peaks was calculated by comparing the 18:1 region of the GLC chromatogram of the isolated t-18:1 with that of the parent FAME mixture prior to AgNO3-TLC fractionation.

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For this purpose, the t-18:1 isomer peaks (6t-11t; peaks 26–27 in Figure 3.1) that were well separated from the c-18:1 isomer peaks served as the internal standard. The total t-18:1 was then calculated, by summing up the proportions of the t-18:1 isomers (12t-16t; peaks 28-30, and 34 in Figure 3.1) that overlapped with the cis isomers and the well-separated t-18:1 isomers. The t-18:1 and c-18:1 isomer distribution was determined by oxidative ozonolysis (34). The positional and geometrical isomers of linoleic acid were determined and identified as described previously (33).

Results and Discussion No significant regional differences in the average fat content and fatty acid profile data of Canadian human breast milk samples were observed in this study. Therefore, only the mean values, standard deviation, and the ranges are presented for the 198 samples (Table 3.1). Usual Saturated and Polyunsaturated Fatty Acids The major fatty acid group was represented by saturated fatty acids (38.5%), approximately one-half of which was 16:0. Sanders and Reddy found that the level of C10–C14 saturated fatty acids was higher in the milk of human vegans and vegetarians than in that of omnivores (30). The mean levels for 10:0, 12:0, and 14:0 in human milk of this study were remarkably similar to the levels found in vegan and vegetarian human milk in the United Kingdom (30). This might reflect that intake of meat by the mothers in the present study was low, although dietary records were not available. Sanders and Reddy have hypothesized that the origin of C10–C14 saturated fatty acids in human milk is not dietary, but most likely derived by de novo synthesis from carbohydrates in the mammary gland (30). Intake of these fatty acids is low in vegans compared with the intake of omnivores, since vegan and vegetarian diets contain more carbohydrates and less fat than those of omnivores (35). The levels of linoleic, α-linolenic acids, and their C20 and C22 metabolites in human milk are of special interest, because of their important physiological significance (22). The levels of linoleic (10.5%) and α-linolenic (1.2%) acids found in this study are similar to those reported in studies of mature human milk from women following ad libitum diets in different regions of the world (27,28,30,36). However, the levels of C20 and C22 n-6 (0.8%) and n-3 (0.3%) LCP were lower than for those in other countries (27,28,30,36) but similar to levels reported for vegans or vegetarians (30). The lower levels of n-6 and n-3 LCP further suggest that a large segment of the lactating women in the present study were vegans or vegetarians. In some of the samples of this study, only trace amounts (99% >93%