Healthful Lipids
Editors Casimir C. Akoh Department of Food Science and Technology University of Georgia Athens, GA, USA
Oi-Ming Lai Department of Bioprocess Technology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia SeI angor, M aI ays ia
%
LIc>cs
PRESS Urbana, Illinois
AOCS Mission Statement
To be a global forum to promote the exchange of ideas, information, and experience, to enhance personal excellence, and to provide high standards of quality among those with a professional interest in the science and technology of fats, oils, surfactants. and related materials. AOCS Books and Special Publications Committee M. Mossoba, Chairperson, U.S. Food and Drug Administration, College Park, Maryland R. Adlof, USDA, ARS, NCAUR, Peoria, Illinois P. Dutta, Swedish University of Agricultural Sciences, Uppsala, Sweden T. Foglia, ARS, USDA, ERRC, Wyndmoor, Pennsylvania V. Huang, Yuanpei University of Science and Technology, Taiwan L. Johnson, Iowa State University, Ames, Iowa H. Knapp, DBC Research Center, Billings, Montana D. Kodali, Global Agritech Inc, Minneapolis, Minnesota T. McKeon, USDA, ARS, WRRC, Albany, California R. Moreau, USDA, ARS, ERRC, Wyndoor, Pennsylvania A. Sinclair, RMIT University, Melbourne, Victoria, Australia P. White, Iowa State University, Ames, Iowa R. Wilson, USDA, REE, ARS, NPS, CPPVS, Beltsville, Maryland Copyright (c) 2005 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-PublicationData Healthful lipids / editors, Casimir C. Akoh, Oi-Ming Lai. p. ; cm. Includes references and index. ISBN 1-893997-51-0 (alk. paper) 1. Lipids in human nutrition. 2. Trans fatty acids. 3. Genetically modified foods. [DNLM: 1. Lipids--chemical synthesis. 2. Genetic Engineering--methods. 3. Lipidsphysiology. 4. Nutrition--physiology. QU 85 H441 20051 I. Akoh, Casimir C., 1955- 11. Lai, Oi-Ming. 111. American Oil Chemists' Society. TX553.L5H43 2005 6 13.2'84--dc22 2004029543
Printed in the United States of America. 1009080706 6 5 4 3 2
In the years to come, the type of fat we consume will become more important than the amount of total fat in our diet. Physiologically and functionally important lipids are the subject of many recent projects and publications. New regulations on the type and amount of fats in various food products are being formulated and the food industry is following or being proactive in reformulation of their products toward healthier alternative products. The consumer is becoming increasingly aware of the importance of a healthier diet that includes beneficial fats. As lipid chemists, nutritionists, and professionals in the food industry, we must provide consumers with what they desire. There is a real need for a book that addresses critical and current regulatory issues and emerging technologies, as well as the efforts made toward the production of healthier lipids. Healthfil Lipids is expected to fill that void, providing a concise, welldocumented presentation of the current state of knowledge of hot issues such as the regulation of trans fatty acids in food. This book examines the latest technological advancements and the emerging technologies in processing and analysis, health-related concerns, and strategies used in the production and application of healthful lipids. An in-depth patent review on enzyme modified and trans-free fats and oils makes this book a valuable reference not only to graduate students and individuals interested in food research, product development, food processing, nutrition, dietetics, quality assurance, genetic engineering of oil crops, oil processing, fat substitutes and lipid biotechnology, but also to food industry professionals seeking background and advanced knowledge in lipids. New firms applying their expertise to lipids or acquisitions will find much information related to the food lipid business. The book is divided into six parts. Part 1 reviews the regulation of trans fatty acids and genetically modified lipids. Processing methods and analysis are covered in Part 2. Nutrition and health effects of healthful lipids and minor constituents of lipids are discussed in Part 3 under nutrition and biochemistry. Part 4 covers patent review and the use of enzymes and genetic engineering for the production and purification of healthful lipids. The causes of oxidation and the ways to stabilize lipids containing highly unsaturated fatty acids are the subject of Part 5 . Finally, the applications of healthful lipids in foods and nutrition are discussed in Part 6 . Efforts have been made to draw contributors for this book from both the academic and industrial sectors.We are grateful to them for lending us their expertise and time in the preparation of this book. We hope that the issues covered in this book will benefit the reader, consumers, students, and the food industry in some ways while stimulating further research toward improving human health and promoting a healthful lifestyle.
Casimir C. Akoh
Oi-Ming Lai September 14,2004 iii
Contents
Preface
..........................................
Part 1:
Current Regulatory Issues
Chapter 1
Trans Fatty Acids in Foods and Their Labeling Regulations Nimal W.M. Ratnayake and C. Zehaluk . . . . . . . . . . . . . . . . . .
Chapter 2
Safety, Regulatory Aspects, and Public Acceptance of Genetically Modified Lipids Ravigadevi Sambanthamurthi, Sharifah Shahrul, and G.K. Ahmad Paweez . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...
111
1
33
Part 2:
Processing and Emerging Analytical Technologies
Chapter 3
Production, Processing, and Refining of Oils Ernesto Hernandez . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
Novel Hydrogenation for Low Trans Fatty Acids in Vegetable Oils Mun Yhung Jung and David B. Min ....................
65
Analysis of Lipids by New Hyphenated Techniques HuilingMu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
Supercritical Fluid Processing of Nutritionally Functional Lipids Jerry W.King . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
99
Short-Path Distillation for Lipid Processing XuebingXu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
127
Fat Crystallization Technology Serpil Metin and Richard Hartel . . . . . . . . . . . . . . . . . . . . . . .
145
Chapter 4
Chapter 5 Chapter 6
Chapter 7 Chapter 8
Part 3:
Nutrition and Biochemistry
Chapter 9
Dietary Fatty Acids and Their Influence on Blood Lipids and Lipoproteins Tilakavati Karupaiah, Mohd Ismail Noor, and KalyanaSundram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
171
Essential Fatty Acid Metabolism to Self-HealingAgents William E M . Lands .................................
204
Chapter 10
V
vi
Contents
Chapter 11
Dietary n-6:n-3 Fatty Acid Ratio and Health Sarah Gebauer, William S . Harris, Penny M . Kris-Etherton, and Terry D. Etherton . . . . . . . . . . . . . . . . . . . 221
Chapter 12
CLA Sources and Human Studies Marianne O’Shea, Margriet Van Der Zee, and IngeMohede . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
249
Lipids with Antioxidant Properties Jan Pokornq and Jana Parkhnyiovh .....................
273
y-Linolenic Acids: The Health Effects Rakesh Kapoor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
301
Phytosterols and Phytosterol Esters RobertA.Moreau ...................................
335
The Effects of Eicosapentaenoic Acid in Various Clinical Conditions Andrew Sinclair, Julie Wallace, Marion Martin, Nadia Attar-Bashi, Richard Weisinger,and Duo Li
361
Chapter 13 Chapter 14 Chapter 15 Chapter 16
........
Part 4:
Enzyme and Lipid Biotechnology
Chapter 17
Lipase Reactions Applicable to Purification of Oiland Fat-Related Materials YujiShimada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
395
Enzymatic Synthesis of Symmetrical Triacylglycerols Containing Polyunsaturated Fatty Acids Tsuneo Yamane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
411
Chapter 18
Chapter 19
Patent Review on Lipid Technology Oi-Ming h i , Seong-Koon Lo, and Casimir C. Akoh . . . . . . . 433
Chapter 20
Genetic Enhancement and Modification of Oil-Bearing Crops G.K. Ahmad Parveez and Ravigadevi Sambanthamurthi . . . . 508
Chapter 21
Genetically Engineered Oils David Hildebrand and Lewamy Mamadou
Part 5:
Oxidation
Chapter 22
Emulsion Technologies to Produce Oxidative Stable Emulsions Containing n-3 Fatty Acids Min Hu, Eric A. Decker, and D.Julian McClements . . . . . . . 547
Chapter 23
Chemistry for Oxidative Stability of Edible Oils Eunok Choe, Jiyeun Lee, and David Min . . . . . . . . . . . . . . . . 558
. . . . . . . . . . . . . . . 526
vii
Contents
Part 6:
Applications of Healthful Lipids
Chapter 24
Structured and Specialty Lipids CasimirC.Akoh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
591
Chapter 25
Lipids in Infant Formulas and Human Milk Fat Substitutes Nikolaus Weber and Kumar D. Mukherjee . . . . . . . . . . . . . . . 607
Chapter 26
Cocoa Butter, Cocoa Butter Equivalents, and Cocoa Butter Replacers Kazuhisa Yamada, Masahisa Ibuki, and Thomas McBrayer
Chapter 27 Chapter 28
Chapter 29
Chapter 30
,,
642
Margarine and Baking Fats Wjai K.S. Shukla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
665
Nutritional Characteristics of Diacylglycerol Oil and Its Health Benefits Noboru Matsuo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
685
Plant Stanol Ester as a Cholesterol-Lowering Ingredient of Benecol@Foods Pia Salo, Anu Hopia, Jari Ekblom, Ritva Lahtinen, and Paivi Luakso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
699
Palm Oil, Its Fractions, and Components Oi-MingLui . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
731
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
vii
Chapter 1
Trans Fatty Acids in Foods and Their Labeling Regulations W.M.N. Ratnayakea and C. Zehalukb aNutrition Research Division and bNutrition Evaluation Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, ON, Canada K1 A OL2
Introduction The relation among dietary intakes of fatty acids (FA), blood cholesterol levels, and risk for cardiovascular disease (CVD) remains an important health issue. Currently, it is well-established that dietary saturated FA raise blood total and low density lipoprotein (LDL) cholesterol (LDL-C) concentrations compared with linoleic and oleic acids (1-3). Over the last 15 years, metabolic studies c o n f iie d that in addition to saturated FA, trans FA also have a negative effect on human plasma lipoprotein profiles and have adverse implications for atherogenesis. The negative effect of trans FA on lipoprotein is caused by increasing total cholesterol,LDL-C, lipoprotein a [Lp (a)], and decreasing high density lipoprotein cholesterol (HDL-C) relative to naturally occurring cis-unsaturated FA (4-1 1). Moreover, the replacement of saturated FA by trans FA was shown to decrease HDL-C in many studies (10-13). Thus, compared with saturated FA and cis-unsaturated FA, the overall effect of increased intakes of trans FA is a less favorable LDL-C/HDL-C ratio (14), which is an additional increase in the risk of CVD (15-17). Some (14,18-24) but not all epidemiologic and case-controlled studies (25-27) showed that high intakes of trans FA increase the risk of CVD, which agrees with the observed effects of trans FA on blood lipids. On the basis of the U S . Nurses’ Health Study, Hu et al. (20) estimated that compared with equivalent energy from carbohydrates, the relative risk of coronary heart disease associated with each increase of 2% energy intake from trans fat would be 1.93, whereas that for a 5% increment in energy from saturated fat would be only 1.17. Furthermore, replacement of 2% of energy from trans fat with energy from cis-unsaturated fats would reduce the risk by 53% (20). In summary, the current scientific literature suggests that replacing saturated and trans FA with unhydrogenated fat has clear beneficial effects on blood lipids and thus provides an alternative strategy for reducing the risk of CVD. Although experts have differed somewhat in their interpretation of the scientific evidence on negative health effects of trans fats (1 l), the recommendations by health organizations such as the Institute of Medicine (2), the Danish Nutrition Council (28), the American Heart Association (29), European Atherosclerosis Society (30), Food and Agricultural Organization (FAO) of the United Nations (31), 1
2
W.M.N. Ratnayake and C. Zehaluk
Canadian Medical Association (32), the Nutrition Committee of the American Heart Association (33), Joint WHO/FAO Consultation on Diet, Nutrition and the Prevention of Chronic Diseases (34), and government health agencies in Denmark ( 3 9 , Canada (36), and the United States (37) to reduce the risk of CVD usually stress the importance of reducing the intake of foods rich in both saturated and trans FA. The food industry is already responding by reducing the trans content of many products, especially in tub margarines. Until quite recently, the negative health effects of trans FA and the phrases ”trans fats” and “partially hydrogenated fats” would have been familiar only to lipid researchers, biochemists, fats and oils technologists, and some health professionals. Although groups such as the Center for Science in the Public Interest have been warning consumers about the adverse health effects of trans fats and the high levels of these in certain commercial food products including fast foods (38), the North American public was largely unaware of the trans FA issue in spite of being the highest consumers of trans FA in the world. In a study conducted in Canada to obtain information on consumer attitudes and behavior related to nutrition labeling, 17% of consumers interviewed claimed to understand well the term “trans fat,” whereas 55% of respondents indicated they had no idea about the meaning of the term (39). The legislation passed by the Danish Government on March 11,2003 (39, which prohibits the sale of foods containing >2% industrially produced trans FA (as a percentage of total fat) and the publication of the amendments to food-labeling regulations in Canada in January 2003 (36) and the United States in July 2003 (37), which require mandatory declaration of trans FA levels in foods, changed all that by introducing the phrases to physicians, nutritionists, dieticians, and other health professionals. The subsequent media reports in Canada and the United States on the levels of trans fats in foods and their potential damaging effect on health brought trans fats to the attention of many health-conscious consumers. This chapter provides information on the origin, structure, and levels in various foods and labeling regulations on trans FA in various countries,
Origin of Trans FA The carbon-carbon double bonds (also known as ethylenic bonds) of natural unsaturated FA, which are widely present in all plant materials and animal tissues, are primarily of cis configuration. Trans FA are also unsaturated FA but contain one or more double bonds in the trans configuration. The hydrogen atoms in the double bond in the trans form are located on either side of the carbon atoms, whereas those in the cis form are located on the same side. Some trans FA isomers occur naturally, although in much less abundance than the cis form. Foods produced from ruminant animals, including meat and dairy products such as milk,butter, and cheese, are the most common natural dietary sources of trans FA. These trans FA are the result of biohydrogenation of dietary cis-unsaturated FA by rumen microorganisms of ruminant animals. Tissues of
Trans Fatty Acids
3
these animals and products derived from them, therefore, contain small amounts of trans FA isomers. Trans FA also occur naturally in green leaves. An example is 3trans-hexadecenoic acid (3t-16:l); although it is a minor ingredient, it is a ubiquitous component of all green leaves. This is an intermediate component in the biosynthesis of saturated FA. Some seed fats (e.g., tung) may contain up to 80% trans FA, such as 9-cis,l l-trans, 13- trans-octadecatrienoic acid (9c,l lt,13t-18:3), although they are not dietary fats. Trans FA are also formed intentionally during the commercial process of hydrogenation that converts liquid vegetable or marine oils rich in cis-polyunsaturated FA (PUFA) into solid fats. Hydrogenation, which is performed by bubbling hydrogen through the liquid oils in the presence of a metal catalyst such as nickel, is usually not allowed to go to completion (hence termed as partial hydrogenation) and results in the conversion of some of the cis double bonds to the trans configuration. The melting point (m.p.) of a trans FA falls between that of the corresponding cis FA and the saturated FA. Food manufacturers prefer partially hydrogenated fats over liquid oils because they provide a solid fat for the manufacture of a variety of food products. Solid fats are essential in making good pastry, cakes, crackers, donuts, and many other bakery products because they contribute tenderness and help incorporate air into the dough or butter. In addition, solid fats are less prone to rancidity; therefore, foods made from solid fats can be stored for a longer period than those made from liquid oils. Partially hydrogenated fats were developed in part to replace the highly saturated solid animal fats such as butter, tallow, and lard previously used for these products. The use of partially hydrogenated vegetable oils (PHVO) in margarines, shortenings, deep frying, bakery products, snacks, fast foods, and other processed foods was thought to provide a more healthful alternative to animal fats because they contain no cholesterol and have less cholesterol-raising saturated FA. However, this thinking has changed over the last 15 years, because of the realization that high trans FA intake may promote arteriosclerosis to a greater extent than do saturated FA.
Structures o f Common Dietary Trans FA In both ruminant and commercial hydrogenation, some of the cis-double bonds of the original cis-unsaturated FA are isomerized. This involves both positional (a shift in position along the hydrocarbon chain) and geometric (changes in geometrical configuration) alterations. Thus, a mixture of trans and unnatural cis isomers is formed. Commercial hydrogenation produces a wide variety of cis and trans isomers, often in concentrations much higher than those with biological processes. In partially hydrogenated fats, the concentration of cis isomers is approximately half that of the trans isomers. In both ruminant fats and PHVO, octadecenoic acid (18:l) represents the major fraction with trans and unnatural cis-unsaturation. The double-bond posi-
W.M.N. Ratnayake and C. Zehaluk
4
tions of both cis and trans-18:l isomeric FA, counted from the carboxylic carbon, usually range from A4 to A16 (40,41). The predominant trans isomers of 18:l in PHVO form a Gaussian distribution that centers around 9t-18:l and lOt-18:l (Fig. 1.1). This isomer distribution is distinctly different from that of fat derived from ruminant milk and meat, which contains trans-vaccenic acid (1 lt-18: 1) as the predominant trans isomer and accounts for -70% of the total trans-18:l (Fig. 1.2). Oleic acid (9c18:1) is always the predominant cis- 18:1 isomer in both dairy products and PHVO (Figs. 1.1 and 1.2). In addition to the 18:l isomers, dietary fats may contain a number of positional and geometrical isomers of linoleic and a-linolenic acids, which are frequently present in low concentrations in both partially hydrogenated and nonhydrogenated dietary fats. PHVO can constitute 15 or more isomers of linoleic acids (42); the major isomers are 9~,13t-18:2,9~,12t-18:2, and 9t,12c-18:2 (Table 1.1). These 18:2 isomers are found in higher amounts (up to 6% of total FA) in mildly hydrogenated vegetable oils, whereas they are scarcely detectable in heavily hydrogenated oils. Small amounts of the linoleic and a-linolenic acid isomers present in nonhydrogenated fats or in many common foods are the result of the exposure of these PUFA to some form of heat treatment, such as steam deodorization or stripping during refining of oils (43) or simple heating in deep-fat frying (44,45). In these processes, the double bonds do not shift in position, but are isomenzed from cis to trans, resulting in the formation of small amounts of geometric trans isomers (46). a-Linolenic acid is more prone to isomerization than linoleic acid, whereas oleic acid is scarcely isomerized at all. In many nonhydrogenated dietary fats, usually the two mono-trans isomers of linoleic (i.e., 9t,12c-18:2 and 9t,12c-18:2) are present at similar levels and very often higher than the all-trans isomer, 9t,12t-18:2. Eight geometric isomers are possible for a-linolenic
8
so
-
40
-
so -40
8
v
v
C
5
b
)
.
30
2
-
m w -
h
r : . co
-m 7
-
v
r : .
Z m m
20-
5
.
F
10
o
l
,
-
Trans Fatty Acids
A
18:i trans 100
,
5
B
18:i cis
Double-bond position
Double-bond position
Fig. 1.2. The distribution of (A) trans and (B) cis-octadecenoic (1 8:l) isomers in bovine milk fat.
acid, but usually only four are present in industrially refined oils (43) or oils subjected to mild heat treatments (44). They have been identified as 9t,12~,15t-l8:3,9t,12~,15t18:3,9t,12~,15~-18:3, and 9t,12c,15c-18:3. TABLE 1.1 Composition of Linoleic Acid Isomers in Hydrogenated Canola Oil Base Stocks with Varying Iodine Values (IV) ~ i i d i t-tyarogenatea y canola oil (IV 92)
Heavily nyarogenatea canola oil (IV 64)
( d l 0 0 g total FA)
18:2 isomer 9c,13t+ 8t,13c 9c,12t 8c,13t 9t,12c 1Ot,l5P+ 9t,15c t@ 9t,12t 8c,13cd 9c,l3cd 9c,14cd 9c,l5c
Moderately nyarogenatea canola oil (IV 80)
1.94 1.09 0.54 1 1.11 0.31 0.36 0.31 0.06 0.03 1.27
0.71 0 0.23 0 0.1 7 0.47 0.28 0.27 0 0 0.48
Tentative identification. of four different tt-18:2 isomers with unknown double-bond positions.
0.08 0 0 0 0 0.1 0 0.1 0 0 0.1
6
W.M.N. Ratnayake and C. Zehaluk
Trans FA Content in Foods Edible Oils. As a result of the refining of oils, the common oils used as salad oils and for general cooking purposes very often contain minor amounts of trans FA (Table 1.2). The predominant isomers are the mono-trans geometrical isomers of linoleic (9 cis, 12 trans-18:2 and 9 trans, 12 cis-18:2) and a-linolenic (9 cis, 12 cis, 15 trans-18:3, 9 cis, 12 trans, 15 cis-18:3, and 9 trans, 12 cis, 15 cis-18:3) acids. Slightly higher amounts of these mono-trans FA are usually present in canola and soybean or any other oil that contains large amounts of linoleic and a-linolenic acids (4648). In some refined canola oil batches, the total amount of trans FA can reach up to 2.4% of the total FA (Table 1.2). The trans FA content in refined oils very much depends on the duration and temperature of refining. The largest quantities of 18:3 isomers were detected in both soybean and canola oils heated at 240°C for 10 h (44). For example, in canola oil heated under these conditions, 38% of the starting linolenic acid was transformed into geometrical isomers. Only minor quantities of these isomers were detected in oils heated at 200°C for 10 h (44). Margarines. Margarine is one of the convenient and readily available sources of the two essential FA, linoleic and a-linolenic acids. However, the presence of large amounts of trans FA in some margarines is a drawback. During the last 10 years, in response to negative health effects of trans FA, margarine manufacturers in Europe and Canada have made some progress in reducing the trans FA content of their margarines (49-61). The reduction of trans FA content was achieved mainly by replacing a portion of the partially hydrogenated fats in the margarine fat blend by unhydrogenated liquid oils. The trans contents of margarines originating from different countries are presented in Table 1.3. In contrast to European products, the margarines sold in Canada and the United States contain quite large amounts of trans fats. The main trans FA in margarines are the 18:1 isomers; usually these isomers account for -85% of the total in vegetable oil-based margarines (Table 1.3). The remaining 15% is composed of trans isomers that originated from linoleic and linolenic acids. The trans,trans18:2 isomer content is of special interest to Canada, because, in 1980, a Health Canada a d hoc Committee on the Composition of Special Margarines determined that the trans,trans-18:2 (tt-18:2) isomers can suppress the blood and tissue levels of arachidonic acid (AA; 20:4n-6) and interfere with the biosynthesis of prostaglandins when diets low in linoleic acid are consumed (74). Consequently, it was recommended that the total level of trans,trans18:2 isomers in Canadian margarines and shortenings should be <1% of total fat. Most of the Canadian margarines meet this recommendation, but there are several margarines with tt-18:2 levels above the recommended 1% limit (Table 1.3). Since 1990, further improvements in the manufacture of margarines in Europe and Canada were made by the complete replacement of partially hydrogenated oils
TABLE 1.2 Trans FA Composition of Some Refined (Unhydrogenated) Edible Oilsa Canola
Soybean
Corn
Sunflower
-
0.04
-
0.03
0.01 0.2 0.1 8 0.09
0.01 0.35 0.31 0.04
-
-
0.2 0.1 5 0.04
0.94 0.1 6 0.84 2.42
0.55 0.09
-
Trans FA 9f-18:l 9t,12t-18:2 9c,12t-18:2 9t,12~-18:2 9t,12~,15t-18:3 9c,12c,l St-18:3 9c, 12t,l5c-18:3 9t,l2c,l 5c-l8:3 X trans FA
Safflower
Rice bran
Coconut
Extra virgin olive
0.03
0.08
0.17
-
-
-
-
-
0.26 0.2 0.02
0.38 0.3
0.02
0.05
-
-
-
-
I 2.
0.1 0.02 0.1 0.74
-
0.34 0.3 0.03 0.1 5 0.02 0.14 1.06
-
-
3
-
-
(%total FA)
0.5 1.89
-
0.08 0.47
-
-
0.02 0.73
-
-
0.19
0.05
2
2 F
B
aBased on analysis in author's (W.R.) laboratory of retail oil samples obtained in 2003 from supermarkets in Ottawa, Canada.
U
TABLE 1.3 Trans FA of Margarines from Various Countriesd Margarine type (n)
Total trans fat
Countrv: vear (Ref.) Austria: 1996 (54)
Bulgaria: 1998 (58)
t-1811
ciYtc-l8:2
fl-18:2
t-18~3
(%total FA) Hard (28) Soft (14) Diet (28)
-
10.0 8.7 0.8 (0.8-8.0)
soft (5)
-
-
-
-
-
(0.4-1.4)
-
-
-
Canada: 1979 (65)
Regular (9)
21.3 (8.7-32.9)
Canada: 1991 (68)
Soft (31) Hard (19)
20.4 (0.5-35.2) 34.4 (20.949.9)
18.2 (0-30.6) 30.5 (17.840.8)
1.2 (0-3.5) 3.1 (1.2-7.6)
0.2 (0-0.8) 0.6 (0-2.8)
0.2 (0-0.8)
soft (79) Hard (30)
18.8 (0.9-46.4) 34.3 (16.343.7)
16.6 (0.2-36.9) 30.0 (14.9-34.3)
1.6 (0.2-6.7) 3.0 (0.6-4.8)
0.4 (0-3.0) 0.9 (0-1 -7)
0.2 (0-1.6) 0.4 (0.2-1.1)
Soft (14) Hard (14)
16.8 (1.144.4) 39.8 (31.144.6)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Canada: 1998 (49) Canada: 1999 (73) Denmark 1996 (55)
Denmark: 1998 (57)
Semi-soft (8) Soft (6) Hard (20) Hard (32) Semi-soft (1 9) Soft (8)
-
4.1 (0-14.2) 3.1 (0-5.8) 0.4 (0-1.9)
1.2 (0-5.8) 1.3 (0-5.6) 4.2 (0-8.2)
(u
Q
c,
9 z$ fi
France: 1995 (53)
Soft (1 2)
-
8.0 (0-1 7.6)
Germany: 1978 (62)
Regular (83)
-
(0.1-53.2)
-
-
-
__
-
-
-
-
-
-
4.9 (1.8-5.6)
0.5 (0.1-0.6)
0.03 (0.0-0.04)
Germany: 1994 (50) Germany: 1996 (56)
Regular (46)
Soft (9) Diet (4)
9.3 (0.2-25.9) 5.0 (0-10.6) 0.2 (0.0-0.4) 5.4 (2.0-6.2)
0.4 (0.1-1.4)
Germany: 2000 (71)
Sunflower (9)
Greece: 1994 (69)
Soft (1 0 )
-
(5.4-9.5)
(0-3.7)
Greece: 2003 (61)
Soft (1 5)
(0-1 5.8)
(0.1-3.2)
Spain: 2000 (60)
soft (12)
(0.1-19) -
(0.15-1 9.8)
0.2-1
NZ: 1996 (99)
Regular (7)
16.4 (12.6-19.7)
14.6 (10.9-1 7.2)
1.2 (0.2-2.0)
0.5 (0.0-0.8)
Table spreads (5)
13.8 (12.5-14.7)
1.1 (0.4-2.5)
0.6 (0.0-1.9)
USA: 1997 (63)
Regular (9)
15.7 (14.3-1 6.9) -
USA: 1997 (64)
Regular (7)
20.4 (6.3-33.6)
-
USA. 1983 (66)
Regular (40)
USA 1985 (67)
Regular (84)
18.4 (6.8-31 .O) -
19.9 (10.7-30.1)
USA: 1997 (701
Regular (6)
USA: 2004 (72)
Regular (7)
(14.9-2 7.7)
dRange is given in parentheses; n is the number of samples.
18.0 (6.9-31.4) -
(0.04-1 3.3) -
.o
~
-
(0-0.3) -
-
P
-?
za
W.M.N. Ratnayake and C. Zehaluk
10
with interesterified liquid oils. The interesterified oils contain no trans FA, but have slightly higher m.p. than unmodified liquid oils. Often, small amounts of palm-kernel oil, palm oil, or coconut oil are added to the margarine fat blend to further increase the map.and to achieve the solid consistency of the margarine. These products, termed “zero-trans margarines,” have a favorable FA composition. The FA composition of zero-trans margarines available in Canada, the United States, and some European countries is shown in Table 1.4. In contrast to products made from partially hydrogenated oils, the zero-trans margarines contain almost no t-18:l. In these, the main trans FA are the geometrical isomers of linoleic (9~,12r-18:2and 9r,12c-18:2) and alinolenic acids (mainly 9~,12~,15r-18:3 and 9t,12c,15c-18:3).The levels and types of these trans PUFA are typical of those found in refined, unhydrogenated canola, soybean, and other common vegetable oils. The saturated fat content of the zero-trans margarines shown in Table 1.4 is comparable to the levels present in margarines prepared using partially hydrogenated oils (49). Furthermore, unlike margarines made from PHVO, the zero-trans margarines contain no unnatural cis positional isomers of oleic acid. There is very little information on the nutritional effects of positional isomers of oleic acid, but a study by Ayagari et aE. (75) found that both cord blood triglyceride trans and cis 18:1 positional isomers were inversely related to cord blood docosahexaenoic acid (DHA), but only the cis isomers were related to AA. DHA and AA, which are the most important metabolites of a-linolenic and linoleic acids, respectively, play a key role in the development of the central nervous system in infants (76).The zero-trans margarines are also a good source of both linoleic and alinolenic acids (Table 1.4). Overall, zero-trans margarines offer an excellent FA profile with regard to the established pathologic and physiologic aspects of dietary fats in human nutrition.
TABLE 1.4 FA Composition of Zero-trans Margarines from Britain, Canada, Germany, and the United States Britaina (n = 1 )
Canadab(n = 8)
18.7 25.4 49.4 1.2 -
18.6 (14.5-21.8) 45.8 (41.3-53.9) 24.5 (18.5-33.8) 7.1 (6.1-7.4) 2.2 (0.9-5.0) 1.l (0.2-2.8) 0.6 (0.3-1.5) 0.01 (0-0.1) 0.6 (0.3-1 .O)
cis-18:1 18:2n-6 18:3n-3 Total trans t-l8:1 Ct/tC-l8:2 tt-1 8:2 t-I 8x3
USAa (n = 1)
17.2 22.7 54.3 1.2 -
28.9 21.7 30
(%total FA)
FA
Saturated
Germanya (n = 1 )
-
aSource: Reference 52. bSource: Reference 49; values are means (range).
2
-
Trans Fatty Acids
11
Processed Foods. Most processed foods such as various bakery products, fast foods, fried foods, dry soup powders, and savory snacks are very often prepared with PHVO and therefore often contain relatively high proportions of trans FA. Reports listing the trans FA level in a number of common processed food items are available from Canada (73,77), the United States (66,70,72,78,79), and Europe (59,80-83). In Canada, it was stated that >60% of the trans fat consumed comes from processed foods, and only 11% comes from margarines (49,84). Similarly, data for the United States suggest that processed foods are likely to be the major, although variable, sources of dietary trans fats (72,79,85). In contrast to Canada and the United States, the use of partially hydrogenated oils is not widespread in European countries. Dairy and animal fats are very often used in Europe in the preparation of processed foods (86,87). In most European countries, animal fats contribute -40-70% of the total fat intake. Therefore, less trans fats are in many processed foods in European countries. In the Mediterranean countries, more than half the intake of trans comes from animal fats (86). The most recent trans FA data for some common processed foods from Canada, the United States, and Europe are shown in Table 1.5. The data show that the amount of trans FA varies widely within a food category; for example, the range of trans FA in 17 brands of crackers sold in Canada was 23-51% total FA, representing differences of 1-13 g trans FNlOO g cracker (73). These variations reflect the differences in the fats and oils used in the food manufacturing or preparation process from one food manufacturer to another. A primary reason for this variability is that processed foods are often prepared using blends of partially hydrogenated oil base stocks with varying levels of trans FA content, and these sometimes are blended with nonhydrogenated oils to obtain the desired physical properties. Furthermore, the use of partially hydrogenated base stocks and nonhydrogenated oils can also be expected to vary with availability and costs of various edible oils. Not only does the amount of trans fat content vary, but there is a considerable variation in the total fat content within a food category. For example, in 13 samples of Canadian donuts analyzed by Innis et al. (73), the total fat content ranged from 3.9 to 21.3%; in 14 brands of crackers, the total fat varied from 2.1 to 27.4% (Table 1.5).These variations in the total fat content and levels of trans FA within a food category can limit the accuracy of estimates of total fat and trans fat intakes when analysis of diet information is made using nutrient databases (59,73). Milk and Dairy Products. Milk, butter, cheeses, and all other forms of dairy products are an important source of dietary fat in the diet of humans, particularly in industrialized countries. For example, in Europe, with the exception of the Mediterranean countries, the consumption of dairy fat from various dairy sources generally exceeds 30 g/d, and it can even reach 40 g/d in France (87). Because milk fat generally contains 3-8% trans fat, dairy products form an important source of trans fats in some diets. The total trans contents of milk and dairy products from several different countries are presented in Table 1,6, The total trans content of whole bovine milk and products made from milk fat generally ranges
12
W.M.N. Ratnayake and C. Zehaluk
I l l
7 h
W
0
12c"
2 N
Chocolate bars Granola bars Potato chips Tortilla chips Donut Peanut butter Meat patty Breaded chicken
soups Sauces and gravy French fries, fast-food restaurant French fries, prefried Popcorn Salad dressings Mayonnaise aRangeis given in parentheses. bSource:Reference 73. cSource: Reference 72. dSource: References 80 and 82.
23.6 (13.430.9) 11.5 (5.1-17.0) 25.1 (21.9-30.6) -
13.5 (3.9-21.3) 43.5 (41.145.9) 16.4 (14.0-19.6) 13.4 (6.6-1 8.1) 8.3 (0.6-1 7.8) 8.7 (0.4-38.3) 5.8 (3.2-10.9)
9.2 (0.1-35.9) 11.3 (5.1-21.7) 5.9 (0.4-25.3) -
-
(1.8-24.5) (25.742.1 ) (2.4-28.2)
-
(0-3.8) (0.2-9.3) (0-17.1)
-
(9.1-23 .O)
29.6 (3.942.7) 4.1 (1.6-6.6) 6.8 (5.6-9.6) 27.4 (1 1.9) 22.4 (1.1-51.6) 33.2 (1.7-60.3) 37.7 (4.9-56.9)
-
(1.1-31.8) -
-
(1.5-1 00) (31.3-86.4)
(0.0-2.2) (0.0-0.4)
(0.5-26.5) (4.4-62.5) (1 0.2-1 8.0)
(6.641.3) (0.2-38.6) (0.5-34.8)
(1.5-1 6.6) (17.0-47.0)
(0.041.5) (0.0-34.8)
22 2 u"
h3
14
W.M.N. Ratnayake and C. Zehaluk
from 1.8 to 7.6%.These different trans FA amounts in milk fat are probably due to a variety of reasons, including variations in the season, feeding systems of cows, and dairy cow breeds. Wolff et al. (92) found that butter produced in spring generally contains slightly higher amount of trans-18:l (4.3%) than that produced in the fall (3.2%). Studies performed in Germany (97) found a higher content in summer milk (4.4%) than in winter milk (1.9%). The seasonal variations might be due to the higher amount of up to 75% C18 PUFA in the pasture fodder and to the lower amount of these FA in the barn fodder (97). The C18PUFA are partially converted to trans FA by bacteria present in the rumen. As mentioned previously, the trans-octadecenoic isomers, in particular vaccenic acid (1lt-18:1), are the predominant trans FA isomer group in dairy fats. In addition to t-18:l isomers, milk fat contains small amounts of trans isomers of 14:1, 16:1, 18:2, and 18:3. Similar to the total trans content, the individual trans isomers also exhibit TABLE 1.6 Total Trans FA in Milk a n d D a i r y Products f r o m Various Countriesa Country: year (Ref.)
Product category (n)
Australia: 1971 (88) Austria: 1994 (89) Canada: 1983 (90) Canada: 1994 (91) Canada: 2003b
Bovine milk (1 16) Bovine milk (1 3) Bovine milk (1 3) Bovine milk (101) Bovine milkC Ice creamC YogurtC Cheese, cheddarC Cheese, cottageC Cheese, processedC ButterC Autumn butter (12) Spring butter (12) Butter (60) Butter (27) Cheese (7) Bovine milk (1 756) Bovine milk (1 32) Cheese (25) Butter (12) Butter (5) Butter (1 0) Bovine milk (4)
France: 1994 (87) France: 1995 (92) Germany: 1992 (93) Germany: 1995 (94) Germany: 1997 (95)
New Zealand: 1996 (99) USA: 1995 (85) USA: 1995 (85)
Total trans fat
(YOtotal FA)
6.0 (4.3-7.6) (1.8-5.2) (4.0-5.7) 3.8 (3.2-4.7) 4.3 4.2 5.4 6.6
5.5 5.9 5.7 3.2 (2.5-3.9)d 4.3 (3.5-5.1)d 3.3 (2.4-4.3)d (2.5-7.9) (1.6-7.5) 3.6 (1.3-6.8) 3.39 4.71 1.9 2.0 6.0(5.1-7.5) 3.5 (2-5) 3.0 (2.7-3.4)
aValues are means (range). bBased on analysis in an author’s (W.R.) laboratory of retail oil samples obtained in 2003 from supermarkets in Ottawa, Canada. CAnalysiswas performed on a composite sample made from 5-10 major brands. q h e values are for total f-l8:1. eAveragetotal trans for milk from cows raised indoors. ‘Average total trans for milk from cows raised in pasture.
Trans Fatty Acids
15
seasonal variation (98). Milk fat from spring milk samples typically has a greater proportion of vaccenic acid (58%) than that of winter milk (48%) (95). Meat and Eggs. The trans FA content in different types of meat and eggs is shown in Table 1.7. Meats from nonruminants such as poultry and pork typically contain very little trans fats (0-1% of total fat), whereas meats from ruminants contain slightly higher levels (1-10%). Among the ruminants, meats from lambs, goats, and kangaroos generally have high levels of trans fats (Table 1.7). In beef fat, the total trans content usually ranges from 2 to 4%. The 18:1 trans isomers are the predominant isomers in meat, and the 18:l isomer distribution is very similar to that of milk fat (93). Eggs contain very little trans fats. Infant Formulas. Published FA composition data from several countries show that infant formulas can have small amounts of trans fats (0-5% of total fat) as a result TABLE 1.7 Trans FA Content of Meat and Eggsa Product category
Total fat
Country: year (Ref.) Canada: 2003b
France: 1995 (1 10) Germany: 1992 (93)
Germany: 1997 (96)
USA: 1995 (85)
Total trans
Trans-18:l
ctltc-l8:2
(% of total fat)
Beef, steak (1 ) Beef, roast (1) Beef, ground (1 ) Veal (1) Lamb (1) Organ meat (1) Pork (1) Chicken (1) Eggs (1 Beef (10) Calf (3) Beef (5) Lamb (3) Sheep (1) Mutton (3) Rabbit (1) Kangaroo (2) Pork (4) Lamb (2) Turkey (2) Beef liver (2) Beef, lean (9) Pork, lean (7) Chicken, lean
8.0 5.9 8.6 1.7 6.0 4.6 5.7 2.4 6.3
-
-
5.2 4.9 3.4
3.2 1.9 3.9 2.9 8.1 3.4 0.5 2.5 1.3
(0.9-1.7) (1.9-3.2) (6.6-8.8) 3.2 (8.2-1 0.6) 0.4 9.8 (0.5-0.7) 9.8 1.2 2.8 4.0 (2-5) 0.2 (0.1-0.3) 1.3 (0.7-1.4)
2.9 1.5 3.3 2.6 7.5 3 .O 0.1 1.1 1.l 2.0 (0.8-3.5) (0.7-1.3) (1.4-2.4) (5.2-7.0) 2.2 (6.0-8.9)
-
7.9 (0.2-0.4) 0.2 0.9 2.3 3.2 (2-5) 0.2 0.9
0.2 0.4 0.3 0.2 0.4 0.3 0.4 1.3 0.2
(0.0-0.2) (0.0-0.3) (0.6-0.9) 0.4 (0.9-1.2) 0.3 0.8 (0.0-0.05) 7.5 0.1 0.03 0.2 (0-0.3) 0 0.3 (0.7-1.4)
dRange is given in parentheses.
bBased on analysis in an author’s (W.R.)laboratory of samples obtained in 2003 from supermarkets in Ottawa, Canada. The results for each product category are for a composite of 5-8 different samples.
16
W.M.N. Ratnayake and C. Zehaluk
of the use of refined vegetable oils and/or milk fats as the source of fat in the formulas (Table 1.8). Infant formulas in Canada (100) and the United States (101) contained trans FA at 0.2-3.1% of total FA. In France (102), the content of trans FA in infant formulas ranged from 0.2 to 5.0%.The predominant trans isomers in Canadian (100) and French infant formulas are those of 18:1, whereas formulas from the United States contain no trans-18:1 isomers. In the United States, the typical geometrical isomers of linoleic and a-linolenic acids, that are commonly present in refined, unhydrogenated vegetable oils, are the predominant trans isomers ( 101). Human Milk. Table 1.9 presents data for the percentage of total trans FA, t-18:1, ct/tc-l8:2, and t-18:3 in human milk fat from different populations consuming habitual diets. Results from human milk studies suggest that the trans FA content of human milk reflects the trans FA content of the mother’s diet on the previous day (103). The variation in the trans fat content of human milk among different countries, therefore, reflects the variations in the trans content in the habitual diets of different countries. Human milk samples from Canada (84,104) and the United States (103,105,106) contain greater amounts of trans FA than do milk samples from Germany (107), Denmark (108), and France (109,111). In two separate Canadian studies (84,104), on average, trans FA made up 7.2% of total FA in breast milk (maximum 18.7%). Trans FA would thus represent an average of 3.5% of energy in the diet of Canadian breast-fed babies. Breast milk from the United States in some studies is also reported to contain high levels of trans fats (103). These data reflect the widespread use of partially hydrogenated oils in adults’ foods in Canada and the United States. Human milk samples from Spain ( l l l ) ,Nigeria (112), China (113), Sudan (114), and Hong Kong (113) have very low amounts of trans FA, indicating a lack of partially hydrogenated fats in the adult diets of these countries. In countries in which there is a widespread use of PHVO, -70-80% of the total trans FA in human milk is monounsaturated isomers (104). The balance includes an assortment of the minor isomers that are normally found in PHVO (Table 1.2).
Baby Foods. At present, few current data on trans FA content in baby foods are available. Those available, however, show that most baby foods contain negligible amounts of trans fats, except small quantities found in beef products (Table 1.10). Holub (115) reported, however, that in Canadian baby cereals and baby biscuits, up to 23 and 37%, respectively, of the fat were trans FA, indicating the use of partially hydrogenated oils as shortening in these products. Fortunately, these products have very little total fat (4 gherving), so that the overall intake of trans FA by those babies consuming moderate amounts of cereals and biscuits is likely insignificant. Regulatory and Nutrition Labeling Considerations
Two recent reports from authoritative scientific bodies have made recommendations with regard to dietary trans FA. In 2002, the Joint WHOPA0 Expert Consultation on
TABLE 1.8 Trans FA Content of Infant Formulasa Country: year (Ref.)
Product category (n)
Canada: 1997 (100)
Powdered formulas (14) Liquid formulas (10) Formulas for: Premature infants (2) 0- to 5-mon-old infants (9) 6- to 10-rnon-old infants (8) Infants up to 10 mon old (1) Liquid formulas (10)
France: 1996 (101)
USA: 1994 (102)
aRange is given in parentheses.
Total trans (YOof total FA)
cf/'tc-l8:2 Trans-1 8:l
(Yo of total fat)
Trans-18:3
1.4 (0.6-2.5) 1.9 (0.9-3.1)
0.64 (0.04-2.26) 0.69 (0.00-2.02)
0.44 (0.1 9-1.41) 0.69 (0.46-1.06)
0.31 (0.05-1.1 3) 0.49 (0.1 1-1.16)
1.9 (1.3-2.5) (0.4-3.1) (0.4-5.0) 3.0 0.8 (0.2-1.3)
(1.02-1.46) (0.21-2.53) (0.234.29) 2.99
(0.140.85) (0.1 7-0.35) (0.10-0.51 ) 0.01 0.31 (0.05-0.43)
(0.1 1-0.16) (0.06-0.1 9) (0.02-0.21) 0.03 0.46 (0.10-0.81)
0
22 E Q $
2-
18
W.M.N. Ratnayake and C. Zehaluk
TABLE 1.9 Trans FA Isomer Content of Human Milk Lipidsa
Country: year (Ref.), n Canada: 1995 (103), 198 Canada: 1999 (84), 103 China: 1997 (1131, 33 Denmark: 1995 (108), 1 France: 1995 (1091, 10 France: 1995 (1lo), 10 Germany: 1988 (107), 15 Hong Kong: 1997 (113), 51 Nigeria: 1991 (112), 10 Sudan: 1995 (114), 1 Spain: 1993 (1111, 38 USA 1977 (1OS), 3 USA: 1985 (1 04), 8 USA: 1985 (106), 52
Total trans (% of total FA)
7.2 (0.1-1 7.2) 7.1 (2.2-1 8.7) 0.22 2.5 2.3
4.4 (2.2-6.0) 0.88 1.2 (0.8-1 0.3) 1.3
4.3
Trans-18:2 Trans-18:l
(% of total fat)
5.9 (0.1-15.4)
0.94 (0.0-2.41)
0.2
Trans-18:3
0.02
-
-
1.9 (1.2-3.0) 2.0 (1.2-3.2) 3.1 (1.5-4.4) 0.7 0.9 (0.5-4.9) 0.4 1 3.1 6.5 (4.2-9.0) 3.7
0.27 (0.124.64)
0.35 (0.2 1-0.74) 0.1 8 0.2 ( 0 . 1 4 4 ) 0.3
aRange is given in parentheses
Diet, Nutrition and the Prevention of Chronic Diseases (34) recommended that populations should limit energy intake from fat to between 15 and 30% of total daily energy intake and shift consumption away from saturated fats and trans FA toward unsaturated fats, with saturated fats providing
Trans Fatty Acids
19
TABLE 1.10 Trans FA Content of Baby Foods in Canada and the U n i t e d Statesa
Country: year (Ref.)
Product category (n)
Canada: 2004b
Cereals (mixed with whole milk) Desserts (1 )c Dinners (cereal + vegetables + meat) ( l ) c
Dinners (meat or poultry + vegetables) (1Ic
Formula, milk-based ( l ) c Formula, soy-based (l)c Fruits (apples + peaches) (1 )c Meat (poultry + beef) (1 )c Vegetables (mixed) Canada: 1999 (1 15)
USA: 1995 (1 16)
Biscuits Cereals Strained vegetables and beef
Total fat (g/lOOg food)
Total trans (Yototal FA)
3.5 0.3
3.6 2.6
0.9 0.6 1.9
0
2.2
0 0
0.05 5.5
0.3
-
-
0 0
5.9 0 Up to 37% Up to 23% 5.1 (4.7-5.6)
aRange is given in parentheses; n is the number of samples. bBased on analysis in W.R.’s laboratory of samples obtained in 2003 from supermarkets in Ottawa, Canada. T h e results of each product category are for a composite of 2-5 different samples.
any incremental increase in trans FA intake increases the risk of CHD.” The Panel recommends “that trans FA consumption be as low as possible while consuming a nutritionally adequate diet .”
Canada. In January 2003, Canada became the first country to require the mandatory declaration of the trans FA content of foods on the labels of most prepackaged foods by December 2005 (36). For the purpose of nutrition labeling and nutrition claims, the Canadian Food and Drug Regulations define “trans fat” as unsaturated FA that contain one or more isolated or nonconjugated double bonds in a trans configuration ( 117). The Canadian “Nutrition Facts” table will include the absolute amount of trans fat in grams per serving of the food as well as the percentage of the daily value (DV) for trans and saturated fat combined. The DV for saturated plus trans fat is 10% of energy based on a 2000 kcal diet or -20 g of saturated plus trans fat. This is considered justifiable in light of the recommendations to decrease the dietary intake of saturated and trans fat (2). Canadian provincial food intake data indicate that intakes of saturated fat in Canada average -1 1-13% of energy ( 1 18). Using provincial food intake data, Health Canada has calculated that intakes of trans fat (as t-18:1) average -3.7% of energy (1 19). A combined DV for saturated and trans fat of 10% of energy represents a reduction from current intakes and is consistent with the “Recommendations for a healthy lifestyle” from the Working Group on Hyper-
cholesterolemia and Other Dyslipidemias (32), which advocated a diet with t10% of total energy from saturated and trans FA.
20
W.M.N. Ratnayake and C. Zehaluk
Further support for the Canadian approach to the declaration of trans fat came from the Committee on Use of Dietary Reference Intakes in Nutrition Labeling of the Institute of Medicine (120). The Committee recommended that for labeling of saturated and trans FA, the amounts should be listed on separate lines in the Nutrition Facts table and that one percentage of DV (%DV) for the two nutrients together be included in the Nutrition Facts table. The Committee noted that consumer research had indicated that %DV is a useful tool for comparing different food products and for determining their relative significance and contribution to a healthy diet. The declaration of separate amounts for saturated and trans FA would allow for the education of consumers about the unique differences between these FA; the combined %DV would be used to educate that neither is desirable in terms of cardiovascular health risk. The Committee recognized that saturated and trans FA are chemically distinct and have different physiologic effects. However, both raise total and LDL-C levels and are potential contributors to CVD risk. The Committee stated that a combined %DV does not suggest that one type of fat is less healthy than the other. In addition, the Committee also stated that such an approach would provide a target for the food industry to utilize when combining saturated and trans FA in product formulations to achieve required functional objectives. Considering saturated and trans FA together was seen as an incentive for food manufacturers to lower both as much as possible. The Canadian Regulations define the level of insignificance for both saturated fat and trans fat as 4 . 2 gheference amount and serving of food (1 17). Higher amounts would result in levels of saturated and trans fats that would be considered to be nutritionally significant and therefore should not be rounded to “0.” For example, consumption of 10 servings of foods containing up to 0.5 g each of saturated and trans fat and declaring “0”g for saturated fat and “0”g for trans fat, could result in an intake of up to 10 g of saturated fat plus trans fat or 50% of the DV. Current analytical methodology allows the determination of levels of saturated and trans fats at ~ 0 . g2 (121). The AOAC Official Methods 996.01 (122) and 996.06 (123) are capable of accurately measuring total saturated fat at levels of 0.2 g/100 g food. A collaborative study that tested the applicability of this method demonstrated that total saturated fat at a level of 0.2 g/100 g food could be measured accurately with very high reproducibility in both inter- (%RSD, 3 S ) and intralaboratory (%RSD, 9.5) analyses. Ali and co-workers (124), who tested this method, also reported a similar high reproducibility and accuracy when it was applied to foods with total saturated fat as low as 2 mg/g food (i.e., 0.2 g/100 g food). In addition, the results of Ali et al. (124) demonstrated that this method is also capable of reliably measuring trans FA at very low levels, i.e., 1-2 mg/g food, and that the method is applicable to all types of food matrices with fat ranging from 0.7 to 97.5 g/100 g food. The Canadian Regulations also define three nutrient content claims for bans fat (I 17). Nutrient content claims are claims that describe the amount of a nutrient
Trans Fatty Acids
21
in a food. The nutrient content claims for trans fat include the claims “trans fat free” and the comparative claims “reduced in trans fat” and “lower in trans fat.” Foods that carry the claim “trans fat free” must contain <0.2 g of trans fatheference amount and serving of food. The food must also be “low in saturated fat.” Foods labeled “reduced in trans fat” or “lower in trans fat” must contain at least 25% less trans fat than the appropriate reference food, and the content of saturated fat must not have been increased in relation to the reference food. Under the Canadian Regulations (1 17), restrictions were introduced on the trans fat content of foods that carry nutrient content claims for saturated FA and cholesterol. Foods labeled “free of saturated fat” must contain <0.2 g of saturated fat and 0.2 g of trans fatheference amount and serving. Foods carrying the claim “low in saturated fat” must contain not >2.0 g of saturated and trans fat combined reference amount and serving and not >15% of energy from saturated and trans fat combined. Foods carrying a comparative claim such as “reduced in saturated fat” or “lower in saturated fat” must contain at least 25% less saturated fat than the appropriate reference food, and the content of trans fat must not have been increased in relation to the reference food. In the case of nutrient content claims for cholesterol, such as “cholesterol free,” “low in cholesterol,” “reduced in cholesterol,” or “lower in cholesterol,” in addition to meeting specific requirements with regard to cholesterol levels, <2 mg cholesterolheference amount and serving, not >20 mg cholesterol/reference amount and serving, and at least 25% less cholesterol/reference amount, respectively, foods must meet the compositional criteria for “low in saturated fat” (117). The Canadian Regulations have made provision for a diet-related health claim with regard to saturated and trans fat: “A healthy diet low in saturated and trans fats may reduce the risk of heart disease. (Naming the food) is [free of] [low in] saturated and trans fat.” The conditions that the food must meet to qualify for this claim include, in addition to the restrictions for “free” or “low” in saturated and trans fat, limits on cholesterol and sodium content. Fats or oils carrying this claim would also have to be a source of n-3 (20.3 gheference amount and stated serving) and/or of n-6 (22 gheference amount and stated serving) FA (1 17). It was considered that nutrition information, together with consumer education would result in consumers making food choices aimed at decreasing their intake of trans fat and that the requirement to declare trans fat content would act as an incentive to the food industry to decrease the trans fat content of foods (36). The evidence is mounting that the intended effect is taking place even before labels are actually required to carry the new Nutrition Facts table. Consumers are increasingly more aware of the trans fat content of foods and of the health effects of trans fat. The food industry has already announced significant changes to the formulation of many foods in categories that are the highest contributors of trans fat to the diet (125-127). The Canadian regulations for mandatory nutrition labeling apply at this time to prepackaged foods. Foods sold in restaurants and other food-service establishments
22
W.M.N. Ratnayake and C. Zehaluk
that are not prepackaged are not required to carry nutrition labeling; however, they may be significant sources of trans fat in the diet. If claims are made for such foods, however, the amount in the food of the nutrient that is the subject of the claim would have to be declared (117).
United States. In July 2003, the U S . Food and Drug Administration (FDA) announced that it was amending its regulations on nutrition labeling to require that trans FA be declared in the Nutrition Facts panel table for most packaged foods and dietary supplements on a separate line immediately under the line for the declaration of saturated FA (37). A DV for trans fat was not established; therefore the Nutrition Facts panel will not include a %DV, which is required for saturated fat (37). Mandatory declaration of trans FA in the United States is effective January 1, 2006. The FDA estimated that 3 years after the effective date, truns fat labeling would prevent from 600 to 1200 cases of CVD and 250-500 deaths each year. Based on this estimate, the FDA calculated that the requirement to label packaged foods with the trans fat content will realize a yearly cost savings of $900 million to $1.8 billion in medical costs, lost productivity, and pain and suffering (128). Under the US. regulations, trans fat may be declared as “0” if it is 4 . 5 gherving (129). The FDA did not define nutrient content claims for trans fat because it was considered that there was insufficient scientific information to establish nutrient content claims for trans fat (37). The compositional criteria for the nutrient content claim “free of saturated fat” require that foods carrying this claim contain ~ 0 . 5g of saturated fat and <0.5 g of trans fat per reference amount customarily consumed and per labeled serving ( 129). As in the Canadian Regulations, the FDA’s regulatory chemical definition for trans FA for the purpose of nutrition labeling is the sum of all unsaturated FA that contain one or more isolated (i.e., nonconjugated) double bonds in a trans configuration (129). Under the Agency’s definition, conjugated linoleic acid would be excluded from the definition of trans fat. Foods served in restaurants and in other establishments in which food is served for immediate human consumption or sold only in such facilities are exempt from nutrition labeling unless a claim is made or other nutrition information is provided (129). Other lurisdictions. Canada and the United States were the first jurisdictions to promulgate regulations requiring the declaration of trans fat in nutrition labeling on prepackaged foods. The Mercosur countries, including Argentina, Brazil, Paraguay and Uruguay, have now recently also passed a resolution that will require mandatory nutrition labeling including declaration of trans fat by 2006 (130). Other countries generally require the content of trans fat to be declared only if claims are permitted and made for trans fat andor other fat components. In 2003, the Codex Committee on Food Labeling agreed that the declaration of trans FA on food labels would be left to the discretion of national authorities under the
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Codex Guidelines for Nutrition Labeling (131). These guidelines state that the amount of a nutrient for which a nutrition claim is made should be listed in the nutrition labeling (132). The Codex Guidelines for Use of Nutrition Claims state that in the case of the claim for “low in saturated fat,” trans FA should be taken into account where applicable and that this provision consequentially applies to foods claimed to be “low in cholesterol” and “cholesterol free” (133). The Codex Guidelines for Use of Nutrition Claims do not define nutrient content claims for trans fat. The Codex also does not have, at this time, a definition for trans fat (131). In the European Union, the Council Directive on nutrition labeling of foodstuffs (134) provides for voluntary nutrition labeling except where a nutrition claim is made. The current rules require that when nutrition labeling is provided, the declaration of energy, protein, carbohydrate, and fat, or of energy, protein, carbohydrate, sugars, fat, saturated fat, fiber, and sodium if a claim for any of sugars, saturated fat, fiber, or sodium is made. Provision is also made for the inclusion of starch, polyols, monounsaturated FA (MUFA) and PUFA, cholesterol and specific vitamins and minerals. There is no provision for the declaration of trans FA. On request from the European Commission, the Scientific Panel of the European Food Safety Authority on Dietetic Products, Nutrition and Allergies, recently published an opinion related to the presence of trans FA in foods and the effect on human health of the consumption of trans FA (135). The panel concluded that, based on prospective cohort studies, a high intake of trans FA is associated with an increased risk of cardiovascular disease and that the effects of trans FA were more pronounced than those of saturated fat. On the other hand, the panel also noted that current intakes of trans FA were 10 times lower than those of saturated FA whose intakes in many European countries exceed dietary recommendations. The Food Standards Agency of the United Kingdom allows claims regarding trans fat, in which case their amount must be declared with nutrition labeling (136). The Australia and New Zealand Food Standards Agency (137) currently requires the declaration of the trans FA content of foods for which a claim is made with respect to cholesterol, or of saturated, trans, PUFA, or MUFA. Claims in relation to the PUFA, MUFA, or n-3/n-6 FA content of food can be made if certain criteria are met, including that the total of saturated and trans FA comprises no >28% of the total FA content of the food. The Australia and New Zealand regulations define trans fat as the total of unsaturated FA in which one or more of the double bonds are in the trans configuration acids and declared as trans fat. South Africa has proposals that will require the mandatory declaration of energy, carbohydrate, total fat, saturated fat, trans fat, dietary fiber, and sodium whenever a nutrition or health claim is made, in addition to a declaration of the nutrient that is the subject of the claim (138). South Africa is proposing to define “trans fat” for the purpose of nutrition labeling as the trans FA with nonconjugated double bonds, excluding conjugated linoleic acid, that occur naturally in meat and dairy products. In 2003, although it had originally considered implementation of regulations to label foods with trans FA content, Denmark chose to limit the amount of trans
W.M.N. Ratnavake and C. Zehaluk
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fat in foods as a way to decrease consumers intake of trans fat (35). Under Order No. 160 of the Foodstuffs Act, as of June 1,2003,it is prohibited to sell oils and fats if they contain >2 g of trans FAD00 g. As of January 1,2004, any oils or fats used as ingedients in foods are prohibited from containing >2 g of fat/100 g of the fat or oil ingredient. The Order does not apply to naturally occurring trans FA in animal products. For the purposes of the Order, “trans FA’ are defined as the sum of all FA isomers with 14,15,18,20, or 22 carbon atoms and one or more double trans; in the case of PUFA, however, only those with methylene-interrupteddouble bonds are included.
Summary Although two different approaches were initiated to decrease the dietary intake of trans fat, i.e., labeling and restriction on level in foods, their potential effect may be similar in terms of the reduction of trans fat in foods and consumer choices of foods with lower trans fat content. As evidenced in Canada and the United States, several major manufacturers have already indicated that they have taken action to remove or decrease the truns fat content of foods. This is happening even before mandatory trans fat labeling goes into effect in Canada and the United States in the case of prepackaged foods. Moreover, even in cases in which foods are exempted from mandatory nutrition labeling, e.g., food service and restaurant foods, the trend is to decrease the trans fat content. Certainly, increased consumer awareness of the health issues associated with dietary trans fat can be credited as a major factor in the move by the food industry to decrease the trans fat content of their products (139). References 1. Kris-Etherton, P.M., and S. Yu, Individual Fatty Acid Effects on Plasma Lipids and Lipoproteins: Human Studies, Am. J . Clin. Nutr. 65 (Suppl.): 16288-1644s (1997). 2. Institute of Medicine, Report of the Panel on Macronutrients of the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, in Dietary Reference Intakes of Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids, The National Academy Press, Washington, 2002, pp. 8-1-8-97. 3. Mensink, R.P., P.L. Zock, A.D.M. Kester, and M.B. Katan, Effects of Dietary Fatty Acids and Carbohydrates on the Ratio of Serum Total to HDL Cholesterol and on Serum Lipids and Apolipoproteins: A Meta-Analysis of 60 Controlled Trials, Am. J . Clin. Nutr. 77: 1146-1 155 (2003). 4. Mensink, R.P., and M.B. Katan, Effect of Dietary trans-Fatty Acids on High-Density and Low-Density Lipoprotein Cholesterol Levels in Healthy Subjects, N . Engl. J . Med. 23: 439-445 (1990). 5. Nestel, P., M. Noakes, B. Belling, R. McArthur, P. Clifton, E. Janus, and M. Abbey, Plasma Lipoprotein and Lp[a] Changes with Substitution of Elaidic Acid for Oleic Acid in the Diet, J . Lipid Res. 33: 1029-1036 (1992). 6. Zock, P.L., and M.B. Katan, Hydrogenation Alternatives: Effects of trans Fatty Acids and Stearic Acid Versus Linoleic Acid on Serum Lipids and Lipoproteins in Humans, J . Lipid Res. 33: 399-410 (1992).
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7. Judd, J.T., B.A. Clevidence, R.A. Muesing, J. Wittes, M.E. Sunkin, and J.J. Podczasy, Dietary trans Fatty Acids: Effects on Plasma Lipids and Lipoproteins of Healthy Men and Women,Am. J . Clin. Nutr. 59: 861-868 (1994). 8. Almendingen, K., 0. Jordal, P. Kierulf, B. Sandstad, and J.L. Pedersen, Effects of Partially Hydrogenated Fish Oil, Partially Hydrogenated Soybean Oil, and Butter on Serum Lipoproteins and Lp[a] in Men, J . Lipid Res. 36: 1370-1384 (1995). 9. Judd, J.T., D.J. Baer, B.A. Clevidence, R.A. Muesing, S.C. Chen, J.A. Westrate, G.W. Meijer, J. Wittes, A.H. Lichtenstein, M. Vilella-Bach, and E.J. Schaefer, Effects of Margarine Compared with Those of Butter on Blood Lipid Profiles Related to Cardiovascular Disease Risk Factors in Normolipedemic Adults Fed Controlled Diets, Am. J . Clin.Nutr. 68: 768-777 (1998). 10. Miiller, H., 0. Jordal, I. Seljeflot, P. Kierulf, B. Kirkhus, 0. Ledsaak, and J.L. Pedersen, Effect of Plasma Lipids and Lipoproteins of Replacing Partially Hydrogenated Fish Oil with Vegetable Fat in Margarine, Br. J . Nutr. 80: 243-25 1 (1998). 11. Judd, J.T., D.J. Baer, B.A. Clevidence, P. Kris-Etherton, R.A. Muesing, and M. Iwane, Dietary cis and trans Monounsaturated and Saturated FA and Plasma Lipids and Lipoproteins in Men, Lipids 37: 123-131 (2002). 12. Sundram, K., A. Ismail, K.C. Hayes, R. Jeyamalar, and R. Pathmanathan, Trans (Elaidic) Fatty Acids Adversely Affect the Lipoprotein Profile Relative to Specific Saturated Fatty Acids in Humans, J . Nutr. 127: 514s-520s (1997). 13. de Roos, N.M., E.G. Schouten, and M.B. Katan, Consumption of a Solid Fat Rich in Lauric Acid Results in a More Favorable Serum Lipid Profile in Healthy Men and Women than Consumption of a Solid Fat Rich in trans-Fatty Acids, J . Nutr. 131: 242-245 (2001). 14. Ascherio, A., E.B. Rimm, E.L. Giovannucci, D. Spiegelman, M. Stampfer, and W.C. Willett, Dietary Fat and Risk of Coronary Heart Disease in Men: Cohort Follow Up Study in the United States, Br. Med. J . 313: 84-90 (1996). 15. Gordon, D.J., J.L. Probstfield, R.J. Garrison, I.D. Neaton, W.P. Castelli, J.D. Knoke, D R J . Jacobs, S. Bangdiwala, and H.A. Tyroler, High-Density Lipoprotein Cholesterol and Cardiovascular Disease: Four Prospective American Studies, Circulation 79: 8-15 (1989). 16. Aro, A., M. Jauhiainen, R. Partanen, I. Salminen, and M. Mutanen, Stearic Acid, trans Fatty Acids, and Dairy Fat: Effects on Serum and Lipoprotein Lipids, Apolipoproteins, Lipoprotein (a), and Lipid Protein Transfer Proteins in Healthy Subjects, Am. J . Clin. Nutr. 65: 1419-1426 (1997). 17. Ballantyne, C.M., J.A. Herd, L.L. Ferlic, J.K. Dunn, J.A. Farmer, P.H. Jones, J.R. Schein, and A.M.J. Gotto, Influence of Low HDL on Progression of Coronary Artery Disease and Response to Fluvastatin Therapy, Circulation 99: 736-743 (1999). 18. Willett, W.C., M.J. Stampfer, J.E. Mason, G.A. Colditz, F.E. Speizer, B.A. Rosner, L.A. Sampson, and C.H. Hennekens, Intake of trans Fatty Acids and Risk of Coronary Heart Disease Among Women, Lancet 341: 581-585 (1993). 19. Ascherio, A,, C.H. Hennekens, J.E. Buring, C. Master, M.J. Stampfer, and W.C. Willett, Trans-Fatty Acids Intake and Risk of Myocardial Infarction, Circulation 89: 94-101 (1994). 20. Hu, F.B., M.J. Stampfer, J.E. Manson, E. Rimm, G.A. Colditz, R.A. Rosner, C.H.
Hennekens, and W,C, Willett, Dietary Fat Intake and the Risk of Coronary Heart Disease in Women, N . Engl. J . Med. 337: 1491-1499 (1997).
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21. Pietinen, P., A. Ascherio, P. Korhonen, A.M. Hartman, W.C. Willett, D. Albanes, and J. Virtamo, Intake of Fatty Acids and Risk of Coronary Heart Disease in a Cohort of Finnish Men, Am. J . Epidemiol. 145: 876-887 (1997). 22. Oomen, C.M., M.C. Ocke, E.J. Feskens, M.A. van Erp-Baart, F.J. Kok, and D. Kromhout, Association Between trans Fatty Acid Intake and 10-Year Risk of Coronary Heart Disease in the Zutphen Elderly Study: A Prospective PopulationBased Study, Lancet 357: 746-751 (2001). 23. Lemaitre, R.N., I.B. King, T.E. Raghunathan, R.M. Pearce, S. Weinmann, R.H. Knopp, M.K. Copass, L.A. Cobb, and D.S. Siscovick, Cell Membrane trans-Fatty Acids and the Risk of Primary Cardiac Arrest, Circulation 105: 697-701 (2002). 24. Clifton, P.M., J.B. Keogh, and M. Noakes, Trans Fatty Acids in Adipose Tissue and the Food Supply Are Associated with Myocardial Infarction, J . Nutr. 134: 874-879 (2004). 25. Aro, A., A.F. Kardinaal, I. Salminen, J.D. Kark, R.A. Riemersma, M. Delgado Rodriguez, J. Gomez-Aracena, J.K. Huttunen, L. Kohlmeier, and B .C. Martin, Adipose Tissue Isomeric trans Fatty Acids and Risk of Myocardial Infarction in Nine Countries: The EURAMIC Study, Lancet. 345: 273-278 (1995). 26. Roberts, T.L., D.A. Wood, R.A., Riemersma, P.J. Gallagher, and F.C. Lampe, Trans Isomers of Oleic and Linoleic Acids in Adipose Tissue and Sudden Cardiac Death, Lancet 345: 278-282 (1995). 27. Bolton-Smith, C., M. Woodward, S . Fenton, and C.A. Brown, Does Dietary trans Fatty Acid Intake Relate to the Prevalence of Coronary Heart Disease in Scotland? Eur. Heart J. 17: 837-845 (1996). 28. Stender, S . , J. Dyerberg, G. Holmer, L. Ovesen, and B. Sandstrom, The Influence of trans Fatty Acids on Health: A Report of the Danish Nutrition Council, Clin. Sci. 88: 375-392 (1995). 29. American Heart Association, Trans Fatty Acids, Plasma Lipid Levels, and Risk of Developing Cardiovascular Disease, Circulation 95: 2588-2590 (1997). 30. European Atherosclerosis Society, Strategies for the Prevention of Coronary Heart Disease, Eur. Heart J . 148: 36-69 (1987). 31. Food and Agriculture Organization of the United Nations, Fats and Oils in Human Nutrition, Report of a Joint Expert Consultation, F A 0 Food and Nutrition Paper 57, Rome, 1994. 32. Fodor, J.G., J.J. Frohlich, J.J.G. Genest, and P.R. McPherson, Recommendations for the Management and Treatment of Dyslipidemia. Report of the Working Group on Hypercholesterolemia and Other Dyslipidemias, Can. Med. Assoc. J . 162: 1441-1447 (2000). 33. Krauss, R.M., R.H. Eckel, B. Howard, L.J. Appel, S.R. Daniels, R.J. Deckelbaum, J.W. Erdman, Jr., P. Kris-Etherton, I.J. Goldberg, T.A. Kotchen, A.H. Lichtenstein, W.E. Mitch, R. Mullis, K. Robinson, J. Wylie-Rosett, S . St Jeor, J. Suttie, D.L. Tribble, and T.L. Bazzarre, Revision 2000: A Statement for Healthcare Professionals from the Nutrition Committee of the American Heart Association, J . Nutr. 131: 132146 (2001). 34. Food and Agriculture Organization of the United Nations. Joint WHO/FAO Expert Consultation on Diet, Nutrition, and the Prevention of Chronic Diseases. WHO Technical Report Series 916, Geneva, 2002. 35. Stender, S., and J. Dyerberg, The Influence of Trans Fatty Acids on Health. A Report from the Danish Nutrition Council, 4th edn., Publication no. 34 (2003).
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73. Innis, S.M., T.J. Green, and T.K. Halsey, Variability in the Trans Fatty Acid Content of Foods Within a Food Category: Implications for Estimation of Dietary Trans Fatty Acid Intakes, J . Am. Coll. Nutr. 18: 255-260 (1999). 74. Ministry of Supply and Services of Canada, Report of the Ad HOCCommittee on the Composition of Special Margarines, Cat, No. H44-46/1980E, Ottawa, Canada, 1980. 75. Ayagari, A., J.M. Peepies, and S.E. Carlson, Relationship of Isomeric Fatty Acids in Human Cord Blood to n-3 and n-6 Status, Pediatr. Res. 39: 304A (1996). 7 6 . Dobbins, J., ed., Developing Brain and Behavior: The Role of Lipids in Infant Formula, Academic Press, San Diego, 1997. 77. Ratnayake, W.M.N., R. Hollywood, E. O'Grady, and G. Pelletier, Fatty Acids in Some Common Food Items in Canada, J . Am. Coll. Nutr. 12: 651-660 (1993). 78. Litin, L., and F. Sacks, Trans-Fatty-Acid Content of Common Foods, N. Engl. J . Med. 329: 1969-1970 (1993). 79. U.S . Department of Agriculture/Agricultural Research Service, USDA Nutrient Database for Standard Reference, Release 14. Nutrient Data Laboratory Home Page, http://www .nal.usda.gov/fnic/foodcomp(2001). 80. van Poppel, G., M.-A. van Erp-Baart, T. Leth, E. Gevers, J. Van Amelsvoort, D. Lanzmann-Petithory, A. Kafatos, and A. Aro, Trans Fatty Acids in Foods in Europe. The TRANSFAIR Study, J . Food Comp. Anal. 11: 112-136 (1998). 81. van Erp-Baart, M.-A., C. Couet, C. Cuadrado, A. Kafatos, J. Stanley, and G. van Poppel, Trans Fatty Acids in Bakery Products from 14 European Countries: The TRANSFAIR Study, J . Food Comp. Anal. 11: 161-169 (1998). 82. Aro, A., E. Amaral, H. Kesteloot, A. Rimestad, M. Thamm, and G. van Poppel, Trans Fatty Acids in French Fries, Soups, and Snacks from 14 European Countries: The TRANSFAIR Study, J . Food Comp. Anal. 11: 170-177 (1998). 83. Wolff, R.L., N.A. Combe, F. Destaillats, G. Boue, D. Precht, J. Molkentin, and B. Entressangles, Follow-Up of the A4 to A16 Trans-18:1 Isomer Profile and Content in French Processed Foods Containing Partially Hydrogenated Vegetable Oils During the Period 1995-1999. Analytical and Nutritional Implications,Lipids 35: 815-825 (2000). 84. Innis, S.M., and DJ.King, Trans Fatty Acids in Human Milk Are Inversely Associated with Concentrationsof Essential all-cis n-6 and n-3 Fatty Acids and Determine Trans, but Not n-6 and n-3, Fatty Acids in Plasma Lipids of Breast-Fed Infants, Am. J . Clin. Nutr. 70: 383-390 (1999). 85. Emken, E.A., Physiochemical Properties, Intake, and Metabolism, Am. J . Clin. Nutr. 62: 6598-669s (1995). 86. Hulshof, K.F.A.M., M.A. van Erp-Baart, M.Anttolainen, W. Becker, S.M. Church, C. Couet, E. Hermann-Kunz, H. Kesteloot, T. Leth, 0. Moreiras, J. Moschandreas, L. Pizzoferrato, A.H. Rimestad, H. Thorgeirsdottir, J.M.M. van Amelsvoort, A. Aro, A.G. Kafatos, D. Lanzmann-Petithory, and G. van Poppel, Intake of Fatty Acids in Western Europe with Emphasis on Trans Fatty Acids: The TRANSFAIR Study, Eur. J . Clin. Nutr. 53: 143-157 (1999). 87. Wolff, R.L., Contribution of Trans-18:1 Acids from Dairy Fat to European Diets, J . Am. Oil Chem. SOC.71: 277-283 (1994). 88. Parodi, P.W., and R.J. Dunstan, The Trans Unsaturated Content of Queensland Milk Fat, Aust. J . Dairy Technol. 26: 60-62 (1971). 89. Henninger, M., and F. Ulberth, Trans Fatty Acid Content of Bovine Milk Fat,
Milchwissenschaft 49: 555-558 (1994).
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90. De Man, L., and J.M. De Man, Trans Fatty Acids in Milk Fat, J. Am. Oil Chem. SOC.60: 1095-1098 (1983). 91. Canadian Nutrient File (Electronic Database), Health Canada, www.healthcanada. cdcnf (2001). 92. Wolff, R.L., C.C. Bayard, and R.J. Fabien, Evaluation of Sequential Methods for the Determination of Butterfat Fatty Acid Composition with Emphasis on Trans-18:1Acids. Application to the Study of Seasonal Variations in French Butters, J . Am. Oil Chem. Soc.
72: 1471-1483 (1995). 93. Pfalzgraf, A., M. Timm, and H. Steinhart, Gehalte von trans-Fettsauren in Lebensmit-tein. 2.Eraehrwiss. 33: 2443 (1994). 94. Precht, D., and J. Molkentin, Trans Fatty Acids: Implications for Health, Analytical Methods, Incidence in Edible Fats and Intake, Nahrung 39: 343-374 (1995). 95. Jahries, G., J. Fritsche, and H. Steinhart, Monthly Variations of Milk Composition with Special Regard to Fatty Acids Depending on Season and Farm Management SystemsConventional Versus Ecological, FettlLipid 98: 356359 (1996). 96. Fritsche, J., and H. Steinhart, Contents of Trans Fatty Acids (TFA) in German Foods and Estimation of Daily Intake, FettlLipid 99: 314-318(1997). 97. Precht, D., Variation in Trans Fatty Acids in Milk Fat,Z. Ernahrung. 34: 27-29 (1995). 98. Wolff, R.L., D. Precht, and J. Molkentin, Occurrence and Distribution Profiles of Trans18:l Acids in Edible Fats of Natural Origin, in Trans Fatty Acids in Human Nutrition, edited by J.L. SCbCdio and W.W. Christie, The Oily Press, Dundee, 1998,pp. 1-33. 99. Lake, R., B. Thomson, G. Devane, and P. Scholes, Trans Fatty Acid Content of Selected New Zealand Foods, J . Food Compos. Anal. 9: 365-374 (1996). 100. Ratnayake, W.M.N., J.M. Chardigny, R.L. Wolff, C.C. Bayard, J.L. SBbBdio, and L. Martine, Essential Fatty Acids and Their Trans Geometrical Isomers in Powdered and Liquid Infant Formulas Sold in Canada, J. Pediatr. Gastroenterol. 25:651-660 (1997). 101. Chardigny, J.-M., R.L. Wolff, E. Mager, C.C. Bayard, J.L. SCbBdio, L. Martine, and W.M.N. Ratnayake, Fatty Acid Composition of French Infant Formulas with Emphasis on the Content and Detailed Profile of Trans Fatty Acids, J . Am. Oil Chem. SOC.73: 15951601 (1996). 102. O’Keefe, S.F., V. Wiley, and S . Gaskins, Geometrical Isomers of Essential Fatty Acids in Liquid Infant Formulas, Food Res. Int. 27: 7-13 (1994). 103. Craig-Schmidt, M.C., J.D. Weete, S.A. Faircloth, M.A. Wickwire, and E J . Livant, The Effect of Hydrogenated Fat in the Diet of Nursing Mothers on Lipid Composition and Prostaglandin Content of Human Milk, Am. J . Clin. Nutr. 39: 778-786 (1984). 104. Chen, Z.Y., G. Pelletier, R. Hollywood, and W.M.N. Ratnayake, Trans Fatty Acid Isomers in Canadian Human Milk, Lipids 30: 15-21 (1995). 105. Picciano, M.F., and E.G. Perkins, Identification of the Trans Isomers of Octadecenoic Acid in Human Milk, Lipids 12: 407-408 (1977). 106. Finley, D.A., B. Lonnerdal, K.G. Dewey, and LE. Grivetti, Breast Milk Composition: Fat Content and Fatty Acid Composition in Vegetarians and Non-Vegetarians, Am. J . Clin. Nutr. 41: 787-800 (1985). 107.Koletzko, B., M. Mrotzek, and H J . Bremer, Fatty Acid Composition of Mature Human Milk in Germany, Am. J. Clin. Nurr. 47: 954-959 (1988). 108. Jorgensen, M.H., A. Lassen, and K.F. Michaelsen, Fatty Acid Composition in Danish Infant Formula Compared to Human Milk, Scand. J . NutrJNaringsforskning 39: 50-54
(1995).
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109. Chardigny, J.-M., R.L. Wolff, E. Mager, J.L. Skbkdio, L. Martine, and P. Juankda, Trans Mono- and Polyunsaturated Fatty Acids in Human Milk, Eur. J . Clin. Nutr. 49: 523-531 (1995). 110. Wolff, R.L., Content and Distribution of Trans-18:lAcids in Ruminant Milk and Milk Fats. Their Importance in European Diets and Their Effect on Human Milk, J . Am. Oil Chem. SOC.72: 259-272 (1995). 111. Boatella, J., M. Rafecas, R. Codony, A. Gibert, M. Rivero, R. Tormo, D. Infante, and F. Sanchez-Valverde, Trans Fatty Acid Content in Human Milk in Spain, J . Pediatr. Gastroenterol. Nutr. 16: 432434 (1993). 112 Koletzko, B., I. Thiel, and P.O. Abiodun, Fatty Acid Composition of Mature Human Milk in Nigeria, 2.Ernaehrwiss. 30: 289-297 (1991). 113. Chen, Z.Y., K.Y. Kwan, K.K. Tong, W.M.N. Ratnayake, H.Q. Li, and S.S. Leung, Breast Milk Fatty Acid Composition: A Comparative Study Between Hong Kong and Chongqing Chinese, Lipids 32: 1061-1067 (1997). 114. Laryea, M.D., M. Leichenring, M. Mrotzek, E.O. el Amin, A.O. el Kharib, H.M. Ahmed, and H.J. Bremer, Fatty Acid Composition of the Milk of Well-Nourished Sudanese Women, Int. J . Food Sci. Nutr. 46: 205-214 (1995). 115. Holub, B.J., Hydrogenated Fats and Serum Cholesterol Levels [Letter to the Editor], N . Engl. J . Med. 341: 1396-1397 (1997). 116. United States Department of Agriculture, Agricultural Research Service, Nutrient Data Laboratory Food Composition Data, Special Purpose Table No. 1: Fat and Fatty Acid Content of Selected Foods Containing Trans-Fatty Acids. Prepared by J. Exler, L. Lemar, and J. Smith, http://www.nal.usda.gov/fnic/foodcomp. 117. Departmental Consolidation of the Food and Drugs Act and of the Food and Drug Regulations (with Amendments to January 1,2003), Ottawa. Minister of Public Works and Government Services Canada, 2002. 118. Report of the Nova Scotia Nutrition Survey, Nova Scotia Department of Health; and Health and Welfare Canada, 1993. 119. Ratnayake, W.M.N., and Z.Y. Chen, Trans Fatty Acids in Canadian Breast Milk and Diet, in Development and Processing of Vegetable Oils for Human Nutrition, edited by R. Przybylski and B.E. McDonald, AOCS Press, Champaign, IL, 1995, pp. 2035. 120. Institute of Medicine, Committee on Use of Dietary Reference Intakes in Nutrition Labeling, Guiding Principles for Nutrition Labeling and Fortification, The National Academies Press, Washington, 2003, pp. 99-101. 121. Ratnayake, W.M.N., Overview of Methods for the Determination of trans Fatty Acids by Gas Chromatography, Silver-Ion Thin Layer Chromatography, Silver-Ion Liquid Chromatography, and Gas ChromatographyIMass Spectrometry, J . AOAC International 87: 523-539 (2004). 122. Association of Official Methods of Analytical Chemists, sec. 996.01: Fat (Total, Saturated, Unsaturated, and Monounsaturated) in Cereal Products. Acid Hydrolysis Gas Chromatographic Method. First Action 1996, AOAC Ofsicial Methods of Analysis, 16th edn., AOAC, Arlington, VA, 2000. 123. Association of Official Methods of Analytical Chemists, sec. 996.06: Fat (Total, Saturated, Unsaturated, and Monounsaturated), Hydrolytic Extraction Gas Chromatographic Method, Revised AOAC Official Methods of Analysis, 16th edn., AOAC, Arlington, VA, 2000.
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124. Ali, L.H., G. Angyal, C.M. Weaver, and J.I. Rader, Comparison of Capillary Gas Chromatographic and AOAC Gravimetric Procedures for Total Fat and Distribution of Fatty Acids in Foods, Food Chemistry 58: 149-160 (1997). 125. Food and Consumer Products of Canada, Members Removing trans Fat, 2004, http:// www .fcpc.cdgovernment/healthy/transfat.html. 126. Pepsico Inc., Press Release, Frito-Lay Eliminates Trans Fats From America's Favorite Salty Snacks: Doritos, Tostitos and Cheetos, Plano, TX, 2002. 127. CanWest News Service, Food Giants Shed Trans Fat, Ottawa Citizen, Ottawa, July 7, 2003. 128. Department of Health and Human Services. U S . Food and Drug Administration. Center for Food Safety and Applied Nutrition. Office of Nutritional Products, Labeling and Dietary Supplements. (2004) Questions and Answers about Trans Fat Nutrition Labelling, http://www .cfsan.fda.gov/-dms/qatrans2.html#s3q7. 129. United States Government. Code of Federal Regulations. Food and Drugs, 21 CFR 101, U S . Government Printing Office, Washington, 2002. 130. ANVISA, National Agency of Sanitary Monitoring Brazil, Mercosul Regulamento TBcnico Sobre Rotulagem Nutricional De Alimentos Embalados, RDC No 360, December 23,2003. 131. Food and Agriculture Organization of the United Nations and the World Health Organization. Codex Alimentarius Commission. Report of the Thirty-First Session of the Codex Committee on Food Labelling. Alinorm 03/22A (2003). ftp://ftp.fao.org/codex/ alinormO3/al0322Ae.pdf. 132. Joint FAOIWHO Food Standards Programme. Codex Alimentarius. Codex Guidelines on Nutrition Labelling. CACIGL 2-1985 (rev. 1-1993).Food and Agriculture Organization of the United Nations and World Health Organization, Rome (1997) http://www .fao.org/ docrep/005N2770ely277OeOO.htm. 133. Joint FAOIWHO Food Standards Programme. Codex Alimentarius. Codex Guidelines for Use of Nutrition Claims. CACIGL 23-1997. Food and Agriculture Organization of the United Nations and World Health Organization. Rome (1997). http://www.fao.org/
docrep/005/y2770E/y277OeOO.htm. 134. Council of European Communities. Council Directive 90/496/EEC on Nutrition Labelling for Foodstuffs of 24 September 1990, OfJicial Journal L 276, October 6,1990. 135. European Food Safety Authority, Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a Request from the Commission Related to the Presence of trans Fatty Acids in Foods and the Effect on Human Health of the Consumption of trans Fatty Acids, Request No. EFSA-Q-2003-022,The EFSA Journal 81: 1 4 9 (2004). 136. United Kingdom Food Standards Agency, Trans Fat Labelling, http://www .foodstandards .gov .uk/healthiereating/asktheexpertlfatsoils/translabel(2003). 137. Food Standards Australia New Zealand, Food Standards Code. Part 1.2 Labelling and Other Information Requirements. Standard 1.2.8 Nutrition Information Requirements (2002). 138. South Africa, Minister for Health, Draft Regulations Relating to Labelling and Advertising of Foodstuffs, R 1055/2002. http://www.doh.gov.zddepartment/index.html. 139. Intini, J., Fat Chance: Stressed About Trans Fat? So Are the Food Giants, Macleans Business, MACLEANS.CA, March 29, 2004. http://www.macleans.ca/topstories/business/ article.jsp?content=20040329-78056-78056.
Chapter 2
Safety, Regulatory Aspects, and Public Acceptance of Genetically Modified lipids Ravigadevi Sambanthamurthi, Sharifah Shahrul, and G.K. Ahmad Parveez Malaysia Palm Oil Board (MPOB), Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia
Introduction Oil crops are important sources of energy for both humans and livestock. They also provide essential fatty acids (FA) such as linoleic and a-linolenic acids and lipid-soluble vitamins such as tocopherols, tocotrienols, and carotenoids. Not only are dietary lipids a valuable source of energy, they also make food taste better and act as carriers for fat-soluble vitamins. Lipids are important constituents of cells and cell membranes; they serve as precursors of certain hormones and act as metabolic regulators. In addition to edible purposes, oil crops also provide the raw material for many industrial products. The value and utility of an oil crop whether for nutritional or industrial purposes depend primarily on the FA composition of its oil. Biotechnology offers many opportunities and challenges in the improvement of oil crops. Scientific advances in molecular biology allow the production of designer oils with an altered composition. A genetically modified organism (GMO) is one that has had its DNA modified by genetic engineering. The word “transgenic” is also often used to describe such an altered organism. A wide range of oil crops has been modified genetically, either to increase the amount or to alter the types of oils they produce; oils with different degrees of “saturation” have different properties. For example, genetically enhanced soybeans that produce oils that are lower in polyunsaturated fats and higher in oleic acid offer better frying stability without further processing (1,2). Some research aims to move the genes that code for valuable oils (e .g., y-linolenic acid) from plants that naturally produce them in small amounts (e.g., borage, or evening primrose) to “oil-factory” plants such as oilseed rape that have the “machinery” to produce oil in vast amounts. The vast majority of vegetable oils is currently used for edible commodities, such as margarines, cooking oils, and processed foods. Only -15% is used for oleochemicals, i.e., industrial products derived from oil crops. The challenge for researchers in the coming years will be to produce greater amounts of higher-yielding oil crops to satisfy increased demands and to increase the spectrum of useful products, whether for edible or industrial use, that can be derived from these crops. Plant breeders have traditionally bred crops with different oils for specific purposes. Breeding, however, is very time-consuming, taking several years to develop a new 33
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variety. Advances in biochemistry and plant genetics revealed the biochemical pathways by which plants make oils and the influence that different FA have on the oil characteristics. Genetic modification now makes it possible to improve the composition and properties of oils from different plants far more quickly and precisely than with traditional breeding. The first genetically modified vegetable oil from canola has a high proportion of lauric acid and is desirable for many food and nonfood applications. It is a critical ingredient in soaps, shampoos, and detergents and is also used in confectionery, icings, crackers, and coffee whiteners. The oil was produced by genetically modifying canola, a variety of oilseed rape. A gene from the California bay laurel tree, which codes for an enzyme involved in the synthesis of lauric acid, was introduced (3-5). It is now used commercially in the United States (6). More recently, a new soybean variety with a less saturated and more heat-stable oil was developed (1).Ordinary soybean oil is often hydrogenated to increase its saturation for use in baked goods and for frying. This process lowers the levels of unsaturated acids. The GM soybean oil obviates the need for hydrogenation and also has a healthier FA composition. These beans are expected to have agronomic yields comparable to those of existing varieties. Biotechnology is a very rapidly moving field, with today’s technical advances becoming tomorrow’s genetically modified products. GM crops, including those modified for oil composition and content, have become an increasingly important economic and fractious world-trade issue. James (6) estimated that 58.7 million hectares of land in the world were under GM crops in 2002. The most dominant crop grown that year was herbicide-tolerant soybeans covering an area of 36.5 million hectares, i.e., 62% of the total area of GM planting. In the case of canola, herbicide-tolerant varieties covered 3.0 million hectares, accounting for 5% of the global transgenic area, mainly in Canada and the United States. A total of 14 countries had GM plantings in 2002, with the United States leading in area, followed by Argentina, Canada, and China. The GM issue has spawned considerable controversy; it is therefore important that the technical advances be backed by appropriate safety and regulatory measures, taking cognizance of public opinion.
Safety Issues Expanding populations, limited land and labor resources, and the promise of better and sustainable yields have all made GMO an attractive option. However, there has been growing concern over their safety. Concerns over GMO were first raised at the Asilomar Conference in 1975 with a call for measures to prevent the accidental release and proliferation of GM bacteria outside the laboratory (7). This alarm was sounded soon after the technique of gene splicing or recombinant DNA was developed at Stanford University (Stanford, CA). “Biosafety” is a term used to describe the policies and procedures adopted to ensure the safe application of modern biotechnology. In relation to biosafety, two important questions must be addressed concerning GMO. The first question is: “Are they safe to be eaten?’ and the second: “Are they safe for the environment?’
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Risk Assessment. An important component of biosafety is risk assessment, the analysis and prediction of risks. Risk assessment is the use of scientific data to estimate the effects of exposure to hazardous materials or conditions. When examining the proposals for release of GMO on an experimental level, risk assessment is required. Risk management is a different activity. It is the process of weighing alternatives to select the most appropriate strategy or action. The first part of risk assessment is risk identification, followed by risk estimation (8). Only after the results are known can the wider release of GMO be considered against other alternatives, the process of risk management. Benefits are a part of risk management but not risk assessment. The key to the issue of risk management is the quality of the scientific data. The molecular genetic characterization of the transgenic organism must be well documented. This includes information on the transformation system, heritability/ transgene stability, DNA insert and protein (translation of the gene) and RNA (expression). A combination of analytical techniques should be used in characterizing the transgenic plants, and a checklist of each of the techniques used is highly recommended. Food Safety. There is no clear evidence that genetic modification, including the modification of lipids, is hazardous to health. However, this does not mean that individual products will never be unhealthy. It means that GMO should be approached on a case-by-case basis. This opinion is supported in the report of the Joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Consultation on foods derived from biotechnology (9), which acknowledged that a safety assessment of GM foods should be conducted on a case-by-case basis. It was agreed in various scientific forums that inquiry into the safety of crops derived from biotechnology should focus on the crops themselves and not on the process used to produce them. The second area of broad consensus was that when a crop derived from biotechnology was demonstrated by sound scientific techniques to be substantially equivalent to the existing nonmodified crops, then the modified crop and products derived from it should be treated as if they were no different from the untransfomed crop and its derivatives. Both of these principles were entrenched in the 1992 U S . Food and Drug Administration (FDA) regulations. In the United States, for the most part, the decision on the safety of human consumable products follows the 1992 FDA Policy. For example, the safety of enzymes used in food processing is determined by investigating their dietary and genetic toxicity, chromosome aberration, eye and dermal irritation, and the effect of intraperitoneal injection. Strategies for assessing the safety of foods produced by genetic engineering were developed by the FAO)/WHO in 1991 and by the Organization for Economic Cooperation and Development (OECD) in 1992. The general strategy for assessment of GM foods is as follows: (i) to obtain and assess information on the characteristics of the genetic modification, including the function and properties of newly inserted genes; (ii) to assess the safety and nutritional
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properties of newly expressed substances in the food; (iii) to identify and evaluate unexpected changes in the composition of the modified product due to insertion of novel genes or suppression of constituent genes; (iv) to evaluate the influence of food processing on the toxicological properties of the new food; and (v) to evaluate foodconsumption patterns of the modified product compared with its conventional counterpart (9). This is scientifically sound and provides a useful historical baseline for judging safety. Regulations in most countries, including the UK, include some variation of substantial equivalence determinations. This concept was recognized by the OECD in 1993, further developed by the FAONHO Consultation in 1996, and recently reaffirmed by both, with particular reference to foods produced by modem biotechnology . Substantial equivalence involves a thorough analysis to demonstrate that the specific GM food product possesses levels and variations in critical nutrients and toxicants similar to the parental plant variety and other conventional varieties of that crop. The presence of novel DNA or protein does not preclude a GM food from being considered substantially equivalent to a conventional food in the United States. Any defined differences are the subject of additional safety assessments, which may include nutritional, toxicological, and immunological testing as appropriate. It may be necessary in certain instances to undertake feeding studies in animals, but practical difficulties are encountered in evaluating the safety of whole foods in conventional toxicology studies. Nevertheless, this targeted approach has been questioned with respect to its ability to detect and evaluate the effect of unintended effects, such as the acquisition of new traits or the loss of existing traits. As the complexity of GM crops increases, profiling techniques, i.e ., DNA microarrays, mRNA profiling techniques, proteomics, and chemical fingerprinting, may be valuable in increasing the probability of detecting unintended effects. The safety considerations for GM food have been considered in many other countries and by international organizations. Health Ministers of Australia and New Zealand approved the sale of foods containing specific varieties of GM soybeans, with an altered balance of oils, after an assessment by the Food Standards Australia New Zealand. This type of soybean was first produced in the United States in 1996. It contains increased amounts of oleic acid, making it a versatile cooking oil. Many GM foods are now being marketed in other countries such as the United States and Canada, following approval by their regulatory authorities. However, these products still must be assessed for safety under the European Union (EU) regulatory framework before they can be marketed in Europe. After such approvals are given, the World Trade Organization (WTO) Sanitary and Phytosanitq Agreement rules prevent countries from taking action to restrict the import of such products into their markets unless evidence of harm subsequently comes to light. The Codex Alimentarius Commission (CAC) adopted a landmark agreement on 9 July 2003 that provided standards for risk analysis and safety guidelines for assess-
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ing both direct and indirect risks of foods derived from biotechnology (10). The guidelines are applicable to premarket safety evaluations, product tracing for recall purposes, and postmarket monitoring. They do not, however, address environmental, ethical, or socioeconomic issues. The guidelines recognize the concept of substantial equivalence. The adoption of these guidelines by the CAC has important implications because the WTO refers to the Codex in the settlement of disputes. Food allergies affect 1-2% of the population (1 1). Virtually all food allergens are proteins. Genetic engineering is capable of introducing allergens into recipient plants (12), but the overall risks of introducing an allergen into the food supply are believed to be similar to those associated with conventional breeding methods. If a GM food contains the product of a gene from a source with known allergenic effects, the gene product should be assumed to be allergenic unless proven otherwise. There are no known cases of allergic reactions caused by marketed foods derived from GM plants. Nordlee et al. (12) showed that 2 s albumin, which is the main allergen in Brazil nuts, can be transferred to soybeans. In that study, a particular variety of soybean engineered with the sulfur-rich protein of Brazil nut by Pioneer Hi-Bred would have had to be labeled under U S . FDA policy. However, the company abandoned development of that variety (13). A decision tree analysis is used to screen proteins from GM foods for possible allergenicity (14). Assessment is normally accomplished by evaluating the source of the gene, the sequence homology of the newly introduced protein to known allergens, the immunochemical reactivity of the newly introduced protein with immunoglobulin E antibodies from the serum of individuals with known allergies to the source from which the genetic material was obtained (if applicable), and the physicochemical properties of the newly introduced protein (effect of pH and/or digestion, heat or processing stability). Genetic engineering can lessen allergenic reactions. Shiseido markets rice without globulin as a health or functional food (15). This provides an alternative for -70% of the people who have allergies. Thus, the future potential of genetic engineering to produce more nutritious and safer food by producing new varieties of crops excluding the naturally occurring toxins and carcinogens that we consume in food should not be overlooked. Extensive safety tests were conducted on several transgenic food products. For example, studies on 20 lines of six different crops of glyphosate-tolerant soybeans showed no adverse health effects. Virus-resistant squash as well as the Flavr Savr (delayed-ripening) tomatoes produced by Calgene had no undesirable health effects even when given in large quantities to rats (16). There has also been concern that because vectors used in cloning often carry antibiotic resistance markers, the resulting GMO may cause antibiotic resistance. This led to the rejection of transgenic maize, modified for resistance to the European corn borer and marketed by Ciba-Geigy, because it carried the ampicillin-resistance gene (17). Of the EU countries, only France supported its introduction. Technically, unanimous disapproval is needed to block a product, but this raises questions concerning interna-
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tional vs. national regulation. Studies in mice and rats of the protein product of the neomycin-resistance marker gene showed it to be safe for consumption (18). A general review of the issues can be found in the Advisory Committee on Novel Foods and Process (19). The Food Linked Agro-Industrial Research (FLAIR) Programme was conducted in the EU from 1990 to 1993 to simulate research in food, including food safety and hazard analysis. Plants engineered with the Bacillus thuringiensis (Bt) gene are capable of producing their own insecticide, thus reducing the need for application of pesticides. In examining its toxicity, the Bt gene protein in concentrations up to 4000 times the maximum likely to be ingested had no harmful effects on the growth of mice. This product is available in >400 products in the United States, and there have been very few instances of harm noted (20). A major concern that is expressed regularly in public surveys and the media is a perception that GM crops are unnatural and insufficiently tested. People want to know whether genetic modification is safe or whether it poses an unacceptable risk. The first joint FAONHO consultation was held in 1990, and one of the fundamental conclusions made by the committee was that “the use of these techniques does not result in food which is inherently less safe than that produced by conventional ones .” GMO foods produced to date have had no overwhelming benefits to the consumer. Attitudes may change when the next wave of GM foods, which have been engineered for consumer-friendly traits such as vitamin content and DuPont’s high-oleic soybean (now awaiting regulatory approval in Europe), reach the market. Producers will gladly label these GMO because their value depends on it.
Environmental Safety. To date, objections to GMO are mainly of two kinds, i.e., the claim that GMO are bad for the environment and the notion that they are bad for health. If GMO are detrimental to the environment, it is likely to be in the same way as normal agriculture. Many GMO require less application of pesticides and herbicides, which should confer significant environmental benefits ( 2I). The rather more insidious environmental fear is contamination of the natural gene pools. GM crops may hybridize with the wild individuals, for example, GM rape with Brassica, leading to a host of problems. Uncontrollable superweeds may be produced with adverse effects on wildlife and biodiversity. It must be appreciated, however, that all man-made interference with the environment, including conventional agriculture, forestry, and urban development, has major effects on biodiversity. Genetic modification takes its place as part of a very long list of such interventions. Thus, the potential environmental risks of GM crops must be framed in the context of current use of conventional technologies. Since 1987, >25,000 field trials of GM plants were conducted in 45 countries with no adverse effects. More than 200 million acres of land were planted with GM crops with no adverse effects (22,23). Nonetheless, as a precaution against possible environmental damage through the introduction of GMO, extensive regulations exist in
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many countries to control what genetic modifications can be made to plants that are to be grown in fields, and how and where they are to be grown. Regulations may be modified as experience demonstrates the safety of particular crops, or if novel risks may be posed by new applications of technology. Regulations in most countries distinguish between experiments in “contained” conditions (i.e., in the laboratory, or enclosed environment) and those in which the GMO is “released to the environment” (i.e., for commercial use in industry or agriculture). In Europe, a BRIDGE (Biotechnology Research for Innovation, Development, and Growth in Europe) program study in five countries and including academic and industry researchers developed materials to allow improved monitoring of environmental safety (24). The study showed that greenhouse tests were useful predictors of environmental behavior but could not predict everything. The relative weediness or fitness of the modified plants did not differ significantly from the corresponding nonmodified plants. Specifically, they found that potato did not transfer genes to weedy relative species, but sugar beet could transfer genes to wild beet species. Oilseed rape did transfer genes to Brassica rapa, but special circumstances were required to transfer to three other weedy species they tested. The rates of transfer decrease rapidly with distance. However, the problem is that weeds often invade crop fields so that distance may not be a major constraint. Pollen is the most likely method of transfer of genes. By the use of traits such as male sterility, it is possible to avoid the risk of pollen transfer. The relevance of the environmental data obtained from small field trials to large-scale sowing on several million acres of land has been questioned. However, as mentioned earlier, 200 million acres of land have been planted with transgenic crops worldwide with no adverse environmental consequences (22,23), A major point of concern is the effect crops engineered to produce pesticides would have on nontarget organisms such as beneficial insects and insects of aesthetic value. The report on the effect of ingestion of pollens from Bt corn by Monarch butterflies reared in the laboratory was highly publicized (25). Bt is the gene for the endotoxin produced by the bacteria, B . thuringiensis,which kills the larvae of certain insects when ingested. There is also concern that the target insects may develop resistance to the Bt endotoxin, with devastating consequences. To delay this process, attempts were made to introduce more than one form of the Bt gene into a crop plant (26); the rationale is that it would take a longer time to develop resistance to both types of endotoxin. The long-term ecological risks and evolutionary consequences of wide-scale release of GMO in the field remain largely unknown. Ecological risk assessment programs are ongoing in most developed countries. The primary concern is whether the GM crops pose any novel threats to the environment in which they are placed. Will modified crops escape cultivation and exhibit “weedy” characteristics as a result of their transformation? For most crops, the likelihood of such an escape is generally believed to be low. Many modified crops such at Bt corn, or herbicidetolerant soybeans can be viewed as isogenic variants of traditionally improved
40
R. Sarnbantharnurthi e t a / .
varieties. Presumably, the performance trials run by the developer can identify any varieties that exhibit phenotypes deemed undesirable or threatening, which, of course, would preclude their release. Crop varieties that are closer to their wild relatives, however, such as the Brassicas or Oryza, may be of more concern. In most cases, field tests involving the time and scale necessary to address our concerns have not and may not be performed. Ecologists and growers alike will have to watch and wait for results of the “experiments” that fast-track deregulation in the United States has inadvertently created.
lnterna tional Regulations In enacting regulations to deal with the issues of GMO, it is important that they be anticipatory and objective rather than reactive and subjective. Since the 1970s, most of the countries that are carrying out GM research have developed their own guidelines and regulations. Because knowledge of the application of genetics is increasing, and the safety record continues to lengthen, guidelines are continually being refined. Most countries have regulations that address application of the research for particular uses, in relation to environmental safety, health and food safety, and ethical aspects. The year 2000 marked a turning point in global biosafety regulation. More than 130 countries adopted the Cartagena Protocol on Biosafety, the first international law to regulate genetic engineering. The Protocol was negotiated under the United Nations Convention on Biological Diversity drawn up at the Earth Summit in Rio de Janeiro in 1992. The protocol provides a framework that makes trade in GMO (mainly crops) possible and is also sensitive to justified concerns about safety and the environment; it addresses the wider political and socioeconomic implications of corporate-driven science and technology. To date, 77 countries, the majority of which are developing countries, have ratified the protocol. The negotiations, which spanned 5 yr, were difficult and divisive. Most developing countries have no biosafety laws or regulations and lack the technological and financial resources to regulate GMO. As public rejection of GMO in Europe and other parts of the world gathered momentum, the fear of becoming dumping grounds for untested and rejected GMO was real. The developing countries, including Asian, some Latin American, and the African countries (the G77), favored a restrictive protocol based on the precautionary principle, i.e., GMO are guilty until proven innocent, and ample testing for risks to human health and to the environment is necessary before the release or commercialization of GMO. The G77 also advocated that transboundary movement of GMO must be practiced on the principle of AIA (Advanced Informed Agreement). According to this principle, before a GMO leaves a country, the receiving country must be informed and agree to its arrival. The nature of the GMO and the producer must also be made known. The G77 called for labeling of all GMO including deregulated GMO. Argentina (currently the second-largest producer of transgenic crops after the United States), Australia,
Genetically Modified Lipids
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Canada, and Mexico (the Miami group) echoed the American view that a protocol that is too tight would restrict international trade. America (part of the Miami group), which is not a party to the Convention on Biological Diversity and hence cannot adopt the Cartagena protocol, obviously has a large influence. The EU, although it called for labeling, was noncommittal at the meeting on the issue of AIA. The final protocol is a compromise. It covers living modified organisms (LMO) and not GMO, i.e., products of LMO were not included. Thus, GM lipids would not be covered by the protocol because they are nonliving products. The principle of AIA was adopted for LMO. The developing countries also called for the inclusion of mechanisms for liability and redress for environmental damage caused by the release of LMO. This issue of liability and redress will be taken up at a later date. Strengthening and rectifying deficiencies in the protocol should be long-term goals. Public Acceptance of GM Lipids Attitude. We are all “public consumers” of biotechnology in one form or another. However, our attitudes toward biotechnology and how it should be used vary widely. These depend on the relative importance we attach to different factors, and our individual beliefs, The GMO debate has become very divided, mainly because of very strong sentiments against GMO in Europe. The concept of substantial equivalence has moved away from the earlier scientific consensus into a demand that the consumer has a right to know and choose. The sound scientific principles originally envisaged for regulatory acceptance are now challenged by consumer activist policy agendas, which are a critical component of public acceptance. Although the opposition is greatest in Europe, it is also true for some ASEAN (Association of South East Asia Nations) countries. In Britain, the anti-GMO sentiment is due mainly to a lack of public trust of regulatory procedures largely because of the government handling of mad cow disease. The cover-ups that characterized the episode made people unwilling to accept any official reassurance on food safety. As a corollary, they are easily swayed against GMO even when extremely thin scientific evidence is presented. Media coverage, which is often inflammatory and contradictory, can confuse the public about the actual facts. A recent scare over the effects of GM potatoes on experimental animals is one such example (27,28). The controversy highlights the need to test in a controlled manner and to draw proper conclusions. In the case of ASEAN and South America, there is the fear that the poorer nations may become “dumping grounds” for GMO. The understanding of public perceptions and consumer attitudes toward genetic modification is important and should be respected. Objective and transparent research on genetic modification offers consumers tools for making decisions about which food product to choose. GM foods are perceived as having brought little direct benefit to consumers. If the public could see or experience tangible benefits in food, attitudes would be more accepting (29-32).
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R. Sambanthamurthi e t a / .
Public Education. Scientific advances related to GM technology are now so rapid and sophisticated that there is a real danger that they will move ahead of society’s understanding. Proponents of biotechnology have failed to inform the public sufficiently about the technology and convince consumers of the benefits. Opponents of biotechnology have raised fears and concerns that have no scientific basis and have not given consideration to the advantages that society might enjoy. In general, governments have failed to articulate a consistent and principled framework for public discussion. The scientific community has a social responsibility to communicate developments to the public. Various strategies have thus been formulated to study public opinion scientifically. The first type is the use of fixed-response questions, i.e., to choose from set answers. These include surveys in the United States and Canada, and the Eurobarometer in all 12 countries of the European Community (8,31,32). There have been several studies in Japan including one among different groups in society, the public, academics, and high school teachers (33). In Australia, Hong Kong, India, Israel, Japan, New Zealand, The Philippines, Russia, Singapore, and Thailand, GMO are viewed positively (33). Less than 10% in all countries saw it as doing more harm than good. In all surveys, plant-plant gene transfers were the most acceptable followed by animal-animal transfers. Animal-plant and humananimal gene transfers were least acceptable (33). In the United States, a recent survey conducted by the University of Arkansas reported that if people see the words “genetically modified” on a food label, they are more likely to buy it if they feel informed about such products (34). A similar survey in three Asian countries indicated that a majority of consumers in Asia recognize that their daily diet almost certainly contains GM food products and they take no action to avoid them. These surveys demonstrate that, in general, the general public remains open-minded and wishes to leam more about genetic modification and its benefits. The problem is that consumers often feel ill-informed about GM food products. This type of survey indicates that companies that produce GM foods and educators such as those at extension services can and should be educating people about these products. With education, public acceptance will no longer be a major issue. Consumer Choice. Some consumers feel that proliferation of GM foods will reduce choice. In the UK, for example, this has led many supermarkets to remove GM products from their shelves. Interestingly,this action has actually deprived UK consumers who may wish to choose GM foods of their right to exercise that choice. For a variety of reasons, some people do not wish to eat foods containing GM material. To be able to make this choice, clear labeling is required. In the EU, the agenda is driven by a consumer’s right-to-know perspective, i.e., the public has a basic right to know the important facts about a food commodity before making a purchasing decision.
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Former U S . President Jimmy Carter endorsed the use of biotechnology as a means of achieving global food security and criticized extremist groups in affluent countries who oppose the use of biotechnology. The public acceptance of GMO crops is related to perception. Anti-GMO sentiments are greatest in developed countries in which there is a food surplus and widespread hunger is not an issue. The situation is very different in developing countries (35). Sahai, Governor of the Gene Campaign in New Delhi, India, concurred with Carter’s views and expressed the opinion that ethical concerns about biotechnology-derived foods and the question of choice are a luxury that only industrialized nations can afford (36). He strongly urged developing countries such as India to exploit biotechnology to address the urgent issue of increasing food production to overcome current suffering from malnutrition, hunger, and starvation, He emphasized that biotechnology should be implemented with high safety standards and that the concerns and debates in each society must be specifically relevant to that society and rooted in its needs and its culture.
Labeling. Applying labels to novel foods seems to be an easy way to balance the opposing wishes of producers and consumers. The reality is more complex. Labeling is neither an easy nor a cheap solution. At present, few processed foods are 100% GMO-free. Testing for GMO is required at every step from the field to the processing plant and can add up to 30% of the cost of the final product (37). The most fundamental problem in labeling GM products relates to the detection of DNA because the measurement of GM material becomes difficult or impossible once the GM product is highly processed. For example, products such as GM oil will not contain any evident GM proteins or DNA. Labeling is quite straightforward for products such as GM tomato puree, but less so for commodity crops such as soybeans because beans from many different sources are usually mixed and processed together. To complicate matters, the regulations on labeling vary around the world. In the United States, regulations do not require mandatory labeling and segregation of GM crops and products. The European Regulations on Novel Foods and Novel Food Ingredients, which came into force in May 1997, indicated that foods must be labeled if they are “no longer equivalent” to those already in existence (38). Foods containing GM soybeans and corn were not covered because they had been approved before this ruling came into force. However, the European Commission recently modified these rules such that all products containing, or possibly containing, GM ingredients must be labeled. This ruling applies retrospectively to include the’approved soybeans and corn. “No longer equivalent” foods would include a food or food ingredient: that differs from its equivalent item such that its composition, nutritional value, or intended use has been changed; that may pose risks to certain sections of the population, for example, those suffering from food allergies; for which there may be ethical concerns;
R. Sambanthamurthi et a/.
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in which GMO are known to be, or may be, present. Voluntary guidelines were also drawn up by food manufacturers and retailers to help implement the European regulations. The European Novel Foods and Food Ingredients regulations require labeling of GM foods if >0.9% of an ingredient is derived from GMO, and this can be detected in the food. If the ingredients are processed from a GM crop, but are no different from ingredients from a conventional crop, they do not have to be labeled. Examples of such ingredients are soybean oil and soybean starch from Roundup ReadyTMsoybeans; because these products are processed and purified, they do not contain either the herbicide-tolerance gene or its protein product. The composition of the oil is identical. However, if DNA or protein from a GM plant is present at a defined threshold level, the food containing that ingredient must be labeled. The threshold level may change as ever more sensitive tests are developed. Regulations will change as knowledge of GM increases, Undoubtedly, the EU is taking the lead to push for more stringent GM foodlabeling regulations for its member countries. The European Parliament of the European Commission recently approved two new proposals for regulating GMO labeling. Under the new proposals, the previously exempted GM foods such as vegetable oils and GM feed will be subject to a new labeling requirement (39). Many people, including food producers, have opposed the use of “may contain” on food labels, saying that it is not believed to be helpful to consumers. Quite apart from what is required by law, many manufacturers and retailers are providing leaflets on genetic modification and, where practicable, information on the products themselves, either on shelves or product labels. However, there is a limit to the amount of information that can be usefully provided for consumers to read concerning ingredients or method of production, i.e., the information has to be straightforward and significant. The labeling of GM products requires segregation of the crop during growing and processing. Producers of commodity crops opposed segregating conventional crops from GM crops, claiming that it would not be economical to do so. However, if consumers are willing to pay the higher price, markets for segregated crops will undoubtedly develop.
Conclusions Biotechnology presents different challenges and offers new opportunities for the 21st century. The many important transgenic crops approved to date provide credibility for biotechnology , not only with the international scientific community but also with the lay public. Rigorous standards will continually be applied to judge biotechnology’s contribution, on the merit of the science, its socioeconomic effect, and its ability to contribute to more sustainable agricultural systems, in which productivity is enhanced and maintained and the environment not put at risk. The pub-
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lic and private sectors will be challenged to work together to implement appropriate new strategies and contribute new knowledge to the global scientific community on this important development. It must be recognized that the needs and priorities of industrial and developing nations are completely different; this calls for the development of need-based and region-specific biotechnology initiatives. Equitable partnerships can be fostered on the basis of national priorities and strengths, for example, the transfer of finished biotechnology products from developed countries and utilization of the wealth of biodiversity of developing nations resulting in mutual advantage.
Acknowledgments The authors thank the Director-General of MPOB, Tan Sri Datuk Dr. Yusof Basiron, for permission to contribute this chapter. The editorial comments of Mr. Andrew Chang are gratefully acknowledged.
References 1. Hitz, W.D., N.S. Yadav, R.S. Reither, C.J. Mauvais, and A.J. Kinney, Reducing Polyunsaturation in Oils of Transgenic Canola and Soybean, in Plant Lipid Metabolism, edited by J.-C. Kader and P. Mazliak, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1995, pp. 506508. 2. Kinney, A.J., Development of Genetically Engineered Oilseeds: From Molecular Biology to Agronomics, in Physiology, Biochemistry and Molecular Biology of Plant Lipids, edited by J.P. Williams, M.U. Khan, and N.W. Lem, Kluwer Academic Press, Dordrecht, The Netherlands, 1997, pp. 298-301. 3. Davies, H.M., L. Anderson, C. Fan, and D.J. Hawkins, Developmental, Induction, Purification and Further Characterization of 12:O-ACP Thioesterase from Immature Cotyledons of Umbellularia californica, Arch. Biochem. Biophys. 290: 31-45 (1991). 4.Voelker, T.A., A.C. Worrell, L. Anderson, J. Bleibaum, C. Fan, D.J. Hawkins, S.E. Radke, and H.M. Davies, Fatty Acid Biosynthesis Redirected to Medium Chains in Transgenic Oilseed Plants, Science 257: 71-74 (1992). 5. Murphy, D.J., Engineering Oil Production in Rapeseed and Other Oil Crops, Trends Biotechnol. 14: 206-2 13 ( 1996). 6 . James, C . (2003). Global Status of GM Crops in 2002. Available at http://www. Isaaa.Org/Kc/Bin/Gstatus/Briefs .Htm. 7. Berg, P., D. Baltimore, S. Brenner, R.0-111. Roblin, and M.F. Singer, Summary Statement of the Asilomar Conference on Recombinant DNA Molecules, Proc. Natl. Acad. Sci. USA 72: 1981-1984 (1975). 8. OTA, U.S. Congress Office of Technology Assessment, New Developments in Biotechnology, 3: Field Testing Engineered Organism, Genetic and Ecological Issues, Washington, U S . G.P.O., OTA-BA-350, May 1988. 9. Food and Agricultural OrganizationKVorld Health Organization (FAO/WHO), Safety Aspects of Genetically Modified Foods of Plant Origin. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology, WHO, Geneva, 29 May-2 June (2000).
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10. Haslberger, A.G., Codex Guidelines for GM Foods Include the Analysis of Unintended Effects, Nature Biotechnol. 21: 739-741 (2003). 11. Anderson, J.A., Allergic Reactions to Food, Crit. Rev. Food Sci. Nutr. 36 (Suppl.): S19-S38 (1996). 12. Nordlee, J.A., S.L. Taylor, J.A. Townsend, L.A. Thomas, and R.K. Bush, Identification of a Brazil-Nut Allergen in Transgenic Soybeans, N . Engl. J. Med. 334: 688-692 (1996). 13. Nestle, M., Allergies to Transgenic Foods-Questions of Policy, N. Engl. J. Med. 334: 726-727 (1996). 14. Metcalfe, D.D., J.D. Astwood, and R. Townsend, Assessment of the Allergenic Potential of Foods Derived from Genetically Engineered Crop Plants, Crit. Rev. Food Sci. Nutr. 36 (Suppl.): ,51654186 (1996). 15. Heissler, A,, and P. Commandeur, The Japanese Biotechnology Industry, Biotechnol. Dev. Monitor 22: 5-6 (1995) 16. Organization for Economic Cooperation and Development, Food Safety Evaluation, OECD, Paris, 1996. 17. Coghlan, A,, Engineered Maize Sticks in Europe’s Throat, New Sci. (6 July 1996) 18. Fuchs, R.L., Safety Assessment of the Neomycin Phosphtransferase I1 (NPTII) Protein, Biotechnology 11: 2543-1547 (1993). 19. ACNFP, Advisory Committee on Novel Foods and Process, Report on the Use of Antibiotic Resistance Markers in Genetically Modified Food Organisms, ACNFP, London, 1994. 20. UNESCO (1997). International Bioethics Committee Report, Food, Plant Biotechnology and Ethics. Available at http://www .biol.tsukuba.ac.jp/-macedfood. 21. Briggs, S.P., and M. Koziel, Engineering New Plant Strains for Commercial Markets, Curr. Opin. Biotechnol. 9: 233-235 (1998). 22. James, C. (1999) Global Review of Commercialized Transgenic Crops. Available at http://www.isaaa.org/Global%20Review%201999/briefs12cj.htm. Accessed: 11-72000. 23. Comer, A.J., T.R. Glare, and J.-P. Nap, The Release of Genetically Modified Crops into the Environment, Plant J . 33: 19-46 (2003). 24. Rudelsheim, P., Safety Assessment of Deliberate Field Use of Genetically Modified Plants. Report of the European Commission BRIDGE Program 1992-1994,1994. 25. Losey, J.E., L.S. Raynor, and M.E. Carter, Transgenic Pollen Harms Monarch Larvae, Nature 399: 214 (1999). 26. Moellenbeck, D.J., Insecticidal Proteins from Bacillus thuringiensis Protect Corn from Corn Rootworms, Nut. Biotechnol. 19: 668-672 (2001). 27. Ghosh, P. (1999). Food Scare Is “Bad Science,” NationalmK. Available at www.sepp. orga/reality/foodscare.html. 28. GM Debate Refuses to Go Away, BBC (UK) Online, June 1 (1999). Available at news8 .thdo.bbc.co.uklhi/EnglishluWnewsid~357000/357987 stm. 29. Gaskell, G., M.W. Bauer, J. Durant, and N.C. Allum, Worlds Apart? The Reception of Genetically Modified Foods in Europe and the US, Science 285: 384-387 (1999). 30. Gaskell, G., N. Allum, and M. Bauer, Biotechnology and the European Public, Nut. Biotechnol. 18: 935-938 (2000).
31. Eurobarometer Survey 39.1 Biotechnology and Genetic Engineering; What Europeans Think About It in 1993. Brussels Commission of the European Communities (1993).
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32. Eurobarometer 46.1:Europeans and Biotechnology, A Complex Relation (2000). Available at http://europa.eu.int/comm/research/press/l997/pr180997.html. Accessed:
10-13. 33. Macer, D., Perception of Risks and Benefits of In Vitro Fertilization, Genetic Engineering and Biotechnology, Soc. Sci. Med. 38: 23-33 (1994). 34.Newswise (2004).Information Gap Influences Consumer Attitudes about Genetically Modified Foods. Available at http://www .newswise.com/p/articles/view/502949/. 35. Carter, J., Forestalling Famine with Biotechnology, Washington Times, 1 1 July, Washington, D.C. (1997). 36.Sahai, S., The Bogus Debate on Bioethics, Biotechnol. Dev. Monitor 30: 24 (1997). 37. The Pitfalls of Food Labeling-Sticky Labels, in The Economist, May 1, 1999,pp. 81-82. 38.Regulation (EC) No. 258197 of the European Parliament and the Council, Off, J . Eur. Communities LA3: 1-7 (1997). 39. Chern, W.S., K. Rickertsen, N. Tsuboi, and T.-T. Fu, Consumer Acceptance and Willingness to Pay for Genetically Modified Vegetable Oil and Salmon: A MultipleCountry Assessment, AgBioForum 5: 105-1 12 (2002).
Chapter 3
Production, Processing, and Refining of Oils Ernest0 Hernandez Texas A&M University, College Station, TX 77843
Introduction The production and consumption of major vegetable oils increased from 60 million tons (MMT) in 1991 to more than 90 million in 2003. This is due mainly to an increasing demand for vegetable protein for animal and human use. The major vegetable oils in the world are soybean, palm, rapeseed, sunflower, peanut, cottonseed, palm kernel, and coconut. Table 3.1 shows the outputs for these oils for the last 10 yr. Soybean oil is the largest in volume with more than 26 MMT. Palm oil is the second largest in volume with 24 MMT/yr produced; together they account for >55% of the total vegetable oil produced in the world. Animal fats are also an important source of fats and oils for edible and industrial purposes; their world production for 2000-2001 was 14 MMT. The United States, Brazil, Argentina, and China account for >70% of the world’s output. Soybeans are the largest and fastest growing oilseed crop worldwide with a production of 172 MMT. This represents >55% of the total oilseed produced and translates into world production of >26 MMT of soybean oil. The production of sunflower oil has decreased due to a shift toward corn, wheat, and soybean crops in the United States, Argentina, Brazil, and the countries TABLE 3.1 World Production of Fats and Oilsa 1991-1 992
1996-1 997
2001-2002
(t x 106) Soybean Palm Rapeseed Sunflower Peanut Cottonseed Coconut Palm kernel Tallow
17.472 13.006 8.393 7.328 3.597 3.644 3.095 1.741 7.51 1
20.369 17.590 10.863 8.561 4.496 3.875 3.583 2.314 8.1 72
aSource: USDA-National Agricultural Statistics 2003.
48
28.867 25.41 8 12.677 7.61 4 4.887 3.815 3.233 3.1 19 8.312
Production, Processing, and Refining of Oils
49
of the former USSR. World production of rapeseed has also suffered a decrease by >4 MMT in 2001 from the previous year as a consequence of reduced planting and/or poor weather conditions in Canada, eastern and western Europe, and China. World production of cottonseed has remained relatively stable; however, production of cottonseed oils has decreased somewhat due to the increasing practice of feeding seed directly to cattle. Production of palm, peanut, coconut, and palm kernel oils has increased steadily over the last 5 yr. Palm oil shows the most dramatic increase, from 16 to 24 MMT, which brings its production, together with palm kernel oil, to similar or slightly higher levels than soybean oil. The price of oilseeds in general has declined over the last 10 yr. This is due mainly to overproduction and an increased yield of new crops. This overproduction has driven down world prices dramatically for most vegetable and animal fats, and oils as well. The cost of soybean oil decreased from 600 USD/t in 1995 to $344 in 2000. Palm oil sold for 651 USDhons in 1995, and the price went down to $309 in 2000. For fats and oils used in specialty applications, the price decrease has not been as dramatic. For coconut oil, the price decreased from $656 in 1995 to $539 in 2000. In 1991-1992, the price of soybean oil in the U S . was $0.20/lb,whereas in 2000-2001 the price decreased to $0.13/lb. The price of the meal on the other hand has remained more stable, i.e., 190 USDItons in 1991-1992 vs. $180 in 2000-2001. Most common oilseeds are rich in oil content with a composition range between 30 and 50%, with the exception of soybean whose fat content ranges between 19 and 22%. Protein content in soybean is the highest, i.e., -40%. The composition of several oilseeds is listed in Table 3.2. Oilseeds usually consist of a kernel or a cotyledon held together by a hull. In the case of soybeans, a dicotyledon and a hypocotyl are held together by the hull. Most of the oil sacks reside in the cotyledon or kernel of the oilseed. The typical composition of crude vegetable oils is described in Table 3.3. The bulk of components in the crude oil is triacylglycerols, which consist of three fatty acids attached to a glycerol backbone. Phospholipids are also present in appreciable amounts; they are especially abundant in soybean oil. The nonsaponifiable components commonly found in vegetable oils include a wide variety of sterols, tocopherols, carotenes, and other minor compounds inherent to the particular oilseed. Table 3.3 shows some of these components. Tocopherols and tocotrienols are present in some vegetable oils. TABLE 3.2 Typical Composition of Several Oilseeds ~
Sunflower
Peanuts
~
Soybeans
Canola
Cottonseed
40 21
22
21
24
25.2
42 25
49 22 6
47 20 7
47.4 19.0
(%I Protein Fat Carbohydrate Moisture
34 10
8
5
E. Hernandez
50
TABLE 3.3 Composition of Crude Vegetable Oils Soybeans
Canola
Sunflower
Peanuts
95-97 1.5-2.5 0.8-1.6 1700-2200 2700
95-98 0.5-0.8 0.5-1 .O 700-1 000 3204
94-97 0.5-1 .O 2000
96-98 0.5-0.8 0.2-1 .o 200-600 -
Cottonseed ~
Triglycerides Phosphatides Unsaponifiables Tocopherols, ppm Sterols, ppm
95-98 0.7-0.9 0.5 900-1 100 3746
The fatty acids found in vegetable oils are 18- and 16-carbon chain length fatty acids. The most common fatty acids found in oilseeds are stearic, oleic, linoleic, linolenic, and palmitic. Table 3.4 lists the fatty acid composition of the major vegetable oils. Most naturally occurring fats have a cis geometric configuration. Catalyzed reactions such as hydrogenation will rearrange this shape into a trans geometry. The position of fatty acids in the glycerol backbone also follows a natural pattern for most vegetable oils. The fatty acid found in the carbon 2 is usually unsaturated, whereas the fatty acids placed in carbons 1 and 3 can be either saturated or unsaturated. The unsaturates in vegetable oil determine the degree of susceptibility to oxidation. The more double bonds found in the fatty acid, the higher the relative oxidation rate, i.e., 1 for stearic, 10 for oleic, 100 for linoleic, and 150 for linolenic acid. The low relative oxidation rate of oleic acid is a driving force in the development of high-oleic genetically modified oils (GMO) either by natural breeding or genetic modification of some of the major oilseeds, i.e., sunflower, canola, soy, and peanut. Currently, the oxidative stability of some edible oils is improved through the selective partial hydrogenation of polyunsaturates,particularly for oils used in frying. Food Products
Fats and oils are prevalent in many foods throughout the world and they are a required component of every diet. Fats and oils are a major source of storage energy; they have important roles in the body’s metabolic processes and the absorption TABLE 3.4 Typical Fatty Acid Composition of Some Commercial Oils
Palmitic, 16:O Stearic, 18:O Oleic, 18:l Linoleic, 18:2 Linolenic, 18:3
Soybean
Canola
11 4 23 53
4 2 56 19 9
a
Cotton
25 2 18 52
0.5
Sunflower
Corn
Peanut
6
12 2 27 54 1 .o
12 3 46 34
4
19 65
-
-
Production, Processing, and Refining of Oils
51
of fat-soluble nutrients, and play an essential role in the processing, quality, and organoleptic and texture properties of food products. It is generally recommended that at least 1 5 2 0 % of adult energy consumption be fat with a higher amount (3040%) for infants. The n-6 and n-3 fats are considered essential for human metabolism. Fats and oils are the primary components of products such as margarines, shortenings, butter fat, fried foods, mayonnaises, salad dressings, baked products, infant formulas, snack, and confectionary products, The major application of cooking oils is in frying, where it functions as a heat transfer medium and contributes flavor and texture to foods. Cooking oils are required to stand heating temperatures of up to 180°C during frying. More saturated oils are preferred as frying and cooking oils because they are less prone to oxidative, thermal, or hydrolytic breakdown. When little or no heating is required, salad oils are normally used in food preparation. Examples of these include pourable salad dressings and mayonnaises. Salad dressings generally consist of an oil-in-water emulsion; basically they are a two-phase emulsion containing 35-65% oil. Mayonnaise and thick salad dressings may contain up to 80% oil. Salad oils are required to remain clear even under refrigerated conditions. One requirement for margarines and shortenings is that they must have the ability to crystallize to maintain a semisolid consistency at refrigerator and room temperatures. The blending in of solid fats and liquid oils is such that a sharp melting of the blend is achieved to prevent a waxy mouth feel. Partially hydrogenated and liquid oils are used in Western countries, and palmbased products are commonly used in Asian countries. The main role of shortenings is to prevent the cohesion of gluten in baked products and to impart stability to the baked product. They also provide flavor, mouth feel, aeration, and a moisture barrier; they are usually a blend of semisolid and solid anhydrous plastic fats used in the preparation of many foods. Most shortenings require the injection of nitrogen to facilitate handling and to improve shelf-life. Types of shortening include the following: general baking, donut shortening, pie crust, biscuit, puff pastry, creme filler, cake and icing, bread and donut frying, liquid, spray, icing stabilizer, glaze, candy, confectionary coating, and lauric fat replacer. Depending on the specific application, shortenings are required to have as wide a plastic range as possible, that is, the melting behavior should remain constant over a specified temperature range, i.e., 2442°C. The edible oil industry is currently going through great changes in the way it looks at oils for general consumption and special applications. The role edible fats and oils play as organoleptic enhancers, bulking agents, or emulsifiers is being reevaluated. Now fats are actually being examined more closely as nutritional supplements and in disease prevention. New techniques in chemical modification, plant breeding, and genetic engineering have been developed to produce new oils with specific composition and functional characteristics. The n-3 oils, for example, were found to play an important role in infant growth, fetal development, and the nutrition of lactating mothers. The fatty acids in triglyceride oils are important in physiologic processes such as hormone and
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prostaglandin synthesis and memory functioning. Special attention is being given in the industry to natural antioxidants such as tocopherols and tocotrienols. These compounds inactivate free radicals and prevent cardiovascular disease. Free radicals are very reactive chemical compounds that are believed to trigger cancer. Lipoproteins and glycolipids, compounds that are also found in nonprocessed oils, may also fight cardiovascular diseases. Currently, the main trend in fats and oils is diversification in composition and applications. New developments include naturally bred and genetically modified oilseeds. Highly stable oils are being developed to replace hydrogenated oils that are high in trans fat. Most of the conventional oils now have high-oleic versions, i.e., soybean, sunflower, and rapeseed oils have a high-oleic version (up to 90%). Some oilseeds, such as organic, non-GMO, or specialty GMO-based oils, have found their way into smaller food niche markets. With new regulations requiring more detailed nutritional labeling of foods and consumers increasingly aware of nutritional aspects of foods, manufacturers are reformulating many food products to minimize undesired components such as saturated and trans fats and also offer a better balance on the essential fats, i.e., n-6 and n-3 fatty acids, as well as other nutrients.
Production of Vegetable Oils Before processing, harvested oilseeds generally must meet specifications for wholesomeness, low presence of foreign matter, and moisture. These specifications are generally achieved through cleaning and drying before processing. Before solvent extraction, oilseeds typically go through thermal conditioning, cracking, flaking and, in some cases, extrusion steps. First the seeds are graded and cleaned of foreign matter and then dried to prevent spoilage and deterioration. Examples of foreign matter found in harvested oilseeds include weeds, pods, stems, leaves, and soil. These materials are commonly removed by sieving or aspiration. After cleaning and drying, the oilseeds are sent to processing, placed in storage, or transported to another location. The most damaging factors for the quality of oilseeds are the combination of moisture and heat, which can trigger deterioration processes such as enzymatic and oxidation activities. A common method of seed drying is the use of countercurrent open-flame grain dryers fueled with natural gas or heating oil. Hot air, not hotter than 76"C, is usually circulated through a bed of beans, with 40-59% recirculation. Generally, ~13% moisture is desirable in oilseeds such as soybeans. Once the seed is dried, it is transported to storage facilities, which can be either steel tanks or concrete silos. Steel tanks are usually used in processing plants and can be of any size with a conical top of 2 7 O , the angle of repose of soybeans. For smaller operations, belt driers are also a common method. Figure 3.1 shows a general diagram of the preparation steps for oilseeds before extraction. One important reason for conditioning the oilseeds before extraction is
Production, Processing, and Refining of Oils
53
r
Cracking
Dehulling Cooking F
Fig. 3.1. Pretreatment of oilseeds.
to prepare the seed and to provide the best quality meal and oil with the best possible yield. After the cleaning step, soybeans are typically conditioned by heating the seed before cracking; this allows the hull to separate easily from the seed and be removed by an air aspirator. It is desired that the cracking roll generate >4 pieces to allow the flaking rolls to produce thinner flakes efficiently. The industry typically uses less than 0 .O 12" but when using expanders, they can be thicker (0.O 16"). One additional processing step in the preparation of seeds for extraction is the use of extruders or expanders. This step consists in subjecting flakes to high shear and heat, which ruptures the oil sac and thermally stabilizes the macerated product. Extrusion of oilseeds such as soybeans is generally done to increase the capacity of the solvent extractors and oil recovery, to inactivate enzymes, and to improve the quality of the extracted oil. This process was introduced into the oilseed industry in the last 15 yr. Its use is now widespread in the solvent extraction operation, and it is beginning to be used as a pretreatment step in expeller operations. When used in conjunction with expellers, the expander also breaks oil sacks in the seed and facilitates the extraction of oil in the pressing. In addition to enzymatic stabilization, it was found that there was higher percolation, lower residual oil in the extracted meal (0.5%),and an increase in the bulk density of the extruded materials (1,2). The basic mechanism when extruding oilseed is to infuse water or water vapor into the material at more than 200 psig pressure in the barrel of the expander. As the material exits the die, the sudden release in pressure causes the complete rupture of the oil cells. Also there is a starch gelatinization and protein denaturation in the extruded material. This causes the material to agglomerate, resulting in pellets with a higher bulk density than the flakes but at the same time with a higher porosity, which allows for higher percolation rates and lower retention of solvent in the marc after extraction. Drainage rates of 30 gal/(min.ft2) for soy extrudates from 9.5
gal/(min*ft2)for flakes were reported (2) with values between 30 and 140 gal/ (min*ft2)for extruded rice bran (3).
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As mentioned above, another important advantage in using expanders is an increase in the porosity of the product, which allows for rapid drainage and less residual solvent and oil in the extracted material (marc). Hold-ups (solvent in the marc) range from 20-17.2% (2) for extruded soybean extracted with hexane; for soybean flakes, residual oil contents between 42.5 and 33% were reported. The presence of moisture in the feed to the extruder lowers the power consumption appreciably. A reduction of power usage in the expanders from 15 K W t to 5 K W t when increasing the moisture in the feed from 12 to 18% was reported (1). When steam is injected into the expander, the load to the motor of the expander is reported to be reduced as reflected by the increase in the capacity of an 8” expander from 60 t/d for a “dry” operation to 300 t/d for extrusion with 750-1000 l b h of injected steam (4). The use of expanders was reported to decrease the amount of nonhydratable phosphatides by inactivating phospholipases during the extrusion process (2). That is to say, the use of expanders allows the extraction of an oil that can be degummed to phosphorous levels comparable to those resulting from processes designed to enable superdegumming such as the Alcon process. The Alcon process was originally developed to generate a soybean oil through heat treatment of the flakes in the stack cooker with steam injection to inactivate enzymes such as phospholipases, ureases, and also trypsin inhibitors (5). Levels of phosphatides in degummed oil obtained from expanded soybean flakes were comparable to those of degummed soybean oil obtained from the Alcon process, i.e., 0.01-0.07% (6). Both the Alcon process and expanders increase the amount of total phosphatides, and to a lesser extent, the amount of free fatty acids (FFA) that end up in the crude (7). This has the consequence of increasing the neutral oil loss. Even though expanders are capable of inactivating enzymes such as lipases and phospholipases and of appreciably increasing extraction, it is still necessary to inactivate ureases and trypsin inhibitors in the toaster desolventizer by injecting steam. The change from extracting flakes to the use of an expander increases the capacity of extraction by 50-100%. The steam required for extraction of soybean pellets is 238 lb/t and 387 lb/t for extraction of flakes. It was also estimated that the electricity required (KWh/t) for extraction of pellets is similar to that required for extraction of flakes (7). The capacity of the flaking rolls can also be increased by 25-50% if the thickness of the flakes is increased to 0.020”. Because the hold-up in the marc is reduced by 30-50%, the steam costs to desolventize the meal are also reduced appreciably, and the drying and milling of the desolventized meal can be eliminated. Table 3.5 summarizes the effects of various pretreatments of soybean oil.
Oil Extraction
Solvent. Currently hexane is the solvent used by the industry. This is not “pure” hexane but a mixture of hexanes with the following typical composition: n-hexane (up to 62%), isohexane (24%), cyclopentane (13%), and dimethylbutane (1%) with
55
Production, Processing, and Refining of Oils
TABLE 3.5 Effect of Pretreatment on the Quality Crude Soybean Oila FFA
Process
(Oh
Expander Alcon Flakine
1
0.8-1 .O 1 .o-2.0 0.3-0.8
Phosphatides
Ca
4
(Old
(PPm)
(PPm)
2.2 6.8 4.0
1.4 5.3 3.5
2.5-4.0 4.0-6.O 1 .O-3.0
NOL (Oh
)
3.5-5.O 5.0-7.0 2.0-4.0
aSources: References 2, 5, 6
a boiling point of 65°C. Other solvents such as isopropanol, ethanol, and supercritical fluids have also been used in the industry on a smaller scale (8). As mentioned above, the main methods for extraction of oil from oilseeds are solvent extraction and mechanical pressing. Figure 3.2 shows a general diagram of the solvent extraction process for vegetable oils. Hot water and steam infusion are used in some isolated cases. Solvent extraction generally consists in a percolation operation in which the solvent, usually hexane, comes in contact with flakes or expander extrudates (pellets). The hot hexane, normally at 140°C, dissolves the lipid materials as it flows through the bed of solids. There are several designs for solvent extractors used in the industry. The most common ones are the following: (i) The Rotary or Deep-bed extractor, also known as Rotacel. It consists of a series of concentrically placed cells that rotate under stationary solvent sprays. A similar version of this system is sold by French Machinery Company in which the cells are stationary and the spray nozzles, bottom screens, and solvent-oil solution (miscella) collection pans rotate on a central shaft; (ii) The Continuous Loop, shallow bed, extractor is commonly used in the United States; it consists of a vertical conveyor that rotates through several extraction stages. The flakes or pellets are fed into the conveyor at the top of the extractor and are then carried to the bottom part where they flow through a series of spray nozzles where hexane percolates through the Solvent
t
Flakes Or Pellets
Solvent
Marc Crude oil
Miscella Extractor
Meal desolventizer
Fig. 3.2. Extraction of vegetable oils.
Oil desolventizer
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solids in a countercunent fashion; (iii) A third type of extractor commonly used in the oil industry is the horizontal bed extractor, which consists of a simple belt conveyor carrying the solid materials countercurrently with a series of spray nozzles. The miscella is pumped countercurrently to the flow of the solids; thus the concentrated miscella is collected at one end of the extractor and the spent solids at the other end. The miscella leaving the extractor is usually desolventized in a two-effect raising film evaporator, and remnants of the solvent in the crude oil are removed by steam stripping. The first effect of the evaporator is heating by the vapors of the solvent generated in the toaster desolventizer (DT) from the meal processing operation. The condensed solvent vapors are then pumped into the solvent work tank. The second effect is usually heating by steam; the bulk of the hexane should be eliminated by the time the oil reaches the steam stripper. The stripping system operates under vacuum (28" Hg and usually 115°C). It is required that the flash point of the oil leaving the steam stripper and drier be 150°C. It is not advisable for the flash point to be much higher because this would mean overheating and possible deterioration of the crude oil. The marc (spent meal-solvent) leaving the last stage of the extractor is desolventized in a DT. The solvent removed in the miscella evaporator and DT is condensed and recycled for further use. The DT consists of 5-6 vertically stacked steam-heated kettles or trays, with mixing by paddles through a central shaft. The desolventized and heated meal flows from tray to tray through level controlled chutes. The spent flakes or meal are also cooked in the DT with steam injection to meet minimal residual solvent requirements (500 ppm) and eliminate factors such as urease and antitrypsin activities. The latest designs in these solventizers combine toaster, drying, and cooling operations in one single piece of equipment. Chemical Reactions in Triglycerides. Once the oil is separated and extracted from the seed, it is susceptible to several chemical changes. Examples of these include the following: (i) Hydrolysis. This is the breakdown of triglycerides into FFA, mono-, and diglycerides. This occurs very commonly in oils exposed to heat and moisture. It also occurs in the seed itself and is due mainly to lipase activity. This is a source of loss of neutral oil in a processing plant. (ii) Oxidation. The presence of metal, air, and heat induces the incorporation of oxygen into the double bonds of triglyceride oils. The sequence of the oxidation reaction usually starts with fatty acids that have one or more double bonds. It can be an unsaturated fatty acid in the triglyceride or an FFA. The incorporation of oxygen into a double bond forms a peroxide; the peroxide then decomposes into carboxylic compounds such as ketones and aldehydes. These are responsible for the development of rancidity and off flavors in the oil. (iii) Saponification. This is the reaction of metals with the acid moiety of the fatty acid. The most common saponification reaction takes place in the refining process in which the FFA are eliminated by saponification with NaOH and centrifugation. (iv) Hydrogenation. This reaction consists of the incor-
Production, Processing, and Refining of Oils
57
poration of hydrogen into the double bonds of unsaturated fatty acids. This is usually done to modify the functional properties of the oil and to improve its oxidative stability, particularly for frying applications. (v) Interesterification. This is the reaction of the esters in the triglyceride with other esters. This can be a rearrangement of esters in the triglyceride also known as randomization. It can also be an exchange of ester with other triglycerides, fatty acids, or alcohols. This operation has many applications in edible and industrial products. (vi) Polymerization. Polymerization of vegetable oils is due to the presence of double bonds and is usually coupled with an oxidation reaction. Polymerization in edible oils is a quality control problem. In industrial applications, polyunsaturated oils are commonly used in paints and varnishes. (vii) Isomerization. The most common isomerization reaction in vegetable oils is the change from cis to trans fatty acids. This occurs during hydrogenation and to some extent during deodorization. Other isomerization reactions occur when the double bond in a fatty acid changes position due to excessive exposure to heat or the presence of a catalyst as in the case of hydrogenation. Mechanical pressing was the method of extraction more widely used in vegetable oil recovery before the introduction of solvent extraction, Pressing was largely abandoned mainly due to lower capacity, higher power requirements, and high residual oil in the press cake. However, this method is still used as a prepress step for high oil content oilseed processing such as for canola, copra, sunflower, and cottonseed. Expellers are also commonly used in small oil processing plants in the United States and abroad where a solvent extraction plant is not economically feasible. A growing market for the use of expellers is in the processing of oilseeds requiring that solvents not be used such as the natural and organic foods markets. Screw presses or expellers were first patented in the U S . by Anderson in 1903. The press consists basically of a horizontal screw mounted on a barrel. These expellers can generate a high amount of heat through friction, hence the need in some cases to have a water-cooled shaft and a water jacket in the cage. The materials fed into the press are usually flaked and heat pretreated in a stacked cooker or heated and extruded in an expander to maximize oil release. The operating temperature of a cooker before the expeller will range between 170 and 190"F, whereas the temperature of an expander will range between 210 and 250°F. The operating temperatures in an expeller range between 240 and 300°F. This depends on the set conditions of the press, i.e., feed temperature, moisture in the feed, and operating pressure. In some cases, the quality of the oil generated from a screw press can be superior to the quality of solvent-extracted oil. When the flakes or meats are pretreated in an expander, for example, this has the effect of rapidly inactivating enzymes such as the lipases and phospholipases that cause the oil to deteriorate. For example, for soybeans, FFA generated in a screw press can be as low as 0.15%, whereas in a solvent-extracted oil, the FFA content of the crude oils will range between O S and 1% for soybean oil.
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Refining Vegetable Oils The refining of edible oils comprises the entire process that generates a final product from a crude oil. This processing step can be done with or without prior degumming of the crude. Degumming or removal of phosphatides is generally done to prepare soybean oil for caustic or physical refining, to avoid settling in the oil during storage or transport, and to produce commercial lecithin. Typical specifications for crude oil are listed in Table 3.6. Figures 3.3 and 3.4 show a general diagram and processing steps involved in the refining of vegetable oils. Figure 3.5 shows the steps for extracting and processing higher-value vegetable oils, for nutritional and other uses, in which minimal chemical and heat treatments are involved. The following are the typical steps followed by the industry in the processing of vegetable oils. Water Degumming. Degumming of crude soybean oil is usually done by adding soft water to the crude in proportion to the measured amounts of phosphatides (% Phosphatides = 30 x phosphorous content) in the crude. However, this method is not as reliable for bleached oils. The amount of water is calculated as 75% of the amount of phosphatides. Typically, for a continuous degumming process, the oil is heated to 70-80°C and water is added through an in-line mixing system; the mixture flows into a retention tank where it is held for 15-30 min and then is centrifuged. The “short mix” method for water degumming, in which little or no retention time is allowed, tends to produce low-quality oil. This method is sometimes preferred because of the savings in processing time; however, it does not completely remove the nonhydratable phosphatidic fraction. The presence of phosphorus in refined oils was found to have a detrimental effect on the quality of the final deodorized oil, causing incomplete bleaching and flavor reversion (9). The “long mix“ method for degumming before refining is recommended to ensure complete removal of phosphatides including the nonhydratable fraction. The removal of nonhydratables can be achieved by adding an acid. Phosphoric or citric acids are the most commonly utilized; they are added at levels between 0.05 and 0.1% to the crude oil, usually at room temperature. Contact times will depend on mixing efficiency. Acid pretreatment is recommended for crude and TABLE 3.6 Analytical Requirements for Crude Vegetable Oila Test Unsaponifiable matter Free fatty acids, e.g., oleic Moisture, volatile matter Insoluble impurities Flash point Phosphorus aSource: Reference 10.
Maximum
Minimum
1 .5%
0.75% 0.5% 0.3%
250°F 0.02%
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Production, Processing, and Refining of Oils
Refining
Bleaching
Deodorizing
Fig. 3.3. Refining of vegetable oils.
degummed oils before refining. Once the acid pretreatment is completed, the water of hydration is added or the caustic solution if the degumming and refining are to be done jointly. Caustic Refining. The majority of the oils consumed in the United States are refined with sodium hydroxide, but others have also been used, including potassium hydroxide, sodium bicarbonate, sodium carbonate, and sodium silicate. As mentioned above, acid pretreatment of either the crude or degummed oils is recommended and the amount of caustic solution to be added is calculated after the acid
I
I
Crude oil
I
Refining
1 v
I I
Bleachina .,
1
1-h
fl
Dewaxing
I
I Hydrogenation I lnteresterification
1
1( Fig. 3.4. Vegetable oil refined/bleached/deodorized (RBD) processing.
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~
Cracking, flaking, conditioning
Finished premium oil
Fig. 3.5. Mechanical pressing of vegetable oils.
has been mixed in. The caustic solution typically is added at 10-20 Be to oil at high speed; the speed is then reduced and the mixture allowed to agitate for 5-10 min. Because the oil has to reach temperature of 150°F, it is passed through a heat exchanger before reaching the separator. The neutralized oil is washed by returning the centrifuged oil back into the vessel; 5-10% hot (deionized) water at 180°F is added and then it is centrifuged again very quickly at 180’F. This operation must be done promptly to prevent emulsification of the water in the oil. The washed oil is then rapidly dried by spraying it into a tank under vacuum (25-29” Hg) at 180°F. This operation removes any water remaining in the oil and must be done immediately after the washing step. Refining Specifications. Typically, a crude oil is considered normal if the FFA are <1%. The phosphatide content in normal oils will range between 1 and 3.2%. Hydratable phosphatides are usually 90% of the total with nonhydratables comprising the difference. Total FFA after refining are usually required to be <0.1%,and 0.05% is the most commonly targeted figure. The level of phosphorus required is usually <200 ppm (10); however, 4 0 ppm is a figure commonly achieved in the industry when good degumming and refining practices are used, If the degumming
is done as a preparatory step for bleaching and physical refining, a level of <3-5 pprn of phosphorus is suggested (1 1).
Production, Processing, and Refining of Oils
61
As mentioned above, the refining step is carried out to remove FFA and gums from crude oils; the separation of the soapstock is usually done by centrifugation. A process was reported in which the soapstock was separated by filtration (12). This process takes advantage of the agglomeration characteristics of sodium silicate during FFA neutralization. The construction of a filter refining system is similar to that of a regular batch refining system with the addition of a filter. Sodium silicate is added in concentrations similar to that of the caustic used in conventional refining. This process removes FFA (<0.02%), residual soaps (to 430 ppm), phosphatides (to <10 ppm), and some color. Filtration of silicate-soapstock can also be performed as a combined operation of refining and bleaching, in which the soapstock is filtered and precipitated with bleaching clay and the use of a filter. The filtration of soapstock can be done using plate and frame or vertical leaf filters. Partial removal of FFA and suspended solids to regenerate frying oils was also reported (13). These methods utilize adsorbents such as bleaching clay blended with caustic silicates. The treatment of used frying oils with adsorbent combinations extends the frying life of oils by reducing FFA and peroxide values. These adsorbent blends consist of a combination of calcium and magnesium silicate powders blended with adsorbents. It was reported that the addition of 8% of these blends to used frying oils lowers the FFA content (from 0.69 to 0.15%) and peroxide values (from 2.55 to 1.27 mEqkg). FFA are only partially removed because of the difficulty of complete dispersion of adsorbents and silicates in the oil. Bleaching of Vegetable Oil. The main purpose for bleaching is to remove color as well as other impurities such as soaps, metals, phosphatide residues, and decomposed peroxides. The main color components to be removed are carotenoids and chlorophyll. The dosage of bleaching clay is dictated to achieve zero peroxides instead of a predetermined color. Normal conditions for vacuum bleaching of soybean oil are as follows: clay dosage = 0.3-0.6%; temperature = 100-110°C; vacuum pressure = 28-29” Hg; processing time = 15-30 min. Silica gels have increasingly been used in the industry to ensure that soaps and phosphatides are removed from the oil during bleaching before deodorization This product is also used to eliminate the water wash step after caustic refining ( 14). The most common filter aid in edible oil processing is diatomaceous earth. However, because of environmental regulations, perlite has become more popular. Precoating is basically depositing a layer of filter aid and/or bleaching clay on the filter medium before filtration. Precoats prevent gelatinous solids from plugging the filter medium and give a clearer filtrate. The precoat is considered to be a part of the filter medium rather than of the cake. In batch filters, the precoat layer is usually thin; in a continuous or discontinuous system, the precoat filter layer is 22” thick. More recently, a countercurrent multistage bleaching system was introduced for edible oil processing operations (15). This system is reported to lower the use of bleaching clay and operating costs and also provide a more efficient operation. In this process, the oil is initially blended with clay that was used in the second stage, and the oil is then filtered.
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In the second stage, filtered oil is retreated with fresh bleaching clay and filtered once more. This process takes advantage of the higher efficiency of countercurrent operations. However, one disadvantage of this process is the requirement of extra equipment and consequently higher initial capital cost. Deodorization of Vegetable Oils. Deodorization of edible oils started with the need to remove undesirable flavor and odor components from cottonseed oil at the beginning of this century. Current deodorization processes are designed to remove impurities from refined and bleached oil such as peroxides, carboxylic compounds, and FFA. The oil being fed to a deodorizer has to meet certain quality requirements from the bleaching operation to prevent damaging the oil at the higher processing temperatures found in the deodorizer. The main function of bleaching the oil is to remove pigments, oxidation products, phosphatides, soaps, and trace metals (16). The removal of metals, soaps, and phosphatides is particularly important before the oil is to be deodorized. Generally the level of phosphorus must be 4 4 ppm. Typical conditions of deodorization of soybean oil are as follows: pressure: 1-6 mmHg, temperature: 210-274"C, holding time: 2-8 hr (Batch) 15-20 min (Continuous). Usually the vacuum system consists of four-stage ejectors. Some systems use three stages, but a fourth stage is required if a vacuum below 3 mmHg is desired. Most deodorizers are constructed of stainless steel 304. For physical refining/deodorization, 316 stainless steel is used to withstand the corrosive effect of FFA in the oil at the higher temperatures. When necessary, nitrogen is used to blanket the deodorized product and to prevent oxidation. The oil is usually cooled down after it has been deodorized to 3849"C, and most operating deodorizers use a heat recovery system so that the incoming oil is preheated and the outgoing oil is heated. This reportedly saves in heating requirements by 4040%. The stripping steam is required to be void of metals or any solids from the boiler; in some cases, superheating of the steam is practiced to prevent any cooling of the oil in the deodorizer (17). Some plants have small boilers assigned to the deodorizers to ensure that steam from pure or deionized water is used. The distillate generated in the deodorizer is usually recovered using scrub coolers in which the vapors from the main body of the deodorizer are in contact with cooled deodorizer distillate. The scrub cooler can be located ahead of the fist ejector of the vacuum system. Up to 90% of the deodorizer vapors can be recovered with this type of system. Recently, the value of these distillates has increased due to a larger demand for natural sources of tocopherols and vitamin E. Therefore, processing plants go to greater lengths to recover tocopherols, FFA, and other nonsaponifiable materials from the deodorizer distillates (18). Furthermore, due to stricter environmental laws, processors are required to minimize the discharge of low-boiling compounds in emissions such as steam ejectors. Most deodorizers operate at temperatures >200"C; the heating medium used in many systems is a thermal fluid. Many U S . processors use Dowtherm A or Thenninol VP-1, a eutectic mixture of diphenyl and diphenyl oxide. The operating pressure of this fluid is only 16 psig at 260°C, and it is relatively easy to handle. However, due to
Production, Processing, and Refining of Oils
63
TABLE 3.7 Fully Refined Vegetable Oil Specifications Test
Maximu m/m i nimum
Flavor Color (Lovibond) Free fatty acids Clear appearance Cold test Moisture and volatile matter Unsaponifiable content Peroxide value StabiIity
Bland Maximum 20Y/2.OR Maximum 0.05% 70-85°F (21-29°C) Minimum 5.5 hr
SO.l% 51.5% s2.0 mEq/kg Minimum 8 h AOM
the toxicity and flammability of these thermafluids, many plants are switching to highpressure steam, which usually operates at 900 psig and 304°C. There are two types of losses in the deodorizer, i.e., by distillation and by entrainment. If the deodorizer is not working properly, losses from polymerization can be a problem. For deodorizers working under normal conditions, the loss of material can be 0.2-1.0%. FFA are distilled off under normal deodorizer conditions along with mono- and diglycerides. Tocopherols and sterols are also distilled off during this operation and are actually considered a loss because tocopherols are desirable components in the final product as antioxidants, Deodorization can reduce the content of tocopherols in the final refined, bleached, deodorized oil by as much as 60% (18). If the oil is not pretreated properly, the high temperatures of deodorization can be detrimental to the oil. Physical Refining. As mentioned above, physical refining of oils consists in the removal of polar compounds, mostly FFA, by distillation under high vacuum (19). Even though no chemical treatment is involved, pretreatment of the oil before refining for the removal of other impurities has to be very thorough to ensure that the final product fulfills the prespecified requirements. If the oil is not pretreated properly, physical refining can actually be detrimental to the oil. The following are some of the contaminants that have to be removed before treatment for physical refining: metals (Fe, Ca, Mg, Cu, S , P, Pb, Ni), excessive peroxides, excessive color, lecithin, air,and residual solvents. Ultimately, whether a finished oil will sell depends on the specifications met at the end of the processing (20). Table 3.7 shows an example of the typical specifications for a finished oil used by the industry for quality control and trading purposes. References 1. Rittner, H., Conditioning of Oil Bearing Materials for Solvent Extraction by Extrusion, J . A m . Oil Chern. Soc. 61: 1200-1203 (1984).
2 . Lusas, E.W., and L.R. Watkins, Extrusion for Solvent Extraction, J . Am. Oil Chem. SOC. 65: 1109-1114 (1988).
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3. Williams, M.A., Preparation of Oil-Bearing Materials for Extraction, in Technology and Solvents for Extracting Oilseeds and Non-Petroleum Oils, edited by P.J. Wan and P.J. Wakelyn, AOCS Press, Champaign, IL, 1997, pp. 121-136. 4. Randall, J.M., R.N. Sayre, W.G. Schultz, R.Y. Fong, A.P. Mossman, R.E. Tnbelhorn, and R.M. Saunders, Rice Bran Stabilization by Extrusion Cooking for Extraction of Edible Oil, J . Food Sci. 50: 361-368 (1985). 5. Kock, M., Oilseed Pretreatment in Connection with Physical Refining, J . Am. Oil Chem. SOC.60: 150A-153A (1983). 6. Watkins, L.R., W.H. Johnson, and S.C. Doty, Extrusion-Expansion of Oilseeds for Enhancement of Extraction, Energy Reduction and Improvement of Oil Quality, in Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, edited by T.H. Applewhite, AOCS Press, Champaign, IL, 1989, pp. 41-46. 7. Erickson, D.R., Practical Handbook of Soybean and Utilization, AOCS Press, Champaign, IL, 1995, pp. 184202. 8. Wan, P.J., and P.J. Wakelyn, eds., Technology and Solvents for Extracting Oilseeds and Non-Petroleum Oils, AOCS Press, Champaign, IL, 1997. 9. Sessa, D.J., H.W. Gardner, R. Kleiman, and D. Weisleder, Lipids 12: 613-619 (1977). 10. National Oilseed Processors Association, Yearbook and Trading Rules, NOPA, Washington, 2003. 1 1 . Seger, J.C., and K.M. van de Sande, in Proceedings of the World Conference on Oilseed Technology and Utilization, edited by D.R. Erickson, AOCS Press Champaign, IL, 1990, pp. 88-93. 12. Hernandez, E., and S. Rathbone, Refining of Glyceride Oils by Treatment with Silicate Solutions and Filtration, U S . Patent 6,448,423 (2002). 13. Lin, S., C.C. Akoh, and A.E. Reynolds, Recovery of Used Frying Oils with Absorbent Combinations: Refrying and Frequent Oil Replenishment, Food Res. Int. 34: 159-166 (200 1). 14. Welch, W.A., J.M. Bogdanor, and G.S. Toeneboehn in Proceedings of the World Conference on Oilseed Technology and Utilization, edited by D.R. Erickson, AOCS Press, Champaign, IL, 1990, pp. 189-202. 15. Transfeld, P., M. Schneider, and G. Borner, U.S. Patent 5,753,103 (1998). 16. Loft, S.C., Deodorization. Theory and Practice, in World Conference Proceedings, Edible Fats and Oils Processing, AOCS Press, Champaign, IL, 1989. 17. Maza, A., R.A. Ormsbee, and L.R. Strecker, Effects of Deodorization and Steam Refining Parameters on Finished Oil, J . Am. Oil Chem. SOC.69: 1003-1008 (1992). 18. Noms, F.A., Deodorization, in Bailey’s Industrial Oil and Fat Products, edited by T.H. Applewhite, John Wiley and Sons, New York, 1985. 19. Zehnder, C.T., Deodorization, in Practical Handbook of Soybean Processing and Utilization, edited by D.R. Erickson, AOCS Press, Champaign IL, 1995. 20. O’Brien, R.D., Fats and Oils. Formulating and Processing Applications, Technomic Publishing, Lancaster, PA, 1998.
Chapter 4
Novel Hydrogenation for Low Trans Fatty Acids in Vegetable Oils Mun Yhung Jungdand David B. Minb aDepartment of Food Science and Technology, Woosuk University, Jeonbuk, Republic of Korea, and bDepartment of Food Science and Technology, The Ohio State University, Columbus, OH 4321 0
Introduction Hydrogenated vegetable oils have been used since the first part of the twentieth century to expand the application of vegetable oils in foods. Vegetable oils are the most important edible oils and represent >70% of edible oils in the world. Hydrogenated vegetable oils impart distinctive flavor, crispness, creaminess, plasticity, and oxidative stability to common foods. But hydrogenated oils contain high quantities of trans fatty acids (TFA), which have been reported to be associated with an increased risk of cardiovascular disease because they raise the level of serum low-density lipoprotein cholesterol and decrease the level of high-density lipoprotein cholesterol (1-6). Desci and Koletzko (7) also reported positive associations between the intake of TFA and the inhibition of arachidonic acid biosynthesis, which is essential for normal growth in children. Due to the increased concerns about TFA, a task force of the federal government suggested that TFA should be added to or included with saturated fatty acids on labels as a separate class and that a threshold proportion of TFA for health claims should be implemented (8). The U.S. legislation of June 2003 requires the Nutrition Facts panel on all food labels to indicate trans fat content by January 1, 2006. Health concerns about TFA have led to interest in interesterification, fractionation, and blending of saturated and polyunsaturated oils as alternate methods to hydrogenation. These alternative methods could be more costly than conventional hydrogenation; in addition, with these methods, it is not easy to produce the desirable physical and chemical properties of oils suitable for the manufacturing of the broad range of oil products such as confectionery, margarines, and shortenings. Therefore, the alternative methods could not easily replace the hydrogenation of vegetable oils. Thus, hydrogenation remains the most viable choice for food manufacture if TFA can be substantially reduced during the hydrogenation processes. New hydrogenation processes such as electrocatalytic hydrogenation (9-14), pre-
cious catalyst hydrogenation (I 5-1 S), and supercritical fluid state hydrogenation (19-21) were reported to reduce the TFA in hydrogenated vegetable oils. This 65
M.Y. /ung and D.B. Min
66
chapter reviews electrocatalytic hydrogenation, precious metal catalyst hydrogenation, and supercritical fluid state hydrogenation to reduce TFA of hydrogenated oils. Elecfrocafalytic Hydrogenation
Conventional hydrogenation uses a nickel catalyst and hydrogen gas at high temperatures (140-230°C). The high hydrogenation temperature produces high TFA in hydrogenated oils. A low-temperature electrocatalytic hydrogenation would be an alternative method for the production of hydrogenated edible oils containing low TFA. Low-temperature electrocatalytic hydrogenation uses an electrically conducting catalyst such as Raney nickel or platinum black as a cathode. Electrocatalytic hydrogenation has been used to produce a variety of organic compounds such as aromatics, phenols, ketones, nitrocompounds, dinitriles, and glucose (22-27). Electrocatalytic hydrogenations were also used successfully to produce hydrogenated oils with low TFA (9-14). The electrochemical reactor contains a cathode for reduction reactions and an anode for oxidation reactions. Atomic hydrogen is generated on the catalytically active cathode surface by the reduction of protons or water molecules through electrolytic reactions in the electrical hydrogenation (Equations 1 and 2). The atomic hydrogen on the catalytically active cathode surface is adsorbed hydrogen (Hads).The electrolytically generated hydrogen atoms react with the double bonds of fatty acids for the formation of saturated fatty acids (Equation 3). The process eliminates dissolution and mass transfer resistances of molecular hydrogen, thereby increasing the reaction rates.
H,O
b--
2H,d,
+ R-CHXH-R
'12 0, + 2H+ + 2e-
-
R-CH,-CH,-R
[I1
[31
The adsorbed hydrogen on the cathode surface is used for the hydrogenation of unsaturated fatty acids. The absorbed atomic hydrogen concentration on the catalyst surface can be controlled by the current applied. This hydrogenation at low temperature and pressures minimizes the isomerization of the cis double bonds to TFA. Molecular hydrogen gas (H,) could be formed either by the chemical combination of two adsorbed hydrogen atoms or by the electrochemical reduction of adsorbed hydrogen (Equations 4 and 5). However, the molecular hydrogen gas
does not participate in reducing the double bonds of fatty acids in the electrochemical hydrogenation. Thus, the formation of molecular hydrogen gas is an unwanted
Hydrogenation of Vegetable Oils
67
side reaction, which consumes current but does not affect the product yield. When the double bonds are nearly hydrogenated, the molecular hydrogen gas evolution rate increases. A common problem of high molecular hydrogen (H2) evolution in electrochemical reactors starts when 2550% of the double bonds in oils is hydrogenated.
Hads+ H++ e-
-
H, (gas)
[51
Yusem and Pintauro (9) and Yusem et al. (10) reported that soybean oil can be electrochemically hydrogenated at atmospheric pressure, a constant current density between 0.1 and 0.45 A/cm2 and a low temperature of 70°C using laboratory-scale axial and radial-flow undivided cells containing a Raney nickel powder cathode. In this electrochemical Raney nickel reactor system, a two-phase reaction medium was used in which soybean oil was dispersed in a waterlt-butanol solvent with tetraethylammonium p-toluenesulfonate as the supporting electrolyte. The hydrogenation current efficiencies, which measure the oil hydrogenation rate relative to the total rate of atomic hydrogen production, are in the range of 50-100% for apparent current densities of 1.0-2.0 x A/cm2 at 2040% oil concentration in a waterlt-butanol solvent with tetraethylammonium p-toluenesulfonate as the supporting electrolyte. The stearic acid and TFA of electrochemically hydrogenated oil are significantly lower than those of conventionally hydrogenated oil with gaseous molecular hydrogen. Yusem and Pintauro (28) designed the radial flowthrough Raney nickel powder electrocatalytic hydrogenation reactor and performed research on a simulation and economic analysis of an electrocatalytic soybean oil hydrogenation. The authors reported that cost (USD 0.13kg) for electrocatalytic hydrogenation was higher than that for a comparable-size chemical hydrogenation with molecular hydrogenation (USD 0.019kg). When the cost of the soybean oil, the starting material (USD 0.68kg), was factored into the economic analysis, the production plus raw material cost of the electrocatalytic process was only 16% greater than that for conventional hydrogenation (28). A drawback of this electrochemical Raney nickel reactor system was the use of nonfood grade t-butanol for stabilizing the emulsion and the supporting electrolyte, tetraethylammonium ptoluenesulfonate, for reasonable ionic conductivity in the water/alcohol phase. An and others (1 1) introduced a different method of electrochemical hydrogenation of edible oils with a solid polymer electrolyte reactor. A solid polymer electrolyte reactor was used in the hydrogenation of organic compounds (29,30). The problem of using nonfood grade t-butanol for stabilizing the emulsion and the supporting electrolyte, tetraethylammonium p-toluenesulfonate, in the electrochemical Raney nickel reactor could be solved by the solid polymer electrolyte
reactor for oil/fatty acid hydrogenation. An and others (11) conducted electrochemical hydrogenation of edible oils in a solid polymer electrolyte reactor, com-
68
M. Y. lung and D.6. Min
posed of a RuO, powder anode and either a platinum-black or palladium-black powder cathode that were hot-pressed as thin films onto the opposing surfaces of a Nafion cation exchange membrane. The solid polymer electrolyte reactor was used for the hydrogenation of soybean oil, canola, and cottonseed oils and with mixtures of fatty acids and fatty acid methyl esters at a constant supplied current density of 0.1 Ncm2 and a temperature between 50 and 80°C. The solid polymer electrolyte reactor consists of separate anode and cathode chambers. The anode and cathode chambers are separated by a thin hydrated cation exchange membrane. The thin hydrated cation-exchange membrane of the precious metal catalyst powder anode and cathode are fixed to opposing faces of the membrane to form a membrane electrode assembly (Fig. 4.1). Water is circulated past the back side of the anode, where water molecules are oxidized to 0, gas and protons (H+), according to [l]. The protons from water oxidation migrate through the ion-exchange membranes under the influence of the applied electric field to the cathode catalyst components of the membrane electrode assembly, where the protons are reduced to atomic and molecular hydrogens (Equations 2, 4, and 5). Because the water and oil phases are separated by a hydrated polymeric cation-exchange membrane, neither solvent nor
Fig. 4.1. Principles of operation of a solid polymer electrolyte reactor for the electrochemical hydrogenation of oil. Source: Reference 11.
Hydrogenation of Vegetable Oils
69
emulsions are required in this reaction system. An and others (1 1) reported that the hydrogenated oils with 60-105 iodine value (IV) had higher stearic acid content and lower TFA than those of conventionally nickel hydrogenated oils. A platinum black powder cathode was more effective for the reduction of trans fats than a palladium black powder cathode at similar IV. The TFA of hydrogenated oil with 66-1 15 IV by a platinum black cathode were <4%, whereas the trans isomer content of hydrogenated oil with 61-105 IV by a palladium black cathode was 6 5 1 0 . 5 % . The TFA of hydrogenated oils with 61-105 IV by a conventional nickel catalyst were 2040%. The stearic acid of hydrogenated oil by a platinum black cathode was slightly higher than that of the hydrogenated oil by a palladium black cathode. An and others (12) further studied the effects of various hydrogenation factors such as cathode catalyst, catalyst loading, the cathode catalyst binder loading, current density, and reactant flow rate on the current efficiency of oil hydrogenation. The currency efficiencies of different cathode catalysts were in the decreasing order of Pd > Pt > Rh > Ru > Ir. The oil hydrogenation current efficiency with a palladium black powder cathode decreased with increasing current density. Current pulsing for frequencies in the range of 0.25-60 Hz had no effect on current efficiencies. The combination of an increased oil feed flow rate and a nickel turbulence promoter inserted into the oil stream increased the current efficiency of oil hydrogenation. An and others (13) also reported the effects of cathode designs and reactor operation conditions on the hydrogenation selectivity. They increased oil mass transfer into and out of the palladium black powder cathode layer by the combination of increasing the porosity of the cathode carbon papedcloth backing material, increasing the oil feed flow rate, and inserting a turbulence promoter into the oil feed flow channel. The increased oil mass transfer in the palladium black powder cathode catalyst layer decreased the concentration of stearic acid and linolenic acid in the hydrogenated oil. The electrodeposited secondary metal (Ni, Cd, Zn, Pb, Cr, Fe, Ag, Cu, or Co) on a palladium black powder cathode increased the linolenate, linoleate, and oleate selectivity and decreased the stearic acid in the hydrogenated oil (13). Warner and others (3 1) reported that oils electrochemically hydrogenated by a solid polymer electrolyte reactor with a Pd cathode catalyst have higher oxidative stability and lower hydrolysis than oils hydrogenated with a Ni catalyst. In room odor evaluations of heated oils at frying temperature at 190°C, soybean oil hydrogenated with a Ni catalyst had the strong, undesirable characteristic hydrogenation aroma of waxy, sweet, flowery, fruity, and/or crayon-like odors. However, the electrochemically hydrogenated soybean oils had only weak hydrogenation aroma, indicating that the hydrogenation aromdflavor would be much less detectable in foods fried in the electrochemically hydrogenated oils.
Mondal and Lalvani (14) introduced a novel electrochemical hydrogenation of vegetable oil using a hydrogen transfer agent of formic acid and a nickel catalyst.
M. Y. lung and D.B. Min
70
An emulsion of oil and water containing formic acid and nickel, placed in the cathode compartment of an electrolysis cell and subjected to an electrical current, was hydrogenated at temperatures as low as 45°C. The TFA content of the hydrogenated canola oil at 45°C was significantly lower than that of the hydrogenated edible oils by conventional Ni hydrogenation at high temperature and high partial pressure of hydrogen gas. Mondal and Lalvani (14) hypothesized that the reduction of double bonds occurred via a chemical reaction between the formate ion and oil at the surface of the hydrogenation catalyst (Equation 6). The oxidized form of the formate ion (bicarbonate ion) was then reduced electrochemically at the cathode, resulting in the regeneration of the formate ion (Equation 7). HCOO-
+ Oil + H,O
HC0,-
+ 2H+ + 2e-
-
Oil-H,
+ HC0,-
161
+ H,O
[71
HCOO-
This hydrogenation induced a slight hydrolysis of the oil due to the low pH (1.5) of the oil/formate/formic acid reaction mixture. This reaction system requires the preparation of an emulsion, and the oil separation should be done at the end of the reaction. Precious Metal Catalyst Hydrogenation
Ni catalysts are most commonly used for vegetable hydrogenation in conventional hydrogenation. Ni catalysts offer high activity, tailored linoleic acid and linolenic acid selectivity, low cost, and easy removal from oils by filtration. The nickel catalyst causes the isomerization of the natural cis double bonds to trans double bonds. The TFA content of vegetable oil hydrogenated by precious metal catalysts was lower than that of oil hydrogenated by nickel catalysts. Low hydrogenation temperature using precious metals produces less TFA than the conventional hightemperature hydrogenation using nickel. Nickel catalysts are not very active below 120°C. Precious metal catalysts, on the other hand, are active at the low temperature of 70°C. Precious metal catalysts at low temperature decreased the TFA in the hydrogenated vegetable oils. Palladium, platinum, and ruthenium are the precious metal catalysts with the most potential for the hydrogenation of vegetable oils. Each metal catalyst has different characteristics in selectivity, reactivity, and trunsisomerization during hydrogenation of vegetable oils. It is generally accepted that the platinum catalyst produces the lowest TFA during hydrogenation. The modified palladium catalysts, which are a partially deactivated palladiumon-carbon catalyst by silver and bismuth, showed improved selectivity over the palladium-on-carbon alone (33). Riesz and Weber (34), using barium sulfate as a carrier for palladium, decreased the trans-isomer formation without greatly affecting the activity or selectivity. Ahmad and others (35) studied the hydrogenation of soybean oil with 1-10% palladium-on-carbon at hydrogen pressures between
Hydrogenation of Vegetable Oils
71
ambient and 70 psi and at temperatures between 80 and 160°C. If palladium is deposited on the exterior of the carbon for the easy access of triglyceride molecules, its selectivity and activity were superior to those of nickel, even at temperatures at which nickel is inactive. Ray (36) reported the effects of the hydrogenation rate, trans-isomer formation, and selectivity on the hydrogenation of soybean oil with palladium-on-carbon catalyst. The author reported that the palladium catalyst did not have any advantage over nickel catalysts on the formation or selectivity. Hsu and others (13, on the other hand, found that palladium black was far superior to nickel catalyst or palladium-on-carbon in lowering the TFA in hydrogenated canola oil. Hydrogenation with 560 ppm of Pd-on-carbon produced 30.2% F A , but that with palladium black produced 19% TFA in hydrogenated canola oil with -68% IV. Hsu and others (37) also reported that hydrogen pressure has the most significant effect on the formation of TFA during hydrogenation with palladium black. The effects of 150 and 750 psi hydrogen on the TFA of canola oil with IV 53 hydrogenated at 90°C and 560 ppm palladium black showed that 150 and 750 psi hydrogen pressure produced 42.8 and 18.7% TFA, respectively. High pressure and low temperature decreased the formation of TFA during the hydrogenation of canola oil with palladium black. Hsu and others (37) reported that the hydrogenated oil of IV 74 with palladium black at 50 psi hydrogen pressure and 70°C reaction temperature had only 9.4% TFA. Wright and others (18) reported the effect of Ni catalyst addition to Pd catalyst on the cis selectivity and the hydrogenation activity for canola oil. The addition of 100 ppm Ni to Pd increased the hydrogenation activity significantly without any effect on cis selectivity of 50 ppm Pd catalyst at 70°C and 754 psi. The 50 ppm Pd catalyst at 70°C and 754 psi comprised the optimum hydrogenation conditions for cis selectivity of canola oil. Wright and others (18) explained that the increased activity of the Pd/Ni system over Pd alone was attributed to the adsorption of catalyst poison from the oil by Ni. Berben and others (16) studied the effects of palladium and platinum catalysts on the formation of TFA and saturated fatty acids during hydrogenation at 60"C, 102 psi, with 50 or 65 ppm precious metal catalysts. The fatty acid compositions of the hydrogenated oil with palladium were similar to those of oils hydrogenated with nickel. The dropping points of the hydrogenated oils with palladium catalyst were higher than those of the nickel-catalyzed products. Palladium catalysts did reduce TFA. However, platinum catalysts lowered trans-isomer formation in the hydrogenated oil. The amount of saturated fatty acids with platinum catalyst hydrogenation was higher than that of oil hydrogenated with nickel or palladium. A platinum catalyst on narrow pore carbon support was more effective in reducing TFA than a platinum catalyst with a wide pore carbon support. The amount of TFA and saturated fatty acids varied with the hydrogenation temperatures in platinum or other catalyst hydrogenation. As the hydrogenation temperature increased, the TFA
increased, but saturated fatty acids decreased, Figure 4,2shows the effects of the hydrogenation temperature on the formation of TFA and saturated fatty acids in
M. Y. lung and D.B. Min
72
hydrogenated oils with 70 IV with platinum on wide pore carbon support. The hydrogenated oil at 68 IV produced at the low temperature of 60°C and high hydrogen pressure of 102 psi contained only 7.7% TFA. Berben and others (17) studied the effects of various precious metal catalysts on the formation of TFA during hydrogenation of soybean oil at 60°C 147 psi hydrogen pressure, and a precious metal loading between 100 and 400 ppm. Both the palladium and the ruthenium on carbon support showed the same amounts of TFA and saturated fatty acids in the hydrogenated oil of 100 IV by Ni. The palladium catalyst had more hydrogenation selectivity with less saturated fatty acids than ruthenium. The platinum catalyst greatly decreased the formation of TFA in the hydrogenated oils. However, the amount of saturated fatty acids in hydrogenated oils with platinum was much higher than with nickel, palladium, or ruthenium. The amounts of TFA and saturated fatty acids in hydrogenated oil with 100 IV by platinum on alumina support were 5.4 and 17.6%, respectively. However, the amounts of TFA and saturated fatty acids in hydrogenated oil with 100 IV by Ni catalyst were 18.8 and 6.8%, respectively. Berben and others (17) produced hydrogenated oil with low saturated fatty acids and TFA by adding ammonia to the catalyst as a reaction modifier. The addition of ammonia to a palladium catalyst formed a very
35
30
25
20
15
10 60
80
I00
120
140
Reaction temperature ("C) Fig, 4.2. Effects of temperature on trans fatty acids and saturated fatty acids during hydrogenation with a platinum catalyst. Source: Reference 17.
Hydrogenation of Vegetable Oils
73
low amount of saturated fatty acids, but allowed a high level of TFA in a hydrogenated oil with 100 IV. The addition of ammonia to a platinum catalyst greatly decreased both saturated fatty acids and TFA. The amounts of TFA and saturated fatty acids in 100 IV hydrogenated oil using a combination of ammonia and platinum on alumina support were only 6.6 and 6.8%, respectively. Figure 4.3 summarizes the amounts of TFA and saturated fatty acids in hydrogenated soybean oil with 70 IV under various hydrogenation conditions and with various catalysts and/or modifiers. Supercritical Fluid State Hydrogenation
Supercritical fluid state hydrogenation was introduced recently to improve the hydrogenation of vegetable oils. Conventional Ni hydrogenation takes place in a three-phase reaction system of hydrogen gas phase, liquid oil phase, and solid catalyst phase. One of the most important hydrogenation rate-determining factors is the mass transfer of hydrogen gas to the liquid oil near the catalyst surface for hydrogenation. Supercritical fluid state improves hydrogen transfer to the catalyst surface during hydrogenation by providing a good homogeneous phase. The improved mass transfer would increase the hydrogenation reaction rate and decrease the for-
Saturates (%) Fig. 4.3. Effects of precious metal catalysts and modifiers on the trans fatty acids and saturated fatty acids of hydrogenated soybean oil with iodine value (IV) 70. Source: Reference 16.
M.Y. lung and D.B. Min
74
mation of TFA. Several investigators showed the efficacy of conducting hydrogenation of oleochemicals and vegetable oils using supercritical carbon dioxide or propane as a solvent. King and others (21) studied the hydrogenation with conventional nickel catalyst under supercritical carbon dioxide at temperatures from 120 to 140°C and hydrogen pressure up to 2000 psi. The supercritical carbon dioxide hydrogenation with nickel catalyst reduced the formation of TFA. The hydrogenated soybean oil with 82 IV under supercritical carbon dioxide conditions contained 6.4% TFA. Macher and others (19) studied the hydrogenation of fatty acid methyl esters of rapeseed oil using supercritical propane with a 3% palladium on aminopolysiloxane in a microscale fixed-bed reactor. The hydrogenated oil with IV 70 produced at 92"C, a hydrogen pressure of 58 psi, and a residence time of 40 ms contained 3.8% TFA. Macher and Holmqvist (20) hydrogenated palm oil in near-critical and supercritical propane using a continuous fixed-bed reactor with a 1% palladium on carbon. The hydrogenated palm oil under the near-supercritical and supercritical conditions had high reaction rates with a residence time of 2 s at 120°C. However, the catalysts showed strong signs of deactivation very early in the hydrogenation, possibly due to the impurities in the feedstock and/or to coke formation. Further data are required to clarify the phase behavior of the reaction mixture and optimal conditions for the minimization of trans-isomer formation in the supercritical fluid state hydrogenation with precious metals (20).
Conclusions Health concerns about TFA have led to interest in interesterification, fractionation, or blending of saturated and polyunsaturated oils as alternatives to hydrogenation. These alternative methods could be more costly and do not readily produce the desirable physical and chemical properties of oils suitable for manufacturing a broad range of oil products. Therefore, the alternate methods could not easily replace the hydrogenation of vegetable oils. Hydrogenation is still the viable choice for food manufacture if TFA can be substantially reduced during the hydrogenation processes. New hydrogenation processes such as electrocatalytic hydrogenation, precious catalyst hydrogenation, and supercritical fluid state hydrogenation have had promising results for the reduction of TFA below the level of 8%. These hydrogenation techniques would be viable alternatives for replacing the conventional Ni catalyst hydrogenation to produce hydrogenated products with low TFA. References 1. Mensink, R.P., and M.B. Katan, Effects of trans Fatty Acids on High-Density and LowDensity Lipoprotein Cholesterol Levels in Healthy Subjects, N. Engl. J. Med. 323:
439-445 (1990).
2. Troisi, R., W.C. Willett, and S.T. Weiss, Trans-Fatty Acid Intake in Relation to Serum Lipid Concentrations in Adult Men, Am. J. Clin. Nutr. 56: 1019-1024 (1992).
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3. Willet, W.C., M.J., Stampfer, and J.E. Manson, Intake of trans Fatty Acids and Risk of Coronary Heart Disease Among Women, Lancet 341: 581-585 (1993). 4. Ascherio, A., C.H. Hennekens, J.E. Burring, C. Master, M.J. Smapfet, and W.C. Willet, Trans Fatty Acid Intake and Risk of Myocardial Infraction, Circulation 89: 94-101 (1994). 5. Judd, J.T., B.A. Clevidence, R.A. Muesing, J. Wettes, M.E. Sunkin, and J.J. Podczasy, Dietary trans Fatty Acids: Effects of Plasma Lipids and Lipoproteins of Healthy Men and Women, Am. J. Clin. Nutr. 59: 861-868 (1994). 6. Aro, A., M. Jauhiainen, R. Partanem, I. Salminen, and M. Mutanen, Stearic Acid, trans Fatty Acids, and Dietary Fat: Effects on Serum and Lipoprotein Lipids, Apolipoproteins, Lipoprotein (a), and Lipid Transfer Proteins in Healthy Subjects, Am. J. Clin.Nutr. 65: 1419-1426 (1997). 7. Desci, T., and B. Koletzko, Do trans Fatty Acids Impair Linolenic Metabolism in Children? Ann. Nutr. Metab. 39: 36-41 (1995). 8. Feldman, E.B., P.M. Kris-Etherton, D. Kritchevsky, and A.H. Lichtenstein, Position Paper on trans Fatty Acids:ASCN/AIN Task Force on trans Fatty Acids, Am. J. Clin. Nutr. 63: 663-670 (1996). 9. Yusem, G., and P.N. Pintauro, The Electrocatalytic Hydrogenation of Soybean Oil, J. Am. Oil Chem. SOC.69: 399-404 (1992). 10. Yusem, G., P.N. Pintauro, P.C. Cheng, and W. An, Electrocatalytic Hydrogenation of Soybean Oil in a Radial Flow-Through Raney Nickel Powder Reactor, J . Appl. Electrochem. 26: 989-997 (1996). 11. An, W., J.K. Hong, P.N. Pintauro, K. Warner, and W. Neff, The Electrochemical Hydrogenation of Edible Oils in a Solid Polymer Electrolyte Reactor. I Reactor Design and Operation, J. Am. Oil Chem. SOC. 75: 917-925 (1998). 12. An, W., J.K. Hong, and P.N. Pintauro, Current Efficiency for Soybean Oil Hydrogenation in a Solid Polymer Electrolyte Reactor, J. Appl. Electrochem. 27: 947954 (1998). 13. An, W., J.K. Hong, P.N. Pintauro, K. Warner, and W. Neff, The Electrochemical Hydrogenation of Edible Oils in a Solid Polymer Electrolyte Reactor. I1 Hydrogenation Selectivity Studies, J. Am. Oil Chem. SOC.76: 215-222 (1999). 14. Mondal, K., and S.B. Lalvani, Electrochemical Hydrogenation of Canola Oil Using Hydrogen Transfer Agent, J. Am. Oil Chem. SOC.80: 1135-1 141 (2003). 15. Hsu, N., L.L. Diosady, W.F. Graydon, and L.J. Rubin, Heterogeneous Catalytic Hydrogenation of Canola Oil Using Palladium, J. Am. Oil Chem. SOC.63: 1036-1042 (1986). 16. Berben, P.H., F. Bominkhof, B.H. Reesink, E.G.M. Kuijpers, Production of Low transIsomer Containing Products by Hydrogenation, in Practical Short Course Series: Vegetable Oils Processing and Modification Techniques, edited by E. Hernandez and K.C. Rhee, Food Protein R&D Center, Texas A&M University, College Station, TX, 2000. 17. Berben, P.H., P.J.W. Blom, and J.C. Sollie, Palladium and Platinum Catalyzed Oil Hydrogenation: Effects of Reaction Conditions on trans-Isomer and Saturated Fatty Acids Formation, in Practical Short Course Series: Vegetable Oils Processing and Modification Techniques, edited by E. Hernandez and K.C. Rhee, Food Protein R&D Center, Texas A&M University, College Station, TX, 2000.
18. Wright, A.J., A. Wong, and L.L. Diosady, Ni Catalyst Promotion of a cis-Selective Pd Catalyst for Canola Oil Hydrogenation, Food Res. Zntl. 36: 1069-1072 (2003).
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19. Macher, M., J. Hogberg, P. Moller, and M. Harrod, Partial Hydrogenation of Fatty Acid Methyl Esters at Supercritical Conditions, FetteLipid 8: 301-305 (1999). 20. Macher, M., and A. Holmqvist, Hydrogenation of Palm Oil in Near-Critical and Supercritical Propane, Eur. J. Lipid Sci. Technol. 103: 81-84 (2001). 21. King, J.W., R.L. Holliday, G.R. List, and J.M. Snyder, Hydrogenation of Vegetable Oils Using Mixtures of Supercritical Carbon Dioxide and Hydrogen, J. Am. Oil Chem. SOC. 78: 107-1 13 (2001). 22. Pintauro, P.N., and J. Bontha, The Role of Supporting Electolytes During the Electrocatalytic Hydrogenation of Aromatic Compounds, J. Appl. Electrochem. 21: 799-804 (1991). 23. Robin, D., M. Comtois, A. Martel, R. Lemieux, A.K. Cheong, G. Belot, and J. Lessard, The Electrocatalytic Hydrogenation of Fused Polycyclic Aromatic Compounds at Raney Nickel Electrodes: The Influence of Catalyst Activation and Electrolysis Conditions, Can. J. Chem. 68: 1218-1227 (1990). 24. Miller, L.L., and L. Christensen, Electrocatalytic Hydrogenation of Aromatic Compounds, J. Org. Chem. 43: 2059-2061 (1978). 25. Cyr, A,, P. Huot, G. Belot, and J. Lessard, The Efficient Electrochemical Reduction of Nirobenzene and Azoxybenzene to Aniline in Neutral and Basic Aqueous Methanolic Solutions at Devarda Copper and Raney Nickel Electrodes: Electrocatalytic Hydrogenolysis of N - 0 and N-N Bonds, Electrochim. Acta 35: 147-152 (1990). 26. Song, Y., and P.N. Pintauro, The Electrochemical Synthesis of Aminonitriles. I. H-Cell Studies with Adiponitriles and Azelanitrile, J. Appl. Electrochem. 21: 21-27 (1991). 27. Park, K., P.N. Pintauro, M.M. Baizer, and K. Nobe, Current Efficiencies and Regeneration of Poisoned Raney Nickel in the Electrohydrogenation of Glucose to Sorbitol, J. Appl. Electrochem. 16: 941-946 (1986). 28. Yusem, G., and P.N. Pintauro, Computer-Aided Electrochemical Process Design: Simulation and Economic Analysis of an Electrocatalytic Soybean Oil Hydrogenation Plant, J. Appl. Electrochem. 27: 1157-1 171 (1997). 29. Ogumi, Z., M. Inaba, S.I. Ohashi, M. Uchida, and Z.I. Takehara, Application of the SPE Method to Organic Electrochemistry. VII. The Reduction of Nirobenzene on a Modified Pt-Nafion, Electrochim. Acta 33: 365-369 (1988). 30. Ogumi, Z., K. Nishio, and S. Yoshizawa, Application of the SPE Method to Organic Electrochemistry. 11. Electrochemical Hydrogenation of Olefinic Double Bonds, Electrochim. Acta 26: 1779-1782 (1981). 3 1. Warner, K., W.E. Neff, G.R. List, and P.N. Pintauro, Electrochemical Hydrogenation of Edible Oils in a Solid Polymer Electrolyte Reactor. Sensory and Compositional Characteristics of Low Trans Soybean Oils, J. Am. Oil. Chem. SOC. 77: 1113-1117 (2000). 32. Zajcew, M., The Hydrogenation of Fatty Oils with Palladium Catalyst 111. Hydrogenation of Fatty Oils for Shortening Stock, J. Am. Oil Chem. SOC.37: 11-14 (1960). 33. Zajcew, M., The Hydrogenation of Fatty Oils with Palladium Catalyst IV. Pilot-Plant Preparation of Shortening Stocks, J. Am. Oil Chem. SOC.37: 130-132 (1960). 34. Riesz, C.H., and H.S. Weber, Catalysts for Selective Hydrogenation of Soybean Oil 11. Commercial Catalysts, J. Am. Oil Chem. SOC.41: 400-4032 (1970). 35. h a d , M.M., T.M. Priestley, and J.M. Winterbottom, Palladium-Catalyzed Hydrogenation
of Soybean Oil, J. Am. Oil Chem. SOC.36: 571-577 (1979).
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36. Ray, J.D., Behavior of Hydrogenation Catalysts. I. Hydrogenation of Soybean Oil with Palladium, J. Am. Oil Chem. SOC.62: 1213-1217 (1985). 37. Hsu, N., L.L. Diosady, and L.J. Rubin, Catalytic Behavior of Palladium in the Hydrogenation of Edible Oils, J. Am. Oil Ckem. Soc. 6.5: 608-688 (1988).
Chapter 5
Analysis of Lipids by New Hyphenated Techniques Huiling Mu BioCentrum-DTU and Center for Advanced Food Studies, The Technical University of Denmark, DK-2800 Lyngby, Denmark
Introduction Fat is one of the major components of the diet; it can be of both plant and animal ongin with great variation in the composition of fatty acids (FA), extending to 30 or more different FA with various chain lengths, degrees of unsaturation, and FA isomers. The nutritional value of lipids depends on both the FA composition and the positional distribution of the acyl groups within the lipid molecules because the stereochemistry of lipid influences its digestion and absorption. In addition to the naturally occumng fats and oils, fats are also manufactured from vegetable oils or animal fats by introducing new FA or by fractionating fats. New technology to interesterify fats using regiospecific lipases may be applied for the production of fats with particular modified structures, i.e., specific structured fats that may have applications in improving fat absorption (1-7). Even though phospholipids and sphingolipids are minor dietary components, they are the major components of biological membranes and their structure and quantity play an important role in the regulation of their biological functions and membrane fluidity. Generally, fats are characterized by gas chromatography (GC),which will provide only the FA profile of fats without any information on species composition or location of FA. High-performance liquid chromatography (HPLC) can be used to separate lipids according to their total FA profile including unsaturation but without separation according to the stereochemistry of the individual lipid molecule. To achieve a complete characterization of lipid molecules, a combination of thin-layer chromatography (TLC), reverse-phase HPLC, Grignard degradation, and chiral phase HPLC is normally required. It is a tedious, time-consuming procedure. An alternative is on-line coupling chromatography with a spectrometric or spectroscopic method; the former can separate lipids into different classes or molecular species, whereas the latter can provide structural information that can be used for identification and quantification. In this chapter, several hyphenated techniques and their applications in lipid analysis are summarized.
Hyphenated Mass Spectrometry
Mass spectrometry (MS) is one of the powerful techniques for structure elucidation. It is nondiscriminatory, that is, it generates data from virtually any ion in an ion source
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in proportion to its concentration or partial pressure. If a mixture is placed in the ion source, one obtains ions from each of the ionizable components in the mixture. One way to alleviate this problem is to use a hyphenated chromatographic technique for sequentially introducing purified compounds from a complex mixture into a mass spectrometer for on-line analysis. These hyphenated techniques are very useful for lipid analysis; chromatographic methods provide excellent separations, and the mass spectrometer works as a universal detector.
Gas Chromatography/Mass Spectrometry Gas chromatography (GC) is the chromatographic method used primarily for the separation of lipids, and especially for FA and FA derivatives. Instrumental, theoretical, and practical guidelines for chromatography can be obtained easily from books and review articles. GC in combination with MS has become one of the powerful tools for lipid analyses; it has been used widely in the analysis of derivatives of various lipid components. Electron impact (EI) ionization and chemical ionization (CI) are two frequently used ionization methods for GCMS. EI is one of the oldest types of ionization techniques, and it is also the most commonly used and highly developed ionization method. EI can produce a quantity of fragment ions, which can be used as fingerprints for the identification of unknowns; however, it generally has the drawback of lacking molecular ions. Rearranged ions can also be obtained as a result of the formation of new bonds between different parts of the molecule. The CI technique was developed in the 1960s and became commercially available in the 1970s. CI results from ion-molecule chemical interactions involving a small amount of sample with an extremely large amount of reagent gas. It is considered to be a soft ionization technique and is a preferred alternative for achieving information about molecular masses. The spectra obtained with CI contain essentially only molecular mass information because fragmentation is either absent or much reduced due to the absence of carbon-carbon cleavage reactions. The small amount of fragmentation of CI provides an increase in sensitivity because most of the ionization is concentrated in the molecular ion. EI and CI complement each other very well; EI provides the fragment ions and information about structures, whereas CI provides information about molecular masses. FA and FA derivatives were the first lipid substances studied in detail by MS. FA are commonly analyzed by GC/MS as their methyl esters; many EI mass spectra of FA methyl esters (FAME) are available through a library search. An alternative approach is to form a special derivative of the carboxyl group to direct mass spectral fragmentation to ascertaining double-bond location. Some special derivatives of FA such as dimethyl disulfide adducts or trimethylsilyl ether derivatives were prepared to obtain information about the position of the double bonds in the molecule. The derivatization of FA to their 4,4-dimethyl oxazoline (DMOX) derivatives proved to be a powerful method in GCMS analysis of FA with a wide range of applicability. Detailed information about MS of FA derivatives can be obtained from a recent review by Dobson and Christie (8). These authors summarized the
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MS of FA derivatives, discussing FAME and nitrogen-containing derivatives, including picolinyl esters, pyrrolidide, and DMOX derivatives. A GCMS method for the determination of trans double bonds in FAME was reported by Mjas and Petterson (9). No difference between the spectra of cis and trans monoenes was observed; however, distinct differences in the EI mass spectra of methylene-interrupted tri- and tetra-unsaturated FAME were observed, and the differences were particularly related to the geometry of the central double bond in trienes or the two central double bonds in tetraenes. Trans geometry in these positions led to a significant decrease of d z 79 [C6H7]+,which was the base peak in the all-cis isomers. However, it is necessary to perform more studies if this method is to be used for quantitative analysis of trans FA. Because CI is a milder ionization technique, fragmentation of sample molecules is minimized. Even though there is no carbon-carbon cleavage in CI, cleavage from some functional groups is still possible. CI MS was used in positive mode for identification of chlorinated FA from fish (10). Ammonium adduct molecular ions of d z 382 ([M+NH,]+) and dechlorinated fragment ions of m/z 293 ([MH-2HCl]+), d z 310 ([M+NH4-2HC1]+), and d z 312 ([M+NH,-ClJ) were observed when ammonium was used as the reagent gas; a monochlorinated fragment ion of d z 346 ( [M-CH30-HCl]+)was also observed with very low intensity. EI MS showed monochlorinated fragment ions of d z 297 ([M-CH30-HCl]+) and dechlorinated fragment ions of d z 292 ([M-2HCl]+) and 261 ([M-CH30-2HC1]+) for both the reference compounds and methyl dichlorooctadecenoates found in the eel sample. Again, EI and CI complemented each other, with EI providing the characteristic fragments ions for the FAME, and CI providing the molecular mass and structural information. With the increased interest in the biological effects of epoxy FA, Wilson and Lyall (11) described a GCMS method for quantitative analysis of hydroxyl and epoxy FA derived from plasma lipids and adipose tissue. FA released from biological samples after hydrogenation and hydrolysis were methylated to FAME, and epoxy groups were quantitatively converted to methoxy-hydroxy groups. The methoxy- and hydroxyl-containing FAME were separated from saturated FAME by solid-phase extraction to eliminate chromatographic interference. Hydroxy and epoxy isomers could be measured in 0.5 mL plasma and 20 mg adipose tissue by EI GCMS after pyrolysis. The authors found that the hydroxyl isomer originated from the oxidation of linoleic acid, whereas the epoxy isomers were characteristic of the oxidation of oleic acid. GC/Combustion/lsotope Ratio Mass Spectrometry (GC/C/IRMS)
Isotope-ratio-monitoring, which was introduced in the 1970s (12,13), offers the advantage of precise quantification of isotopes without radiation hazards. When the enrichment of a stable isotope is >1%, several MS techniques can be used for distinguishing isotopes. The isotope ratio (IR) MS is used for detecting low level of tracers,
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but it is restricted to low-molecular-weight stable gases, and it is a compound-specific analysis. Samples have to be converted to a suitable gas. Isotope-ratio-monitoring after on-line combustion (C) is a new method for GCMS. It allows the direct measurement of isotope ratios of individual molecular components of mixtures. For GC/C/IRMS, the GC is used to separate the sample mixture into individual components, which are converted to CO, in the combustion interface. Quantitative combustion is the most important key for accurate analysis of 13C in organic material. The mass spectrometer has an EI ion source and a multiple Faraday cup collector, which is used to measure the isotopomer ion currents simultaneously. GC/C/IRMS proved to be a good method for studies of the metabolism of 13Clabeled lipids. A short review on measurement and application of stable isotopes in FA was given recently by Scrimgeour (14). The metabolism and interconversion of dietary lipids were also studied by GC/C/IRMS (15-17). After intake of 13C 16:O triacylglycerol (TAG), enrichment of 13C was detected for both nonesterified FA (NEFA) and TAG in plasma by GC/C/IRMS, and maximal values of mole percentage excess (MPE) of 13C 16:O were significantly higher in NEFA than in TAG (15). This method allowed accurate and reproducible measurements of enrichment as low as 0.009 MPE in a range between 0 and 0.65 MPE. Croset et al. (17) studied the metabolism of 13C 22:6n-3 TAG in more detail by separating different lipoproteins and lipid classes. Blood samples were taken from healthy subjects at various periods after ingestion of a yogurt containing tracer TAG, and different lipoproteins were separated from the platelet-rich plasma. Total lipids were extracted from plasma, platelets, and lipoproteins and separated on TLC; the FA from different lipid fractions were studied by GC/C/IRMS. Maximal labeling was observed in the TAG of the VLDL + chylomicron fraction 2 h after ingestion; concomitant with the TAG utilization of this fraction by lipoprotein lipase from tissues, unesterified 13C 22:6n-3 appeared in the plasma albumin. 13C 22:6n-3 bound to albumin was present in NEFA mainly before 12 h postingestion; after that period, lysophosphatidylcholine (lysoPC) bound to albumin carried higher 13C 22:6n-3 concentrations. Thus, 13C 22:6n-3 esterified in TAG was rapidly absorbed and redistributed within plasma lipoproteins. A retroconversion of 13C 22:6n-3 was also detected in HDL PC in a similar study (18) by the appearance of 13C22511-3 and 13C 20511-3. Structured lipids can be designed and produced for different groups of consumers; for example, they can be used in infant formulas and low-energy structured lipids (19-21). Specific structured TAG containing essential FA and medium-chain FA were considered to be special nutrients in medical applications for patients with cystic fibrosis or short bowel syndrome (4,20,22). GC/C/IRMS was used in a study on the absorption of structured lipids. The lymphatic recoveries of intragastrically administered L*L*L*, M*M*M*, ML*M and ML*L* (*=13C-labeled FA; L, longchain FA; M, medium-chain FA) in rats were examined. Lymph lipids were separated into lipid classes and analyzed by GC/C/IRMS; the results demonstrated a tendency toward faster lymphatic recovery of long-chain FA after administration of specific structured TAG compared with long-chain TAG (23).
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Liquid Chromatography/MS and Tandem MS
Liquid chromatography (LC) is another frequently used chromatographic method in lipid analysis, especially for the separation of intact lipid molecules. It includes both TLC and HPLC. Normal-phase TLC and HPLC can be used for separation of the lipid classes and purification of lipids, whereas reverse-phase HPLC is used for separation of molecular species for FA, TAG, and phospholipids. On-line coupling of MS with HPLC is a fast and sensitive method for elucidation of intact lipid structures. A proper interface is required to solve the incompatibility problem of HPLC and MS. A monodisperse aerosol-generation interface, a moving-belt interface, direct inlet introduction, and atmospheric pressure ionization (API) interface were used for LCNS. In API LCNS, ions are formed from a liquid flow introduced into a source region maintained at atmospheric pressure. The coupling of a liquid flow inlet to an API source offers some advantages over other approaches; for example, it avoids problems associated with the introduction of a liquid flow directly into a high vacuum, thus making it possible to introduce the conventional HPLC methods directly into LC/MS. The two ionization methods frequently associated with API for L C N S are electrospray (ES) and atmospheric pressure chemical ionization (APCI). ES is a technique based on liquid-phase ionization and comprises three basic steps, i.e., nebulization and charging, desolvation, and ion evaporation. The HPLC effluent containing the analyte ions emerges from the tip of the nebulizing needle and is nebulized. The needle is at the ground potential surrounded by a semicylindrical electrode to which high voltage is applied. The potential difference between the nebulizer and the counter electrode produces a strong electric field that charges the surface of the emerging liquid and forms a fine spray of charged droplets. A high-pressure gas flow assists the nebulization. The charged droplets are attracted toward the capillary sampling orifice through a counterflow of heated nitrogen drying gas, which shrinks the droplets and carries away uncharged material. The droplet continues to shrink until the repulsive electrostatic forces exceed the droplet cohesive forces leading to droplet explosions. This process is repeated until the analyte ions are ultimately desorbed into the gas phase. The ions are driven by strong electric fields on the surface of the microdroplets. The emerging gas-phase ions are then passed through the capillary sampling orifice into the low-pressure region of the ion source, and on to the mass analyzer. The APCI process begins with gas-assisted nebulization into a hot, typically 25O40O0C, vaporizer chamber that serves to rapidly evaporate the spray droplets. The results are a gas-phase HPLC solvent and analyte molecules. The gas-phase solvent and analyte molecules are ionized by the discharge from a corona needle. Similar to the processes encountered in positive CI for G C N S , the protonated solvent transfers a proton to the analyte if the proton affinity of the analyte is greater than that of the solvent. The analyte ions are then transported to the mass analyzer. The APCI process may be considered as an evaporation followed by ionization;
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thus, it is useful only for those samples that can be vaporized. The APCI process usually results in an ion with a single charge. API is a relatively soft-ionization technique, producing primarily pseudomolecular ions. Collision-induced dissociation (CID), a process of colliding ions with neutral gas molecules to cause fragmentation, is helpful for both qualitative analysis and quantification. Qualitatively, structural information about the molecule is revealed; quantification specificity is increased by the presence of confkmatory ions. Using high potentials, CID can provide a fingerprint spectrum that is characteristic for the molecular structure; however, it will also result in the loss of the molecular ions. Therefore, it is necessary to optimize the LCMS system for special applications to obtain the best ionization stability, sensitivity, and fragmentation. CID is also compound dependent; thus, the degree of fragmentation should be optimized experimentally. Tandem MS such as a triple quadrupole has special advantages in lipid analysis because it can identify and quantify co-eluting components. For a triple quadrupole, three quadrupoles are situated in series between the ion source and the ion detection system; the first quadrupole is used for scanning or selective monitoring of parent ions, the second quadrupole is used for dissociation of parent ions into smaller fragments, and the third quadrupole is used for scanning or selective monitoring of the daughter ions. Tandem MS may also be used directly in lipid analysis without chromatographic separations.
Analysis of Neutral Lipids. There are a number of studies on the identification and quantification of TAG from different dietary sources and biological samples. Laakso and Manninen (24) summarized mass spectrometric techniques for the analysis of TAG. Recently Dorschel (25) characterized TAG species of peanut oil by HPLC combined with tandem MS; a total of 168 TAG species were identified even though only 27 species was presented in the report. APCI LCMS was used in the identification of diacylglycerol (DAG) and TAG molecular species in a structured-lipid sample. The most distinctive differences between the DAG and TAG molecules were found to be the pseudomolecular ions and the relative intensity of monoacylglycerol fragment ions (26). An ammonium adduct molecular ion [M+NH,]+ was observed for all TAG; protonated molecular ions were produced for TAG containing unsaturated FA, and the intensity increased with increasing unsaturation. DAG fragment ions were also formed for TAG. The ammonium adduct molecular ion was the base peak for TAG containing PUFA, whereas the DAG fragment ion was the base peak for TAG containing saturated and monounsaturated medium-chain and long-chain FA. The most abundant ion for DAG, however, was the pseudomolecular ion [M- 17]+, and the relative intensity of the monoacylglycerol fragment ion was also higher than that for TAG. Those distinctive differences between the DAG and TAG spectra were utilized for rapid identification of the acylglycerols in structured-lipid samples (26). Hsu and Turk (27) studied TAG as their lithiated adducts by EI MS using lowenergy CID on a triple quadrupole. The ES tandem spectra contained [M+Li-
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R,CO,H]+, [M+Li-R,,CO,Li]+, and [R,,CO]+ions, which permitted assignment of the masses of fatty acyl groups. Relative abundances of these ions reflect positions on the glycerol backbone to which fatty acyl groups were esterified; fragment ions reflecting neutral loss of the sn-2 fatty acyl groups either as a free FA or as a lithium salt were less abundant than the corresponding ions reflecting such losses of either the sn-1 or the sn-3 fatty acyl groups. Tandem MS based on ammonia negative ion CI was applied in an analysis of the regioisomeric structure of different structured TAG (28,29). CID of parent TAG with argon was used to produce daughter ion spectra with appropriate fragmentation patterns for structure determination. Fatty acyl groups were identified according to [RCO,]- ions in the daughter ion spectra. [M-H-18:2-100]- and [M-H8:0-100]- ions ( d z 225 and 361) were detected for both 8:O-18:2-8:0 and 8:O-8:O18:2, but the ratio of the intensities of these ions increased by a factor of 18 when 8:O18:2-8:0 was compared with 8:O-8:O-18:2. This increase indicated the easier formation of [M-H-8:0-100]- fragment ions when 8:O was located at the primary positions in the TAG. By using the standard curve for the ratios of [M-H-RCO,H-100]- ions corresponding to each [RCOJ ion determined with known mixtures of sn-1/3 and sn-2 regioisomers of structured TAG, it was possible to determine the proportions of different regioisomers in unknown samples. The ratio of [M-H-18:2-100]-/[M-H-8:0-100]ions as a function of the molar proportion of 8:O-8:O-18:2 (MML) and 8:O-18:2-8:0 (MLM) was determined on the basis of CID spectra. A linear relation between the ratio of ion intensities and the molar proportions of MLM-MML-type TAG isomers in the mixture was observed, which enabled the quantification of isomers in unknown mixtures (29). APCI L C N S was also used in the identification of TAG molecular species in lymph samples from rats given either a structured lipid or safflower oil (30). The TAG composition of lymph varied significantly between structured TAG and safflower oil. The lymph TAG were identified from their ammonium adduct molecular ions and DAG fragment ions. In addition to the intact MLM-type structured TAG, MLL- and LLL-type TAG were also identified in lymph. Therefore, the absorption pathway of MLM-type structured TAG was suggested to be similar to that of conventional longchain TAG, i.e., they were hydrolyzed into 2-monoacylglycerol and medium-chain FA, which were then used for resynthesis of TAG. One of the methods often used in stereospecific analysis of TAG is to separate the diastereomeric naphthylethylurethanes (NEU)of DAG derived by Grignard degradation on normal-phase HPLC. Recently Agren and Kuksis (31) reported an LC/MS method for the analysis of diastereomeric DAG NEU. Even though sn-1,2- and sn-2,3diastereomers were not resolved completely, ES MS could complement the resolution by allowing minor unresolved components to be identified. Both ammonia adduct ions [M+18]+and sodium adduct ions [M+23]+were formed when ES was used in the positive mode. DAG-like ions [M-NEU]' were also observed.
Analysis of Polar Lipids. Phospholipids and sphingolipids occur in cell membranes as mixtures of molecular species; their complexity depends on the size and
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degree of unsaturation of the acyl groups according to the biological source. The composition and content of polar lipids are altered in physiologic and pathologic cellular processes. Therefore, it is important to study the structures of these lipids. HPLC equipped with a diol column and plasmaspray tandem MS was used in a study of phospholipids (32). DAG- and monoacylglycerol-derived fragment ions observed in the positive mass spectra were used for quantification, whereas CID of DAG fragments revealed the FA composition of the native lipid. Two-dimensional (2D) analysis of phospholipids from cultured cells by capillary L C N S was reported by Taguchi et al. (33). To analyze a very small amount of phospholipids, a capillary silica column was selected. ES was selected as the ionization method, and it was used in both positive and negative modes. Spectra obtained under low CID showed molecular ions, whereas spectra obtained under high CID showed fragment ions. In negative mode, fragment ions originated from fatty acyl groups, and polar head groups of phospholipids were detected at specific elution times. The combined information was used in the identification of phospholipids. There are many studies on the molecular species of PC by L C N S or tandem MS. Dobson and Deighton (34) studied the molecular species of PC from soybean, egg yolk, and bovine liver by LCNS. APCI was used in the positive mode to study the DAG nicotinate derivatives of phospholipids; UV was used for quantification, whereas MS was used for identification. The structure of PC was deduced from pseudomolecular ions [MH-123]+and monoacyl fragment ions; acyl chain regioisomers were distinguished by the ratio of [MH-RCHCO]+ions. The most abundant ions in the negative ion spectra of PC and phosphatidylethanolamine (PE) were the sn-1 and sn-2 carboxylate ions (35,36). Nucleophilic attachment of the anionic phosphate onto the C-1 or the C-2 of the glycerol to which the fatty acyl groups attached expelled the sn-1 or sn-2 carboxylate anion, respectively, and this pathway was more favorable at sn-2 than at sn-1 (35,37). Vernooij et al. (35) utilized the difference in abundance to assign the acyl chain positions and determine the composition of positional isomers. A different(AQ5) result was reported for diacylglycerophosphatidic acids (PA) by Hsu and Turk (38), who studied charge-driven fragmentation processes in PA upon low-energy CID. They suggested that the abundance of [R,CO,]- > [R,CO,]- for PA could be attributed to the fact that the [M-H-RCO,H]- and the [M-H-RCH=C=O]- ions might undergo further fragmentation under the applied CID after they were formed (38). Another study by Hsu and Turk (39) on the characterization of phosphatidylinositol (PI), PI monophosphate (PI-P) and PI bisphosphate (PI-P2) with tandem MS suggested that relative intensities of the [RCO,H]- ions did not reflect their positions on the glycerol backbone because further dissociation of [M-H-RC02H-inositol]-, [M-H-RCO,H]-, and [M-H-RCH=C=O]- also yielded carboxylate anions, whose abundance was affected by the collision energy applied. Therefore, the determination of regiospecificities based on intensities of carboxylate anions was not reliable (39). Ekroos et al. (37) further examined the way in which the intensity of fragments depended on the applied collision energy and found that with increasing collision energy, the inten-
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sity of precursor ions decreased concomitantly with increasing intensity of acyl anion fragments. The intensity of acyl anions decreased when the collision energy was too high, presumably because it compromised focusing and steering of the ion beam in the mass spectrometer. They found a linear regression (R2 = 0.998) between the ratio of intensities of the demethylated lysoPC fragment ions rendered via neutral loss of fatty acyl groups as ketenes (Lee, [M-15-RCH=C=O]-) and the abundance of isomeric species. The intensity of other fragments or a combination of intensities of fragments observed in the MS3 spectra did not correlate well with the mol% of the isomers, most likely because the yield of fragments from other fragmentation pathways was less position specific than that of ketene loss. Moreover, they also found that lysoPA fragments formed by the loss of ketene from the sn-2 position enabled accurate estimates of the mol% in mixtures of PA16:0/18:1 and PA18:1/16:0 (37). The bioactivities of sphingolipids are of great interest and depend on their structures. Sphingosine (SPH) comprises the backbone of sphingolipids and is a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. HPLC with tandem MS was used for the quantification of SPH and spinganine (SPA) from crude lipid extracts (40). ES in the positive mode was used for sphingosine analysis. The production spectrum of SPH showed fragments of m/z 282, 264, and 252, resulting from a loss of one water molecule, two water molecules, and one water and formaldehyde molecule, respectively. A nonnaturally occurring species, SPH17:O, was added as an internal standard before lipid extraction and used for quantification. Sphingomyelin (SM) also formed highly abundant [M+H]+ions (41). A specific ion of m/z 184 was formed for SM by cleavage of the phosphorylcholine head group. Kerwin et al. (42) identified molecular species of SM and phospholipids using ES MS and tandem MS. Ions corresponding to the head group itself or the loss of the head group from the molecular adduct ions formed in positive ion ES provided information on the nature of the head group, whereas [RCOOI- ions formed in negative ion ES provided information on acyl constituents. The nature of alkyl or alkenyl substituents in PE molecular species was identified from residual ions after the loss of ethanolamine plus the loss of the acyl groups at the sn-2 position, and cyclization of phosphate oxygen with C-2 of glycerol. However, they could not provide any information on the position of fatty acyl groups by using ES MS, and the system was not capable of differentiating in all instances between alkyl-acyl and alkenyl-acyl substituents without earlier separation of these lipid subclasses. Recently, Isaac et al. (43) used a capillary LC/MS with ES in positive mode to analyze PC and SM molecular species from brain extracts. Protonated molecular ions and sodium adduct molecular ions were used in the identification. Tandem MS spectra of selected PC and SM ions were used to confirm their structural assignments. Both PC and SM could be detected in the low fmol range. Postle et al. (44) studied phospholipid molecular species from different mammalian lung surfactants by tandem MS. The molecular species of phospholipids in
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lipid extracts of surfactants were analyzed on a triple quadrupole mass spectrometer with ES. PC and SM were detected under positive ionization conditions, whereas phosphatidylglycerol (PG) and PI were detected under conditions of negative ionization. Structural identities of individual phospholipid species were determined by tandem MS. Fatty acyl compositions were determined as product ions under negative ionization with CID. The fractional concentrations of individual molecular species within a phospholipid class (PC, PG, PI) were calculated from their ion current response relative to that of the relevant internal standard after correction for the contribution from the 13C isotope effect. They found that the dominant PC specie was PC 16:0/16:0, and only minor variations existed across the animal species, i.e., humans, rabbits, rats, and guinea pigs; however, there were wide variations of PG and PI concentrations and compositions (44). PC16:0/16:0 is generally accepted as an important pulmonary surfactant in the mammalian lung because of the rigidity of the two saturated palmitoyl moieties. It was proposed that the enrichment of PC16:0/16:0 within the surface film makes the surfactant capable of withstanding the high surface pressures generated at the aidliquid interface of the mammalian lung. The instrument responses for different phospholipid classes are different, depending on the head group and the solvent used. It should also be noted, however, that not all molecular species of the same phospholipid class are detected with equal efficiency. Koivusalo et al. (45) studied the effects of acyl chain length, the level of unsaturation, and lipid concentration on instrument response. They found that the instrument response for both saturated and unsaturated phospholipid species decreased with an increase in acyl chain length, and this effect became increasingly prominent with increasing overall lipid concentration. The degree of acyl chain unsaturation also had a significant effect on instrument response, but it diminished with progressive dilution. Because of the high sensitivity of ES MS for lipid analyses, multiple ES tandem MS techniques have been developed and used extensively for the analyses of various classes, subclasses, and individual molecular species of lipids from biological sources (46,47). Murphy et al. (48) summarized the development and applications of ES MS in a recent review. The major advantage of multiple ES MS in lipid analyses is the fast analysis because it can be used directly in analyzing crude lipid extracts without chromatographic separations. For instance, ES MS was used in both positive and negative modes to determine the alterations in individual molecular species of human platelet phospholipids during thrombin stimulation (49). PI and PS were analyzed in diluted platelet extracts in the negative mode; PC was analyzed in the positive mode, and PE in the diluted platelets was also analyzed in the negative mode after the addition of NaOH. The individual molecular species of different phospholipids were quantitated by comparison of the intensity of molecular ions with that of internal standards. The authors found that plasmenylethanolamines were the major storage depot of arachidonic acid in resting platelets and the major source of arachidonic acid mobilized after thrombin stimulation of human platelets. Recently, we studied spleen lipids of rats using ES MS and found significant differences in polar
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lipid composition between diabetic rats and that of control group (Mu et al., unpublished results). These results suggest that ES MS may be used in the future as a quick method for clinical diagnosis. Polar lipids of plant chloroplasts were identified by ES tandem MS by Welti et al. (50,51). Polar lipids in each class were identified by precursor or neutral loss scanning of head group-specific fragments. Different head group scans were conducted sequentially to obtain a complete polar lipid molecular species profile. The uncharged galactoglycerolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, were studied in the presence of sodium acetate, which promotes the formation of sodium adducts; the negatively charged sulfoquinovosyldiacylglycerol and all phospholipids were analyzed in the presence of ammonium acetate. Analysis of Lipid Oxidation Products. FA hydroperoxides are labile key intermediates in lipid metabolism. They are normally analyzed by G C N S after derivatization. Schneider et al. (52), however, used HPLC with ES tandem MS in a direct analysis of FA hydroperoxides. Ammonium adduct molecular ions were formed in the presence of ammonium acetate and used for identification of FA hydroperoxides. Low-energy CID of the ammonium adduct molecular ions resulted in the loss of hydrogen peroxide and the formation of characteristic ions, which were used in the identification of 9and 13-regioisomeric FA hydroperoxides. This method was approved as a rapid method for identification of FA hydroperoxides, and it opened a versatile analytical approach for the structure-specific determination of labile lipid mediators in biological samples (52). Oxidative damage of biological tissues is often determined by analyzing degradation products of lipid peroxidation. L C N S , however, can provide the possibility for characterization of intact oxidized phospholipids in oxidatively stressed mammalian cells. It is also able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. Therefore, it allowed the detection of monohydroperoxides of PC16:0/18:2 and PC18:0/18:2 in U937 and HL60 cells after treatment in vitro with butylhydroperoxide + Fe2+; the membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used (53). Another study on phospholipid oxidation using L C N S was reported by Jerlich et al. (54), who studied the oxidation of LDL by HOC1, which is a highly toxic oxidant produced by myeloperoxidase in phagocytes. They detected chlorohydrin products from phospholipids containing 18:2n-6 and 20:4n-6 by LCMS, thereby providing the first direct evidence that lipid chlorohydrins rather than peroxides were the major products of HOC1-treated LDL phospholipids. Analysis of Fat-Soluble Vitamins. Vitamin E is the most potent lipid-soluble antioxidant in vivo, and it is normally analyzed by HPLC with UV or fluorescence detection. L C M S is a better alternative for the analysis of vitamin E because it is more sensitive and can also provide structural information. To study the delivery of vitamin E to human skin, Vaule et al. (55) used L C M S to trace deuterated a-
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tocopherol. APCI was chosen as the ionization method and used in the negative mode. LC/MS was used for quantification of deuterium-labeled and -unlabeled aand y-tocopherols at their d z [M-ll-, using single ion recording. They found a linear range for a-tocopherol from 1 to 100 pmol with a detection limit of 93 fmol (40 pg). The method was twice as sensitive for y-tocopherol as for a-tocopherol because of greater ionization efficiency for y-tocopherol. Another important fat-soluble vitamin is vitamin A, which is essential for vertebrate survival. It includes all compounds that possess the biological properties of the parent compound, retinol. The most extensive biological influences of vitamin A occur via the oxidation of retinol to retinoic acid, the transcriptionally active retinol metabolite. McCaffery et al. (56) reported an HPLC method with tandem MS for the analysis of retinoic acids from rat liver. The retinoids were separated by normal-phase HF'LC; APCI was applied as the ionization method for MS. Each retinoid was detected by a unique series of MS" function sets at selected CID. All-trans 9-cis, and 1 3 4 s retinoic acid isomers were separated. Chithalen et al. (57) studied the metabolites of all-trans-retinoic acid using HPLC with tandem MS. The retinoic acids and their metabolites were separated on a C18 column, and tandem MS was performed using ES in the negative mode. Characterization of retinoid metabolites was based on the following three criteria: HPLC retention time, UV spectra, and mass spectra. A prominent molecular ion [M-11- was observed for retinoic acid and its metabolite; its daughter fragment ions were used for further characterization.
GC/Fourier Transform Infrared Spectroscopy Infrared spectroscopy (IR) can be used in the determination of FA configurations and thereby provide useful structural information about functional groups. On-line coupling of IR with chromatography allows the measurement of IR spectra for individual compounds and isomers with additional chromatographic data such as retention times; the retention time is used for identification of FA by comparing the time with that of a standard, whereas the spectra are used to confirm their geometric configurations. For example, it can measure the absorption of trans double bonds in oils and fats at -967 cm-', and the principle is used in several standard analytical procedures for determination of trans FA. Fourier Transform Infrared Spectroscopy (FTIR) is the preferred method for infrared detection of chromatographically separated species because of its rapid scanning features. Details about the instrumentation and interfaces of chromatographyFTIR are available in a book by White (58). Applications of IR in lipid analysis were summarized in a review by Ismail et al. (59). There are also reviews on the applications of GCETIR in lipid analysis by Le QuCrC (60) and Mossoba et al. (61). Hydrogenation of vegetable oils often leads to the isomerization of naturally occurring cis unsaturated FA to trans isomers, which was shown to interfere with
lipid metabolism. Both GCDR and GC/FTIR were used in the analysis of trans FA in hydrogenated oils and margarines made from partially hydrogenated oils
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(62-66). Mossoba et al. (63) studied FAME isomers of 18:3 and 18:2. The FAME of isomerization products of 18:3 and different hydrogenated soybean oils as well as margarine were separated on a CP-Sil-88 capillary column. Co-eluting of positional isomers with the same configuration or different geometric isomers from margarine and hydrogenated soybean oil could not be excluded. The authors observed gradual variations in spectral features between the isomers; the relative intensity of the cis =CH out-of-plane deformation vibrations increased, whereas it decreased for trans =CH with a progressive increase in cis character and decrease in trans character. They found that the 18:3 constituents of partially hydrogenated soybean oil were dependent on hydrogenation conditions. At an iodine value of 111, methyl linolenate and its tn-trans, di-trans mono-cis, and mono-trans di-cis geometric isomers were identified. At a higher iodine value of 123, the tri-trans isomer was not observed, whereas at a lower iodine value of 96, tri-cis isomer was no longer found. They also found tri-cis and mono-trans di-cis configurations for the margarine analyzed. A similar method was used in confirmation of the doublebond configuration of conjugated 18:2 isomers (67). Recently Mjos and Pettersen (68) analyzed the FA composition of partially hydrogenated fish oils and partially hydrogenated vegetable oils using GCAR with a lightpipe interface; IR was the only detector used in their analyses. There was no separation of trans and cis isomers on the GC column because they wanted to quantify the isomers by their IR spectra. The strongest signal at 3025 cm-' was used for quantification of cis double bonds, and the signal at 970 cm-' for quantification of trans double bonds. The numbers of cis and trans double bonds were predicted by multivariate partial least-squares regression of the IR spectra. The method was validated by summing the values to a total trans value and total unsaturation and comparing these values with that determined by AOCS methods; the GCAR provided a 10% overestimation of trans FA. Some of the cyclic FA are degradation products found in heated fats and are potential sources of dietary toxicity (69). A double bond configuration in cyclic FA can be determined by GClFTIR (70). GClMS is normally used together with GC/FTIR to confirm the molecular mass and the position of double bonds. Even though G C M S has been widely used in the analysis of unsaturated FA and identification of the position of double bonds, it cannot be used in the identification of cisltrans isomers without reference substances. A combination of G U M S with FTIR has an advantage in this respect. Wahl et al. (71) studied cisltrans isomers of FAME and DMOX of unsaturated FA using GC/FTIR/MS. Molecular mass and the degree of unsaturation of FA were determined from mass spectra of the FAME and DMOX derivatives, and the position of the double bonds was determined from the mass spectra of DMOX. Information about cisltrans isomers was obtained from their FTIR spectra by analysis of bands arising from C-H out-of-plane bending; both FAME and DMOX derivatives gave a band near 720 cm-' and a bond at 3012 cm-' for cis isomers, whereas they gave a band near 967 cm-' for trans isomers.
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Hyphenated Nuclear Magnetic Resonance Spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy is one of the powerful spectroscopic techniques for structure elucidation of unknown compounds, and it has been used also in lipid analyses. An NMR spectrum typically provides two kinds of information, i.e., the chemical shift of each signal and their relative intensities. The former can be used for elucidation of structures; the latter provides quantitative information of analytical value. The instrumentation and the underlying theory of NMR spectroscopy are well described in books devoted to NMR spectroscopy. Several reviews on the 13C NMR of FA and lipids are also available such as the book chapter by Gunstone (72). Hyphenated NMR is an alternative approach that can be used in the characterization of components of a mixture. Schiller et al. (73,74)combined several analytical methods such as MS, NMR, and IR spectroscopy in their analyses to obtain reliable information on detailed lipid composition. However, they concluded that separation by chromatographic methods is necessary (73). Hyphenated NMR with chromatography provides a fast analytical method and further explores the advantages of the individual techniques. It is also a suitable method for the analysis of unstable compounds because both light and oxygen may be excluded. A recent review by Albert et al. (75) summarized the on-line coupling of separation techniques to NMR including HPLC, gel permeation chromatography, and supercritical fluid chromatography. The review offers a detailed description about continuous-flow and stopped-flow NMR as well as the coupling techniques for different chromatographies and NMR. Even though LC/NMR has been widely used in the investigation of complex mixtures of organic compounds in polymer, pharmaceutical, and biomedical research, only limited information is available regarding its application in lipid analysis because the aliphatic region is difficult to resolve. Kleinwachter et al. (76) studied 2-trans,4-cis 10:2 as a microbial secondary metabolite from the genus Agromyces. To obtain rapid preliminary structure information, on-line LC/NMR and LC/MS analyses were performed on the lyophilized crude product. Only a few signals were observed in its 'H NMR spectrum because of the aliphatic skeleton, and two conjugated double bonds were identified. The 13C NMR spectrum suggested a carboxyl group conjugated to double bonds. Therefore the metabolite was identified as 2-trans,4-cis 10:2. The molecular mass was confirmed by LC/MS. The different isomers of vitamin E, a natural antioxidant, have different antioxidant abilities. As mentioned previously, they are often separated and quantitated by normal-phase or reverse-phase HPLC. Strohschein et al. (77) reported an LC/NMR method for analysis of a-,p-, y-, and 8-tocopherols with the advantage of NMR for structural identification and recognition of co-eluting peaks. The structure differences among these isomers could be monitored by the 'H NMR signals between 2,O and 2,2 ppm of the methyl groups attached to the aromatic ring or by examining the aromatic 'H NMR signals between 6.4 and 6.5 ppm. LC/NMR was
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also used in structure elucidation of p-carotene isomers (78). Reisomerization of the separated isomers was inhibited by the on-line coupled LC/NMR technique. A C30 bonded phase column was used in the study of thermally isomerized pcarotene to improve the separation of cis/trans isomers. Five dominated isomers were examined using 'H NMR. Both the 1D and 2D NMR spectra were recorded in the stopped-flow mode for the selected chromatographic peaks. Structure information was obtained by comparing simulated 1D spectra with the experimental data and also by analysis of the proton-proton connectivity in the 2D spectra. The isomerization shift of signal groups (A6 = 6cis - Gtrans) provided useful information for the assignment of structures. Deuterated solvents, which are conventionally applied for NMR spectrometry, are expensive, and that limits the application of such materials in conventional LC/NMR. Reductions in solvent consumption as well as higher separation efficiencies require miniaturized chromatographic separation techniques. A capillary LC/NMR method was reported by Albert et al. (79) to derive structural information about constituents of a mixture of vitamin A derivatives. They demonstrated the progress in capillary LC/NMR coupling and the possibility of obtaining a 2D NMR spectrum in the nanoliter scale. A novel hyphenated technique such as LC/NMR/MS offers a new approach for the structural elucidation of different compounds. It represents a comprehensive analytical system providing the complementary information of both NMR and MS in a single chromatographic separation. Sample molecules are separated by HPLC and characterized by their mass spectra, and their NMR spectra provide more detailed structural information especially important for isomers of identical molecular mass. The technique has been applied mainly to pharmaceutical drug metabolism research (80) and analyses of natural products (81-83). Wilson et al. (82) studied polyhydroxy steroids, which are widely distributed in plants. Because of the variety of similar ecdysteroids that can be encountered in plant extracts, it is often valuable to obtain both NMR and MS data to ensure unambiguous identification. In their study, HPLC was coupled in parallel to NMR and MS after UV detection with a split ratio of 95 to 5 for NMR and MS. Both on-flow and stopped-flow NMR detection were used; the former was used in screening, whereas the latter was used to obtain further NMR data on the peaks of interest. Integristerone A, 20-hydroxyecdysone, 2-deoxy20-hydroxyecdysone, and 2-deoxyecdysone were identified by this method. LC/NMR/MS was also used in the characterization of sesame oil extracts with a split ratio 1:20 (vol/vol) for MS and NMR (84). Sesame lignans such as sesamin and sesamolin are naturally occurring antioxidants in sesame oil. They were separated on a C18 column and identified by APCI MS. The spontaneous loss of a water molecule was observed for both sesamin and sesamolin; a deoxygenated fragment ion was also observed for sesamolin. Significant differences in chemical shifts were observed in their 'H NMR spectra; the spectrum of sesamolin was more complex than that of sesamin due to the loss of the symmetry axis in the molecule. This method enabled the characterization of sesame oil-derived lignans within a few minutes.
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Summary Chromatography is a powerful tool for the separation of lipids. GC and HPLC complement each other; GC is better for the separation of FA and FA derivatives, whereas HPLC is better for the separation of lipid classes and individual molecular species within a class. On-line coupling of GC or HPLC with spectrometric or spectroscopic methods allows a fast identification and quantification of lipid classes or individual species. GC/C/IRMS is a suitable method for studies of lipid metabolism using 13Clabeled lipids. GCMS is better for identification of unknown FA and the location of double bonds, whereas LCMS can be used for the analyses of intact lipid molecules such as TAG and phospholipids. The positional distribution of acyl groups in TAG and phospholipids can be determined by their spectra according to the different intensities of selected fragment ions. FTIR or NMR can complement MS by providing more information about the geometric distributions of double bonds. References 1. Mu, H., and C.-E. Hpry, Intestinal Absorption of Specific Structured Triacylglycerols, J. Lipid Res. 42: 792-798 (2001). 2. Hpry, C.-E., and H. Mu, Intestinal Metabolism of Interesterified Fat, in Intestinal Lipid Metabolism, edited by C. Mansbach, P. Tso, and A. Kuksis, Kluwer AcademicPlenum Publishers, 2000, pp. 383401. 3. Jandacek, R.J., J.A. Whiteside, B.N. Holcombe, R.A. Volpenhein, and J.D. Taulbee, The Rapid Hydrolysis and Efficient Absorption of Triglycerides with Octanoic Acid in the 1 and 3 Positions and Long-Chain Fatty Acid in the 2 Position, Am. J. Clin. Nutr. 45: 940-945 (1987). 4. Ikeda, I., Y. Tomari, M. Sugano, S. Watanabe, and J. Nagata, Lymphatic Absorption of Structured Glycerolipids Containing Medium-Chain Fatty Acids and Linoleic Acid, and Their Effect on Cholesterol Absorption in Rats, Lipids 26: 369-373 (1991). 5. Mu, H., and C.-E. Hpry, Effect of Medium-Chain Fatty Acids on Lymphatic Absorption of Essential Fatty Acids in Specific Structured Lipids, Lipids 35: 83-89 (2000). 6. Hay, C.-E., and H. Mu, Effects of Triacylglycerol Structure on Fat Absorption, in Fat Digestion and Absorption, edited by A. Christophe and S. de Vriese, AOCS Press, Champaign, IL, 2000, pp. 218-234. 7. Straarup, E.M., and C.-E. Hpry, Structured Lipids Improve Fat Absorption in Normal and Malabsorbing Rats, J. Nutr. 130: 2802-2808 (2000). 8. Dobson, G., and W.W. Christie, Spectroscopy and Spectrometry of Lipids-Part 2, Eur. J. Lipid Sci. Technol. 104: 3 6 4 3 (2002). 9. Mjas, S.A., and J. Pettersen, Determination of Trans Double Bonds in Polyunsaturated Fatty Acid Methyl Esters from Their Electron Impact Mass Spectra, Eur. J. Lipid Sci. Technol. 105: 156-164 (2003). 10. Mu, H., C. WCsen, P. Sundin, and E. Nilsson, Gas Chromatographic and Mass Spectrometric Identification of Tetrachloroalkanoic and Dichloroalkenoic Acids in Eel Lipids, J. Mass Spectrom. 31: 517-526 (1996).
11. Wilson, R., and K. Lyall, Simultaneous Determination by GC-MS of Epoxy and Hydroxy FA as Their Methoxy Derivatives, Lipids 37: 917-924 (2002).
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12. Sano, M., Y. Yotsui, H. Abe, and S. Sasaki, A New Technique for the Detection of Metabolites Labelled by the Isotope I3C Using Mass Fragmentography, Biomed. Mass Spectrom. 3: 1-3 (1976). 13. Matthews, D.E., and J.M. Hayes, Isotope-Ratio-Monitoring Gas Chromatography-Mass Spectrometry, Anal. Chem. 50: 1465-1473 (1978). 14. Scrimgeour, C.M., Measurement and Applications of Stable Isotopes in Fatty Acids, Eur. J. Lipid Sci. Technol. 104: 57-59 (2002). 15. Binnert, C., M. Laville, C. Pachiaudi, V. Rigalleau, and M. Beylot, Use of Gas Chromatographyfisotope Ratio-Mass Spectrometry to Study Triglyceride Metabolism in Humans, Lipids 30: 869-873 (1995). 16. Rhee, S.K., A.J. Kayani, A. Ciszek, and J.T. Brenna, Desaturation and Interconversion of Dietary Stearic and Palmitic Acids in Human Plasma and Lipoproteins, Am. J. Clin. Nutr. 65: 451-458 (1997). 17. Croset, M., N. Brossard, C. Pachiaudi, S. Normand, J. Lecerf, V. Chirouze, J.P. Riou, J.L. Tayot, and M. Lagarde, In Vivo Compartmental Metabolism of 13CDocosahexaenoic Acid, Studied by Gas Chromatography-CombustionIsotope Ratio Mass Spectrometry, Lipids 31: S 109-S 115 (1996). 18. Brossard, N., C. Pachiaudi, M. Croset, S. Normand, J. Lecerf, V. Chirouze, J.P. Riou, J.L. Tayot, and M. Lagarde, Stable Isotope Tracer and Gas-Chromatography Combustion Isotope Ratio Mass Spectrometry to Study the In Vivo Compartmental Metabolism of Docosahexaenoic Acid, Anal. Biochem. 220: 192-199 (1994). 19. Fomuso, L.B., and C.C. Akoh, Enzymatic Modification of Triolein: Incorporation of Caproic and Butyric Acids to Produce Reduced-Calorie Structured Lipids, J. Am. Oil Chem. SOC.74: 269-272 (1997). 20. Haumann, B.F., Structured Lipids Allow Fat Tailoring, Inform 8: 10041011 (1997). 21. Auerbach, M.H., P.W. Chang, R. Kosmark, J.J. O’Neill, J.C. Philips, and L.P. Klemann, Salatrim: A Family of Reduced-Calorie Structured Lipids, in Structural Modified Food Fats: Synthesis, Biochemistry, and Use, edited by A.B. Christophe, AOCS Press, Champaign, IL, 1998, pp. 89-1 16. 22. Bell, S.J., and B.R. Bistrian, Structured Triglycerides and Their Medical Applications, in Structural Modified Food Fats: Synthesis, Biochemistry, and Use, edited by A.B. Christophe, AOCS Press, Champaign, IL, 1998, pp. 189-196. 23. Vistisen, B., H. Mu, and C.-E. H@y,Recoveries of Rat Lymph Fatty Acids After Administration of Specific Structured 13C-Triacylglycerol, Lipids 38: 903-91 1 (2003). 24. Laakso, P., and P. Manninen, Mass Spectrometric Techniques in the Analysis of Triacylglycerols, in Spectral Properties of Lipids, edited by R.J. Hamilton and J. Cast, Sheffield Academic Press, Sheffield, 1999, pp. 141-190. 25. Dorschel, C.A., Characterization of the TAG of Peanut Oil by Electrospray LC-MS-MS, J. Am . Oil Chem. SOC.79: 749-753 (2002). 26. Mu, H., H. Sillen, and C.-E. Hay, Identification of Diacylglycerols and Triacylglycerols in Structured Lipid Sample by Atmospheric Pressure Chemical Ionization Liquid Chromatographyhlass Spectrometry, J. Am. Oil Chem. SOC.77: 1049-1059 (2000). 27. Hsu, F.-F., and J. Turk, Structural Characterization of Triacylglycerols as Lithiated Adducts by Electrospray Ionization Mass Spectrometry Using Low Energy Collisionally Activated Dissociation on a Triple Stage Quadrupole Instrument, J. Am. SOC. Mass Spectrorn. 10: 587-599 (1999).
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28. Mu, H., J.-P. Kurvinen, H. Kallio, X. Xu, and C.-E. Hgy, Quantitation of Acyl Migration During Lipase-Catalyzed Acidolysis, and of the Regioisomers of Structured Triacylglycerols Formed, J. Am. Oil Chem. Soc. 78: 959-964 (2001). 29. Kurvinen, J.-P., H. Mu, H. Kallio, X. Xu, and C.-E. H@y,Regioisomers of Octanoic Acid Containing Structured Triacylglycerols Analyzed by Tandem Mass Spectrometry Using Ammonia Negative Ion Chemical Ionization, Lipids 36: 1377-1382 (2001). 30. Mu, H., and C.-E. H@y,Application of Atmospheric Pressure Chemical Ionization Liquid Chromatography/Mass Spectrometry in Identification of Lymph Triacylglycerols, J. Chromutogr. B 748: 4 2 5 4 3 7 (2000). 31. Agren, J.J., and A. Kuksis, Analysis of Diastereomeric DAG Naphthylethylurethanes by Normal-Phase HPLC with On-Line Electrospray MS, Lipids 37: 613-619 (2002). 32. Valeur, A , , P. Michelse, and G. Odham, On-Line Straight-Phase Liquid ChromatographyPlasmaspray Tandem Mass Spectrometry of Glycerolipids, Lipids 28: 255-259 (1993). 33. Taguchi, R., J. Hayakawa, Y. Takeuchi, and M. Ishida, Two-Dimensional Analysis of Phospholipids by Capillary Liquid Chromatography/Electrospray Ionization Mass spectrometry, J. Mass Spectrom. 35: 953-966 (2000). 34. Dobson, G., and N. Deighton, Analysis of Phospholipid Molecular Species by Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry of Diacylglycerol Nicotinates, Chem. Phys. Lipids 111: 1-17 (2001). 35. Vernooij, E.A.A.W., J.F.H.M. Brouwers, J.K. Bosch, and D.J.A. Crommelin, RPHPLCESI MS Determination of Acyl Chain Positions in Phospholipids, J. Sep. Sci. 25: 285-289 (2002). 36. Brouwers, J.F.H.M., E.A.A.W. Vernooij, A.G.M. Tielens, and L.M.G. van Golde, Rapid Separation and Identification of Phosphatidylethanolamine Molecular Species, J. Lipid Res. 40: 164-169 (1999). 37. Ekroos, K., C.S. Ejsing, U. Bahr, M. Karas, K. Simons, and A. Shevchenko, Charting Molecular Composition of Phosphatidylcholines by Fatty Acid Scanning and Ion Trap MS3 Fragmentation, J. Lipid Res. 44: 2181-2192 (2003). 38. Hsu, F.-F., and J. Turk, Charge-Driven Fragmentation Processes in Diacyl Glycerophosphatidic Acids Upon Low-Energy Collisional Activation. A Mechanistic Proposal, J. Am. Soc. Muss Spectrom. 11: 797-803 (2000). 39. Hsu, F.F., and J. Turk, Characterization of Phosphatidylinositol, Phosphatidylinositol-4phosphate, and Phosphatidylinositol-4,5-bisphosphateby Electrospray Ionization Tandem Mass Spectrometry: A Mechanistic Study, J. Am. Soc. Mass Spectrom. 11: 986-999 (2000). 40. Lieser, B., G. Liebisch, W. Drobnik, and G. Schmitz, Quantification of Sphingosine and Sphinganine from Crude Lipid Extracts by HPLC Electrospray Ionization Tandem Mass Spectrometry, J. Lipid Res. 44: 2209-2216 (2003). 41. Sullards, M.C., Analysis of Sphingomyelin, Glucosylceramide, Ceramide, Sphingosine, and Sphingosine 1-Phosphate by Tandem Mass Spectrometry, Methods Enzymol. 312: 32-44 (2000). 42. Kerwin, J.L., A.R. Tuininga, and L.H. Ericsson, Identification of Molecular Species of Glycerophospholipids and Sphingomyelin Using Electrospray Mass Spectrometry, J. Lipid Res. 35: 1102-1 114 (1994).
43. Isaac, G., D. Bylund, J.E. Mansson, K.E. Markides, and J. Bergquist, Analysis of Phosphatidylcholine and Sphingomyelin Molecular Species from Brain Extracts Using
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48. 49.
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56. 57.
58. 59.
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Capillary Liquid Chromatography Electrospray Ionization Mass Spectrometry, J. Neurosci. Methods 128: 111-1 19 (2003). Postle, A.D., E.L. Heley, and D.C. Wilton, A Comparison of the Molecular Species Compositions of Mammalian Lung Surfactant Phospholipids, Comp. Biochem. Physiol. 129: 65-73 (2001). Koivusalo, M., P. Haimi, L. Heikinheimo, R. Kostiainen, and P. Somerharju, Quantitative Determination of Phospholipid Compositions by ESI-MS: Effects of Acyl Chain Length, Unsaturation, and Lipid Concentration on Insttument Response, J. Lipid Res. 42: 663-672 (2001). Han, X., and R.W. Gross, Structural Determination of Picomole Amounts of Phospholipids Via Electrospray Ionization Tandem Mass Spectrometry, J. Am. SOC.Muss Spectrom. 6: 1202-1210 (1995). Hsu, F.-F., A. Bohrer, and J. Turk, Formation of Lithiated Adducts of Glycerophosphocholine Lipids Facilitates Their Identification by Electrospray Ionization Tandem Mass Spectrometry, J. Am. SOC.Mass Spectrom. 9: 516-526 (1998). Murphy, R.C., J. Fiedler, and J. Hevko, Analysis of Nonvolatile Lipids by Mass Spectrometry, Chem. Rev. 101: 479-526 (2001). Han, X., R.A. Gubitosi-Klug, B.J. Collins, and R.W. Gross, Alterations in Individual Molecular Species of Human Platelet Phospholipids During Thrombin Stimulation: Electrospray Ionization Mass Spectrometry-Facilitated Identification of the Boundary Conditions for the Magnitude and Selectivity of Thrombin-Induced Platelet Phospholipid Hydrolysis, Biochemistry 35: 5822-5832 (1996). Welti, R., W. Li, M. Li, Y. Sang, H. Biesiada, H.-E. Zhou, C.B. Rajashekar, T.D. Williams, and X. Wang, Profiling Membrane Lipids in Plant Stress Response, J. Biol. Chem. 277: 31994-32002 (2002). Welti, R., X. Wang, and T.D. Williams, Electrospray Ionization Tandem Mass Spectrometry Scan Modes for Plant Chloroplast Lipids, Anal. Biochem. 314: 149-152 (2003). Schneider, C., P. Schreier, and M. Herderich, Analysis of Lipoxygenase-Derived Fatty Acid Hydroperoxides by Electrospray Ionization Tandem Mass Spectrometry, Lipids 32: 331-336 (1997). Spickett, C.M., N. Rennie, H. Winter, L. Zambonin, L. Land, and A. Jerlich, Detection of Phospholipid Oxidation in Oxidatively Stressed Cells by Reversed-Phase HPLC Coupled with Positive-Ionization Electroscopy MS, Biochem. J. 355: 449-457 (2001). Jerlich, A., A.R. Pitt, and R.J.S.C.M. Schaur, Pathways of Phospholipid Oxidation by HOCl in Human LDL Detected by LC-MS, Free Radic. Biol. Med. 28: 673-682 (2000). Vaule, H., S.W. Leonard, and M.G. Traber, Vitamin E Delivery to Human Skin: Studies Using Deuterated a-Tocopherol Measured by APCI LC-MS, Free Radic. Biol. Med. 36: 456-463 (2004). MaCaffery, P., J. Evans, 0. Koul, A. Volpert, K. Reid, and M.D. Ullman, Retinoid Quantification by HPLCMS", J. Lipid Res. 43: 1143-1 149 (2002). Chithalen, J.V., L. Luu, M. Petkovich, and G. Jones, HPLC-MS/MS Analysis of the Products Generated from All-Trans-Retinoic Acid Using Recombinant Human CYP26A, J. Lipid Res. 43: 1133-1 142 (2002). White, R., Chromatography/Fourier Transform Infrared Spectroscopy and Its Applications, Dekker, New York, 1991. Ismail, A.A., A. Nicodemo, J. Sedman, F.R. van de Voort, and I.E. Holzbaur, Infrared
Spectroscopy of Lipids: Principles and Applications, in Spectral Properties of Lipids, edited by R.J. Hamilton and J. Cast, Sheffield Academic Press Ltd., Sheffield, 1999, pp. 235-269.
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60. Le Qutrt, J.N., Gas Chromatography-Fourier Transform Infrared Spectrometry in the Analysis of Fatty Acids, in New Trends in Lipid and Lipoprotein Analyses, edited by J.L. StbCdio and E.G. Perkins, AOCS Press, Champaign, 1995, pp. 232-241. 61. Mossoba, M.M., R.E. McDonald, M.P. Yurawecz, and J.K.G. Kramer, Application of On-Line Capillary GC-FTIR Spectroscopy to Lipid Analysis, Eur. J. Lipid Sci. Technol. 103: 826-829 (2001). 62. Mossoba, M.M., R.E. McDonald, J.-Y.T. Chen, D.J. Armstrong, and S.W. Page, Identification and Quantitation of trans-9,trans- 12-Octadecadienoic Acid Methyl Ester and Related Compounds in Hydrogenated Soybean Oil and Margarines by Capillary Gas Chromatography/Matrix Isolation/Fourier Transform Infrared Spectroscopy, J . Agric. Food Chem. 38: 86-92 (1990). 63. Mossoba, M.M., R.E. McDonald, D.J. Armstrong, and S.W. Page, Identification of Minor C18 Triene and Conjugated Diene Isomers in Hydrogenated Soybean Oil and Margarine by GC-MI-FT-IR Spectroscopy, J. Chromatogr. Sci. 29: 324-330 (1991). 64.Mossoba, M.M., R.E. McDonald, and A.R. Prosser, Gas Chromatographic/Matrix Isolation/Fourier Transform Infrared Spectroscopic Determination of TransMonounsaturated and Saturated Fatty Acid Methyl Esters in Partially Hydrogenated Menhaden Oil, J. Agric. Food Chem. 41: 1998-2002 (1993). 65. Mossoba, M.M., R.E. McDonald, J.A.G. Roach, D.D. Fingerhut, M.P. Yurawecz, and N. Sehat, Spectral Confirmation of trans Monounsaturated C18 Fatty Acid Positional Isomers, J. Am. Oil Chem. SOC.74: 125-130 (1997). 66. Ratnayake, W.M.N., R. Hollywood, E. O’Grady, and J.L. Beare-Rogers, Determination of cis and trans-Octadecenoic Acids in Margarines by Gas Liquid ChromatographyInfrared Spectrophotometry, J. Am. Oil Chem. SOC.67: 804-810 (1990). 67. Mossoba, M.M., Application of Gas Chromatography-Infrared Spectroscopy to the Confirmation of the Double Bond Configuration of Conjugated Linoleic Acid Isomers, Eur. J. Lipid Sci. Technol. 103: 594-632 (2001). 68. Mjas, S.A., and J. Pettersen, A Rapid Method for the Analysis of Hydrogenated Fats by GC with IR Detection, J. Am. Oil Chem. SOC.80: 839-846 (2003). 69. Mossoba, M.M., M.P. Yurawecz, J.A.G. Roach, H.S. Lin, R.E. McDonald, B.D. Flickinger, and E.G. Perkins, Rapid Determination of Double Bond Configuration and Position Along the Hydrocarbon Chain in Cyclic Fatty Acid Monomers, Lipids 29: 893-896 (1994). 70. SCbtdio, J.L., J.L. Le Quere, E. Semon, 0. Morin, J. Prevost, and A. Grandgirard, Heat Treatment of Vegetable Oils. I1 GC-MS and GC-FTIR Spectra of Some Isolated Cyclic Fatty Acid Monomers, J. Am. Oil Chem. SOC.64: 1324-1333 (1987). 71. Wahl, H.G., S.Y. Habel, N. Schmieder, and H.M. Liebich, Identification of cisltrans Isomers of Methyl Ester and Oxazoline Derivatives of Unsaturated Fatty Acids Using GC-FTIR-MS, J. High Resolut. Chromatogr. 17: 543-548 (1994). 72. Gunstone, F.D., High Resolution 13C NMR Spectroscopy of Lipids, in Advances in Lipid Methodology-Two, edited by W.W. Christie, The Oily Press Ltd., Dundee, 1993, pp. 1-68. 73. Schiller, J., 0. Zschornig, M. Petkovic, M. Miiller, J. Arnhold, and K. Arnold, Lipid Analysis of Human HDL and LDL by MALDI-TOF Mass Spectrometry and 31P-NMR, J. Lipid Res. 42: 1501-1508 (2001).
74. Schiller, J., R. Sub, M. Petkovic, G. Hanke,A. Vogel, and K. Arnold, Effects of Thermal Stressing on Saturated Vegetable Oils and Isolated Triacylglycerols-Product
Analysis by
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MALDI-TOF Mass Spectrometry, NMR, and IR Spectroscopy, Eur. J. Lipid Sci. Technol. 104: 496-505 (2002). 75. Albert, K., M. Dachtler, T. Glaser, H. Hhdel, T. Lacker, G. Schlotterbeck, S. Strohschein, L.-H. Tseng, and U. Braumann, On-Line Coupling of Separation Techniques to NMR, J. High Resolut. Chromatogr. 22: 135-143 (1999). 76. Kleinwachter, P., K. Martin, I. Groth, and K. Dornberger, Use of Coupled HPLC/'H NMR and HPLCESI-MS for the Detection and Identification of (2E,4Z)-Decadienoic Acid from a New Agromyces Species, J. High Resolut. Chromatogr. 23: 609-612 (2000). 77. Strohschein, S., M. Pursch, D. Lubda, and K. Albert, Shape Selectivity of C30 Phases for RP-HPLC Separation of Tocopherol Isomers and Correlation with MAS NMR Data from Suspended Stationary Phases, Anal. Chem. 70: 13-18 (1998). 78. Strohschein, S., M. Pursch, H. Handel, and K. Albert, Structure Elucidation of pCarotene Isomers by HPLC-NMR Coupling Using a C30 Bonded Phase, Fresenius J. Anal. Chern. 357: 498-502 (1997). 79. Albert, K., G. Schlotterbeck, L.-H. Tseng, and U. Braumann, Application of On-Line Capillary High-Performance Liquid Chromatography-Nuclear Magnetic Resonance Spectrometry Coupling for the Analysis of Vitamin A Derivatives, J. Chromatogr. 705: 303-309 (1996). 80. Corcoran, O., and M. Spraul, LC-NMR-MS in Drug Discovery, DDT 8: 624-63 1 (2003). 81. Wolfender, J.-L., S. Rodriguez, and K. Hostettmann, Liquid Chromatography Coupled to Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy for the Screening of Plant Constituents, J. Chromatogr. 794: 299-3 16 (1998). 82. Wilson, I.D., R. Lafont, J.P. Shockcor, J.C. Lindon, and J.K. Nicholson, High-Performance Liquid Chromatography Coupled to Nuclear Magnetic Resonance Spectroscopy and Mass Spectrometry Applied to Plant Products: Identification of Ecdysteroids from Silene otites, Chromatographiu49: 374-378 (1999). 83. Fritsche, J., R. Angoelal, and M. Dachtler, On-Line Liquid-Chromatography-Nuclear Magnetic Resonance Spectroscopy-Mass Spectrometry Coupling for the Separation and Characterization of Secoisolariciresinol Diglucoside Isomers in Flaxseed, J. Chromatogr. 972: 195-203 (2002). 84. Dachtler, M., F.H.M. van de Put, F. v. Stijn, C.M. Beindorff, and J. Fritsche, On-Line LCNMR-MS Characterization of Sesame Oil Extracts and Assessment of Their Antioxidant Activity, Eur. J. Lipid Sci. Technol. 105: 488-496 (2003).
Chapter 6
Supercritical Fluid Processing of Nutritionally Functional Lipids Jerry W. King Supercritical Fluid Facility, Los Alamos National Laboratory, Chemistry Division, C-ACT Group, Los Alamos, N M 87545
Introduction A synergism exists between the development of healthy foods and the use of critical fluid (CF) technology in their production. CF, especially carbon dioxide (CO,), offer an alternative medium with which to extract, enrich, react, and analyze foodstuffs, without resorting to the use of organic solvents that are of concern to consumers in the final products they ingest. Benign, low-temperature processing conditions are possible by using either liquid (LCO,) or supercritical carbon dioxide (SC-CO,) and similar fluids for the extraction, enrichment, and conversion of the lipid moieties that appear in foods and natural product matrices (1). Critical fluids such as CO,, propane, and GRAS (Generally Recognized as Safe) cosolvents have found commercial niches in the development of new food products, and a plethora of commercial products now exists that even notes the use of this technology on ingredient labels. Environmentally acceptable manufacturing processes have been the focus of a new technology, commonly called “green chemistry and engineering” ( 2 ) . Among the technologies prominent in this field are supercritical fluid (SF) technology and its many variants, namely, supercritical fluid extraction (SFE), supercritical fluid fractionation (SFF),supercritical fluid chromatography (SFC), and reactions conducted in supercritical fluid media (SFR). The nature of CF processing has also changed over the last 30 y because it is no longer limited only to SFE for the isolation of specific components from natural matrices. As shown in Figure 6.1, fractionation and reaction modes utilizing SF are becoming more common, and coupling them with SFE can be used to produce a customized end-product or fraction that has implications in maintaining a healthy lifestyle. Traditionally the first processing step using CF, as illustrated in Figure 6.1, was the use of SFE to produce a desired end-product, e.g., the removal of caffeine from coffee to produce a decaffeinated product, or isolation of a hops extract for beer flavoring. Today, processes will frequently use an SFF process, either initially or coupled with SFE, to produce value-enhanced foods and nutritionally enhanced
products. The recent literature (3) also demonstrates that SFR offers a unique processing approach to modifying food ingredients with respect to their composition 99
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Fig. 6.1. Process sequences
utilizing supercritical fluid media for isolating or synthesizing desired end products.
of flavor, for example, and this is especially true for lipid moieties because of their relative high solubilities in compressed CO, and propane (4). The application of SF and similar media for the processing of agricultural or natural products traditionally focused on the extraction mode utilizing CO,. Examination of alternative fluids such as subcritical water expanded the “natural” fluid base available to the processor of foodstuffs. Therefore, it should be possible to process foods in a “natural” way, utilizing a series of pressurized fluids as suggested by the sequence below: SC-CO, or LCO,
5. SC-CO,/ethanol or H,O
5. Pressurized H,O or H20/ethanol The above sequence suggests that some degree of selective solvation of food-related solutes should be possible, i.e., SC-CO, or liquid CO, for extracting nonpolar solutes followed by the enhanced extraction of more polar solutes via the addition of ethanol to the CO,. Processing with pressurized water, i.e., subcritical H,O (sub-H20) expands the range of extractable solutes into the “polar” range, with the selectivity in this case controlled by the extraction temperature or addition of ethanol. Combining these fluid media with the processing options mentioned previously results in a powerful array of possibilities that can be used for enriching higher-value foodstuffs and phytonutrients via alteration of their lipid content. Examples of the use of this approach include eggs with reduced cholesterol con-
tent ( 5 ) , low-fat nut products (6), pesticide-free products (7), as well as meats with a reduced cholesterol concentration (8). There is no doubt that the development of
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such products, coupled with a proper commercial marketing campaign, provides a powerful stimulus for the consumer to sample such products. This expanded “all natural” processing approach is illustrated in Figure 6.2. On one end of the solvent (fluid) scale lies SC-CO, and LCO,, whereas pressurized water on the other end is available for isolating polar moieties. Sequential fluid processing of a natural product, e.g., soybeans, permits removal of nonpolar components such as carotenoids, triterpenes, or phytosterols by CO,. This can be followed by extraction with a C0,kosolvent combination that can remove the more polar components. Finally, after removal of the above compounds, subcritical water can be applied to isolate the isoflavones or phytates, for example. It should be recognized that some targeted compounds may occur in each of these “green” solvent combinations. An additional benefit from the process depicted in Figure 6.2 is that leftover residual proteinaceous meal is available for further use, devoid of any solvent residues. This is an appealing extraction andor fractionation scheme that can be accomplished with commonly used high-pressure processing equipment as noted by King (9). Enrichment of a lipid ingredient to a sufficient purity or concentration cannot always be accomplished by using only an extraction step, and this can necessitate the use of one or more fractionation methods. A popular approach for fractionating natural products containing desired lipid components is the use of a fractionating tower with an internal packing, imposing a temperature gradient along the length of the column, and facilitating concurrent or countercurrent contact between the fluid phase and those containing the lipid moieties (10). Chromatographic-based fractionation has also been utilized (1 1); examples of these chromatographic options will be discussed later for the enrichment of steryl esters and phospholipids
Neutral lipids Carotenoids Triterpenes Phytosterols
’
“Green Solvents”
CO, extractable
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(PL). Recently, extraction and other processing options were noted that can be accomplished with the aid of an expeller, i.e., the CF is introduced into the expeller barrel where it attains supercritical conditions under the applied temperature and pressure (12 ) . This mode of processing not only allows extraction/fractionation to be accomplished on a continuous feed of raw material, but also permits the introduction through selective dissolution of a lipid ingredient into the fluid, and their impregnation into a product matrix. This addition of CO, into the barrel of the extruder, where it becomes a SF due to the heat and pressure generated during the extrusion process, not only facilitates solubilization of lipid materials from the substrate being processed, but also enhances the fluidity of the potential extract, e.g., a nutraceutical-based oil. Healthful lipid ingredients frequently occur at very low levels in natural products and oils, and their enrichment or isolation, either by traditional methods, e.g., vacuum distillation, or CF-based techniques, frequently yields extracts with considerable extraneous lipid material. Indeed extracting or enriching a desired solute from the natural product matrix is somewhat akin “to finding a needle in a haystack.” Combinations of these healthy lipid ‘‘needles’’ within these oily “haystacks” are cited in Table 6.1, It is possible by employing SFF methods to obtained mixtures enriched in active ingredients, such as sterols or steryl esters from vegetable oils (13). Similarly, fractionation schemes were reported for removing objectionable lipid matter, e.g., fatty acids (FA), from triglyceride-based oils or by-product streams (14). However fractionation is not always necessary because in some SFE lipid-enriched products, e.g., saw palmetto, there appears to be a synergistic effect between the lipid components such as the FA and steryl esters found in SC-CO,-processed saw palmetto extract. In this chapter, we will approach the application of CF technology from the perspective of yielding a “healthful” lipid-containing product. Individual sections document how the proper application of CF in their super- and subcritical states can achieve the following:
Reduce the toxicity of a lipid extract Adjust beneficially the composition of lipid mixture Enrich nutritionally beneficial ingredients, i .e., nutraceuticals Provide a better lipid delivery technology TABLE 6.1 Natural L i p i d Matrices with Critical Fluids for Their Nutraceutical Components Natural lipid
Nutraceutical component
Rice bran Marine-derived oils Wheat germ Alfalfa leaf protein Evening primrose Barley Saw palmetto
Phytosterols n-6, n-3 FA Tocopherols Carotenoids y-Linolenic acid-enriched FA Tocotr ieno I s Steryl esters + FA
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Provide integrated environmentally benign processing schemes Yield commercially documented products for the consumer Lipid-rich matrices are particularly amenable to processing using SC-CO, and LCO, as was documented previously in the literature (15). The properties of CO,, i.e., low toxicity, nonflammability, and low cost, are the basis of what will be discussed in the section that follows. Reduction in Toxicity
One of the early initial attractive features of SC-CO, and LCO, processing was the promise of an alternative extraction method to replace hexane and similar solvents for extracting vegetable oils from their seeds (16). The SFE approach was described in the literature and summarized in the work of King and List (15). Essentially, the driving force was to develop an extraction process devoid of the dangers associated with extraction using commercial hexane. Research showed that SC-C02-processed oils had many superior properties, no solvent residuals, and voided the need for commercial degumming refining steps, but were susceptible to oxidative deterioration relative to conventionally processed vegetable oils (17). However, the early SFE process was judged to be capital-intensive for commercial implementation, at least with regard to commodity oil extraction, and could not provide the throughput associated with processing plants in the soybean or corn industries. Niche applications were developed over the years for certain specialty high-value oils, as noted by some examples given in Table 6.1. In recent years, with the nutraceutical “explosion,” C0,-extracted oils have become commercially viable as judged by such label declarations as “Supercritical Extraction-The Technology for the New Millennium” and “Extracted with the Natural Effervescence of Nature.” Such declarations were countered with opposing statements, specifically from the oil “pressing” industry who feel that CO, is still a chemical approach relative to mechanical expression of the oil. Interestingly, extraction of vegetable oils with sub- and supercritical propanes was studied and implemented commercially for specific targeted products, The well-known Solexol process (18) is based on dissolution of oils such as fish oil in propane, and such commercial processes are not as capital-extensive as extraction with SC-CO,. However, even subcritical propane extraction or fractionation takes place at higher temperatures than those used in CO,-based extractions, and the threat of explosion due to propane’s flammability remains a processing issue. Propane is fairly abundant and from a GRAS perspective, and it is also approved for use with foodstuffs; however, the persistence of a propane “taint” in extracted oils could be a health issue for the informed consumer. In recent years, propane extraction was applied to lower the fat content of various food products and as a hydrogenation reaction medium; these topics will be discussed in a later section.
C02-based processing of commodity and specialty oils is used to remove objectionable solvents from conventionally extracted oils. Usually this is done by
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employing a packed fractionated tower in which the hexane-extracted vegetable oil is countercurrently contacted with either SC- or L-CO, (19). Figure 6.3 shows a similar option in which LCO, is concurrently contacting with LCO, to reduce hexane to very low ppm levels. Here the oil is injected into the bottom sector of the
Fig. 6.3. Schematic of the packed-column fractionation system for stripping hexane
from soybean oil using liquid (L)CO,. RD, rupture disk; PG, pressure gauge; MMV, rnicrornetering value; DTM, dry test meter; F, filter; R, rotorneter.
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packed column, and the hexane partitions into the CO, during its transit up the column. This avoids the need to heat the oil to affect stripping of the hexane. Similar columnar approaches were also used in fractionating lipid moieties, such as glyceride mixtures (20), tocopherols (21), and steryl esters (13), yielding final products devoid of any chemical solvent. Purification of used frying oils is also possible using this continuous approach. Reactions can also be conducted in SC-CO,, thereby avoiding the use of organic solvents. Two types of reactions having implications in the oleochemical industry are enzyme-catalyzed and hydrogenation reactions. The coupling of enzyme catalysis with critical fluids such as CO, is particularly attractive because both can be viewed as “natural” agents, allowing lipid food components to be manufactured without introducing a solvent or catalyst residues into the final product. Such enzymatic-initiated transformations are usually conducted in the presence of a lipase, and can include transesterification (22), glycerolysis (23), hydrolysis (24), and randomization of fats (25). These reactions are sensitive to the chosen reaction temperature, pressure, and presence of water in the CF system. Reactions of this type can be conducted in either batch or flow systems; the latter are attractive for the continuous production of a desired product. Of note is the extensive use of Novozym 435, a lipase derived from Cundidu anturctica, in affecting the above lipid transformations in SC-CO,. Additional details on this approach will be discussed in the following section on the alteration of lipid content. Hydrogenation in the presence of SF attracted considerable attention recently for both the production of food and industrial lipid products. From an engineering perspective, the use of propane/H, over CO,/H, mixtures is advocated (26) due to the attainment of higher lipid solubilities, i .e., and consequently higher production throughput, as well as lower operating pressures when using these binary fluid mixtures. This preference was cited by Harrod and Moller (27) for the synthesis of lipids intended for industrial use. However this approach does not necessarily follow in producing lipid-based products intended for food use, where CO, would be preferred over propane from a toxicological perspective. As with the enzymatic conversions cited above, SF hydrogenation is used to alter the composition of lipids preferentially, and will be covered in more depth in the next section. Alteration in Lipid Content
The alteration in lipid content using CF is usually accomplished by employing SFF and/or SFR, or occasionally SFE. These alterations in molecular composition of a lipid mixture are usually enacted to make a physical or chemical change in the targeted lipid or mixtures thereof, thereby changing its physical and chemical properties. SFF techniques can be used to enrich a specific component in a complex mixture of lipids or change the overall composition of a mixture. The previously mentioned columnar fractionation techniques can be employed for this purpose to adjust for example, the mono-, di-, or triglyceride composition in a mixture. Early studies by
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Brunner and Peter (28) as well as others (29) focused on the purification of mixed glyceride mixtures usually to achieve a high purity level of the mono- or diglyceride specie. However, the principle of columnar fractionation using CF, which is based on each glyceride’s respective solubility in the CF and its attendant vapor pressure, also allows for customizing a desired glyceride composition having certain physical properties intended for food use. Thus, by using this method, glyceride species can be sorted and fractionated according to their class functionality, e.g., mono-, di-, and also their respective chain length. This can also be accomplished by fractionation using SFE and varying the pressure, temperature, and time of extraction enacted on a lipid mixture, but the fractionation efficiency is much poorer relative to results attainable using a packed column. The molecular composition of an oil or fat can also be changed using an SFR approach. There is a historical precedent for conducting specific reactions under supercritical conditions, such as the polymerizations or isomerizations. In these types of reactions, it is often simply the application of pressure as a thermodynamic variable that permits the reaction equilibria to be shifted, yielding the desired end products. Since the early 199Os,there has been an increasing awareness that by conducting reactions in CF media, one can control not only the equilibrium position of a reaction, but also the product distribution and end properties, i.e., melting point, color, or morphology. For example, mass transfer rates of reactants and products are substantially improved in SF because transport properties, e.g., diffusion coefficients, are superior to those found for solutes in the liquid state. Alteration of the density of the fluid also allows for subtle control of the reactant or product solubility in these dense reaction media, and this can be used to control the molecular composition of the final product. It is also possible in some specific cases to conduct reactions at low temperatures, and in a nonoxidative environment, i.e., CO,, thereby protecting compounds that would be altered in more thermally intensive reaction environments. Reaction catalysts can also be regenerated with the aid of SF after completion of an SFR. Reactions on lipid substrates were achieved successfully in CF (Table 6.2). These include basic esterifications, transesterifications, glycerolysis, interesterification, hydrogenation, and hydrolysis reactions. With respect to using lipases as catalysts in the presence of SC-CO,, it is critical that conditions be optimal with respect to maintaining the enzyme’s activity and extending its lifetime. It should be noted that the catalytic activity of many enzymes can be restored after extended use in CF. Typically, pressures from 15 to 40 MPa and temperatures ranging from 50 to 80°C are consistent with sustaining lipase activity and extended use. Also critical for the use of these enzymes in the presence of SC-CO,, is the maintenance of a low level of water to allow the enzyme to sustain its active conformation in the presence of SC-CO,. For example in the methanolysis of soybean oil, a level of 0.1 volume % of water in SC-CO, at 24 MPa and 50°C is sufficient to prevent denaturization of the enzyme. Additional water, however, can prove inhibitory to facili-
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TABLE 6.2 Reactions on Lipid Substrates Conducted in Critical Fluid Media Type of reaction
Reaction medium Catalyst
Esterification Esterification Transesterification Transesterification Transesterification Glycerolysis GIycerol ysis Interesterification Hydrogenation Hydrogenation Transesterification + H, Hydrolysis
sc-co,
sc-co, sc-co, sc-co,
sc-co, sc-co, sc-co, sc-co, SC-COJH, SC-CO,/H, SC-C02/H2 Sub-H,O
Lipid substrates
Lipase Lipase Lipase Lipase Lipase Lipase None Lipase Ni T-4489a T-4489/lipase None
Simple alcohols/acids Fatty acids/alcohols Soybean oil/methanol Ferulate estedtriolein Chiral esters Soybean, corn oils Vegetable oils Soybean, palm oils Soybean oil Soybean oil Soybean oil Vegetable oils
dChromium-free catalyst (United Catalysts, Louisville, KY).
tating esterification or transesterification reactions, and it is fortuitous that water has a low but predictable solubility in SC-CO, ( 2 2 ) . Recently, the production of sterol esters was accomplished using Chirazyme L-1 with SC-CO, (30). As illustrated in Figure 6.4, FA of various chain lengths can be reacted with sterols, such as cholesterol and sitostanol. Recorded ester yields were consistently >90% for C,-,, saturated FA reacting with the sterols at 27.6 MPa and 50°C. Of particular interest were the >98% yields achieved for the
C8:O
C1O:O
C12:O
C16:O
C18:O
Fatty acid Fig. 6.4. Product Chirazyme L-I.
yields for fatty acids reacting with cholesterol or sitostanol using
I08
J.W. King
CI6 and C,, sitostanol esters, which could be utilized in functional food margarine formulations to lower cholesterol levels in humans. Jackson et al. (25) demonstrated that lipases could also effectively randomize vegetable oils to yield potential products for incorporation into margarine formulations. The degree of randomization attained on the vegetable oils dissolving in SCCO, was a function of extraction/reaction pressure, the flow rate of the SC-CO,, and the quantity of enzyme utilized. Dropping point and solid fat index (SFI) data of the products randomized in SC-CO, were compared with oils randomized by conventional methods, and the agreement between these two differently synthesized products was encouraging. As shown in Figure 6.5, for a randomized palm olein (PO) as well as a genetically engineered soybean oil also randomized in SCCO,, high stearate (HS)-1, both products had an SFI as a function of temperature similar to those exhibited for hydrogenated blends of vegetable oils. It was further found that the fat dropping point was dependent on the catalyst concentration, exhibiting an inverse dependence on throughput of the dissolved vegetable oil across the supported lipase bed. HPLC analysis of the randomized and initial oils showed only small changes in the triunsaturated, diunsaturated, disaturated, and trisaturated glyceride species.
Temperature ("C) Fig, 6,5, Solid fat content of palm olein (PO) and high-stearate (HS)-1 soybean oil before and after randomization.
Supercritical Fluid Processing
109
As shown in Figure 6.6, there was a change in relative composition of the glyceride components for PO, particularly in the increase of the LOP, SOO, and PO0 triglycerides (TG) in randomized PO, relative to the levels found in PO. Figure 6.7 compares the HPLC profiles for the HS soybean oil with the randomized product after passage through the lipase reactor bed. Again, subtle changes are in the TG composition that have a considerable effect on the physical properties of the oil, before and after interesterification. Both of the above oils showed losses in the disaturated glyceride along with a concomitant increase in tri- and diunsaturated TG. The loss of disaturate functionality was shown to aid in fat crystallization. This is a clear illustration of how a customized oleochemical-derived product can be synthesized using a combination of enzyme and CF technology.
Palm olein
Q LL
Randomized palm olein
I N IIII
I 25.00
II
I
I
ASOP
gm'F8m I I I 1 t~' II
35.00
45.00
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55.00
1 s~n II
I
I
I
@
65.00
Retention time (min) Fig, 6,6, RP-HPLC of palm olein fraction before and after randomization, FID, flameionization detection; P, palmitic acid; 0, oleic acid; S, stearic acid; L, linoleic acid.
J.W. King
110
%S
0
s:
?!?
Q
LL
Randomized
118118111
25.00
35.00
118111111
45.00
1111)1111
55.00
1 8 1 8 1 1 1
65.00
Retention time (min) Fig. 6.7. RP-HPLC of high-stearate (HS) soybean oil before and after randomization. FID, flame ionization detection. P, palmitic acid; 0, oleic acid; S, stearic acid; L, linoleic acid; Ln, linolenic acid.
As noted previously, hydrogenation in the presence of SF media has attracted considerable interest, based, to a large extent, on the superior mass transfer characteristics achieved when using hydrogen mixed with SC-CO,. Due to several factors, contact of the dissolved hydrogen with catalyst and substrates is better facilitated in both flow and batch reactor systems using an H,/SC-CO, mixture. Recently, we conducted experiments in batch reactor systems at slightly elevated pressure (up to 13.8 MPa) in which hydrogen is admixed with CO, in a vessel containing a Ni commercial catalyst and soybean oil (31). Using mixtures of H, and CO, at equal pressures and temperatures between 120 and 140°C, a variety of hydrogenated oils was produced that had varying iodine values (IV), % trans fatty acid, and SFI. It is interesting to note that the achieved SFI are relatively invariant with respect to temperature and can be adjusted by varying the ratio of H2 to CO, pressure. Thus, it appears possible using this approach to produce equivalent or better hydrogenated oil basestocks for use in mar-
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111
garine or shortenings, relative to those obtained using conventional technology. As shown in Table 6.3, oils with lower trans FA are possible using the above technique, relative to an equivalent commercial product of similar IV value produced by conventional hydrogenation. Modeling studies of these hydrogenation reactions conducted under these supercritical conditions have been reported (32). The mechanism for the hydrogenation of specific TG moieties is a complex one; however, the addition of H, to CO, at 120°C results in selective hydrogenation between 0.34 and 0.69 MPa, and the nonselective mode at H2 pressures of 3.45 MPa. These experimentally produced margarine basestock substitutes, i.e., hardened in the presence of H, + SC-CO,, have a slightly higher IV and stearic acid content than their conventionally produced counterparts; however, the % trans fatty acid content of the H2/SC-C02-hardenedproduct is much lower than the conventional product. Recently the Recasens research team (33) hydrogenated sunflower oil in a continuous recycle reactor using 2% palladium on carbon in the presence of SC-propane/H2 mixtures. The conditions used, 430490K, 20 MPa, provided consistently for the existence of only one fluid phase in which to conduct the hydrogenation of the vegetable oil. Some of these results in terms of the IV and % trans of the final product are plotted in Figure 6.8 for the conditions noted on the inset. Here it can be seen for the same degree of hydrogenation that the % trans obtained is considerably lower than that obtained in either conventional process or the above method of King and co-workers (31). The stearic acid content as a function of IV appeared to be slightly larger than that obtained via the conventional hydrogenation process, but similar in value to those published by King et al. (31). This again points to the considerable potential that hydrogenation conducted in the presence of an SF has for synthesizing oils having healthy and desired end properties. Enrichment of Ingredients
Before the mid- 1980s, CF-processed products were largely derived using SFE, either by selecting a given fluid density that would yield the desired product, alterTABLE 6.3 Properties of Potential Vegetable Oil-Derived Margarine/Shortening Using Hydrogen/ Carbon Dioxide Mixtures During Hydrogenation Margarine basestock (D.P.a 32-39°C) Oil property ~
Shortening basestock (D.P. 45-52°C)
Conventional
Experimental
Conventional
Experimental
6-9 11-30 90-1 10
7-1 1 1-3 108-114
11-13 15-20 85-90
13-24 3-8 88-1 02
~~
% Stearic acid
Trans Iodine value
Oh
aDroppingpoint.
J.W. King
112
140
x l 7 M P a H 2 1 1 7MPaC02.393K x 0 3 MPa H2 383 K. 950 P oil (ConventionalProcess) *O 9 MPa H2 448 K moi% 1 4 95 105 radh A 1 2 MPa H2 488 K mol% 1 6 93 157 radfs 1 5 MPa H2 473 K mol% 1 8 91 105 radis m2MPaH2 458K mol% 11089 157radls 00 9 MPa H2 47816 mol% 1 4 95 105 radls A 1 5 MPa H2 445 K mol% 1 8 SS 105 radls
130 120
a H2 431 K mol% 1 6 93 157 radls a H2 458 K mol% 1 6 93 157 radls
110
King 81 el. experlmentaldata
100 90
-- x I
0
5
I
I
10
15
20
YO trans (wt) Fig. 6.8. Trans C,, formed vs. reduction in iodine value (IV) for hydrogenation of sunflower oil on Pd/C. Initial IV = 130, data from King e t a / . (31) lie within the dashed region.
ing the extraction fluid density as a function of processing time, or in some cases, selectively decreasing the pressure after the extraction stage to achieve the desired extract. Useful separations of lipid moieties were attained using the above techniques, but largely between compounds differing significantly in their physicochemical properties, e.g., molecular weight, vapor pressure, or polarity. In the last decade, fractionation processes utilizing CF media were improved by combining principles utilized in supercritical extraction with other separation techniques. These improved methods often make use of fractionating columns or scaled-up chromatography to yield improvements in the resolution of lipid mixtures. The fractionating column or tower approach is somewhat analogous to operating a distillation column, but there are differences when using CF media. For an understanding of the fundamentals involved in using this technique, one should consult the works of Clifford (34) and Brunner (35). Figure 6.9 illustrates the components and principles involved in fractionating using a single column operating in concurrent flow mode. In this case, SC-CO, is brought to the desired pressure and then directed to flow upward inside the fractionating column, which usually contains a packing to encourage contact between the SC-CO, and the lipid components being separated. The components to be separated are injected with a pump into the flowing SC-CO,, before its entry into the column. This is very similar to the hexane-stripping operation illustrated in Figure 6.3, except
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Fig. 6.9. Schematic of the packed-column thermal gradient fractionation system.
in this case, the column is held under conditions of a thermal gradient rather than operated isothermally. The fluid-solute mixture then enters the first heated zone in the vertical fractionating column, and the separation process is initiated. The SCC02/solute mixture ascends the column, usually encountering zones of increasing temperature, which facilitate the separation of solutes based on their relative solubilities in SC-CO, and their respective vapor pressures. In effect, the column is operating under a density gradient because the fluid is kept isobaric. The described fractionating column can be operated in either the batch or semicontinuous mode with concurrent flow of the solute and SF streams. When operating these columns
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in either the concurrent or countercurrent mode, there is a trade-off between throughput and resolution of the lipid mixture. Usually higher lipid throughput is associated with higher operating pressures because higher pressure increases the lipid solubility in SC-CO, (29). Fractionating efficiency is an acute function of throughput, but optimal fractionation occurs at lower pressures. As an example, consider the fractionation of crude rice bran oil to remove unwanted FA without a significant loss of oryzanol or TG content using a semibatch fractionating concurrent column mode (13). Extracts with the highest free fatty acid (FFA) content (36.6%) were achieved at 20.5 MPa and 80°C by operating the fractionating column in an isobaric mode. Raffinate samples had increased amounts of TG and sterols, and reduced levels of FFA (2.9-5.1%). Column fractionations conducted over an extended time period further resolved the FFA moieties from the TG, yielding extracts after 8 h of operation of almost 60% FFA content. This is an 8-fold enrichment relative to the FFA content of the starting rice bran oil which was 7% FFA. Using this technique, it was possible to produce a raffinate fraction containing 95% TG, <1% FFA, 0.35% free sterol, and 1.8% oryzanol content, at 13.6 MPa and 45°C. This composition compares favorably with the content of commercially refined rice bran oil or high-oryzanol rice bran oil; in addition, it contains three times as much oryzanol. A more striking result can be achieved by superimposing a thermal gradient on the fractionating column. For example, the FFA content of crude rice bran oil can be reduced from 7 to 0.5% at 13.6 MPa using a four-zone thermal gradient of 40/60/70/80"C on the column pictured in Figure 6.9. The raffinate of this initial fractionation step can then be further fractionated at 20.5 MPa using the above thermal gradient to yield a product whose steryl ester content exceeds that found in a commercially available, steryl ester-enriched margarine. This two-step fractionation process is summarized in Figure 6.10. Similar results were achieved using corn fiber oil as a starting substrate in which the phytosterol content is increased from 6 to 19% using the above two-step enrichment process. Again, the result is a lipophilic-based composition that is similar to a commercially available phytosterol-enriched margarine having a low FFA content (
95%), steryl esters (>95%), FFA (>95%), pigments, i.e., carotenoids (>90%), sterols (32-61%), tocopherols and tocotrienols (3547%). For this reason, various investigators apply fractionation techniques such as those described above to enrich nutritionally beneficial compounds from vegetable oil distillates (36,37). Figure 6.1 1 shows the effect of the column fractionating pressure on the composition of rice bran deodorizer distillate (DD), with the wax fraction removed, at 45°C. As noted in Figure 6.11, selection of fractionation pressure allows one to obtain an extract of specific composition in terms of FFA, phytosterol, and TG
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FFA
115
Phytosterol-enrichedTG
t High P & T
TG + sterol esters
i TG
Fig. 6.10. Schematic diagram of a two-step columnar fractionation process for steryl ester enrichment from vegetable oils. TC, triglyceride; FFA, free fatty acid.
content. Therefore, depending on the pressure selected, 13.6 MPa vs. 27.2 MPa, one could reduce the FFA content by 20%, double the phytosterol content, and increase the TG content eight-fold in the resultant extract. This ability to customdesign extracts using a columnar SFF technique is particularly attractive in achieving an optimal therapeutic composition in a nutraceutical extract. A two-step enrichment process, 13.6 MPd45"C followed by a 34.0 MPd80"C step, can produce extracts with >30% phytosterols and reduced FFA from rice bran or soybean oil deodorizer distillates. Another SFF option is to employ chromatography in the preparative mode, coupled perhaps with a preliminary SFE-enrichment stage. Using this approach, it is possible to enrich moieties such as tocopherols, PL, or steryl esters from vegetable oils, seeds, and by-products of the milling or vegetable oil refining processes (3840). Figure 6.12 illustrates the specific case for the separation, enrichment, and fractionation of PL from vegetable oils (40). Here soybean flakes are initially extracted with SC-CO, to remove the oil from flakes, followed by extraction of the PL from the deoiled flakes with an SC-CO,/ethanol mixture. The second extraction step produces an extract enriched in PL because they are not appreciably soluble in neat SC-CO,, but can be removed selectively from the flake matrix with the aid of ethanol as a cosolvent. As shown in Table 6.4, the second SFE using SC-C02/ethanol produces an extract containing 43.7% PL by weight. This is a considerable enrichment relative to the concentration of the PL in the starting oil or seed matrix. Further PL enrichment is facilitated as noted above by transferring this extract enriched in PL to an alumina preparative SFC column, where SC-CO, modified with a 5-30 volume %, 9:l ethano1:water eluent is used to elute and fractionate the PL. In the case of the SFC enrichment step, eluent fractions can be collected as a function of time and their PL content quantitated. As indicated by the program given in Table 6.4, collection of dis-
crete fractions during the SFC process can produce PL purities in excess of 75% for the individual PL, namely, phosphatidylcholine and phosphatidylethanolamine. It
J.W. King
116
12
4
0 13.6
20.4
27.2
Pressure (MPa) Fig. 6.11. Effect of temperature on the composition of the extract fraction collected during the supercritical fluid fractionation of rice bran deodorizer distillate at 45°C. TG, triglyceride; St, phytosterol; FFA, free fatty acid.
should be noted in the described process that in the SFC steps, only GRAS solvents were used for the enrichment process. Recently, a similar SFE/SFC process was used to isolate sterols and phytosterol esters from agricultural by-products such as corn bran and fiber (40,41). For example, by using both SFE and SFC, it was possible to isolate a fraction containing up to 53% by weight of a cholesterol-lowering ferulate phytosterol esters (FPE) from corn fiber oil. Sterols and steryl esters can also be produced by SFE/SFC from a similar substrate, corn bran. In the case of the corn bran oil, it was determined via a combinatorial experimental approach with the aid of the automated analytical SFE instrument that two sets of conditions, 69 MPd80"C and 34.5 MPd 40"C, were optimal for enriching the steryl esters. The resultant extracts contained 1.25% FPE. Using an aminopropyl silica-based sorbent, the initial extract from the corn bran oil was fractionated using a program similar to that described in Table 6.4. Initially, the SFE-derived extract was chromatographed at 69 MPa and 80°C to remove most of the interfering TG. Then the pressure and temperature were lowered to 34.5 MPa and 40°C, respectively, and the ethanol cosolvent added stepwise
to achieve fractions enriched in phytosterols. Using this approach, ferulate phytosterol esters could be enriched to a content of 14.5 g/lOO g.
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Soybean flakes
SC-CO2 Extraction
Extract
Defatted Soybean Flakes
SC-CO2lEtOH Extraction
I
PPL=Enriched Fraction
SC=COdEtOH/H20 Chromatography
8
Exhausted Oil-Cake
Pure Phospholipid Fig. 6.1 2. Process for the enrichment of phospholipids by supercritical fluid extraction (SFE)/supercritical fluid chromatography (SFC). PPL, porcine pancreatic Iipase.
As in the case of the deodorizer distillates, starting with a substrate containing a higher level of the phytosterols can be advantageous. For this reason, the SFE/SFC process was also applied to corn fiber oil, which has been reported to contain up to four times as much FPE as the previously mentioned corn bran oil. However, corn fiber itself contains a low percentage of oil; in fact SFE at 34.5 MPa and 40°C yielded only 0.56 g/100 g based on the weight of the corn fiber. Using the stepwise gradient program given in Table 6.4, it was found that the first fraction collected contained 15 wt% g FA sterol esters and 85 wt% g TG. Further elution of the starting substrate resulted in a final fraction consisting mainly of FPE, FFA, and TABLE 6.4 Supercritical Fluid Chromatography Fractionation of Lecithin on Silica Gel Fraction collecteda #1 #2 #3 #4 #5
#6 #7
Eluent parameters
Principal compounds
350 bar, 350 bar, 350 bar, 500 bar, 500 bar,
Triglyceride oil
50°C, 50"C, 50"C, 50T, 80T,
CO, CO,/M CO,/M CO,/M CO,/M
500 bar, 80T, CO,/M 500 bar, 8OoC, C02/M
Phosphatidylethanolamine Phosphatidylinositol + phosphatidylcholine Phosphatidylcholine Phosphatidylcholine
aFraction #2 modifier is 10% ethano1:water (9:l);fractions #3-7 modifier is 25% ethano1:water (9:l).
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1. W. King
sterols, and minor amounts of diglycerides. The proportion of FPE in this fraction was 53%, twice the amount found in the starting corn fiber oil. It should be noted that for both the corn bran and corn fiber SFE/SFC studies, that the average mass balance for all of the corn fiber or bran components was close to 100% recovery. Such enriched lipid concentrates have the potential to be used as additives in a functional food formulation, which bodes well for the use of preparative SFC for these purposes. Delivery Technology
Numerous CF processes exist for producing fine powders and encapsulated products that use or contain lipid materials. Space does not permit a thorough discussion of these various processes, but the reader is referred to a recent publication by York et al. (42), which is an excellent reference source. Generically, these processes consist of spraying a lipid material dissolved in CF-CO, or a suitable solvent, i.e., ethanol, into a pressurized vessel via a narrow capillary tube/orifice, which facilitates the production of unique particle sizes and/or morphology. The formed particles have narrow particle size ranges, i.e., 1-2 pm, and this can be extended down into the nano-particulate range. Alternatively, precipitation of the dissolved lipid moieties can be augmented by using compressed CO, as an antisolvent, which provides yet another mechanism for producing microparticulate powders. Such small particles of lipid materials can facilitate more rapid dispersion in food formulations, accelerated delivery of biologically active lipids into the bloodstream, and in certain cases, enrobe active ingredients in a lipid sheath, i.e., liposomes, for further use. If such particle formation processes are collectively viewed as a combination of CF as well as particle formation technology, i.e., CF-PT (particle formation technology), then a coupled process similar to an SFESFF or SFE-SFC scheme results. This would suggest that CF-PT could be combined to advantage with SFE to permit not only the extraction of a targeted compound, but also allow consecutive formation of a desired morphology of the lipid compound. Table 6.5 lists some examples of CT-PT for producing lipid nutraceutical-type ingredients. These are based on examples collectively cited in the Proceedings of the 6th International Symposium on Supercritical Fluids (43). The controlled powder formation listed (CPF) process seems particularly promising for lipid-related nutraceutical and natural product extracts. The CPF technique usually employs carrier materials that are biologically compatible with their absorption into the human body. Various starches, silicic acids, celluloses, sugars, and emulsifiers serve as carriers in the CPF technique. As an example, by using the appropriate carrier in the CPF process, powderized lipophilic paprika extracts can be prepared for use as water dispersions. The combination of using the CPF process with an appropriate carrier containing an additive results in materials that can be protected from oxidation and have their particle size regulated (increasing shelf-life); these affect redispersion and sustained release properties into food matrices. Further examination of Table 6.5 shows additional applications of CT-PT that have implications for the nutraceutical and/or functional food industry. These
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TABLE 6.5 Materials Processing Using Critical Fluid Media with Implication or Direct Application in the Nutraceutical or Functional Food Industry Process
Application
Referencea
Concentrated powder form (CPF), jet dispersion
Dispersion of paprika soup powders
E. Weidner pp. 1483-1 495
Semicontinuous gas antisolvent process
Cholesterol morphology and precipitation
P. SubrafA. Vega pp. 1629-1 634
Rapid expansion of supercritical solution
Phytosterol micronization
S. Jiang eta/. pp. 1653-1 658
DELOS crystallization
Crystallization and control of stearic acid morphology
N. Ventosa eta/. pp. 1673-1 676
CPF process
Controlled release of flavors and vitamins
F. Otto eta/. pp. 1707-1 712
Rapid expansion of supercritical solution
Encapsulation of p-sitosterol in low M W polymer matrix
M. Turk eta/. pp. 1747-1 752
Supercritical antisolvent process
Incorporation of cholesterol or proteins in biodegradable matrix
Pellikaan pp. 1765-1 770
PCA and GAS processes
p-Carotene precipitation
F. Miguel eta/. pp. 1783-1 788
Particle from gas saturated solution (PCSS)
Lipid micronization of phosphatidylcholine and tristearin
N. Elvassore eta/. pp. 1853-1 858
Supercritical antisolvent process
Biodegradable polymer precipitation studies
A. Vega-Conzalez pp. 1877-1 882
aThe articles listed all appear in Reference 43
include specific lipid chemicals that are utilized in nutraceuticals, such as antioxidants, steryl esters, carotenoids, PL, and FA. As was noted previously, the high value and application of PL-containing materials, such as lecithin or PL-based liposomes, as functional food ingredients and throughout the food industry, merit a more detailed examination. The early research of Wagner and Eggers in Germany (44,45) involving the jet extraction of lecithin from soybean oil provides powdered extracts somewhat analogous to the lecithin- or PL-based powders noted in Table 6.5. The basis of jet extraction is that it facilitates the dispersion of a thin filet of lecithin into a highly turbulent jet of SC-CO,. This is made possible by the use of two overlapping capillary jets. The lecithin is fed into the inside capillary, whereas the CO, enters into the larger outside capillary encasing the smaller diameter capillary tube. The compressed CO, then mixes with the lecithin extradite in a mixing chamber under conditions of high turbulence, which affect the deviling of the lecithin and formation of a powdered product. The physicochemical basis of this separation process in
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J. W. King
terms of its effect on lecithin viscosity and interfacial tension was reported by Eggers and Wagner (44,45), and resulted in the conceptualization of a continuous processing plant based on the above principle. Other notable advancements that use CF for producing ym-size, PL-based powders as well as the formation of PPL-based liposomes were reported in the literature. For example, Castor at Athos Corporation patented several concepts, called the CFL Process (46,47), which permit the encapsulation of hydrophobic drugs as well as naturally derived drugs, such as taxol. The basis of such patents is that PPL deposit out at the phase boundary of the PPL-drug/aqueous phase, or multilamellar vesicle, when the mixture undergoes decompression in the CF atmosphere. The resulting liposomes formed in the presence of SC-CO, and other alternative fluids showed stability life times >6 mon, and this could be extended by the inclusion of a-tocopherol in the PLL matrix, which provides prophylactic protection for extending the lifetime of the resultant liposome. Similar studies were also performed by the research group of Charbit in Marseilles (48,49) in which fine PPL particles were formed by decompression using the SAS, or Supercritical Antisolvent Process. The focus of this research was to develop drug delivery systems, but it would be equally applicable to the functional food area. Typically, a 2 g/100 g solution of soy-derived lecithin is dissolved in an ethanol solution, which is subsequently injected into a vessel containing SC-CO,. Typical precipitation temperatures and pressures were 35°C and 8-1 1 MPA, respectively. The micronized-PPL particles tended to be in the range of 15-60 microns, amorphous in nature, and coalescent when they were exposed to air. Other schemes cited in the literature employ PL-based materials and SF for fine particle formation or encapsulation. Weber et al. (50) recovered lecithin from egg yolk extracts and induced crystallization by implementing the gas antisolvent process. Similarly, Frederiksen et al. (51) developed a new method of preparing liposomes to encapsulate water-soluble substances with the aid of ethanol. In that study, liposomes with 40- to 50-nm dimensions could be formed from phosphatidylcholine. A mixing process, the ESMIC Process (52) using SF, was also reported. The precipitation is conducted in a stirred autoclave to produce an embedded pharmaceutical preparation in a lecithin matrix. Final particle size is partially controlled by the milling process taking place in the stirred autoclave containing the SF medium. In summary, the above-mentioned studies and processes show the versatility of utilizing CF media in conjunction with lipids for producing powdered lipid compositions, i.e., nutraceuticals for functional food use. In the particular case of PPL, the above-cited studies illustrate how they could be prepared via CF technology for facilitating more rapid dissolution in food formulations, or to encapsulate them for sustained delivery purposes. This is an exciting future area for research and the development of new product concepts, and could supplant or augment developments in the pharmaceutical industry that use CT-PT. This, of course, would open up additional markets for lipid-based materials.
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Commercial Development
The nutraceutical and functional food industry continues to provide a powerful incentive for the use of CF technology, i.e., the resultant products are targeted toward the health-conscientious consumer. Public knowledge in many countries has increased to the point that product labeling often refers to SFE or cold-pressing techniques as evidence that such products have been isolated in a “natural” or “green” manner. Depending upon the definition of a nutraceutical, the potential marketplace is conservatively estimated to be $3.15-4.6 billion in the United States and ranges from U.S. $1.05 to 1.6 billion in Europe. If one broadens this definition to also include the definition of “functional” food, which embraces lipid moieties, then the value of this market expands to between U S . $14.2 and 17.6 billion. A more liberal definition of healthy foods, i.e., if one assumes that 50% of the food selected for consumption is based on health considerations, then the estimated value of the market expands to U S . $250 billion. This is a tremendous economic incentive to apply CF technology for the processing of lipids into the nutraceutical, functional food, or natural pharmaceuticals markets. Table 6.6 lists typical commercial products produced by CF technology; this includes several lipid compositions. These companies are mutually dependent on a number of key processors or toll refiners, including the following: U.S. Nutraceuticals, Eustis, FL; Croda-Lee, United Kingdom; Norlac, Edmonton, Canada; Flarex, Repligen, Germany; Hitex, Vannes, France; Arkopharma, Carros, France; Aromtech, Tornio, Finland; NATECO,, Wolnzach, Germany; and GreenTek 21, Seoul, South Korea. Other commercial production plants are located in New Zealand, several in mainland China, Taiwan, Japan, Italy, and India. Some of the producers directly market their own extracts; for example, Aromtech retails sea buckthorn and blackcurrant oils, U S . Nutraceuticals markets astaxanthin, and Arkopharma markets bee pollen and pumpkin seed extract. Somewhat absent in the production of nutraceutical extracts are largerscale producers, such as the hops manufacturers whose plants and vessel sizes tend to be larger than currently needed for nutraceutical production. TABLE 6.6 Commercial Retailers of Nutraceuticals and Natural Product Extracts for the Personal Health Market in the United States Company
Location
Product
Buckton, Scott Inter-Cal NuturNutra Sage V Foods Primal Essence Prostate Rx CIA Herbs New Chapter
Fairfield, NJ Prescott, AZ Piscataway, NJ Los Angeles, CA Oxnard, CA Naples, FL Brevard, NC Brattleboro, VT
Lutein esters Saw palmetto Borage, oil C0,-defatted products Spices Saw palmetto Oregano oil Assorted products
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1.W. King
It is worth noting that many current oil extraction and refining methods remove valuable nutraceutical components from the oil in the name of appearance and flavor. By-products and streams from these milling and refinement steps often contain preconcentrated sources of healthy lipids, e.g., deodorizer distillate, residual protein meals, fibrous materials. Sources such as corn gluten meal, corn brand fiber, and alfalfa leaf protein concentrate were extracted successfully with SC-CO, for their sterols, FA, and pigment content. Another processing agent that is a concentrator for nutraceutical components comprises the bleaching sorbents used by the vegetable oil processing industry. King and co-workers (53) showed that very high oil yields can be obtained from clay bleaching earths; however, there the resultant extracts must be analyzed for their specific contents. CF extraction has been applied for sometime for the extraction of specialty oils, such as evening primrose, borage, blackcumant, and flax. These moieties contain y-linoleic acid, a component that was implicated favorably for the treatment of several medical conditions. Other specialized oils that were also extracted with SCCO, include wheat germ, avocado, sea buckthorn, sorghum brand/germ oat, and amaranth. Recently, some studies employing CF were used to obtain oil from fungi or marine sources, such as spirulina, which are devoid of cholesterol. Partial deoiling was also performed using SC-CO,, more with respect to developing a functional food ingredient that has less fat (oil) or cholesterol content, i.e., low-caloric pecans, almonds, or peanuts. Such deoiling or defatting can be done using either SC-CO, or propane, and still meet the GRAS or “natural” criterion for use in functional food products. As was stated previously, PL and many of the herbal medicine-type components are amenable to CF extraction provided that G U S cosolvents are employed along with SC-CO,. Purified phosphatidylcholine and phosphatidylserine, obtainable using CF as was demonstrated in this chapter, are finding widespread use, the latter in improving cognitive function. Other natural extracts derived via CF technology that are commercially available include the following: chamomile, paprika, feverfew, peppermint, chia, lemongrass, garlic, and ginger, most of the common spice oils, mint oils, as well as a commercially available extract of rosemary. However, their extraction and refinement are beyond the scope of this chapter.
Concluding Remarks In this chapter, we surveyed the applicability of CF technology for producing “healthy” lipid products employing various techniques that embrace extraction, fractionation, reaction, and fine particle production. Today, there are close to 75 plants producing commercial CF-derived products, ranging from the large facilities devoted to coffee decaffeination and the isolation of hops extracts to smaller units specializing in the extraction, fractionation, and reaction modes to produce an array of products. For the products mentioned in the last section, CF technology faces stiff competition from molecular (vacuum) distillation techniques, a time-
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honored technique, despite the fact that greater molecular selectivity is potentially available utilizing CF-based methods. Aiding the adoption of CF-manufacturing processes as applied to food and agricultural matrices is the fact that CO,-derived extracts exhibit superior attributes, e.g., extended shelf-lives due to the prophylactic action of the residual, nonoxidative CO, atmosphere. Future trends in this area will see CF technology coupled with other types of processing and/or alternative fluid combinations as was suggested at the beginning of this chapter. Of considerable promise is the coupling of membrane processes in conjunction with CF for both the SFE and SFR modes. This was already demonstrated as a feasible coupling for the enrichment of PL. Smith and co-workers (54) recently showed that SC-CO, can be used to remove the lipophilic components from cashew shells; however, subcritical water was also employed in this study to selectively isolate the bioactive phenolic constituents, in a manner similar to that illustrated in Figure 6.2. With respect to the extended shelf-life of SC-C0,-treated products noted above, the merger of ultrahigh-pressure food processing with CF processing should open up a large array of possibilities for improving food products, not originally envisioned >30 yr ago when CF technology was first exploited commercially (55). References 1. King, J.W., Critical Fluid Options for Isolating and Processing Agricultural and Natural Products, in Proceedings of the 1st International Symposium on Supercritical Fluid Technology for Energy and Environmental Applications (Super Green 2002), Suwon, South Korea, November 3-6,2002, pp. 61-66. 2. Anastas, P.T., L.G. Heine, and T.C. Williamson, eds., Green Engineering, American Chemical Society, Washington, 2001. 3. Jessop, P.G., and W. Leitner, eds., Chemical Synthesis Using Supercritical Fluids, VCH-Wiley, Weinheim, 1995. 4. Stahl, E., K.-W. Quirin, and D. Gerard, Dense Gases for Extraction and Refining, Springer-Verlag, Heidelberg, 1987. 5. Froning, G.W., R.L.Wehling, S.L. Cuppett, M.M. Pierce, L. Niemann, and D.K. Siekman, Extraction of Cholesterol and Other Lipids from Dried Egg Yolk Using Supercritical Carbon Dioxide, J . Food Sci. 55: 95-98 (1990). 6. Passey, C.A., Commercial Feasibility of a Supercritical Extraction Plant for Making Reduced-Calorie Peanuts, in Supercritical Fluid Processing of Food and Biomaterials, edited by S.S.H. Rizvi, Blackie Academic, London, 1994, pp. 223-243. 7. King, J.W., Development of New Critical Fluid-based Processing Methods for Nutraceuticals and Natural Products, Proceedings of the 2nd International Symposium on Supercritical Fluid Technology for Energy and Environmental Applications (Super Green 2003), Nagoya, Japan, November 9-12,2003, pp. 47-56. 8. MacLachan, C.N.S., and O.J. Catchpole, Separation of Sterols from Lipids, World Patent 90/02788 (1990).
9 , King, J,W,, Coupled Processing Options for Agricultural Materials Using Supercritical Fluid Carbon Dioxide, in Supercritical Carbon Dioxide: Separations and Processes,
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edited by A. Gopalan, C. Wai, and H.J. Jacobs, ACS Symposium Series No. 860, American Chemical Society, Washington, 2003, pp. 104-129. 10. King, J.W., Supercritical Fluid Technology for Lipid Extraction, Fractionation, and Reactions, in Lipid Biotechnology, edited by T.M. Kuo and H.W. Gardner, Marcel Dekker, New York, 2002, pp. 663-687. 11. King, J.W ., Sub- and Supercritical Fluid Processing of Agrimaterials: Extraction, Fractionation, and Reaction Modes, in Supercritical Fluids: Fundamentals and Applications, edited by E. Kiran, Kluwer, Dordrecht, 2000, pp. 451-488. 12. Foidl, N., Device and Process for the Production of Oils or Other Extractable Substances, U S . Patent 5,939,571 (1999). 13. Dunford, N.T., and J.W. King, Using a Supercritical Carbon Dioxide Fractionation Technique for Phytosterol Enrichment in Rice Bran Oil, J . Food Sci. 65: 1395-1399 (2001). 14. Dunford, N.T., and J.W. King, Thermal Gradient Deacidification of Crude Rice Bran Oil Utilizing Supercritical Carbon Dioxide, J . Am. Oil Chem. SOC.78: 121-125 (2000). 15. King, J.W., and G.R. List, eds., Supercritical Fluid Technology in Oil and Lipid Chemistry, AOCS Press, Champaign, IL, 1996. 16. Friedrich, J.P., G.R. List, and A.J. Heakin, Petroleum-Free Extraction of Oil from Soybeans with Supercritical CO,, J . Am. Oil Chem. SOC.59: 288-292 (1982). 17. List, G.R., and J.P. Friedrich, Processing Characteristics and Oxidative Stability of Soybean Extracted with Supercritical Carbon Dioxide at 50°C and 8,000 psi, J . Am. Oil Chem. SOC.62: 82-84 (1985). 18. Dickinson, N.L., and J.M. Meyers, Solexol Fractionation of Menhaden Oil, J . Am. Oil Chem. SOC.29: 235-239 (1952). 19. Reverchon, E., M. Poletto, L. Sesti Osseo, and M. Somma, Hexane Elimination from Soybean Oil by Continuous Packed Tower Processing with Supercritical CO,, J . Am. Oil. Chem. SOC.77: 9-14 (2000). 20. King, J.W., E. Sahle-Demessie, F. Temelli, and J.A. Teel, Thermal Gradient Fractionation of Glyceride Mixtures Under Supercritical Fluid Conditions, J . Supercrir. Fluids 10: 127-137 (1997). 21. Ssuss, D., and G. Brunner, Countercurrent Extraction with Supercritical Carbon Dioxide: Behaviour of a Complex Natural Mixture, in Proceedings of the GVC-Fachaussschuss Hochdruckve&hrenstechnik, Karlsruhe, Germany, 1999, pp. 189-192. 22. Jackson, M.A., and J.W. King, Methanolysis of Seed Oils in Flowing Supercritical Carbon Dioxide, J . Am. Oil Chem. SOC.73: 353-356 (1996). 23. Jackson, M.A., and J.W. King, Lipase-Catalyzed Glycerolysis of Soybean Oil in Supercritical Carbon Dioxide, J . Am. Oil Chem. SOC.74: 103-106 (1997). 24. Turner, C., J.W. King, and T. McKeon, Selected Uses of Enzymes with Critical Fluids Anal. Chem. Znt. 87: 797-810 (2004). in Analytical Chemistry, J . Assoc. Off. 25. Jackson, MA., J.W. King, G R . List, and W E . Neff, Lipase-CatalyzedRandomization of Fats and Oils in Flowing SupercriticalCarbon Dioxide, J . Am.Oil Chem. SOC.74: 635-639 (1997). 26. Rovetto, L.J., S . Pereda, S.B. Bottini, and C.J. Peters, Phase Equilibria in Mixtures of Hydrogen, Propane, and Fatty Oil Derivatives at Supercritical Conditions, in Proceedings of the 6th International Symposium on Supercritical Fluids, Versailles, France, April 28-30,2003, Vol. 2, pp. 825-830. 27. Harrod, M., and P. Moller, Hydrogenation of Fats and Oils at Supercritical Conditions, in High Pressure Chemical Engineering, edited by P.R. von Rohr and C. Trepp, Elsevier, Amsterdam, 1996, pp. 4 3 4 8 .
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28. Brunner, G., and S. Peter, Zum Stand der Extrraktion mit Komprimierten Gasen, Ger. Chem. Eng. 5: 181-188 (1992). 29. Sahle-Demessie, E., Fractionation of Glycerides Using Supercritical Carbon Dioxide, Ind. Eng. Chem. Res. 36: 49064913 (1997). 30. King, J.W., J.M. Snyder, H. Frykman, and A. Neese, Sterol Ester Production Using Lipase-Catalyzed Reactions in Supercritical Carbon Dioxide, 2. Lebensm-Unters.Forsch. A 212: 566-569 (2001). 31. King, J.W., R.L. Holliday, G.R. List, and J.M. Snyder, Hydrogenation of Vegetable Oils Using Mixtures of Supercritical Carbon Dioxide and Hydrogen, J. Am. Oil Chem. SOC. 78: 107-1 13 (2001). 32. Holser, R., G.R, List, J.W. King, R.L. Holliday, and W.E. Neff, Modeling of Hydrogenation Kinetics from Triglyceride Compositional Data, J. Agric. Food Chem. 50: 7111-7113 (2002). 33. Ramirez, E., F. Recasens, M. Fernandez, and M.A. Larrayoz, Sunflower Oil Hydrogenation on Pd/C in SC Propane in a Continuous Recycle Reacto, AZChE J. 50: 15451555 (2004). 34. Clifford, T., Fundamentals of Supercritical Fluids, Oxford University Press, Oxford, 1999. 35. Brunner, G., Gas Extraction, Springer-Verlag, Berlin, 1994. 36. Ibanez, E., F.J. Palacios, G. Senorans, G. Santa-Maria, J. Tabera, and G. Reglero, Isolation of Tocopherols from Olive By-products with Supercritical Fluids, J. Am. Chem. SOC. 77: 187-190 (1977). 37. King, J.W., and N.T. Dunford, Phytosterol-Enriched Triglyceride Fractions from Vegetable Oil Deodorizer Distillates Utilizing Supercritical Fluid Fractionation Technology, Sep. Sci. Technol. 37: 451462 (2002). 38. King, J.W., F. Favati, and S.L. Taylor, Production of Tocopherol Concentrates by Supercritical Fluid Extraction and Chromatography, Sep. Sci. Technol. 31: 1843-1857 ( 1996). 39. Taylor, S.L., J.W. King, L. Montanari, P. Fantozzi, and M.A. Blanco, Enrichment and Fractionation of Phospholipid Concentrates by Supercritical Fluid Extraction and Chromatography, Ital. J. Food Sci. 12: 65-76 (2000). 40. Taylor, S.L., and J.W. King, Optimization of the Extraction and Fractionation of Corn Bran Oil Using Analytical Supercritical Fluid Instrumentation, J. Chromatogr. Sci. 38: 91-94 (2000). 41, Taylor, S.L., and J.W. King, Preparative Scale Supercritical Fluid Extraction/ Supercritical Fluid Chromatography (SFE/SFC) of Corn Bran, J. Am. Oil Chem. SOC. 79: 1133-1 136 (2002). 42. York, P., U.B. Kompella, and B.V. Shekunov, eds., Drug Delivery and Supercritical Technology, Marcel Dekker, New York, 2004. 43. Proceedings of the 6th International Symposium on Supercritical Fluids, Versailles, France, 2003, Vol. 3. [See Table 7.51. 44. Eggers, R., and H. Wagner, Extraction Device for High Viscous Media in a High-Turbulent Two-Phase Flow with SC-CO,, J. Supercrit. Fluids 6: 31-37 (1993). 45. Wagner, H., and R. Eggers, Entolung von Ssojalecithin mit Uberkritischen Kohlendioxid in Hochturbulenter Zweiphasestromung,Fat Sci. Technol. 95: 75-80 (1993).
46. Castor, T.P., and L. Chen, Method and Apparatus for Making Liposomes Containing Hydrophobic Drugs, U.S. Patent 5,776,486 (1998).
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47. Castor, T.P., Methods and Apparatus for Making Liposomes, U.S. Patent 5,554,382 ( 1996). 48. Magnan, C., H. Commenges, E. Badens, and G. Charbit, Fine Phospholipid Particles Formed by Precipitation with a Compressed Fluid Anti-Solvent, in Proceedings of the GVC-Fachaussschuss Hochdruckverfahrenstechnik, Karlsruhe, 1999, p. 223. 49. Magnan, C., E. Badens, N. Commenges, and G. Charbit, Soy Lecithin Micronization by Precipitation with a Compressed Fluid Anti-Solvent-Influence of Process Parameters. J . Supercrit. Fluids 19: 69-77 (2000). 50. Weber, A., C. Nolte, M. Bork, and R. Kummel, Recovery of Lecithin from Egg YolkExtracts by Gas Anti-Solvent Crystallization, in Proceedings of the 6th Meeting on Supercritical Fluids: Chemistry and Materials, Nottingham, 1999, pp. 181-184. 51. Frederiksen, L., K. Anton, B.J. Barrat, P. VanHoogevest, and H. Lenenberger, in Proceedings of the 3rd International Symposium on Supercritical Fluids, Strasbourg, France, 1994, Vol. 3, pp. 235-240. 52. Heidlas, J., and Z . Zhang, New Approaches to Formulate Compounds Using Supercritical Gases, in Proceedings of the 7th Meeting on Supercritical Fluids, Antibes, France, 2000, Vol. 1, pp. 167-172. 53. King, J.W., G.R. List, and J.H. Johnson, Supercritical Carbon Dioxide Extraction of Spent Bleaching Clays, J . Supercrit. Fluid 5: 38-41 (1992). 54. Setianto, W.B., R.L. Smith, H. Inomata, and K. Arai, Processing of Cashew Nut (Anacardium occidentale L.) and Cashew Nut Shell Liquid with Supercritical Carbon Dioxide and Water, in Proceedings of the 6th International Symposium on Supercritical Fluids, Versailles, France, April 28-30,2003, Vol. 1, pp. 41-46. 55. Zosel, K., Process for the Separation of Mixtures of Substances, U S . Patent 3,969,196 ( 1976).
Chapter 7
Short-Path Distillation for Lipid Processing Xuebing Xu Food Biotechnology and Engineering Group, BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
Introduction Distillation is one of the most important thermal separation methods. Short-path distillation (SPD) is a continuous separation process working under vacuum conditions, in which low evaporating temperature and short residence time allow distillation of thermosensitive products with minimal thermal stress. The separation mechanism of distillation arises from the difference in boiling points (b.p.) of the compounds. If the difference in b.p. of two compounds is sufficient, in principle, the separation can be made by distillation. To use distillation as a method for separation, the compounds must be sufficiently volatile before the compounds are decomposed or polymerized at high temperatures. The reductions in operating temperature and residence time through the use of SPD will extend the separation possibility of those bioactive compounds. SPD also refers to molecular distillation. The process is termed “molecular distillation” when the distance between the evaporator and condenser is less than the mean free path of the vapor molecules. The operating system is set up in such a way that the free path of the molecules is longer than the distance between the heated and cooled surfaces. Therefore, the probability of a vapor molecule colliding with another molecule is smaller than the probability of traveling without collision to the cold surface. For this reason, the technology was initially named molecular distillation. In most current publications, the two terms often refer to the same technology. Lipids are various natural compounds. Oils and fats represent a large group of natural and artificial materials in the food industry as well as in the cosmetic or chemical industries. As a milder separation technology, SPD has been used in oil and fat industry for the separation of monoacylglycerols (MAG), polyunsaturated fatty acids (PUFA) from fish oil derivatives, carotenoids, and tocopherols, With the increasing awareness of nutritional functionality of some lipids and the possible ill effects of chemically or structurally changed compounds, the use of SPD for the processing of lipids, especially the functional lipids, is increasing, Many process and engineering studies have been made on distillation technology. Also numerous theoretical and engineering studies on the understanding and development of SPD have been made (1-6). The purpose here is not to thoroughly evaluate 127
x. xu
128
the progress of these theoretical backgrounds. Instead, the chapter focuses on the practical applications of SPD in the processing of lipids. Process, Apparatus, and Operation Effect
A simple one-stage SPD process scheme is depicted in Figure 7.1. The process is considerably simpler than packing column distillation or other distillation concepts, The central part of the process is the short-path evaporator. An illustration of the evaporator is depicted in Figure 7.2. The short-path evaporator with an internal condenser combines an evaporator and a condenser in a single apparatus. The distance between evaporation and condensation is extremely short. This eliminates pressure losses caused by piping (2). In a normal distillation evaporator, the evaporator and condenser are two separate components connected by nozzles for vapor transfer. The vacuum pump system is connected to the condenser. The ultimate pressure of the design is limited to some millibars by the pressure drop through the connection between evaporator and condenser. Even a larger suction capacity of the vacuum pump will not lower
U 1 Feeding pump
exchanger J-~ilbath
n
+ vacuum P-P
trap-vacuum Pumps (for condenser)
U
U
Fig. 7.1. Process scheme of short-path distillation.
SPD for Lipid Processing
129
Fig. 7.2. Illustration of the evaporator. Source: Reference 7.
the pressure in the evaporator. This limitation is caused by the conductance of the connecting pipe, which is conceptually longer than SPD. In an SPD system, the distance between the evaporator and condenser is only a few centimeters. Actually there is no pressure drop between the evaporator surface and the internal condenser. This makes very low pressure distillation possible (1). A typical pressure range is the “fine vacuum range,” i.e., between 1 and 0.001 mbar or even lower. At this pressure, the required evaporation temperature will
decrease so far that during the short residence time, no thermal decomposition of the product can occur. This makes it possible to use low temperatures to minimize
130
x. xu
damage to thermally labile molecules that would be decomposed by boiling at higher temperatures. The feed liquid is admitted into the evaporator under vacuum, immediately spread into a very thin film, and forced quickly down the evaporation surface. Heated walls and a high vacuum drive the more volatile components (distillate) to the closely positioned internal condenser as the less volatile components (residue) continue down the cylinder. The resulting fractions, thus separated, exit through individual discharge outlets. Depending on the application, the desired product is either the distillate or the residue fractions. The commonly used roller-wiper system consists of a cylindrical “basket” composed of guide rods on which rollers are fixed (2). The system is driven by a motor from outside the distillation chamber. It optimizes the efficiency of the evaporators. The product film on the surface of the evaporator is mixed continuously. The wiper system minimizes the concentration gradient inside the thin film. In fact, the evaporator surface is also increased by the surface of the rollers. The most significant advantage of an efficient roller-wiper system is its self-cleaning characteristic. Centrifugal force presses these rollers into the liquid film flowing downward on the evaporator wall. Because there is no mechanical load on the evaporation surface, scratches and “fouling” are avoided. The open construction of the wiper basket, with its large distances between the individual roller strands, guarantees a direct discharge of the vapors with very little pressure loss. A good roller-wiper system guarantees a uniform, turbulent mixing of the film on the entire evaporator surface. So-called “dead-zones’’are avoided due to overlapping of the rollers as well as wiping on the unheated areas. At the same time, the liquid film on the rollers themselves is continuously renewed, resulting in a very low residence time for the evaporating distillate on the evaporator surface. Build-up of material deposited on the evaporator surface and on the rollers should be avoided completely (2). The speed of the roller-wiper is one of the important parameters that affect the separation efficiency. It not only affects the thickness of the falling film and flow mixing, but also the splashing of the residual to the distillate (6) as shown in Figure 7.3. The speed of the roller-wiper should not be too low or too high as discussed in our previous results (8). The feed from the feed tank is first heated to a higher temperature by a heat exchanger. It is reported that the temperature of the liquid entering the evaporating cylinder of an evaporator is one of the important technological parameters that determine an evaporator’s operation (5). The surface temperature of the liquid along the downward flow can be described by Equation 1:
where T,(z) is the surface temperature (K) along the coordinate parallel with evaporator cylinder (m); T, is the wall temperature (K); h(z) is the film thickness (m)
131
SPD for Lipid Processing
Splashing
Condensed quantity
Evaporated quantity
Resistance flow Evaporator
Condenser
Fig. 7.3. Illustration of the flows in the evaporator. Source: Reference 6.
along the coordinate parallel with the evaporator cylinder; AevpHis the heat of evaporation (J/mol); and k is the rate of evaporation [mol/(m2.s)]. In general, it is useful to preheat the feed to a temperature close to the asymptotic temperature in an appropriate front-end heat exchanger before it enters the evaporator. The materials are sent to a degasser before flowing into the evaporator. Degassing is a very important step in large-scale operations because the air and low-molecule volatiles will strongly interfere with the vacuum stability in the evaporator and affect the distillation efficiency. Many other factors can affect the separation efficiency. Feeding rate and evaporator temperature are the most important factors as shown in the previous study (8). An increase in feeding rate will increase the productivity of the process, but will reduce the residence time of the fluids in the evaporator and affect the separation efficiency. The high volatility of the evaporation at a high feeding rate will increase the loss of heat. Therefore, the evaporator temperature and the efficiency of heat transfer of the evaporator are also related to the feeding rate. These factors interact with each other; however, too high an evaporation temperature will reduce the separation efficiency even though the volatility can be increased. The Rayleigh equation is used to determine the separation efficiency in the case of distillation on continuous-film evaporators (Equation 2 ) (6): ln[xw(l - xF)/xF(l - x w ) ] a=1+
In[ W( 1 - xw)/F(1 - xF)]
PI
where a is the relative volatility, which represents the separation efficiency of the process; xw and xF are mole fractions of the more volatile components in the residual and in the feed, respectively; and W and F are residual (mol/s) and feed flow (molh), respectively. The loss of the nonvolatile fraction to the distillate flow can be seriously affected by the choice of suitable or optimal operation conditions such
x. xu
132
as evaporator temperature (6). This can be expressed by the molar fraction in the distillate flow (Y)as in Equation 3: Y=
X0(1 - U K ) 1-XoUK-
[31
( 1 -X0)UK+1
where X and Xo are mole fractions in the residual flow and in the feed, respectively; K is the constant below -1; and u is given in Equation 4: u=-
a. = Xo/(l - Xo)- Xo(l -x> a X(1-X) X(1-X0)
[41
(E)
can be simply described
With reference to this description, the recovery yield as follows:
5.=1-uK
[51
According to the volatility of the two components in the evaporator, absolute separation of the two components is normally impossible. Theoretically, there will exist volatile components will be in the residual flow and less volatile components in the distillate flow in a single pass of the evaporator. It is possible only to optimize the conditions to obtain the best performance for both residual and distillate flows. The limit of the molar fraction of the distillate flow can be described in Equation 6:
Therefore a better separation requires more than one step. This was proven in our previous operations in the pilot plant and laboratory processes (8).
Applications for Lipid Processing Many lipids can be separated and processed by SPD as the sole process or as a part of the process setup. In principle, lipids that have sufficiently different b.p. can be separated by SPD. The relation between vapor pressure and temperature for some lipids is given in Figure 7.4. Different pressure units are used in the literature. For convenience in comparing the results, a table is provided for the change rates between different units (Table 7 . 1 ) . The separation possibility is largely dependent on the difference in vapor pressures between two components at certain temperatures. The larger the difference, the better the separation that can be expected by SPD. In the following sections, typical applications in lipid processing are provided.
SPD for Lipid Processing
133
1000
100
10
1
0.1
0.01
0.001 1
Fig. 7.4.
I
I
I
I
I
I
I
1
I
1
Temperature ("C) Relation between vapor pressure and temperature for different lipids.
Separation of FFA from Oils and Fats. When large amounts of FFA are present in oils and fats, the traditional methods such as alkali refining or physical refining are not commonly applied. Traditional refining is normally applied to FFA 4%. In the system of enzymatic production of structured lipids in which acidolysis is commonly used for the reaction between oil and an FA, relatively large amounts of FFA exist in the product mixture after reaction. Evaporator conditions depend highly on the b.p. of the FA separated (8). Figure 7.5 demonstrates the effects of the interaction between evaporator temperature and feeding rate (pump scale) on the contents of oleic acid in rapeseed oil in a model operation. In general, the lower feeding rate and higher evaporator temperature favor a lower FFA content in the residuals. To reduce the FFA content to O S % , for example, a range of possible
conditions can be used as shown in Figure 7.6 (black zone). This again indicates that the two parameters are closely interrelated (8). SPD is widely used for the
x. xu
134
TABLE 7.1 Pressure Units Used in the Literature and Their Conversion Ratesa mbar
1
mbar
0.01 1.33322 68.947
Pa torr/mmHg
psi
Pa
100 1 75.06 6894.7
torrImmHg
0.75006 0.0075006 1 51.71 5
psi
0.01 45 0.0001 45 0.01 9337 1
1013.25; mbar = 76; cmHg = 1.01325 x lo6; dyne/cm2 = 33.8985; ftH,O = 29.9213; inHg = 406.782 in H,O = 1,03323;kg-force/cm* = 101.325; kPa = 760; mmHg = 14.6859; psi = 760; torr = 101 325; a1 atm =
Pa = 1013.25 hPa.
purification of structured lipids in laboratory studies or in pilot-scale operations. Some of the references that demonstrate the actual uses are presented (9-15). Fractionation of FA. Figure 7.4 demonstrates the differences in vapor pressure under different temperatures. Some FA clearly can be separated due to their different b.p. A typical application is the fractionation of PUFA such as EPA and DHA in fish oils, which are particularly sensitive to temperature and other harsh conditions. The fractionation of EPA and DHA by SPD is normally conducted using their ethyl esters, which have even lower b.p. The FA compositions of fish oils are considerably more complicated than those of common vegetable oils and fats such
c C
8C
8
Fig. 7.5. Interaction effects of evaporator temperature ("C) and pump scale on the optimization of the short-path distillation separation of oleic acid from rapeseed oil
for FFA content (wt%) in the residuals. The relation between volume feeding rate and pump scale is illustrated in Figure 7.6. Source: Reference 8.
SPD for Lipid Processing
135
Pump scale Fig. 7.6. Optimal parameter zone (black) to obtain the FFA content in the residual of 0.5 wt% for the short-path distillation separation of oleic acid from rapeseed oil. The relation between volume feeding rate and pump scale is illustrated in the inside figure. Source: Reference 8.
as rapeseed and soybean oils. The SPD fractionation can normally remove the FA with a chain length <20 carbons. Most of 20:0, 20:1, and 22:l will be difficult to remove from EPA and DHA by a simple SPD operation. In general, EPA and DHA contents in the residuals can be increased to 5 0 4 % from a material with 20-30% EPA and DHA in a single distillation under optimal conditions. The higher the EPA and DHA content expected in the residuals, the higher the loss of EPA and DHA to the distillates because a relatively higher temperature must be used to remove as much of the other FA present as possible. Therefore, the recovery of EPA and DHA will be reduced in an effort to increase the content of EPA and DHA in the residuals. This phenomenon was described in a pilot study with enzymatically produced ethyl esters from salmon oil (unpublished data) as shown in Figure 7.7. Higher EPA and DHA enrichment as well as their recovery can be obtained through multiple distillation steps under optimal distillation conditions. The theoretical background was discussed earlier. This will eventually improve the separation efficiency. Under the conditions given in Figure 7.7, two further distillations at 140°C for both the distillate and residual from the distillation at the same temperature can improve the recovery from 65%
in the figure to 90% with similar EPA and DHA content in the product (6045%). A large amount of work has been conducted to date for the SPD fractionation of EPA
x. xu
136
100
I00
--8 80 2
80
4 E In
-
P !2
2
.G
rn
60
60
Q)
I D
C 0
3
C
4-
0
a 40
4o
I
n 0 C
m
2
20
20
w 0
0
90
100
110 120 130 140 150
Evaporator temperature ("C)
8
-
.;!
Fig. 7.7. EPA (+) and D H A (0) enrichment in
the residuals (YO) and EPA and D H A recovery (%) under different evaporator temperatures ("C). Operation conditions: UIC KD6; feeding rate 3.8 kg/h; vacuum <0.001 mbar; heat exchanger temperature 90°C; and condenser temperature 30°C (unpublisheddata).
and DHA. The technology has been used in industry. A few publications and patent documents are included for further reading (16-19). SPD processing of other FA was also reported (20). Separation of Partial Glycerides. Monoglycerides, diglycerides, and triglycerides with similar FA compositions have different b.p. This is obvious because they contain different numbers of FA in the molecules. Few detailed studies or data exist that show the difference in the vapor pressures for different FA compositions under different temperatures. However, SPD has been used for a long time in industry for the purification of monoglycerides as food emulsifiers. Actually, very few publications are available on SPD separation of monoglycerides. Most of the detailed information on the industrial technology remains unknown to the public. For those glycerides containing pure saturated FA, the b.p. of the corresponding monoglyceride, diglyceride, and triglyceride differ by >50°C under different vacuum conditions. This difference makes the separation much easier. Purity up to 90% can be obtained easily in a single-step distillation in our pilot operation. Commercial products with purity up to 95% from many international companies claim to use purification by SPD. In a mixture containing different glycerides (40% monoglycerides) produced from linseed oil, SPD was used for the separation of the monoglycerides. Purity 295% with a recovery >90% was obtained at 160°C (unpublished data). A detailed demonstration is given in Figure 7.8. In a more complicated case for the produc-
tion of diglycerides from a glycerolysis mixture produced from butterfat, a product with up to 85% diglycerides was obtained after SPD separation (21). The FA com-
SPD for Lipid Processing
m
a m CrJ
w
C
a
ea
a
Evaporator temperature ("C)
137
Fig. 7.8. Short-path distillation separation for the recovery of monoglycerides from a mixture containing -40% monoglycerides, 40% diglycerides, and 20% triglycerides. Conditions: UIC KD6; heating exchange temperature 80°C; condenser temperature 40°C; feeding rate 2,500 mVh; and vacuum <0.001 mbar (unpublished data).
position of the product was not significantly changed compared with butterfat (Table 7.2). The effect of the evaporator temperature on the distillate glycerides profile is given in Figure 7.9. By considering the amount of monoglyceride in the distillates, optimal conditions can be selected. Notably, conditions for the different scales may vary greatly depending on the efficiency of heat transfer and other process setups. The conditions noted in the literature should not be extrapolated to another specific situation. Optimizations should be made individually. Some related reports are provided for further reading (22-24).
Fractionation of Oils and Fats Oils and fats can be fractionated by many different methods such as chromatography, crystallization, adsorption, and distillation on the basis of the differing physical and chemical properties of the various components in oils and fats. Crystallization has been used in industry for the fractionation of palm oil and some other fats, using m.p. as the basis for differentiation. Distillation is not commonly used for oil and fat fractionation because the b.p. of different triglycerides do not differ greatly for most common oils and fats. For certain oils and fats containing short- or medium-chain FA, the difference in b.p. is larger and can be applied for the separation into two fractions. Butterfat is a typical case. Under certain temperatures, butterfat can be separated into two parts. The distillates will definitely con-
tain more short-chain FA such as 4:0,6:0,8:0 as well as 10:0 and 12:O (Table 7.3). This was demonstrated in two earlier publications (25,26). The different fractions
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TABLE 7.2 Proportion of Acylglycerols and FA Composition of DAG Product Produced from Butterfat from SPD Separationa(b Acylglycerols
(8/100 g)
MAG 1,2(2,3)-DAC 1,3-DAG TAG
7.0 35.0 51 .O 7.0
FA 4:O 6:O 8:O 1o:o
12:o 12:l 14:O 14:l 15:O 16:O 16:l 17:O 18:O 18:l n-9 18:2n-6 18:3n-3 20:o
( moI%)
6.78 4.65 2.54 5.10 5.00 0.21 13.22 1.06 1.31 28.7 1.20 1.27 9.00 17.1 1 1.19 0.39 0.12
aSource:Reference 2 1 . bAbbreviations: MAG, monoglycerides; DAC, diglycerides; and TAG, triglycerides.
with different FA compositions will have different properties and can be used for different applications. The distillates containing a higher content of short- and medium-chain FA can be used as structured lipids for nutritional purposes. Palm kernel oil and coconut oil can be fractionated using the same principle. Deodorization of Oils and Fats. Deodorization is performed to remove smelling/ odor compounds from oils and fats to meet the sensory quality requirement. Smelling compounds are normally low-molecule volatiles commonly formed by oxidation. For certain sensitive oils and fats, milder deodorization in terms of lower temperature and operation time is used. For fish oils, SPD has been considered for deodorization as well as for some other nutritional oils (27,28). Even though SPD is more efficient in removing volatiles than a normal deodorizer, SPD is not as effective as traditional batch deodorization, in which steam stripping is used (8). Sensory evaluations for the products of SPD deodorization and batch deodorization are given in Figure 7.10. The SPD product had a considerably poorer sensory evaluation than the batch deodorization product. As an explanation for this outcome, Figure 7.3 demonstrates the occurrence of a resistance flow during the distillation. SPD has been implemented industrially for the deodorization of red palm oil to preserve the carotenoids and tocopherols for nutritional considerations
(29). Crude palm oil contains 600-700 ppm carotenes and 800-900 ppm tocopherols. Normally refined and deodorized palm oil contains very little carotenes
SPD for Lipid Processing
MAG
1,2 (2,3)-DAG
139
1,3-DAG
Fig. 7.9. Effect of temperature on the glyceride profiles of the distillates from shortpath distillation of a glyceride mixture from butterfat. Conditions: UIC KD4; heat exchange temperature 80°C; condenser temperature 80°C; feeding rate 200-250 mL/h; and vacuum <0.001 mbar. Abbreviations: MAG, monoglyceride; DAG, diglyceride; TAG, triglyceride. Source: Reference 2 1.
(close to zero) and 500-600 ppm tocopherols. After SPD deodorization, red palm oil contains 500-550 ppm carotenes and -700 ppm tocopherols. Purification or Separation of Unsaponifiable Substances in Oils and Fats.
Unsaponifiable matters in oils and fats represent a large group of compounds even though their content in oils and fats is very low. In general, 0.1-1.0% unsaponifiable matters are found in oils and fats. In most cases, these compounds are nutritionally valuable; therefore, they should be recovered if possible. In other cases, such as the cholesterol in butterfat or lard, they should be removed. A few reports exist. One report discussed the fractionation of squalene from amaranth seed oil in which squalene concentration was increased about sevenfold, with a squalene
recovery of 76% in the distillate when degummed amaranth seed oil was fractionated at 180°C under a 0.003 tom vacuum (30). Another report discussed the reduc-
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TABLE 7.3 Yield of Distillates (YO) and FA Composition (% Area) of Native Butterfat and Distillates Obtained by Short-Path Distillation at Different Evaporator Temperaturesa,b Evaporator temperature ("C)
FA
4:O 6:O 8:O 1o:o 12:o 14:O 14:l 15:O 16:O 16:l 18:O 18:l 18:2 18:3 20:o
Butterfat
125
150
180
190
200
225
250
3.3 2.6 1.5 3.3 3.5 10.8 1.8 1.3 27.6 2.9 11 .o 25.1 3.2 0.8 1.1
4.4 3.1 2.2 4.6 5.1 13.0 2.1 1.6 25.2 2.8 8.5 20.7 2.9 1.8 2.0
10.7 7.1 5.6 9.3 8.0 15.1 1.7 1.5 21.6 2.0 4.1 10.5 1.6 0.7 0.4
12.5 7.4 5.5 9.4 8.2 14.3 1.7 1.7 21.7 1.9 4.0 9.1 1.4 0.8 0.3
9.7 4.1 7.6 0.1 16.9 2.0 1.8 30.7 2.4 5.6 11.5 1.2 0.5 0.3
9.0 5.1 3.6 7.0 7.0 16.0 1.8 1.6 26.7 2.3 5.5 12.0 1.5 0.5 0.5
8.1 4.8 2.9 5.5 5.7 14.4 1.9 1.4 27.8 2.6 6.7 15.3 1.9 0.5 0.4
4.8 3.4 2.0 4.0 4.3 12.4 1.9 1.4 28.8 2.8 9.1 20.5 2.7 1.0 1.0
0.5
1
2
8
10
20
43
Yield of distillates ( d l 0 0 g)
5.7
aSource: Reference 25. bPope laboratory short-path distillation still, feeding rate 9.8 g/min, vacuum 0.01-0.02 mbar, and blade rota tion speed 20 on the dial. The oil was first degassed at 100°C and 0.1 mbar.
tion of cholesterol in butterfat and lard (3 1). Cholesterol was almost completely removed with minimal loss of low-M.W. TAG at 185°C under a <0.001 torr vacuum. Similar results were obtained for lard at 250°C without significant modification of the TAG composition. A third example discussed the recovery of carotenoids from palm oil instead of preserving them in the oil as discussed earlier (32-36). A product containing 30,000 ppm carotenes was obtained from an ethyl ester material containing 600 ppm carotenes by distillation at optimal conditions. Tocopherol recovery is also an important consideration for SPD separation (37-40). This process has been applied in industry in combination with other process steps. Phytosterol purification and separation have also received attention (37,38,41). In a particular case, SPD was used for the purification of wheat germ oil to preserve the large amount of unsaponifiable material (42). Sesame oil is important in terms of its sesamol and sesamin content. A process involving the use of SPD was proposed for the recovery of these compounds (43). Apart from oils and fats, the pigments in carrot can be enriched by SPD processing (44).
Furthermore, chlorinated insecticide compounds in milk fats, possibly coming from polluted farm fields, can be removed by SPD (45).
SPD for Lipid Processing
141
4SPDRi
2y
BDR1
SPDAv SPDR2
0-2 -
BDAv
-4BDR2
6-
PC1
X-eXpl: 55%,30%
1.0 -
0.5 -
0-
9.5 -
-1.0
I
Fig. 7.10. Sensory evaluation of randomized structured lipids from fish oil and tricaprylin that was either batch-deodorized (BD) or deodorized by short-path distillation (SPD). (A) Scores plot and (B) loadings plot of primary component (PC)1 and PC2 from primary component analysis (PCA). Three PC were validated, together explaining 95% of the variation. A = aroma, F = flavor. The following sensory attributes were used: train oil (train), rancid (ranc), dusty (dust), metallic (metal), green, acidic (acid), synthetic (synt), rubber-like (rubb), nutty (nutt), pricks on the tongue (pric), bitter (bitt),
and miscellaneous (misc). R refers to pseudo-replicates calculated as the mean of half of the assessor data. Source: Reference 8.
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Concluding Remarks SPD has played an increasingly important role in lipid processing especially for heat-sensitive compounds. It has the important advantages of lower temperature and shorter residence time. This is especially useful in the treatment of nutritional materials. Many lipid substances can be separated, fractionated, or purified by SPD technology by itself or as part of a multistep process. More and more interesting applications will b e seen in industry in the future. On the other hand, SPD is not applicable to those lipids that will decompose at high temperatures. It is also not as effective as traditional methods for the deodorization of oils and fats. Thus, more studies are warranted to examine these problematic areas. More studies are also needed to examine internal phenomena to improve efficiency for different applications in lipid processing. A new mechanism to improve the above-mentioned problems might be achievable. Acknowledgments Bert Nielsen and Preben B. Hansen did an excellent job in the application of SPD for lipid processing in our pilot plant. Support from the Danish Technological Research Council (STVF) and Center for Advanced Food Studies (LMC) is acknowledged.
References 1. Maa, J.R., and S.Y. Tsay, Separation Efficiency of Molecular Distillation, J . Vacuum Sci. Technol. 10: 472-477 (1973). 2. Erdweg, K.J., Molecular and Short-Path Distillation, Chem. Znd.: 342-345 (1983). 3. Nguyen, A.D., and F. LeGoffic, Limits of Wiped Film Short-Path Distiller, Chem. Eng. Sci. 52: 2661-2666 (1997). 4. Micov, M., J. Lutisan, and J. Cvengros, Balance Equations for Molecular Distillation, Sep. Sci. Technol. 32: 3051-3066 (1997). 5. Cvengros, J., J. Lutisan, and M. Micov, Feed Temperature Influence on the Efficiency of a Molecular Evaporator, Chem. Eng. Sci. 78: 61-67 (2000). 6. Lutisan, J., J. Cvengros, and M. Micov, Heat and Mass Transfer in the Evaporating Film of a Molecular Evaporator, Chem. Eng. Sci. 85: 225-234 (2002). 7. Brownilie, R., Distillation Takes a Short Cut, Process Eng.: 31 (1999). 8. Xu, X., C. Jacobsen, N.S. Nielsen, M.T. Heinrich, and D.Q. Zhou, Purification and Deodorization of Structured Lipids by Short-Path Distillation, Euro. J . Lipid Sci. Technol. 104: 745-755 (2002). 9. Kawashima, T., Y. Shimada, T. Nagao, A. Ohara, T. Matsuhisa, A. Sugihara, and Y. Tominaga, Production of Structured Triglycerides Rich in 1,3-Dicapryloyl-27-linolenoyl Glycerol from Borage Oil, J . Am. Oil Chem. Soc. 79: 871-877 (2002). 10. Yankah, V.V., and C.C. Akoh, Batch Enzymatic Synthesis, Characterization and Oxidative Stability of DHA-Containing Structured Lipids, J . Food Lipids 7: 247-261 (2000). 11. Willis, W.M., and A.G. Marangoni, Assessment of Lipase- and Chemically Catalyzed Lipid Modification Strategies for the Production of Structured Lipids, J. Am. Oil Chem. SOC.76: 4 4 3 4 5 0 (1999).
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12. Lee, K.T., and C.C. Akoh, Characterization of Enzymatically Synthesized Structured Lipids Containing Eicosapentaenoic, Docosahexaenoic, and Caprylic Acids, J . Am. Oil Chem. SOC.75: 495-499 (1998). 13. Nielsen, N.S., X. Xu, M. Timm-Heinrich, and C. Jacobsen, Oxidative Stability During Storage of Structured Lipids Produced from Fish Oil and Caprylic Acid, J . Am. Oil Chem. SOC.81: 375-384 (2004). 14. Timm-Heinrich, M., X. Xu, N.S. Nielsen, and C. Jacobsen, Oxidative Stability of Milk Drinks Containing Structured Lipids Produced from Sunflower Oil and Caprylic Acid, Eur. J . Lipid Sci. Technol. 105: 4 5 9 4 7 0 (2003). 15. Jacobsen, C., X. Xu, N.S. Nielsen, and M. Timm-Heinrich, Oxidative Stability of Mayonnaise Containing Structured Lipids Produced from Sunflower Oil and Caprylic Acid, Eur. J . Lipid Sci. Technol. 105: 449-458 (2003). 16. Breivik, H., G.G. Haraldsson, and B. Kristinsson, Preparation of Highly Purified Concentrates of Eicosapentaenoic Acid and Docosahexaenoic Acid, J . Am. Oil Chem. SOC. 74: 1425-1429 (1997). 17. Goffic, F.L., A.D. Nguyen, and C . Khayat-Frydman, Separation and Recovery of Individual Long-Chain Fatty Acids from Natural Oils by Transesterification, Short-Path Distillation to Form Two Fractions, Transesterification of Fraction Containing Lower Alkyl Esters, Purification and Recovery, GB Patent 2350610-A (2000). 18. Zigerlig, M., High-Concentration Polyunsaturated Fatty Acid (Ester) Mixtures Preparation from Animal or Vegetable Oil, by Alkaline Hydrolysis, Acidification, Extraction and Molecular Distillation, Used as Dietetic Alimentary Additives, etc ., GB Patent 2218984-A (1989). 19. Liang, J.H., and L.S. Hwang, Fractionation of Squid Visceral Oil Ethyl Esters by ShortPath Distillation, J . Am. Oil Chem. SOC. 77: 773-777 (2000). 20. Cermak, S.C., and T.A. Isbell, Pilot-Plant Distillation of Meadowfoam Fatty Acids, Ind. Crops Products 15: 145-154 (2002). 21. Yang, T., X. Xu, H. Mu, and A.J. Sinclair, Production of Butterfat Diacylglycerols by Glycerolysis and Short-Path Distillation, J . Am. Oil Chem. SOC.81, in press. 22. Kaplon, J., K. Minkowski, and E. Kaplon, Concentration of Monoglycerides by Molecular Distillation, Inzynieria Chemiczna I Procesowa 22: 627-632 (2001). 23. Peter, S . , E. Weidner, B. Czech, and U. Ender, Fractionation of Glycerides by LowPressure Gas Extraction, Fat Sci. Technol. 95: 4 7 5 4 8 2 (1993). 24. Szelag, H., and W. Zwierzykowski, The Application of Molecular Distillation to Obtain High-Concentration of Monoglycerides, Fette Seifen Anstrichm. 85: 443-446 (1983). 25. Campos, R.J., J.W. Litwinenko, and A.G. Marangoni, Fractionation of Milk Fat by Short-Path Distillation, J . Daily Sci. 86: 735-745 (2003). 26. Arul, J., A. Boudreau, J. Makhlouf, R. Tardif, and T. Bellavia, Fractionation of Anhydrous Milk-Fat by Short-Path Distillation, J . Am. Oil Chem. SOC.65: 1642-1646 (1988). 27. Marschner, S .S ., and J.B. Fine, Simultaneous Deodorization and Cholesterol Reduction of Fat or Oil-esp. Fish Oil, by Deaerating, Mixing with Steam, Heating, Flash Vaporizing, Thin-Film Stripping with Counter-Current Steam and Cooling, WO Patent 8802989-A1 (1988). 28. Basu, H.N., A.J. Del Vecchio, and F. Kincs, Production of Micronutrient Enriched Deodorized Seed Oil Involves Subjecting the Extracted and Degummed Seed Oil to Short-Path Distillation, and Removing the Free Fatty Acids and Odor Producing Compounds, WO Patent 2000491 16-A (2000).
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29. Nagendran, B., U.R. Unnithan, Y.M. Choo, and K. Sundram, Characteristics of Red Palm Oil, a Carotene- and Vitamin E-Rich Refined Oil for Food Uses, Food Nutr. Bull. 21: 189-194 (2000). 30. Sun, H., D. Wiesenborn, K. Tostenson, J. Gillespie, and P. Rayas-Duarte, Fractionation of Squalene from Amaranth Seed Oil, J. Am. Oil Chern. SOC.74: 413-418 (1997). 31. Lanzani, A,, P. Bondioli, C. Mariani, L. Folegatti, S. Venturini, E. Fedeli, and P. Barreteau, A New Short-Path Distillation System Applied to the Reduction of Cholesterol in Butter andLard,J.Am. 0ilChern.Soc. 71:609414(1994). 32. Batistella, C.B., E.B. Moraes, R. Maciel, and M.R.W. Maciel, Molecular Distillation Process for Recovering Biodiesels and Carotenoids from Palm Oil, Appl. Biochem. Biotechnol. 98: 1149-1159 (2002). 33. Batistella, C.B., and M.R.W. Maciel, Recovery of Carotenoids from Palm Oil by Molecular Distillation, Cornput. Chem. Eng. 22: S53-S60 (1998). 34. Lenfant, C., and F.C. Thyrion, Extraction of Carotenoids from Palm Oil 2. Isolation Methods, Oleagineux Corps Gras Lipides 3: 294-307 (1996). 35. Ooi, C.K., Y.M. Choo, S.C. Yap, Y. Basiron, and A.S.H. Ong, Recovery of Carotenoids from Palm Oil, J. Am. Oil Chern. SOC.71: 423-426 (1994). 36. Tou, G.P., and P.T. Gee, Recovery of Minor Components, e.g., Carotene, and Refining of Vegetable Oils and Fats Comprise Removal of Polar Components Using Alcohol, U S . Patent 2002115876-A1 (2002). 37. Eloy, M.A., Extracting Purified Tocopherols and Sterols, for Chemical or Pharmaceutical Use, from Mixtures Containing Fatty Acids, by Esterifying the Acids with Trimethylol Propane and Two-Stage Molecular Distillation, WO Patent 200200640-A (2002). 38. Schwarzer, J., W. Johannisbauer, B. Bmegel, and M. Nitsche, Method of Concentrating Tocopherol(s) and/or Sterol(s) -by Fractional Distillation and Molecular Distillation of Mixtures of Fats and/or Fat Derivatives, Especially Rape Oil, Sunflower Oil or Soya Oil, DE Patent 19652522-A (1965). 39. Bracco, U., J.L. Viret, and J. Rehacek, Antioxidant-Containing Fraction Preparation from Vegetable Materials -by Distillation of Mixture of Solvent, Extract, Distillation Vector and Oil, Used in Foodstuffs and Cosmetics, CH Patent 641829-A (1984). 40. Green, J., and P.R. Watt, The Concentration of Tocopherols from Natural Sources by Molecular Distillation, J. Sci. Food Agric. I : 157-162 (1950). 41. Norinobu, S., N. Seo, F. Sato, S. Kaneko, and M. Mankura, Manufacture of Sterol Fatty Acid Ester for Foodstuff, Involves Hydrolyzing Fatty Acid Ester in Deodorized Scum Oil, Removing Generated Fatty Acid, and Reacting Sterol Fraction with Lipid Degrading Enzyme and Refining, JP Patent 3 19241 1-B1 (2001). 42. Singh, L., and W.K. Rice, Purification of Wheat Germ Oil-by Degumming, Bleaching with Activated Clay and Molecular Distillation, WO Patent 8002100-A (1980). 43. Ozaki, T., Y. Hoshi, H. Matsueda, and Y. Hoshii, Refining Sesame Seed Oil with Production of Sesamin Fraction by Steam Stripping Sesame Seed Oil and Molecular Distillation of Fraction, EP Patent 449436-A (1991). 44. Wu, Y., and G. Zhong, Molecular Distillation Extraction for Carrot Colored Pigment in Orange Fragrant Oil, CN Patent 1107007-A (1995). 45. Bills, D.D., and J.L. Sloan, Removal of Chlorinated Insecticide Residues from Milk Fat by Molecular Distillation, J. Agric. Food Chern. 15: 676-681 (1967).
Chapter 8
Fat Crystallization Technology Serpil Metina and Richard Hartelb aCargill, Incorporated, Wayzata, MN 55391 and bUniversity of Wisconsin, Madison, W I 53706
Introduction Foods can be accepted as functional and nutritional if they contain bioactive components that reduce the risk of disease andlor promote good health. Significant research has been conducted in the area of the diet-health relation. For more than a decade, there has been a substantial demand for the production of functional foods by manufacturers and for the consumption of healthy foods by health-conscious customers to promote good health. Fats and oils, which are essential components of our diet, have also garnered much interest from researchers, food manufacturers, and consumers. Although fats and oils have been considered an undesirable ingredient in our diet for some time, recent research findings emphasize their necessity for our body functions and good health. Many fats and fatty acids (FA) are linked to good health. For example, fish oil is linked to the prevention of cardiovascular disease and to mental development, and phospholipids are linked to brain development and the prevention of Alzheimer’s disease. Even though many healthy fat components are available in the form of tablets, consumers prefer to consume healthy lipids in food products because multiple tablet intake is generally required to consume the suggested amounts for daily requirement. Thus, modification and purification technologies are increasingly important, an area in which crystallization plays a large role. Recent activities in the area of positive aspects of fats and oils, and their components include: (i) the development of trans-free margarines, shortenings, and spreads with desired functional properties; (ii) the production of fats and oils with low saturated fatty acid (SFA) content and high monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA) contents; (iii) structuring lipids either to enhance the absorption of healthy components such as n-3 FA, phosphatidylserine, palmitic acid, phytosterols, conjugated linoleic acid (CLA), or to decrease their energy contents; and (iv) the development of high-stability oils that have greater resistance to oxidation than regular oils. Fats and oils perform a wide range of functions in foods. The functionalities include emulsion formation, lubrication, imparting and carrying flavor, protection of
flavor and nutrients, providing texture and taste, and serving as a heat transfer medium. For example, fats and oils play a crucial role in the formulation and production 145
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of many bakery items. Their ability to become softer and melt is an important functionality during baking. In addition, baking fats must resist the hydrolytic effects of water. At the same time, their wetting abilities help them cling to the wall surfaces of dough and act as an emulsifier or surfactant. Moreover, fats also remove energy from the dough during cooling through their latent heat of crystallization. This is required for setting up a product, especially in flaky pastries. Baking fats also act as shorteners, i.e., shortenings. This functionality allows fat to reduce binding between protein and carbohydrate, resulting in a softer and more tender texture in baked goods. This functionality also helps when chewing and swallowing a baked good. Another functionality of shortening is to trap air during the whipping process, helping the cell structure in the baked foods (1). Fat crystallization is an important determinant in food quality, texture, and shelf-life. Understanding and controlling the crystallization behavior of fats are required for the development of the desired functional properties, texture, and stability. Fat and oil modification is a rapidly growing area in the food industry, which requires economic and efficient technologies to produce healthy oils, fats, and food products. Knowledge of fat crystallization may be required in many applications; thus, in this chapter, fat crystallization and its applications for healthy foods will be reviewed.
Crystallization of Fats Nucleation, Growth, and Polymorphism
Natural fats are composed of a mixture of lipid materials. They are made up primarily of triacylglycerols (TAG), which may constitute from 95 to 98% (or higher) of the fat. Minor components in natural fats, depending on the degree of refining, also include diacylglycerols (DAG), monoacylglycerols (MAG), free fatty acids (FFA), phospholipids, sterols, stanols, and a wide range of other more polar lipidlike components (e.g., sphingolipids, glycolipids, or waxes). Although TAG dominate crystallization behavior in natural fats, minor lipid components can have a significant effect. Within the TAG components of a natural fat, there is generally a wide diversity of chemical composition. The FA esterified to the glycerol molecule come in a range of chain lengths (from C4:oto C24:o)and degrees of unsaturation (from saturated to up to three or more double bonds). Furthermore, if the fat has been hydrogenated, trans isomers of unsaturated FA may also be found. These diverse FA are then arranged on the glycerol molecule by the particular plant or animal source, usually not in a completely random pattern, to provide specific properties of the oil for the plant or animal. Some natural fats are relatively simple, with only a few TAG that dominate the crystals that form. Cocoa butter is a good example of a relatively simple crystallizing fat because typically there are three molecules that form the bulk of the
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147
crystalline lattice. Milk fat, on the other hand, is probably the most complicated of natural fats, with >300 different TAG species, none of which are present at >4-5% (molar basis) (2). Thus, milk fat crystallization is quite complex (3), and significant co-crystallization of different TAG molecules occurs within the lattice. To initiate crystallization of a fat, the crystallizing molecules must be supersaturated in some way. Crystallization of a single TAG molecule simply requires lowering the temperature of the molten fat below its melting point (m.p.). The degree of supersaturation, or the undercooling in this case, is simply the difference between the m.p. temperature (equilibrium condition) and the actual temperature. The lower the temperature, the higher the driving force for crystallization. In natural fats, a similar approach to defining undercooling is generally used, although in this case, with much less accuracy due to the range of compositional factors. In a natural fat, the m.p. is taken as the temperature at which the highestmelting crystals are completely melted. Because of the range of m.p. of individual TAG that make up the natural fat, in reality this is a melting profile, in which the amount of crystalline fat decreases as temperature increases over the melting temperature range. Cocoa butter, with its relatively simple composition (few dominant TAG), has a sharp melting profile somewhat similar to a single TAG (pure component), with melting over a fairly narrow range of temperatures from -25-35°C. Milk fat, on the other hand, has a very broad melting profile, sometimes stated to be from -40 to 40°C, due to the wide range of TAG that comprise it. When a natural fat, such as cocoa butter or milk fat, is cooled below its m.p., only certain TAG are undercooled by AT. Other TAG may actually be above their m.p.. For this reason, defining a true undercooling for a natural fat is nearly impossible. The empirical approach of using AT based on the final m.p. temperature is generally accepted, although it is recognized as being incorrect. At the moment, no better methods of defining the thermodynamic driving force for crystallization of a given species of TAG within a mixture in a natural fat have been developed. In some situations, as in solvent fractionation of fats, an organic solvent is used to control crystallization of fats. In this case, some of the fat crystals dissolve in the solvent, in the same manner as sugar dissolves in water. Supersaturation is then taken as the difference in concentration between the crystallizing state and the equilibrium solubility concentration (in the same way as sugar in water). When a natural fat is cooled sufficiently to be supersaturated, TAG with the highest m.p. come together and organize into a nucleus, the smallest state of aggregation with a crystal lattice ordering. During nucleation, molecules in the liquid state give up energy (in the form of latent heat) and associate in a lattice structure with other similar molecules (degree of saturation and chain length), as seen schematically in Figure 8.1. This step is called nucleation. The particular molecules that nucleate together and the form of the lattice structure depend on the conditions (e.g., temperatures, cooling rates, agitation rates) during nucleation. Typically, for conditions that give slow crystallization (cooling is slow, temperature is high), only the most similar molecules crystallize together (purest crystals),
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early
Fig. 8.1, Schematic representation of nucleation of triacylglycerols. Source: Reference 5. and the attractive interaction within the crystal lattice structure is the highest. On the other hand, when crystallization is rapid (temperature is low, cooling rate is fast), the nuclei formed contain more diverse lipid molecules and the crystal lattice energies are slightly less, indicative of a more disorganized crystal lattice. The different crystal lattice structures that can form under different crystalliza-
tion conditions are indicative of polymorphism, a common phenomenon in lipid crystallization. Polymorphism may be defined as the ability of certain molecules to
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form into different crystal lattice structures. That is, TAG molecules can arrange in several different crystal lattice orientations, each of which is recognized as being a distinct crystal structure. The most tightly oriented arrangement of TAG molecules in a crystal lattice gives the crystal structure with the lowest energy state (most stable polymorph). Typically, the most stable polymorph is termed a P polymorph, as indicated by a distinct X-ray scattering pattern associated with a particular lattice packing. The p polymorph has the lowest energy state, the highest density, the highest m.p., and releases the greatest amount of latent heat of all of the polymorphs. In lipids, monotropic polymorphism occurs, in which lower-stability polymorphs tend to crystallize easily and then transform into more stable polymorphs. If cooling is rapid and to a temperature below the m.p. of the a polymorph, the cx form is the one that generally crystallizes first. Because the a form is not the most stable polymorph, it quickly transforms to the more stable P' polymorphic form. If the 6' polymorph is not the most stable for a given fat, it will eventually convert to the most stable P form. The time scale for polymorphic transformation depends on numerous factors, including temperature, molecular diversity in the fat, and the presence of emulsifiers. The relative rates of formation of the different polymorphs and the rates of transformation to the most stable form are critical pieces of information for controlling lipid crystallization in foods. If formation of the desired polymorph in a food is not controlled during crystallization, undesired changes in the crystal structure may occur during storage that result in undesirable changes in the product over time. Some natural fats tend to form the most stable polymorphs (P-forming), whereas other fats remain in a less stable form @'-forming). In general, the more molecularly simple a fat, the more likely it will be found in the 6 polymorph. Cocoa butter, a relatively simple fat, easily forms the P polymorph. On the other hand, milk fat, the most molecularly diverse of the natural fats, is almost never found in the /3 polymorph, with the most stable form being the p' polymorph. Palm kernel oil (PKO) is intermediate. The 6' polymorph remains stable for months, even as long as a year under certain storage conditions, but eventually it transforms to the P polymorph. Once a nucleus forms, crystals continue to grow until the crystalline state reaches an equilibrium with the remaining liquid fat by either adding additional TAG molecules or through aggregation of crystalline clusters. At the same time, polymorphic transformation may be taking place. Frequently, nucleation, growth, and polymorphic transformation occur simultaneously during processing of a fat. Operating parameters that affect these phenomena include crystallization temperature, cooling rate, subsequent release of latent heat during crystallization, agitation rate or shearing, and whether the fat is in emulsion form or a bulk liquid. Furthermore, natural variations in composition (both TAG and minor lipids) in nat-
ural fats cause variations in these crystallization phenomena. Thus, it is often very difficult to control the formation and development of crystals in a natural fat.
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Despite the difficulties in controlling lipid crystallization, the size, shape, polymorphic form, and network interactions (all elements of the crystalline microstructure) of fat crystals are critical parameters that determine the physical properties of the material. A significant amount of work is currently underway to try to better understand the relations among crystallization conditions, the crystalline microstructure that is developed, and the physical properties of the resulting product. Some general principles relating lipid crystalline microstructure and physical properties are fairly well understood. For example, when natural fats are fractionated for specific uses, crystallization must be controlled to generate a crystal population that is easily separated from the liquid phase with little liquid entrainment. Ideally, large and uniformly shaped crystals that do not entrap liquid fat within the branches of individual particles should form in the fractionation process. When crystals are formed correctly during crystallization, the liquid fat can easily be pressed out to give efficient separation between liquid and solid fat. A second example in which controlling crystallization is important is in margarines and spreads. Here, the fat crystals provide the body that gives the spread its solid-like characteristics. There should be the right amount of fat (solid fat content; SFC), with crystals in the correct size and shape, and in the proper polymorph. The fat crystals also help to stabilize fine water droplets to provide a uniform texture. Usually, the p’ polymorph is desired in spreads because it gives the desired properties. Formation of a polymorph in margarine generally leads to a grainy product because these crystals are larger and more easily detected by the palate. Thus, controlling crystallization to generate the desired crystalline microstructure for each specific product is critical to product quality. Experimental Techniques Used in Fat Crystallization
There are well-established methods to determine FA and TAG composition as well as to determine overall crystallization (nucleation, growth, polymorphism, morphology). Numerous articles and books have been published on the principles and practical aspects of fat crystallization. More detailed information on experimental techniques and principles of fat crystallization can be found elsewhere ( 4 3 . The chemical composition of a fat should be known to determine the effects of major and minor components on crystallization. Chromatography methods such as high-pressure liquid (HPLC), gas (GC), gas-liquid (GLC) column, and a chromatography-mass spectroscopy (MS) combination are the most common techniques used to determine FA and TAG components of a fat. The FA composition of fats and oils is commonly determined by conversion of FA in TAG to either methyl or butyl esters, followed by separation of these esters with GC or GLC. Care should be taken to prevent losses of FA during analysis. For example, the high volatility of methyl or butyl esters of short-chain FA makes them vulnerable to losses during analysis. HPLC is also used for the determination of FA composi-
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tion of fats and oils. TAG composition of fats and oils is generally determined using a combination of different techniques. The most common techniques are HPLC coupled with GC, a combination of silver-ion HPLC, reversed-phase chromatography and GC, reverse-phase LC coupled with GC, reverse-phase HPLC coupled with MS, and a combination of GC, GLC, and MS (6). More comprehensive information about the isolation, separation, identification, and structural analysis of glycerolipids, including triacylglycerols, phospholipids, sphingolipids, and their various hydrolysis products, can be found in Christie (7). One of the most important characteristics of a fat is its final m.p. temperature, or the temperature at which the last, highest-melting crystal melts. However, several different methods were developed and commercialized for the final m.p. temperature. The m.p. of a fat is generally expressed as clear, cloud, slipping, softening, dropping, or cloud points. Each of these values represents a slightly different indicator of the final melting temperature. Standard analysis methods for determination of these points can be found in both AOCS (8) and AOAC (9). Because of its molecular diversity, natural fats melt over a range of temperatures. This melting profile is given by the change in SFC at different temperatures. The determination of SFC is one of the most important experimental parameters utilized by the fats and oils industry to indicate functionality and stability of fats and oils. In the past, dilatometry was used to characterize the percentage of crystallized fat at a given temperature, based on the extent of volume expansion upon melting of a crystal. However, because natural fats are never completely (100%) crystallized, even at very low temperatures, the solid fat measured with dilatometry is only a relative number (assuming 100% crystalline). This measurement gives a solid fat index (SFI). More recently, the amount of crystalline fat in a sample can be measured very accurately by using pulsed nuclear magnetic resonance (pNMR) spectroscopy. Because the actual crystalline content can be measured directly, pNMR gives a true value of SFC. Thus, SFC has slowly taken the place of SFI in the fats and oils industry. Differential scanning calorimetry (DSC) can also be used for determination of SFC; however, it is generally considered to be less accurate than pNMR. In the case of polymorphism, X-ray diffraction (XRD) spectroscopy is the most definitive method for determining which polymorph is found in a sample. XRD analysis can also provide detailed crystallographic information regarding the orientation of TAG molecules in the crystalline lattice. Recently, synchrotron XRD methods were developed to follow changes in crystallinity and polymorphic form in real time. However, access to synchrotron XRD is still limited to a select few laboratories around the world. Once peaks in a DSC melting curve have been identified as due to a particular polymorphic form in a specific fat, subsequent DSC analysis based on melting peaks can be used to differentiate polymorphs. However, because DSC peaks may also arise due to melting of different compound crystals in the same polymorphic form, the DSC is generally not used for definitive determination of polymorphs.
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Numerous methods have been developed to study crystallization of fats; however, measurement of and differentiation between nucleation and crystal growth are major challenges in fat crystallization studies (10). Light-scattering techniques, pNMR, DSC, and turbidimetry are commonly used to monitor nucleation and growth. However, many of these techniques are not sensitive enough to distinguish between nucleation and crystal growth. Wright et al. (10) compared light scattering, pNMR, microscopy, and turbidimetry techniques for measurement of induction times (i.e., nucleation event) and crystal growth. The results showed that the choice of experimental technique should be dependent on the application. The pNMR technique has been the most reliable method to characterize overall crystallization behavior. Turbidimetry and light-scattering techniques are very sensitive in early stages of crystallization; however, their sensitivity decreases at the latter stages of crystallization because highly saturated signals are obtained at this stage. Temperatures and cooling rates are easily controlled in pNMR, light scattering, and microscopy. Although microscopy is a cumbersome methodology, the combination of microscopy with image analysis gives information about crystal morphology as well as crystallization kinetics. More discussion of measurement techniques for characterizing crystallization is given in Hartel (5).
Use of Crystallization in Modification and Production of Nutritionally and/or Functionally Enhanced Oils and Fats Modification Techniques The edible-oil industry modifies fats and oils to increase their stability and functionality for a range of application areas and nutritional benefits. Blends of fats and oils may yield a fat mixture with different characteristics than that of the individual components. The range of food applications of the fats and oil or their blends may be increased by application of a modification technique or combination of modification techniques (1 1). Several processes are used to modify the properties of fats and oils. The most common processes are hydrogenation, interesterification, and fractionation. Hydrogenation. Hydrogenation, or the saturation of FA containing double bonds, has been a widely used process to improve the plasticity of oils. During the hydrogenation process, double bonds migrate along the carbon chain, resulting in positional isomerization. Additionally, some double bonds change their natural cis configuration to the trans configuration, which is known as geometric isomerization (1 1). However, recent concerns about trans FA produced during partial hydrogenation make it a less desirable process for the improvement of plasticity of fats. During partial hydrogenation, process parameters such as pressure, catalyst type,
concentration, and temperature can be controlled to decrease the amount of trans acids in the final product; however, to date, no process conditions, other than full
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hydrogenation, have successfully produced trans-free (<0.5%)or low-trans (4%) oil blends (11). Recently, fully hydrogenated fats were blended with oils to produce fat mixtures with no trans acids for different food applications. However, the high amount of SFA in these oil-fat blends can still make them a less desirable ingredient for healthy food applications. Esterification. Chemical and enzymatic interesterification processes are becoming more favorable for improvement of fats and oils based on their use in final food products. In chemical esterification, the FA within the TAG of a natural fat are either rearranged randomly among themselves or rearranged in the presence of another fat or oil to provide new mixtures of FA or TAG. Chemical esterification is inexpensive and easy to scale up; however, chemical esterification has some disadvantages for the production of structured lipids for medical, nutraceutical, and food applications because only random esterification of FA on a glycerol backbone is achieved. In short, the reaction lacks specificity; thus, there is no or little control over the positional distribution of FA in the final products. Chemical esterification is generally utilized for production of random mixtures of short-, medium-, and long-chain FA. For example, a mixture of medium- and long-chain TAG is hydrolyzed to obtain medium- and long-chain FA. After random mixing of medium- and long-chain FA, a chemical ester-exchange process is applied to obtain randomly mixed TAG containing both medium- and long-chain FA. This reaction is catalyzed by alkali metals or metal alkylates at high temperatures under anhydrous conditions. In this process, numerous unwanted products are also obtained in addition to the desired randomized TAG. The unwanted products may include FFA, mono- and diglycerides, which may be difficult to remove (12). Lipase-catalyzed enzymic esterification has the advantage of greater control over positional distribution of FA in the final products; however, it is usually more expensive than chemical esterification. Lipases catalyze both the hydrolysis of TAG into MAG, DAG, FFA, and esterification products based on the reaction conditions. Therefore, it is necessary to control reaction conditions. In addition to the esterexchange reactions, lipases can catalyze direct esterification, acidolysis, and alcoholysis reactions (12). Lipases, as used for the production of structured lipids for healthy oil applications, are usually most active between 30 and 40°C. As the temperature increases above 40°C, the stability of lipases decreases, although immobilization of the enzyme generally increases their thermal stability. Commercial lipases with nonspecific as well as sn-1 and sn-3 positional selectivity are available, but none are available with sn-2 positional selectivity. Enzymic esterification has been utilized for the production of nutritionally enhanced TAG, fats, and oils. For example, stearic acid and high-laurate canola oil were enzymatically esterified to produce trans-free margarine (13). Furthermore, structural lipids containing both long- and medium-chain FA were esterified enzymatically to achieve simultaneous delivery of the FA for enteral and parenteral nutrition.
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Fractionation. Fractionation of fats into different groups of TAG is typically done to provide certain textural properties in foods. Fractionation requires controlled crystallization of fats because solid and liquid fractions should be separated from each other easily. Thus, a large crystal size is desired to decrease the specific surface area. In fractionation, the different TAG in a fat are separated on the basis of either m.p. or solubility in a solvent to produce new fractions with different physical, chemical, and melting properties. The solid fractions are usually called stearins, whereas the liquid fractions are called oleins. The crystallization and subsequent separation process can be repeated to obtain more fractions with different melting properties. A variety of fractionation processes exists, including dry fractionation, solvent fractionation, fractionation by distillation and supercritical fluid extraction; however, only dry and solvent fractionation processes are based on crystallization of fats and oils. Dry fractionation, the most common fractionation technique due to its simplicity, cost efficiency, and nonuse of solvents, is based on the solidification properties of components of fats and oils. In dry fractionation, a fat is completely melted at a high temperature before controlled crystallization at a lower temperature so that the fat can be partially solidified. After completion of crystallization, the liquid portion is separated from the solid portion by a separation technique, usually filtration. A range of different fat fractions with different chemical compositions and melting properties can be produced by a multistage fractionation process. If a single-stage fractionation is applied, one solid and one liquid fraction are obtained. Controlling crystallization conditions is one of the most important criteria for dry fractionation. If crystallization is not controlled, and improper crystallizer and separation systems are utilized, fractionation will not be successful. The composition of the starting fat should be known to control crystallization during dry fractionation. Controlling crystal size and distribution is extremely important for efficient separation of crystals from liquid portion. Nucleation and growth must be controlled so that all nuclei reach the desired size and number at the same time so that they all grow in a uniform fashion in a crystallizer. Controlling crystallization temperature is also required for uniform crystallization and obtaining the desired polymorphic form ( 5 ) . The design of a crystallizer is of utmost importance. Heat transfer between the starting material and the cooling system dictates the local supersaturation and nuclei formation. Mixing and shear also affect the mass and heat transfer in a crystallizer. Many types of crystallizers are available commercially in the market differing in the nature of heat exchange surface, cooling system, and agitation system (14). As mentioned previously, fractionation includes two steps, i.e., crystallization of high-melting lipid components and subsequent separation of the solid from the liquid. Different separation techniques can be used including vacuum filtration, pressure filtration, and centrifugation. The separation of the solid and liquid fractions should be conducted at the same temperature as crystallization to prevent melting of crystals because even a small increase may cause melting of some solidified fat and a loss of
solid fraction yield (1 1). If there were no interactions between low- and high-melting components of the fat, separation efficiency would be very high. However, this is usu-
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ally not the case in natural fats, and mixed crystals (co-crystallization of low- and high-melting components) are usually formed, resulting in a decrease in separation efficiency. The amount of liquid fat trapped in the solid fractions affects their properties. In general, dry fractionation can also be classified into two crystallization processes, suspension crystallization and layer crystallization. Suspension crystallization is generally conducted as a batch process in which crystal suspension is filtered to obtain the crystalline fraction. Layer crystallization is also performed as a batch process either in static mode or in dynamic mode (falling film). Falling film layer crystallization (FFLC) involves growth of crystals on a heat-transferring surface by progressive cooling of the molten fat (15,16). Solvent fractionation has higher efficacy than dry fractionation; however, it is more expensive and requires a solvent separation step, which also removes lipid flavors. Solvent fractionation applies the same basic principles as dry fractionation except that a solvent solution is used for crystallization. The addition of a solvent promotes crystallization and improves separation efficacy. The chemical composition of the fats and fractionation temperatures are important parameters in solvent fractionation, as in dry fractionation. Commonly used solvents include acetone, hexane, ethanol, pentane, and isopropanol. The type of solvent affects the crystallization temperature as well as the crystallization behavior of fats (6,ll). Usually, scraped-surface heat exchangers are used as a crystallizer for solvent fractionation. Compared with dry fractionation, the crystallization residence time for solvent fractionation is much shorter (1 1). In solvent fractionation, a molten fat is dissolved in a solvent to form a solution, which is cooled to a desired temperature to start crystallization. After reaching equilibrium, high-melting glycerides solidify, whereas liquid fat remains in the solvent. The washing stage during separation is critical for removal of entrapped liquid fat in the solid cake. Vacuum filtration and centrifugation may be used for separation. Separation of the solid fraction from the liquid fraction should be followed by removal of solvent from the fractions for food applications. Evaporation, vacuum, or steam distillation can be used to remove any remaining traces of solvent (6,14). The formation of mixed crystals in natural fat systems is not as much a concern for solvent crystallization because of weaker interactions between solid and liquid components of a fat in a solvent solution. Therefore, fractions obtained from solvent fractionation have more distinctive chemical compositions than the fractions obtained from dry fractionation, allowing a better separation of the solid fraction from the liquid fraction (6,ll). Production and/or Modification of Fats for Enhanced Health and Functional Benefits
The ideal fat or oil should have excellent oxidative stability, the desired functional-
ity, and nutritional benefits, Fats and oils should be stable at ambient temperatures for excellent oxidative stability. In addition, fats and oils should have enough solid
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fat for use as margarines, shortenings, spreads, and frying fats for desired functionalities. To have nutritional benefits, fats and oils should contain less SFA and more PUFA and/or essential fatty acids (EFA). However, no single fat or oil fully satisfies the ideal fat requirements (17). To enhance oxidative stability, functionality and nutritional benefits, various modification techniques are employed by the oil industry such as hydrogenation, esterification, and fractionation. In some cases, use of a single modification technology may not give the desired properties to the final fat. Therefore, many applications include multiple techniques, such as both interesterification and fractionation processes together, to obtain the desired fats for food application. In the following section, examples of fat and oil products are reviewed to provide insight into the use of crystallization processes to improve nutritional and functional properties. Low or No-Trans FA Products. Hydrogenation of vegetable oils increases their m.p., plasticity and stability; thus, hydrogenation of vegetable oils has been commonly used to produce suitable ingredients for margarines, shortenings, and bakery fats. However, hydrogenation of TAG produces trans double bonds from cis double bonds, and the double-bond locations can change, resulting in numerous isomers. The trans-containing polyunsaturated fats cannot be substituted for EFA, and they are not converted to biologically active prostaglandins (18). The presence of trans FA in our diet has been correlated with increasing risk of heart disease (19). Because of the link between a diet containing trans FA and health, much research has been ongoing to decrease or remove the trans FA content of oils and fats; as a result there are many publications and patents in this area. Market demand for alternatives to trans fats is moving in parallel to the rising health concerns. Interesterification has found many applications in the area of low or no-trans fatty acid-containing oils and fats especially for the production of margarines, spreads, and shortenings. In addition, both dry and solvent fractionation are used to decrease/remove trans FA levels for these food products. For example, in a patent application of Cargill, Inc. (18), the dry fractionation process was employed to reduce trans double bonds in vegetable oils. In this patent application, solid flakes of low-melting glycerides of soybean oil were used as a seeding agent. The oil was crystallized in the presence of the solid flakes at temperatures of 5-15°C by agitation. After crystallization of the oil in the presence of the seeding agent, dry fractionation was applied to separate the high-melting fraction from the low-melting fraction. The low-melting fraction can be used as a salad oil and oil in salad dressing with the advantage of having low trans FA content. In the present invention, the low-melting fraction was also completely hydrogenated, mixed with the highmelting fraction, and then interesterified. This interesterified composition was blended with vegetable oils to produce shortening stocks, cooking oils, margarine stocks, and liquid shortening with a reduced amount of trans FA. The other developing area in the reformulation of food products with transfree fats is the use of palm oil. The FA composition of palm oil comprises 10%
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PUFA, 40% MUFA, and 50% SFA (20). Palm-oil formulations were shown to be very compatible in functionality with table margarines and bakery shortenings produced using partially hydrogenated oils. Fat crystallization plays an important role for the physical properties of margarines and shortenings, as discussed previously. Fractionation of palm oil results in the production of different fractions with different melting properties and FA composition. For example, palm mid-fraction, a POP (palmitic-oleic-palmitic)-rich fraction, can be mixed with another oil fraction to produce cocoa butter substitutes (20). Palm-olein fractions and palm oil can be used in frying applications due to their high stability at high temperatures, based on low levels of linoleic and linolenic acids, which tend to oxidize easily. Due to the increase in demand for trans-free fats, palm stearins obtained by fractionation are the most economic source of saturated fat for the interesterification process. Interesterification between palm stearin and an oil can give the functionality required for margarines and shortenings in food products. Fractionation conditions can be adjusted to obtain different yields of both palm olein and palm stearin. In addition, fractionation conditions can be controlled in a way to produce palm mid-fractions. Today, a range of palm oleins and stearins is available commercially by means of fractionation processes. The physical and chemical characteristics of palm stearin differ significantly from the mother oil, palm oil, and palm olein, and it is available in a wide range of chemical composition (i.e., FA and TAG). Palm oil tends to crystallize in the p’ form, but when it is diluted with liquid oil, its tendency to crystallize switches to the p form (21). Palm oil can be fractionated (see Fig. 8.2) to obtain different fractions with different crystallization properties, melting profiles, and chemical composition. Palm stearin is the solid fraction of palm oil obtained through the fractionation process. It is separated from the liquid fraction (palm olein) using filtration techniques. Palm stearin is a cost-effective natural vegetable hard fat, whereas palm olein is a more expensive product. The diverse FA profile of palm stearin leads to the formation of the p’ polymorph, the form desired for margarines and shortenings. However, the presence of a high level of liquid oil in soft margarines alters its polymorphic nature to the p form, which is not desirable in margarines. Thus, palm stearin by itself is not a good hard stock for margarines. However, interesterification of palm stearin with other oils and fats may yield a good hard stock with desirable properties for margarines (21). Fractionation can be utilized to produce a range of palm-stearin fractions suitable for different applications. Interesterification of palm stearin with palm kernel olein, a fraction of PKO, can produce a very good hardstock for margarines (21). Interesterification has become very popular for the production of low-trans or truns-free fats. Interesterification is one of the best answers to solve the trans FA issue in margarines and shortenings; however, the process is -20% more expensive than regular shortening production based on hydrogenation (22). Fractionation is
less expensive than interesterification, but fractionation can produce some undesirable qualities in fats to be utilized in margarine and shortening production (22). For
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Palm Fruit
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Fig. 8.2. Fractionation of palm oil and palm kernel oil into several fractions with different chemical composition and crystallization behavior.
example, palm stearin obtained from palm oil causes a waxy mouthfeel. The use of a combination of interesterification and fractionation allows production of oils and fats with desired properties for margarine and shortening production. For example, depending on the desired characteristics of the final product, the process would involve fractionation of fully hydrogenated palm oil blended with soybean or canola liquid oil, then interesterification and fractionation again. However, this process is more costly than interesterification alone (22). Norizzah et al. (23) suggested that chemical interesterification of mixtures of palm stearin with palm kernel olein results in a series of fat blends that can be used for the production of margarine, shortenings, nontempered types of confectionery fats, whipped creams, and similar products. Melting thermograms showed the disappearance of the high-melting glycerides of palm stearin when it was interesterified with PKO. In addition, the fat crystal morphology of the blends dramatically changed after interesterification.
Another attempt to prepare heat-stable, low-fat spreads without using hydrogenated fats was disclosed in a Unilever patent application (24). A mixture of two
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fats (Fat A and Fat B) in ratios from 1:9 to 4:6 by weight was formulated as a hardstock for the spreads. Fat A contained at least 50% saturated TAG (at least 80% palmitic or stearic acids), whereas Fat B had up to 5% lauric or myristic FA. Fat A was prepared by blending two fats with the ratio of palmitichtearic as 75:25 to 25:75. The fat was then interesterified enzymatically and dry fractionated. The same fat was also produced in the following order of processes: dry fractionation, enzymatic interesterification, and dry fractionation. The low-fat spread was prepared by emulsifying 50-80 wt% of the aqueous phase with 20-50 wt% of the fat phase and cooling. The saturated fat content of the spread was claimed to be <25% by weight. A range of solutions is available today for the production of trans-free fats. A first approximation for replacing trans fats in margarine, shortenings, and frying fat formulations is to produce trans-free fats with the same SFC of the original fat. However, matching the SFC alone usually does not result in a successful fat replacement. Overall crystallization properties of the replacement fat (trans-free fat), such as crystallization kinetics, polymorphism, crystal size and number, and melting properties, should be taken into account. It was shown that even though fats may have similar SFC over a relevant temperature range, their crystallization behavior, microstructure, and mechanical properties may be substantially different (25). For example, crystallization of a solution of 10% hardened palm oil in sunflower oil at 25°C results in the formation of a spreadable solid-like dispersion with small crystals. If the same solution is crystallized at 38OC, a dispersion with the same SFC at room temperature with large spherulitic crystals, which can be a pourable product at room temperature, is obtained (26). The difference in the crystallization kinetics at the two temperatures resulted in a difference in crystal size and the arrangement of crystals in the same fat solution. Thus, different textural properties in a food product can be experienced if a substitution or replacement is based solely on the SFC of a former fat. Understanding the relations among crystallization behavior, microstructure, and mechanical properties of fats is necessary for a successful replacement or substitution of one fat with another (25,26). Singh et al. (25) demonstrated how the microstructure of a fat plays a key role in determining mechanical properties. A chemically interesterified hydrogenated palm oil (IHPO) and a partially hydrogenated palm oil (PHPO) were structured on the basis of their microstructure to have the same mechanical properties. These fats showed different SFC and rheological properties in a particular temperature range. The Avrami index, which describes nucleation and growth kinetics of crystals, was used to match the crystallization behavior of IHPO and PHPO. The Avrami indices of the two fats were matched, resulting in similar hardness and spatial distributions of mass in the two fats. Dairy and Confectionery Fats, Dairy fats in our diet provide vital nutrients including EFA and fat-soluble vitamins. However, dairy and confectionery fats are
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typically classified as highly saturated fats. It is often suggested that consumption of dairy and confectionery fats should be avoided because of their high levels of saturated fat. However, this assumption is controversial. For example, Gurr (27) showed that consumption of dairy and confectionery fats does not necessarily result in high plasma cholesterol levels or increase the risk of cardiovascular diseases in human studies when these fats were utilized in diets. Milk fat is perceived as a natural and high-quality product with desirable flavor; however, the use of milk fat has declined because of its high saturated fat level and cholesterol content. Milk fat includes -70% SFA, from C8:oto C,o:o, However, FA with e l 2 carbon atoms are found to be neutral or may even decrease cholesterol. Stearic acid is actually found to decrease the cholesterol level (28). The main SFA in milk fat are myristic and palmitic acids, which are both considered to have a cholesterol-raising effect. Because of public awareness of the health-diet link, alternative strategies were investigated to reduce the elevating effect of milk fat on plasma cholesterol. These involve the modification of milk fat composition either by altering the cow’s feeding regimen or by fat fractionation (or both). The dairy industry has made technological advancements by modifying the chemical and physical properties of milk fat. Modification of the FA profile of milk fat is possible through feeding practices for dairy cows (29). Modification of milk fat can also be accomplished through physical processing. In an effort to increase the utilization of milk fat in many food applications. different modification techniques were used, including hydrogenation, interesterification, and fractionation. Hydrogenation has recently suffered from the drawbacks of trans FA formation. Interesterification is successfully used to modify the TAG structure of milk fat; however, a considerable flavor loss is often experienced because of the high process temperatures (16). Fractionation of milk fat is a more common process to improve functionality and to tailor fats for specific applications. Fractionation of milk fat was reviewed extensively by Kaylegian and Lindsay ( 6 ) .A typical fractionation process for milk fat is shown in Figure 8.3. In this process, four different fractions, two solid and two liquid fractions at room temperature, with different physical and chemical properties can be obtained through multistage fractionation. Changing FA andlor TAG composition and distribution of FA on a TAG affect crystallization and the melting behavior of milk fat. The ratio of SFA to unsaturated FA, the ratio of short-chain to long-chain FA, and the ratio of trisaturated TAG to mono- and polyunsaturated TAG change the physicochemical and functional properties of resulting products. For example, milk fat fractions can be combined with other fractions or with anhydrous milk fat to produce cold-spreadable butter (30). A spreadable butter at refrigeration temperature was accomplished by blending high- and low-melting fractions of milk fat; however, the butter turned to liquid at room temperature (30). A considerable increase in the unsaturated FA content of milk fat, either through feeding practices or fractionation procedures, may result in the formation of a butter that is too soft. Therefore, when composi-
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I Melted Anhydrous Milk Fat
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Solid High-Melting Fraction (HMF)
Liquid Fraction
Solid Middle-Melting Fraction (MMF)
Liquid Fraction
Fig. 8.3. Fractionation of anhydrous milk fat into several fractions with different chemical composition and crystallization behavior.
tional changes are pursued in milk fat, research on crystallization behavior should be combined with performance evaluation in the final food products. Scott and co-workers (31) studied the functionality and nutritional aspects of dairy creams formulated with natural dairy products, fractionated milk fat, and milk-derived components. Melting-range characteristics of butter-oil fractions influenced the FA profile of formulated creams. Incorporation of a low-melting butter-oil fraction lowered the amount of long-chain saturated acids and slightly increased unsaturated acids such as oleic and linoleic acids compared with the addition of medium-melting butter-oil fraction. Cholesterol was lower in creams formulated with a medium-melting butter-oil fraction than those with a low-melting butter-oil fraction. The incorporation of fractionated butter-oils changed the chemical composition of these creams, with possible nutritional benefits. The nutritional effects of fractionated milk fats on human plasma lipid and lipoprotein responses were reported by Jacques et al. (32). Milk fat was modified by removal of cholesterol by short-path distillation and melt crystallization. In this study,
the nutritional effects of the modified milk were compared with a regular milk fat and nonhydrogenated margarine. The SFC of the modified milk fat in the temperature
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range from 0 to 40°C was higher than that of regular milk fat and nonhydrogenated margarine. Based on the results, the modified and regular milk fats did not change plasma total and LDL cholesterol, whereas the margarine reduced them. However, the modified milk fat significantly reduced total- and VLDL cholesterol concentrations compared with regular milk fat and margarine. The authors stated that consumption of modified milk fat with a low cholesterol content might be responsible for this positive effect; a possible explanation is that this was due to lower intestinal fat absorption. The modified milk fat had a high solid fat level; thus, it had high viscosity at body temperature, and as a result, intestinal adsorption decreased. Thus, the authors suggested that the consumption of the modified milk fat might prevent the onset of hypertriacylglycerolemia. Complete understanding of the physiologic action of this modified milk fat requires further animal and human studies focusing on the effects of the fat on intestinal fat absorption, TAG production, and plasma postheparin lipoprotein lipase activity. Nutritional enhancement of butter was described in an Ault Food Limited patent (33) in which a plastic fat was produced via crystallization of a mixture of water and fat (butter, butter oil and renovated butter). The fat was heated to a temperature to retain stable crystals in the fat while melting all unstable crystals. An emulsion was prepared by the addition of 30-70% water by weight. The emulsion was cooled to crystallize a substantial amount of the liquid fat to obtain the desired plastic structure. It was claimed that the amount of fat in the final product was -30-50% by weight compared with regular butter, with 80% fat by weight. There has been a growing interest in CLA for its preventive role in cancer. CLA is formed during the biohydrogenation of PUFA in the rumen of cows and is found in milk and beef tissues (27). The CLA content of dairy foods can be enriched through dietary feeding practices by fortification with full-fat rapeseed and soybean oils, and by milk fat fractionation (34). The effects of dry fractionation on the CLA content of bovine milk fat were investigated by O'Shea et al. (35). Different cooling rates and crystallization temperatures were selected to determine the effects of cooling rate and temperature on the CLA content of the fractions. Molten milk fat was fractionated into hard and soft fractions at 19, 15, and 10°C at four cooling rates, 0.58, 0.78, 1.17, and 2.8"C/h. A soft fraction obtained at the 0.58"C/h cooling rate contained 63.2% more CLA (22 g/100 g FAME) and more PUFA and vaccenic acids compared with the parent fat. Vaccenic acid, the major trans FA in milk fat, is considered to be a nutritional FA because it is converted in vivo to CLA by the A9-desaturase enzyme. In comparison, vegetable oil spreads were found to contain more trans FA and lower levels of CLA than milk fat-containing products. The authors also stated that agitation after completion of fractionation has adverse effects on the CLA yield. Confectionery fats include cocoa butter, palm oil, PKO, coconut oil, and others. Cocoa butter is the main fat in chocolate products. Although cocoa butter has a high content of SFA, it has been accepted as a neutral fat in terms of its cholesterol-raising effect. This neutral effect is often explained as resulting from its high stearic acid content (27). The lauric oils, PKO and coconut, also include high lev-
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els of SFA. To address the controversial findings about the relation between high dietary intake of SFA and elevated plasma cholesterol levels, Gurr (27) published a well-documented article containing several research findings on the effect of consumption of dairy fats and confectionery fats on plasma cholesterol and TAG levels, and cardiovascular diseases. High-melting fractions of coconut and PKO are used as cocoa butter substitutes (CBS). These fats are known as lauric fats, and they solidify in the p’-form (the stable form) without tempering, whereas cocoa butter should be tempered to obtain the stable form @-form) for chocolate products for ease of release from molds, desired gloss and snap properties, shelf-life, and bloom prevention. However, lauric fats suffer from hydrolysis into soapy products and low compatibility with cocoa butter; they also initiate bloom. Because of these drawbacks, nonlauric and nontempering CBS alternatives were produced from soybean oil, sunflower oil, palm oil, and other vegetable oils. However, these products are transhydrogenated and suffer from the presence of trans FA, which are believed to have adverse health effects such as increasing blood cholesterol level and coronary diseases. Additionally, they have a slower crystallization rate than lauric CBS. There are also cocoa butter equivalents (CBE), which are trans-free, nonlauric alternatives such as palm oil and Illipe butter. CBE have polymorphism similar to that of cocoa butter; thus, they have to be tempered to obtain the desired stable crystals in the final products. Tempering is an expensive and complex process. If tempering is not accomplished properly, these fats suffer from the problem of fat bloom. To increase process efficiency and minimize the drawbacks of tempering and fat bloom, many researchers have pursued nontempering and non-trans fats. For example, the patent application of Aarhus Oliefabrik (36) describes the application of randomization and dry-fractionation processes to produce a nonlauric, non-trans, and nontempering confectionery fat. It was claimed that this fat crystallizes in a stable polymorphic form without tempering.
EFA and Vegetable Oils with Biologically Active Components EFA are those that the body cannot produce and have functions in our body that cannot be replicated by alternative means (37). EFA such as the n-3 and n-6 FA have gained enormous popularity, particularly in infant development; maternal, joint, and mental health; and prevention of cardiovascular disease. In addition, consumption of a diet rich in n-3 FA was shown to decrease blood pressure and blood lipids, and the risk of blood clotting. The key members of the n-3 FA are a-linoleic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Both EPA and DHA are found in fish oils, and ALA is found in flaxseed and many vegetable oils and nuts (37). Most EFA are very sensitive to heat, light, and oxygen; thus, they oxidize eas-
ily and become rancid. As a result, they are prone to produce off-flavors and free radicals that may be hazardous to human health. EFA cannot be hydrogenated
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because their health benefits cannot be preserved when their double bonds are saturated. Antioxidants are commonly added to prevent oxidation. However, it is still difficult to incorporate EFA into food products because of their sensitivity to processing conditions, particularly high-temperature conditions. When EFA are incorporated into food products, they should be added at the end of the process to preserve their efficacy. Encapsulation is also employed to fish oils to increase their resistance to processing conditions. Spray-drying can also be a method of choice for protection of EFA against oxidation (37). Crystallization can be used for the purification of EFA. In the following, several examples will be given in which crystallization was used as a main step in the purification of EFA. A patent of Lipozen, Inc. (38) describes the use of crystallization to isolate and purify certain unsaturated FA from a mixture. Crystallization is enhanced by the addition of urea at low temperatures to raise the purity level of oleic or linoleic acid to 99%. Safflower oil, corn germ oil, or olive oil is subjected to two-step crystallization using methanol and urea, followed by crystallization of the concentrated unsaturated FA using solvent at temperatures of -5 to -10°C. The patent also disclosed a method for isolating EPA at a purity level of at least 99% from sardine oil. The sardine oil is crystallized in two steps using urea and methanol to obtain an EPA-rich concentrate. Liquid chromatography using an Agsilica or Ag-alumina column is then used to purify the concentrate. Purification of PUFA can utilize crystallization as a processing step. Vali et al. (39) utilized a single-cell oil (Arasco of Martek Biosciences Corporation) to obtain high-purity arachidonic acid (AA). A three-step process was used with low-temperature solvent crystallization as a first step, resulting in a FA fraction containing 75.7% AA with 97.3% yield. The second step involved enrichment of AA content via lipase-catalyzed selective interesterification of the FA fraction with lauryl alcohol. A solvent extraction procedure was used as the third step to enrich AA from the FA fraction dissolved in n-hexane. The researchers were able to increase the AA content in the single-cell oil from 38.8 to 95.3% with a total yield of -71%. Vegetable oils and fats contain a large number of biologically active components in addition to TAG. The biologically active components are polar lipids such as phospholipids, sphingolipids, and galactolipids, and semipolar to nonpolar lipids such as phytosterols, triterpene and aliphatic alcohols, tocopherols, and tocotrienols. The unsaponifiable part of the oil, including nonpolar to semipolar lipids, hydrocarbons, and waxes, can be extracted by petroleum ether or diethyl ether after alkaline hydrolysis. For example, the solvent fractionation of a vegetable oil at low temperatures to obtain one or more solid fractions for confectionery applications as well as a liquid fraction rich in unsaponifiable biologically active components was described in a Karlshamms AB patent (40). In this patent, a mixture of shea butter and refined rapeseed oil having a m.p. of 32-55°C was blended with a solvent in a ratio of 1:3-7 (wthol). This blend was heated to transparency, and then cooled at a rate of O,l-l,S"C/min to a first fractionation temperature of -5 to +lO"C, The solid fractions were found suitable for use in confectionery applications. The liquid frac-
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tion was mixed with additional solvent and cooled at a rate of O.l-l"C/min to a second fractionation temperature of -30 to -15"C, and the resulting solid fraction was removed. The remaining liquid fraction was distilled to remove the solvent to give a liquid fraction in which unsaponifiable, biologically active components were enriched by at least a factor 2. Plant sterols, stanols, and their esters were shown to be very effective in lowering the plasma cholesterol level. They have been used in pharmaceuticals and cosmetics, and recently have become important additives to food products. Recently, margarines and spreads with added stanol and sterol esters became available commercially, targeting plasma cholesterol reduction (4 1). Sterols of plant origin are sometimes referred to as phytosterols. The major phytosterols are campesterol, stigmasterol, and 6-sitosterol. The sterols, tocopherols, and hydrocarbons are the unsaponifiable materials in oils. Processes such as solvent extraction, chemical treatment, crystallization, and molecular distillation are employed for recovery of sterols and tocopherols from deodorizer distillate (DOD), which is the by-product of deodorization during oil refining (41,42). Crystallization is commonly used as a processing step to purify sterols from DOD. Saponification is also a common process to concentrate tocopherols and sterols. The sterols are then separated from the concentrate by crystallization. Recently, a combination of molecular distillation with crystallization was used to concentrate tocopherols and sterols. Esterification andor transesterification is used before molecular distillation to increase the separation efficacy. However, this step makes the whole process both labor- and cost-intensive. Lin and Koseoglu (42) developed a simple, efficient, and economical process to concentrate tocopherols and sterols from DOD. They used crystallization because of its simplicity and because it prevented heat damage to tocopherols. Crystallization was used mainly to separate sterols from tocopherols and squalene. DOD was dissolved in different solvents (1:3, wdvol) and crystallized at -10 or -20°C. The sterol crystals were then removed from the solution by centrifugation at low temperature, followed by filtration and washing. The optimum conditions for crystallization were determined to be a temperature of -20°C for 24 h using a mixture of acetone and methanol. Over 90% of the tocopherols and squalene in DOD was recovered in the filtrate fraction, and 80% of total sterols remained in the cake fraction. Because phytosterols are effective in decreasing serum cholesterol levels and are inexpensive by-products of food processing, their utilization in food products was investigated rigorously by many researchers. The main problem with the use of phytosterols in foods is their poor solubility; they are poorly soluble in fats and insoluble in water. Therefore, several strategies were proposed by different investigators to increase the solubility and bioavailability of phytosterols. For example, a US. patent by Raisio, Finland (43) describes methods of producing FA esters of phytosterols to increase the solubility in a fat. Another Raisio patent application by Wester (44) uses unsaturated FA of sterol andor stanol as a replacement for a substantial portion of all of the undesirable saturated and trans-unsaturated fats in
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margarines, mayonnaise, cooking oils, cheeses, butter, and shortenings. This invention was based on the formation of a crystal network by stanol and sterol FA esters with properties similar to conventional hardstock triglycerides. An increase in the nutritional value of a fat blend containing stanol and/or sterol esters was claimed because only the FA portion is digested or absorbed (the sterol part is not). Thus, fats including these esters would result in a decrease in the total energy ingested when consumed in food products. MUFA and PUFA. These FA have been used in food applications for both their positive health effects and their wide range of functionalities. Consumption of MUFA and PUFA derivatives in the human diet was linked to reduced plasma cholesterol level. In addition, these FA are widely used as an intermediate raw material for emulsifiers such as monoglycerides and diglycerides, and an intermediate raw material for other sorts of industrial products. These FA are usually produced by hydrolysis of a vegetable oil or a decomposition method utilizing enzymes. However, sometimes fractionation of unsaturated and SFA may be desired for certain applications. Generally, a fractionation process utilizing solvents is employed for the separation of SFA and unsaturated FA. Although, solvent fractionation gives high efficiencies of separation, it is a costly process. Dry fractionation has become the most common approach when crystallization is used to separate desired FA or FA groups. For example, a European patent application by Kao Corporation (45) shows how to use the dry-fractionation process for the separation of SFA from unsaturated FA in a mixture of FA using polyglycerol esters of FA. The function of a polyglycerol ester of FA is to increase the crystal size of SFA; thus, separation (filtration) becomes very efficient. They also provided a formula to predict efficient separation, given as: 0 . 3 8 ~+ 13 s y s 0 . 5 4 ~ + 44, where x is a ratio (% by mass) of SFA (CI2-C2J in raw FA, and y is the clear m.p. ("C) of a polyesterglycerol of FA. When the clear m.p. of the polyglycerol ester of a FA is out of range as given by the above formula, the size of crystals becomes fine and filtration becomes impossible because of clogging. The properties of a fractionated fat can be changed by fractionation temperature, time, agitation, and the sequence of fractionation steps. For example, the trisaturated (SSS) fraction of palm oil is obtained by fractionation at 3640"C, whereas fractionation at 28-30°C enhances the disaturated-monounsaturated (SSU) content, and fractionation at 18-20°C results in a fraction with enhanced monosaturated-diunsaturated (SUU) content (11). By careful selection of fractionation temperature and time, a series of fractions with different properties can be obtained. A U.S. patent by Foglia and Lee (46) describes the production of MUFA- and PUFA-esters from menhaden oil and partially hydrogenated menhaden oil via either a one-step solvent fractionation or two-step (dry then solvent) fractionation. The partially hydrogenated menhaden oil was fractionated in solvent at a temperature range of 0 to 4 0 ° C for 24 h, and the solid fraction separated from the liquid fraction by centrifugation, cold pressing, or vacuum filtration at the same temperature. This process
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resulted in enrichment of both MUFA (0.5-24% by weight) and PUFA (340% by weight) in the liquid fraction compared with the original oil. In the two-step fractionation process, both menhaden oil and partially hydrogenated menhaden oil were dry fractionated at -10 and 30°C, respectively. After separation of the solid fraction from the liquid fraction, the liquid fraction was solvent fractionated. This method resulted in the use of less solvent for the enrichment of MUFA and PUFA in the resulting liquid fraction.
High-Stability Oils (HSO). HSO are liquid at room temperature, oxidatively stable, and preferably clear; applications include use as spray oil, carrier of flavors and vitamins, moisture barrier, gloss enhancer, and frying of products requiring long shelflife. HSO have low levels of SFA and high levels of unsaturated FA; they are liquid at room temperature. However, this combination of fatty acids typically would mean that these oils are very unstable. Specific processing steps andor genetic modifications are required to make them stable (47). Traditional HSO products use commodity oils. These oils are relatively inexpensive, but require a reasonable amount of processing to provide required oxidative stability because of poor oxidative stability of the starter oils. Hybrid oils (nontransgenic) offer alternative products. They have lower saturated and trans fatty acids. These oils are more costly than their commodity counterparts, but processing costs are reduced, resulting in similar final pricing for both HSO oils (17,47). The physical properties of HSO are dependent on the properties of the unmodified oil and the processing steps such as refining, bleaching, deodorization, and chemical and physical modification of the oils (47). Production of HSO generally includes refining, bleaching, deodorization, light or partial hydrogenation, and dry/ solvent fractionation. Hydrogenation is generally applied to increase the trans unsaturated FA because they have higher oxidative stability than their cis unsaturated counterparts. However, health issues related to trans FA will likely influence the use of hydrogenation in these oils. Fractionation based on crystallization behavior of the FA of these oils is used to separate the high-melting TAG because they impart the solid behavior at room temperature. In addition to crystallization, PUFA are replaced with MUFA to improve stability while maintaining liquidity at room temperature (47). HSO are highly functional and convenient to use. They are most commonly used as oils sprayed onto the surface of foods as a moisture barrier, gloss enhancer, lubricating agent, or antidusting/anticaking agent. Nonspray applications include use as a nut-roasting oil, a frying oil, and a viscosity modifier (47). List (17) stated the expectation of increased use of HSO in foods to reduce the fat content and improve shelflife and nutritional properties.
Summary The properties of many lipid-based foods can be altered through the proper use of crystallization-based technologies. Fractionation, either dry or solvent, separates lipids
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based on m.p. and solubility. When used in conjunction with other fat-modification technologies, fractionation can b e used t o produce a w i d e range of fats with improved health characteristics. Controlling crystallization can result in lipid products with modified saturation levels a n d l o w trans FA concentrations, with enhanced levels of EFA, and with high oxidative stability.
References 1. Stier, R.F., Finding Functionality in Fats and Oils. http://www,preparedfoods.com/ pf/cda/articleinformation/features (posted 11/23/2003). 2. Gresti, J., M. Bugaut, C. Maniongui, and J. Bezard, Composition of Molecular Species of Triacylglycerols in Bovine Milk Fat, J. Dairy Sci. 76: 1850-1869 (1993). 3. Kaylegian, K.E., and R.W. Hartel, Advances in Milk Fat Fractionation: Technology and Applications, in Crystallization of Fats and Lipid Systems, edited by N. Garti and K. Sato, Marcel Dekker, Inc., New York, 2001, pp. 381427. 4. Sato, K., Crystallization of Fats and Fatty Acids, in Crystallization and Polymorphism of Fats and Fatty Acids, edited by K. Sat0 and N. Garti, Marcel Dekker Inc., New York, NY, 1988, pp. 227-266. 5. Hartel, R.W., Crystallization in Foods, Aspen Publishers, Inc., Gaithersburg, MD, 2001. 6. Kaylegian, K.E., and R.C. Lindsay, Handbook of Milkfat Fractionation Technology and Applications, AOCS Press, 1995, Champaign, IL. 7. Christie, W.W., Lipid Analysis, The Oily Press Ltd., 2003, Dundee, Scotland. 8. American Oil Chemists’ Society, Official Methods of the American Oil Chemists’ Society, American Oil Chemists’ Society, 1973, Champaign, IL. 9. Association of Official Analytical Chemists, Official Methods of Analysis of the Association of Official Analytical Chemists, AOAC, 1990, Arlington, VA. 10. Wright, A.J., S.S. Narine, and A.G. Marangoni, Comparison of Experimental Techniques Used in Lipid Crystallization Studies, in Crystallization and Solidijkation Properties of Lipids, edited by N. Widlak, R.W. Hartel, and S. Narine, AOCS Press, Champaign, IL, 2001, pp. 120-131. 11. Allen, D., Fat Modification as a Tool for Product Development. Part 1. Hydrogenation and Fractionation, Lipid Technol. 10: 29-33 (1998). 12. Osbom, H.T., and C.C. Akoh, Structured Lipids-Novel Fats with Medical, Nutraceutical and Food Applications, Comp. Rev. Food Sci. Technol. 1: 93-103 (2002). 13. Fomuso, L.B., and C.C. Akoh, Enzymatic Modification of High-Laurate Canola to Produce Margarine Fat, J. Agric. Food Chem. 49: 4482-4487 (2001). 14. Illingworth, D., Fractionation of Fats, in Physical Properties of Lipids, edited by A. Marangoni and S. Narine, Marcel Dekker Inc., New York, 2002, pp. 41 1-448. 15. Erjawetz, S.P., J. Ulrich, M. Tiedtke, and R.W. Hartel, Milk Fat Fractionation by SolidLayer Melt Crystallization, J. Am. Oil Chem. SOC.76: 579-584 (1999). 16. Belkacemi, K., P. Angers, 0. Fischer, and J. Awl, Fractionation of Milk Fat by Falling Film Layer Crystallization, Sep. Sci. Technol. 38: 31 15-3131 (2003). 17. List, G.R., Decreasing Trans and Saturated Fatty Acid Content in Food Oils, Food Technol. 58: 23-31 (2004). 18. Cargill, Inc., Patent Application, International Publication Number WO 03/077665 A1
(2003).
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19. Juttelstad, A., Trans Fats: Status and Solutions, Food Technol. 58: 20-22 (2004). 20. Malaysia Palm Oil Promotion Council. Malaysian Palm Oil, Palm Oil Information Series, American Palm Oil Council (2003). 21. deMan, J.M., Use of Palm Stearin as a Component of Interesterified Blends. http:// www.soci.org. SCI Lecture Papers Series, 1-2 (2000). 22. Wade, M.A., Ingredients in Use: Margarines and Oils, http://www.preparedfoods.com/pf /cda/articleinformation/features (posted 02/01/2004). 23. Norizzah, A.R., C.L. Chong, C.S. Cheow, and 0. Zaliha, Effects of Chemical Interesterification on Physicochemical Properties of Palm Stearin and Palm Kernel Olein Blends, Food Chem. 86: 229-235 (2004). 24. Unilever N.V., International Patent Application WO 03/084337 A1 (2003). 25. Singh, A.P., C. Bertoli, P.R. Rousset, and A.G. Marangoni, Matching Avrami Indices Achieve Similar Hardness in Palm Oil-Based Fats, J. Agric. Food Chem. 52: 1551-1557 (2ow. 26. Kloek, W., P. Walstra, and T. van Vliet, Crystallization Kinetics of Fully Hydrogenated Palm Oil in Sunflower Oil Mixtures, J. Am. Oil Chem. SOC.77: 389-398 (2000). 27. Gurr, M.I., Health Aspects of Dairy and Confectionery Fats, in Production and Application of Confectionery Fats, Selected papers from a conference organized by the Society of Chemical Industry Oils and Fats Group held in London, UK, on 15-16 October 1996. Paper No. 10, edited by W. Hamm and R.E. Timms, P.J. Barnes and Associates, 1997, Bridgewater, UK. 28. Ney, D.M., The Role of the Nutritional and Health Benefits in the Marketing of Dairy Products, J. Dairy Sci. 74: 40024009 (1991). 29. Palmquist, D.L., A.D. Beaulieu, and D.M. Barbano, Feed and Animal Factors Influencing Milk Fat Composition, J. Dairy Sci. 76: 1753-1771 (1993). 30. Kaylegian, K.E., R.W. Hartel, and R.C. Lindsay, Applications of Modified Milk Fat in Food Products, J. Dairy Sci. 76: 1782-1796 (1993). 31. Scott, L.L., S.E. Duncan, S.S. Sumner, K.M. Waterman, and K.E. Kaylegian, Influence of Emulsifying Component Composition on Creams Formulated with Fractionated Milk Fat, J. Agric. Food Chem. 51: 5933-5940 (2003). 32. Jacques, H., A. Gascon, J. Arul, A. Boudreau, C. Lavigne, and J. Bergeron, Modified Milk Fat Reduces Plasma Triacylglycerol Concentrations in Normolipidemic Men Compared with Regular Milk Fat and Nonhydrogenated Margarine, Am. J. Clin. Nutr. 70: 983-991 (1999). 33. Ault Food Limited, US.Patent 5,096,732 (1992). 34. Stanton, C., A Health-Promoting Component of Animal and Milk Fat, Dairy Products Research Center, Project Report No. 26. School of Biology, Dublin, Ireland (1999). 35. O’Shea, M., R. Devery, F. Lawless, K. Keogh, and C. Stanton. Enrichment of Conjugated Linoleic Acid of Bovine Milk Fat by Dry Fractionation, Int. Dairy J. 10: 288-294 (2000). 36. Aarhus Oliefabrik A/S,International Patent Application. WO 03/037095 A1 (2003). 37. French, S., Formulating with EPAs, Nutraceuticals World, April, 34,36,38 (2004). 38. Lipozen, Inc. (Gyeonggi-Do, Korea), U.S. Patent 6,664,405 (2003). 39. Vali, S.R., H.Y. Sheng, and Y.-H. Ju, An Efficient Method for the Purification of Arachidonic Acid from Fungal Single-Cell Oil (ARASCO), J. Am. Oil Chem. SOC.80:
725-730 (2003).
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40. Karlshamms AB, U.S. Patent 6,552,208 B1 (2003). 41. Weber, N., P. Weitkamp, and K.D. Mukherjee, Cholesterol-Lowering Food Additives: Lipase-Catalysed Preparation of Phytosterol and Phytostanol Esters, Food Res. Znt. 35: 177-181 (2002). 42. Lin, K.-M., and S . S . Koseoglu, Separation of Sterols from Deodorizer Distillate by Crystallization, J. Food Lipids 10: 107-127 (2003). 43. Raisio Inc., U.S. Patent 5,502,045 (1996). 44. Wester, I., Fat Compositions for Use in Food, A Raisio Patent Application, U.S. Patent Application 0175402 A1 (2003). 45. Kao Corporation, European Patent Application, EP 1 371 717 A1 (2003). 46. Foglia, T.A., and K.-T. Lee, U.S. Patent 6,492,537 (2002). 47. Lampert, D., High-Stability Oils: What Are They? How Are They Made? Why Do We Need Them? in Physical Properties of Fats, Oils and Emulsifiers, edited by N. Widlak, AOCS Press, Champaign, IL, 1999, pp. 238-246.
Chapter 9
Dietary Fatty Acids and Their Influence on Blood Lipids and Lipoproteins Tilakavati Karupaiaha,Mohd lsmail Noora, and Kalyana Sundramb aDepartment of Nutrition and Dietetics, Faculty of Allied Health Sciences, National University of Malaysia and bMalaysian Palm O i l Board, Bandar Baru Bangi, Malaysia
Introduction Recommendations for lipid-lowering diets have traditionally focused on fat quantity and composition as a means of lowering total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), which are independent risk factors for coronary heart disease (CHD). To achieve these end-points, dietary modulations require not only a decrease in fat content per se, but also a decrease in saturated fatty acids (SFA) and an isoenergetic increase in polyunsaturated fatty acids (PUFA) and/or monounsaturated fatty acids (MUFA). Thus, dietary manipulations require a control over the fat composition in the diet, by striking a balance between the various fatty acid (FA) classes. Given the complexity of lipoprotein metabolism, the approach to dietary modulation for the last 40 yr has remained focused on the steady state of LDL-C generation and catabolism, through an understanding that saturated fats promote increased LDL-C production by downregulating LDL receptor (rLDL) activity. Conversely, replacing these saturated fats with unsaturated fats upregulates rLDL activity, thereby increasing the catabolism of LDL. This approach was substantiated by considerable evidence from human and animal studies. However, it has also been documented that most lipid-lowering diets designed to achieve decreases in TC and LDL-C also simultaneously cause decreases in high-density lipoprotein cholesterol (HDL-C). Thus, traditional dietary advice concentrates on the endogenous fat transport pathways, whereas it ignores the exogenous fat transport and reverse cholesterol transport systems, which are also components of lipoprotein metabolism. The ratio of TC and HDL-C is considered a more specific marker of CHD risk than LDL-C alone. This is in line with evidence correlating an increase in HDL-C concentration with a lower risk of CHD. Another aspect to consider about lipid-lowering diets is that the premise on which recommendations are based, i.e., the predictive regression equations, accounts for the behavior of only limited fat variables and FA, and oversimplifies lipoprotein response to these variables. The wider matrix today includes the adverse effects of trans FA and the recognition that plasma HDL-C and triacylglycerol (TAG) also constitute risks for CHD especially when there exists a constellation of risk factors for “low metabolic capacity.” For purposes of discussion, the Step 1 and Step 2 diets referred to in the 171
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subsequent sections are the widely advocated diets of the National Cholesterol Education Program (NCEP) and adopted by the American Heart Association as dietary measures for treating abnormal blood cholesterol levels (1). Rationale for Decreased Consumption of SFA
Dietary therapy for elevated LDL-C limits SFA consumption to ~ 1 0 % of energy intake as a moderate measure in the Step 1 diet and <7% of energy in the Step 2 diet for a more rigorous intervention (1). Cross-population studies established strong correlations between SFA intake and CHD incidence (2). In a cross-sectional survey of healthy men aged 40-49 yr in four European countries by Riemersma et al. (3), the highest incidence of mortality from CHD (212/100,000 in North Karelia men, Finland) was associated with the highest SFA content in adipose tissue (38% by weight) compared with the lowest rates in Italian men (26% SFA by weight with CHD mortality incidence of 43/100,000). Controlled feeding trials predict that a major reduction in SFA would also lower plasma TC by as much as 10-15% or 0.8 mmol/L (4). Intervention studies such as the Dietary Alternatives and DELTA trials demonstrated the benefit of lowering LDL-C to as much as 10% by reducing SFA energy intake to 7% (5,6). Yu-Poth et al. (7) postulated, with multiple regression analyses of 37 selected studies involving free-living subjects, that for every 1% decrease in energy consumed from dietary SFA, TC decreased by 0.056 mmol/L and LDL-C by 0.05 mmoVL. Clarke et al. (4) showed in the metabolic ward situation (by meta-analysis of n = 395 experimental human studies) that an overall 10% isoexchange of SFA led to reductions in plasma TC by 0.63 mmol/L and LDL-C by 0.52 mmol/L. That fat quality made the difference was clearly shown by a randomized, doubleblind study in which reduction of dietary fat energy intake from 37 to 30% did not lower TC and LDL-C concentrations unless accompanied by a substantial decrease in SFA from 16 to 9% of energy (8). Results from that study agreed with the predictive equations of both Keys (2) and Hegsted et al. (9), suggesting that SFA content had a major effect on TC and LDL-C levels. The meta-analysis of Clarke et al. (4) based on the metabolic ward studies also supported the conclusion that SFA, and not total fat reduction, made the difference in lipid lowering. The mechanism for change in LDL-C levels, affected by diets differing in fat saturation, may be explained by shifts in the steady-state levels of plasma TC, either as free or esterified forms predominantly in LDL and less so in very low density lipoprotein (VLDL) and HDL. This may reflect a change in lipoprotein structure, resulting in a lower TC to LDL ratio. The suggestion from metabolic studies involving normal humans is that any manipulation of dietary fat saturation leading to structural changes in LDL affects LDL-C metabolism (10). This likely involves both LDL generation and LDL catabolism. Goldstein and Brown (1 1) demonstrated in their landmark study that LDL levels in plasma are controlled by the activity of rLDL present in liver and peripheral
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cells, and rLDL determine LDL clearance. Following lipoprotein kinetics, such as with 1251-LDLin normal humans, indicated that reducing SFA increased the fractional clearance rate of LDL without a change in its synthesis, but with an associated fall in apolipoprotein (apo) LDL concentration. The ability of SFA to increase LDL-C levels by decreasing or “downregulating” rLDL-mediated catabolism has been mainly consistent. The study of Hara et al. (10) with normocholesterolemic humans showed that reducing dietary SFA increases or “upregulates” rLDL activity; the authors suggested that alterations in LDL composition promote increased receptor clearance. Woollett et al. (12) indicated that SFA independently regulated rLDL and LDL production rate with SFA, causing a dose-dependent increase in LDL-C production rate, whereas the “downregulation” mechanism occurs through depressed rLDL mRNA expression and decreased membrane fluidity. One of the consequences of decreasing dietary fat saturation is a concomitant decrease in HDL-C concentrations. A meta-analysis of 37 intervention studies in free-living subjects using either the Step 1 or Step 2 diets found that HDL-C decreased as much as 7% ( P = 0.05) in response to Step 2, but not Step 1 dietary intervention (7). The DELTA Study (a multicenter, randomized, crossover-design trial with 103 healthy adults inclusive of both sexes, Caucasian and Blacks, and pre- and postmenopausal women) showed that HDL-C levels fell in response to decreasing dietary saturation, suggesting a dose-dependent relation despite optimal dietary cholesterol control (5). In nonhuman primates, the reduction in HDL-C due to decreasing dietary fat saturation is thought to occur through a delay in the clearance of HDL apo A1 from the circulation (13). When a reduction in dietary cholesterol accompanies the reduction in total dietary fat and SFA, decreased HDL apo A1 production and hepatic apo A1 mRNA expression are thus anticipated (14). Plasma TAG levels were reported in the DELTA Study to increase -9% when dietary saturation was dropped from 15 to 9% energy from SFA, but did not increase any further despite a further reduction in saturation to 6% energy from SFA (5). Such an increase may be a consequence of an isoenergetic exchange of carbohydrates at the expense of fat, and will be discussed further in the section concerning carbohydrates. Of recent interest is dietary fat modulation on the activity of cholesteryl ester transfer protein (CETP). The role played by CETP in atherosclerosis is controversial because there are both pro- and antiatherogenic associations during reverse cholesterol transport. Human studies associate saturated fat with increased CETP activity, and an isoenergetic substitution of a high-SFA diet with an NCEP Step 1 diet is linked to decreased plasma CETP concentrations (15,16). Interestingly, in this study, an additional enrichment of 10% of energy by MUFA did not substantially add to the decrease in plasma CETP concentrations. Rationale for Increasing PUFA
The reduction of saturated fat to lower the risk of CHD raises the question concerning which type of fat substitution is desirable in achieving clinically relevant
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results for lipid lowering. The assessment of food variables in a cross-population study such as the Eighteen Countries Study found a negative correlation between PUFA intake and CHD mortality (17). Adipose tissue levels of linoleic acid, an indicator of dietary PUFA adequacy, were the lowest (7.36% by weight) in men from North Karelia, Finland, who had the highest incidence of CHD mortality (212/100,000) in contrast to Italian men who had the highest adipose tissue levels of linoleic acid (13% by weight) and the lowest CHD mortality incidence (43/100,000), in a survey of healthy men aged 40-49 yr from four European countries (3). The regression equations of Keys (2) and Hegsted et al. (9) identified PUFA as a critical component in cholesterol lowering. The NCEP guidelines, given in Table 9.1, suggest increasing energy from PUFA to match energy from SFA, as a moderate measure in the Step 1 diet or an even greater increase compared with SFA energy [polyunsaturated/saturated (P/S) =1.4] for a more intensive intervention (1). The PUFA family of dietary fats comprises the n-6 and n-3 cis FA. Of these, the n-6 family is reported to be more effective in improving plasma lipid profile, whereas the n-3 family is reported to give protection against fatal CHD events (18). Obviously, different FA mediate benefits differently in reducing CHD risk. Prospective studies support a beneficial relation between PUFA intake and CHD mortality. Howell et al. (19), from a meta-analysis of 224 published studies, estimated that a 1% change in energy from PUFA would produce a 0.023 mmol/L, (or 0.9 mg/dL) reduction in serum TC. Clarke et al. (4) showed in the metabolic ward situation, from a meta-analysis of 395 human studies, a 5% isoexchange of SFA reduction with PUFA was able to lower plasma TC by 0.39 mmol/L and LDL-C by 0.11 mmol/L. Blood cholesterol predictive equations indicated that n-6 PUFA have the ability to decrease TC and LDL-C levels by approximately twice the cholesterol-raising ability of SFA, i.e., a function of the expression 2s-P where S represents energy from SFA and P represents energy from PUFA as a percentage of total energy (20). When an n-6 PUFA (predominantly linoleic acid or 18:2) replaces SFA in the diet, the major portion of cholesterol lowering is said to occur in the LDL fraction; TABLE 9.1 National Cholesterol Education Program Dietary Recommendationsa Nutrient recommendations Amount of energy Dietary cholesterol Energy from fat OO / Energy from saturated fatty acids Polyunsaturated: saturated ratio O/O
aSource: Reference 1
Step 1
Step 2
Sufficient to achieve desirable weight <300 rng <30 <10 1 .o
Sufficient to achieve desirable weight <200 mg <30 <7 1.4
Dietary Fatty Acid Influences on Blood Lipids
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because there is a concomitant fall in LDL-apo B concentrations (21), this is likely through a reduction in the number of LDL particles in circulation. Exchanging PUFA for SFA in the diet also yields larger more buoyant LDL particles. Substitution of unsaturated FA for SFA results in increased rLDL activity and LDL transport rate in hamsters (12,22). An increase in rLDL activity and LDL transport raises the fractional clearance rate of LDL, contributing to increased LDL catabolism and decreased plasma LDL-C concentration. A passive effect is suggested by SFA reduction because of alterations in LDL composition when PUFA are exchanged for SFA, which perhaps causes the upregulation of rLDL (10). However, this last effect is also mediated by a MUFA diet. Woollett et al. (12) showed that the upregulation of rLDL by PUFA is an independent effect and occurs in a dose-dependent manner. Whether this is also true for MUFA is not known. The ability of 18:2 to reduce TC is nonlinear (23) and flattens out at 5% of energy. This effect was hypothesized by Hayes and Khosla (23) to mimic the 18:2 thresholds reported in humans, which means the upregulation of rLDL by 18:2 is maximal between 5 and 6%. Beyond this threshold, any further increase in 18:2 will no longer affect rLDL upregulation. On this basis, maximal benefit from increased intake of 18:2 was suggested to occur at 6-7% of energy. However, Hegsted et al. (24), in a meta-analysis of experimental trials, observed that blood response to 18:2 was essentially linear up to 20% of energy, and this included the relevant range in human diets and specifically the recommended levels in the Step 1 and Step 2 diets, i.e., 0-12% of energy. A negative association of increasing n-6 PUFA in the diet is reported with increased susceptibility of LDL particles to oxidation. Oxidized LDL particles are hypothesized to be associated with atherothrombosis (25). In vivo studies with LDL particles either incubated with test fats in the test tube (26) or harvested from subjects consuming test fats (27) demonstrated increased susceptibility to oxidation with n-6 PUFA compared with MUFA-rich oils. However, in vitro LDL oxidation, arising from dietary fat unsaturation and development of clinical end-points related to CHD risk, has not been established. Replacement of saturated by unsaturated FA may result in lowering TC and LDL-C concentrations but have an undesired effect in also decreasing the HDL-C fraction. Animal studies using cholesterol-enriched diets but substituting PUFA for SFA also demonstrated the effect of lowering both the HDL-C and non-HDL-C fractions (28). B m et al. (8) conducted their study by keeping PUFA concentrations constant across treatment groups and varying only the SFA content, yet they obtained the same effect of HDL-C lowering with decreasing saturation. Their study indicated that PUFA were not implicated in reducing HDL-C response when low-SFA diets were consumed. Other studies showed no changes in HDL-C concentrations despite increasing PUFA content (29). Harris (30) reviewed the effect of n-3 PUFA on serum lipoproteins in both animal and human studies. These included plant-derived n-3 FA [a-linolenic acid or
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T. Karupaiah e t a / .
(18:3)] and marine-derived n-3 FA [eicosapentaenoic (205) and docosahaexenoic (22:6) acids]; of these, the marine-based FA exert a more powerful effect on human lipoprotein metabolism. In humans, marine-based n-3 FA do not affect TC; they cause LDL-C to rise by 5-lo%, HDL-C to rise by 1-3%, and decrease TAG by 25-30%. The marine n-3 FA lipoprotein and lipid effects differ between normal and hypertriacylglycerolemic individuals. The plasma TAG-lowering effect is 25% in normal subjects but enhanced to 34% in hypertriacylglycerolemic individuals. The LDL-C-increasing effect is 4.5% in normal subjects but 10.8% in those with hypertriacylglycerolemia. The potency of marine n-3 FA, as a hypotriacylglycerolemic agent, is estimated at 3 g (1% energy) to reduce serum TAG levels by 30%. Plant-derived n3 FA, in general, exert effects on lipids and lipoproteins similar to those of n-6 FA. Their hypotriacylglycerolemic effect, unlike marine n-3 PUFA, can be exerted only when present in very large amounts, i.e., at nonphysiologic doses. It is unclear from animal studies what effects are potentiated by n-3 PUFA. TAG-lowering effects do not occur with marine n-3 PUFA, and there appears to be a marked reduction in HDL-C. According to Harris (30), underlying species differences in lipoprotein metabolism and the tendency to feed animals large amounts of the target FA, even in comparison with supplemental amounts consumed by humans, make it difficult to elucidate the biochemical basis for the hypotriacylglycerolemic effects reported in human studies. The Role of MUFA
Determining which fat type is a suitable substitute for SFA in the diet has led to much debate over MUFA and PUFA as ideal substitutes, given conflicting results from human dietary studies. Support for the beneficial role of MUFA came from the Seven Countries Study, which showed that subjects following a Mediterranean diet had a low incidence of CHD (2). The n-9 cis unsaturated FA commonly referred to as monounsaturates [MUFA (18:1)] are considered to be neutral in behavior. Blood cholesterol predictive equations demonstrate that MUFA potentiate either a neutral effect, or are hypocholesterolemic but with less potency than PUFA. Ginsberg et al. (3 1) demonstrated this in a randomized double-blind study, by feeding a Step 1 diet (SFA:PUFA:MUFA = 10:10:10% of energy) or a Step 1 MUFA-enriched diet (SFA:PUFA:MUFA = 10:18:10% of energy) to two groups of subjects after standardization by consumption of an average American diet (SFA:PUFA:MUFA = 18:10:10% of energy). MUFA, added in an amount equal to the quantity of SFA removed, significantly reduced plasma TC levels. The authors reported that the addition of MUFA did not substantially add to the decrease in plasma TC attributable to the Step 1 diet, i.e., MUFA had no independent effect on plasma cholesterol levels, but merely in replacing SFA. No advantage to plasma HDL-C levels was reported for either PUFA or MUFA, as a choice to decrease dietary saturation. A human study by Dreon et al.
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(29) compared PUFA and MUFA diets in which SFA and dietary cholesterol were held constant and overall fat, energy, and cholesterol content were in compliance with the Step 1 diet guidelines. Exchanging PUFA or MUFA did not affect TC and LDL-C levels, and both also lowered HDL-C levels. However, the exchange of MUFA by PUFA significantly increased plasma HDL,-C levels and HDL, mass, accompanied by a decrease in plasma HDL,-C levels and apo B. In contrast is the meta-analysis of metabolic ward studies by Clarke et al. (4) discussed earlier. The authors were able to separate the effect of a 5% isoexchange of SFA with either PUFA or MUFA, and in doing so showed that PUFA replacement had a greater effect on lipid lowering. Their analysis indicated that MUFA had no significant effect on plasma TC and LDL-C, with TC reduced by 0.39 mmol/L and LDL-C by 0.11 mmol/L with PUFA, whereas TC was reduced by 0.24 mmol/L and LDL-C by 0.04 mmol/L with MUFA. A study by Howard et al. (32) that evaluated a reciprocal dose-response comparison in isoenergetic substitutions for PUFA and MUFA keeping all other cholesterol-influencing dietary factors constant, found greater benefit in TC lowering with PUFA substitutions than with MUFA. Animal models that offer a way of measuring atherosclerotic changes as opposed to simply lipoprotein end-points in human studies were able to compare PUFA and MUFA diets in terms of which fat type is a suitable SFA replacement. Primate studies showed a MUFA diet to be more atherosclerotic than either a SFA or PUFA diet despite a more favorable plasma lipid profile (33). MUFA diets also produced larger LDL particles because of the increased oleic acid content (33). Lada and Rude1 (34) linked macrophage foam cells containing either SFA or MUFA with cholesteryl esters (CE) in a more rigid liquid crystalline state to atherogenicity compared with PUFA enrichment. The ordered structure within macrophage cells slows down the rate of hydrolysis and cholesterol efflux, which continues further foam cell accumulation and progression of atherosclerotic plaque buildup. The Rationale for Reducing Dietary Cholesterol
Dietary cholesterol has been considered to be second to SFA in its ability to raise blood concentrations of cholesterol. Epidemiologic studies support a strong, independent correlation between dietary cholesterol and the risk of CHD. Predictive equations before 1990 estimated that a 100-mg increase in dietary cholesterol increased blood cholesterol levels by 0.259 mmol/L, but most meta-analyses did not allow for baseline dietary cholesterol concentrations or consider individual variability in response (35). Since then, a number of predictions yielded a narrower range of 0.035-0.069 mmol/L as reviewed by Howell et al. (19). By including sensitivity analyses, these authors estimated that consumption of 100 mg dietary cholesterol/d would produce a 0.057 mmol/L change in blood cholesterol levels. Although dietary cholesterol was shown to cause marked hypercholesterolemia in many animal models including nonhuman primates (33), this effect is
J. Karupaiah e t a / .
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not consistent in humans, and the degree of response varies from individual to individual (4,35). Plasma TC response to dietary cholesterol is considered linear until at higher intakes (400-600 mg/d), the response reaches a plateau (35). Hopkins (35), who described a hyperbolic shape to the dose-response relation of dietary cholesterol intake and blood cholesterol levels, suggested that individual degrees of response may be mediated by many mechanisms. Differences in cholesterol absorption efficiency, neutral sterol excretion, conversion of hepatic cholesterol to bile acids, or modulation of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, or other key enzymes involved in cholesterol efflux between intracellular and extracellular tissue compartments will ultimately up- or downregulate rLDL, thereby influencing LDL-C levels in the circulation (35). Data from a meta-analysis (n = 395 human studies) by Clarke et al. (4) in the metabolic ward situation suggested that dietary cholesterol minimally affects human lipoprotein. Data from their study are presented in Table 9.2. The data clearly show that compared with the isoenergetic replacement of 5% SFA by PUFA or MUFA or carbohydrates, a reduction in dietary cholesterol elicits a smaller response in lowering lipoproteins. It was suggested that individual FA augment the effects of dietary cholesterol in elevating plasma LDL-C levels (23,36). Although the plasma TC and LDL-C response to dietary cholesterol eventually reaches a plateau as discussed above (35), in individuals with impaired lipoprotein metabolism arising from clinical, TABLE 9.2 Comparison of Dietary Cholesterol with lsoenergetic Equivalent Replacements of 10% Saturated Fatty Acids (SFA) or 10%Total Fat with Carbohydrates, Polyunsaturated Fatty Acids (PUFA), and Monounsaturated Fatty Acids (MUFA) by Multivariate Analysesa Change in blood cholesterol levelsb TC
LDL-C (mmoliL)
HDL-C
Replacement of 10% SFA energy with carbohydrates Replacement of 10% total fat energy with carbohydrates Replacement of 10% SFA energy with 5% PUFA + 5% carbohydrate energy Replacement of 10% SFA energy with 5% MUFA + 5% carbohydrate energy
-0.52 (0.03)
-0.36 (0.05)
-0.1 3 (0.02)
-0.20 (0.05)
-0.12 (0.06)
-0.10 (0.02)
-0.65 (0.02)
-0.47 (0.03)
-0.1 0 (0.01)
-0.50 (0.03)
-0.40 (0.02)
-0.10 (0.01)
Overall SFA exchange Reduction in dietary cholesterol by 200 mr!
-0.63 -0.13 (0.02)
-0.52 -0.10 (0.02)
-0.08 -0.02 (0.01)
Dietary change
dSource:Reference 4. bValues are means
(SE).
Dietary Fatty Acid influences on Blood Lipids
179
biological or genetic causes, the cholesterol-response threshold will be lowered because of depressed rLDL activity and the fractional clearance rate of LDL (37). Overall variance in lipoprotein set-point is suggested to explain hypo- and hyperresponders to 18:1, 18:2, or 16:O in normocholesterolemic and hypercholesterolemic nuns (38) or when elderly men with moderate-to-severe hypercholesterolemia were segregated into tertiles (39). The Role of Carbohydrates
Carbohydrates were also suggested for isoenergetic partial replacement of both SFA and total fat in the diet and many low-fat, high-carbohydrate (LF-HC) diets evolved as interventions for lipid lowering. Parks and Hellerstein (40) defined LFHC diets as total energy derived from 530% energy as fat, 255% energy as carbohydrate, or both. Both the Step 1 and Step 2 diets met these criteria. Subsequent discussions in this section are in reference to whole-food diets and not to liquid or purified diets that contain high glycemic index carbohydrates capable of exaggerated lipemic response in their own right as reviewed by Parks and Hellerstein (40). Controlled interventions with high-carbohydrate energy were effective in reducing TC and LDL-C but at the same time reduced plasma HDL-C and raised TAG levels (41,42). In a different meta-analysis (n = 60 trials) with the end-point as the TC:HDL-C ratio, Mensink et al. (43) were able to show that 10% isoenergetic exchange of any type of fat with carbohydrates produced the greatest increase in the TC:HDL-C ratio and at the same time contributed to an increase in fasting TAG levels. “Carbohydrate-induced lipemia” is distinguishable from “fat-induced lipemia” in primary hyperlipidemia because the latter disappears with dietary fat reduction but carbohydrate-induced lipemia does not. In contrast, decreasing fat without increasing carbohydrate nullified the TAG effect. That the effect of an LF-HC diet is not transient was shown in a study by Knopp et al. (6). In the year-long Dietary Alternatives Study, which examined long-term cholesterol-lowering effects of four grades of fat restriction in free-living men with different types of hyperlipidemia, hypertriacylglycerolemia (22-39% increase in plasma TAG) was induced with aggressively lower-fat diets (<25% energy as fat) and progressively higher intake of carbohydrates. The general observation was that “. . . the greater the fat reduction in the diet, the greater the increase in plasma TAG.” Plasma TAG increased in a dose-dependent manner, with increases starting with as little as 10%increases in energy from carbohydrates (42). The American Heart Association’s Phase 3 Diet, described as providing 20-25% of energy as fat with SFA providing 6%, carbohydrates providing 52-59%, cholesterol
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As little as 10% isoenergetic replacement of fat by carbohydrates is sufficient to alter the composition of VLDL in humans (45). Alterations in lipoprotein composition unique to increased carbohydrate feeding indicated an increase in the mass of all fractions of VLDL (S, c 400), as well as intermediate density lipoprotein (IDL) (Sf 14-20) (46). Increased size of VLDL and an increase in TAG content in VLDL, which is 8-13% of total TAG content of lipoproteins, were also apparent (47). An increase in VLDL represents endogenous synthesis, as shown by Mancini et al. (47), who fed subjects only 1 g dietary fat/d and took blood samples from fasting subjects. Additionally, an increase in apo B48 particles, which indicative of chylomicron presence in the fasting state, was suggested by Park and Hellerstein (40) as either an increased generation of chylomicrons in the postprandial state or a less efficient clearance. Further support for changes in VLDL composition comes from shifts in apolipoprotein metabolism, which include an increase in apo C3 content (48), change in apo E isoforms (45) and increases in apo BlOO particles in VLDL (48). Mittendorfer and Sidossis (49), in a recent metabolic study using in vivo isotopically labeled VLDL-TAG tracers in six healthy subjects, found accelerated VLDL-TAG secretion occurring after a high-carbohydrate feeding. The increased secretion of VLDL-TAG came from increased hepatic FA availability resulting from reduced hepatic FA oxidation. Apart from changes in VLDL, carbohydrate induction is said to affect LDL particle size. Dreon et al. (50) examined healthy men categorized into phenotype A (larger LDL particles, which are more buoyant and carry less atherosclerotic risk) or phenotype B (smaller LDL particles, which are more dense and carry an increased atherosclerotic risk) groupings. Both groups were switched from a high-fat (4046%) to an LFHC (20-24% fat) diet over 6 wk. A major finding was that one third of phenotype A subjects converted to phenotype B (developed smaller LDL particles) as a result of the carbohydrate induction, whereas the rest of the phenotype A and all of the phenotype B subjects were unaffected. The group later tested extreme fat reduction (10%) on the same group of men with phenotype A (large) LDL particles (51). Of those who did not convert to phenotype B with the 20-24% LF-HC diet, 32% did convert to phenotype B with additional restriction to a very low-fat (10% of energy), high-carbohydrate diet. A shift to phenotype B (small dense LDL particles) did not lower LDL-C. Instead, hypertriacylglycerolemia and reduced HDL-C concentrations developed. The metabolic basis for this conversion is not known. A regression prediction from this study was that two thirds of the men would express phenotype B with a 10% energy as fat/HC diet. Meta-Analyses on Behavior of Individual FA
Natural dietary fats are composed of mixtures of FA that are highly characteristic for their dietary source (Table 9.3). In recent years, the individual FA components of dietary fats were investigated for their effect on lipid and lipoprotein levels. Predictive equations based on human trials led to an early understanding of their effects in relation to cholesterol metabolism (Table 9.4). It was thus assumed that the SFA, including lauric acid (12:0), myristic acid (14:0), and palmitic acid (16:O)
TABLE 9.3 Fatty Acid Composition of Selected Dietary Fats and Oilsa Fatty acid carbon no.
Palm oil
Palm olein
Palm kernel
Corn oil
Soybean oil
-
-
0.1 trace 9.7 0.3 2.7 37.0 48.7 0.5 0.4
0.1
oil
<12:0 12:o 14:O 16:O 16:l 18:O 18:l 18:2 18:3 Other
0.3 0.8 39.5 0.3 4.3 43.1 10.5 0-0.5 0.5
-
0.2 0.8
37.2 0.4 4.2 43.6 11.5 0.1-0.6 0.3
aSource:Sundram ef a/. (unpublished data).
8.2 49.6 16.0 8.0 2.4 13.7 2.0 0.1
Olive oil (g/lOO g fat)
-
trace
-
8.9 0.3 3.6 20.6 57.2
13.0
-
0.3
-
2.5 74.0 9.0 0.5 0.1
Butter oil 9.2 3.1 17.7 26.2 1.9 12.5 28.2 2.9 0.5
-
Cocoa butter
0.1 25.8 0.3 34.5 35.3 2.9 -
1.1
Safflower oil
6.5 -
2.5 11.5 79.0 0.5
Coconut oil 14.9 48.5 17.6 8.4 2.5 6.5 1.5 -
0.1
Beef Lard
fat
0.1 0.1 1.4 24.8 3.1 12.3 45.1 9.9 1.1 3.0
0.1 0.1 3.3 25.5 3.4 21.6 38.7 2.2 0.6 4.6
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TABLE 9.4
Predictive Equations of Fatty Acid Class vs. LipoproteinsaCb Lipoprotein
ATC ALDL-C AHDL-C
Equation
0.0522A12:O to 16:O - 0.0008A18:O - 0.0124AM - 0.0248AP 0.0378A12:Oto 16:O + 0.001 8A18:O - 0.01 7AM - 0.0248AP 0.016A12:O to 1610 - 0.001 6A18:O + 0.0101 AM + 0.0062AP
aSource: Reference 52. bM, monounsaturated fatty acid; P, polyunsaturated fatty acid.
had equal cholesterol-raising properties. Hegsted et al. (9) found 14:O to be more cholesterolemic than 16:0, but 12:O had little or no effect. Medium-chain FA (8:O-1O:O) and stearic acid (18:O) were also considered to have little effect or were neutral (9,24). MUFA (predominantly oleic acid, 18:1) was speculated to be either neutral (9,24) or half as potent as PUFA in lowering blood cholesterol levels (52). PUFA, predominantly linoleic acid (18:2), were estimated to have twice the capacity to lower blood cholesterol that SFA had in raising them. Meta-analyses identified 14:O as the most hypercholesterolemic FA (4,9,52,53). Its hypercholesterolemic potential was variously assessed by investigators to be significantly more powerful than either 12:O or 16:O (9). There is agreement that changes in TC are paralleled by changes in LDL-C, and there is also similarity in their predictive coefficients. In contrast, the recent meta-analysis by Mensink et al. (43) found that TC:HDL-C ratio decreased with increasing saturation from 12:O to 18:O. A basis for comparing the capacity of individual FA to modulate blood cholesterol levels was derived from regression coefficients developed through these predictive equations. This was calculated as 1% of energy equivalent to 6 g of carbohydrates, or 2.7 g of FA for an average man or woman consuming a total daily energy of 2400 kcal(l0 MJ). Regression coefficients for raising TC by a 1% isoenergetic exchange of carbohydrate for the respective FA were quoted by Mensink et al. (43) to be 0.021 mmol/L for 12:0, 0.123 mmol/L for 14:0, 0.034 mmol/L for 16:0, 0.030 mmol/L for 18:0, -0.007 mmol/L for 18:1, and -0.016 mmol/L for 18:2. Clarke et al. (4) quoted 0.045 mmol/L for 12:0,0.071 mmol/L for 14:0,0.053 mmol/L for 16:0,0.015 mmol/L for 18:0, 0.02 mmol/L for 18:1, and -0.13 mmoVL for 18:2. Both analyses found 14:O to be the most hypercholesterolemic of the saturates. Although the analyses by Clarke et al. (4) found that 12:O and 16:O were similar in their potential to raise blood cholesterol, the analysis by Mensink et al. (43) found 16:O to be more hypercholesterolemic than 12:O. Both groups based their study on experiments conducted under metabolic conditions. Other investigators also found 12:O to be less hypercholesterolemic than 16:O (52,53). Early clinical trials did not allow satisfactory predictive equations for the effects of individual FA on HDL-C (24). This was because diet-induced changes in HDL-C were within a small range, and day-to-day within-person fluctuations were relatively
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large (54). There is now recognition that fats have the capacity to raise HDL-C. However, the degree to which HDL-C levels rise is dependent on chain length and saturation, with SFA being most potent, PUFA the least, and MUFA intermediate. Among the SFA, 12:O and 16:O were shown to increase HDL-C (52), but opinion on 14:O is divided between ‘‘. . . no effect” (52) and “. , . positive” (53). Just as 18:O is neutral toward TC levels, it appears to have no effect on HDL-C concentrations (53). The recent meta-analysis of 35 studies by Mensink et al. (43) demonstrated that HDL-C concentrations decreased with increasing chain length of the various SFA. The investigators quoted the following regression coefficients for 1% isoenergetic exchange with carbohydrates: 0.027 mmoVL for 12:0, 0.018 mmolL for 14:0, 0.010 mmol/L for 16:0, and 0.002 mmol/L for 18:O. They also examined data using a unique end-point, the TC:HDL-C ratio, and found that although 12:O greatly increased TC, much of its effect was on HDL-C. Consequently, oils rich in 12:O beneficially decreased the ratio of TC:HDL-C; 14:O and 16:O had little effect on the ratio, whereas 18:O reduced this ratio slightly. TC:HDL-C ratios were also beneficial for 18:1 (-0.026 mmol/L) and 18:2 (-0.032 mmolL) but less compared with 12:O (-0.037 mmoVL). Very few meta-analyses developed prediction equations for serum TAG levels. Mensink et al. (43) in their study found that the TAG regression coefficients were negative for all saturates with the greatest decrease for 12:O (-0.019 mmoVL) and similar decreases for 14:0, 16:0, and 18:O (-0.017 mmol/L). Two new parameters additionally reported were apo B and apo A l . Apo B was positive for the SFA 12:O (5.6 mg/L), 14:O (1.9 mg/L), and 16:O (4.2 mgk), but increasingly negative for 18:O (-3.8 mg/L), 18:1 (-4.8 mg/L), and 18:2 (-7.7 mg/L). Apo A1 decreased with increasing saturation from 12:O (13.8 mg/L), 14:O (10.4 mg/L), 16:O (7.5 mg/L), and 18:O (-1.6 mg/L), as well as increasing unsaturation (5.2 m g k for 18:1 and 2.2 mg/L for 18.2). Individual SFA The following review will examine individual SFA, from 12 to 18 carbon chain lengths, for their ability to mediate negative, positive, or neutral behaviors associated with blood cholesterol levels. MUFA, predominantly 18:1, and PUFA, predominantly 18:2, were reviewed extensively earlier.
Lauric Acid (72.9). Lauric acid, which is of intermediate carbon chain length, is hypothesized to be absorbed through the portal circulation, similar to mediumchain TAG (8-10 carbon atoms), and through the small intestine, which is favored by the longer-chain TAG such as 14:0, 16:0, and 18:O. Few studies have tested lauric acid separately for its hypercholesterolemic properties because it co-occurs naturally with 14:O in coconut oil, palm kernel oil (PKO), and milk fat. Grundy and Denke (55) referred to the influence of lauric acid as uncertain, in view of both its intermediate chain length and its occurrence together with 14:O in
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natural fats, Nevertheless, the investigators synthesized a lauric acid-rich test fat by a base-catalyzed rearrangement of sunflower oil and trilaurin and tested this in comparison with natural oils (56). They then used liquid formula diets to compare the effects of lauric acid (12:O) with palmitic (16:O) and oleic acids (18:l). The base composition of the diets was without any dietary cholesterol, but contained isoenergetic distributions of fat:carbohydrate:proteinat 40:40:20%, and differed only in their FA compositions. Isoenergetic exchanges of 12:O for 16:O were at 17%, and for 18:l at 13%. These diets were fed to 14 men in random order for 3 wk/diet, The results of this study indicated that the 12:O diet was more hypercholesterolemic than 18:l (1 1% for TC and 12% for LDL-C), but did not raise TC and LDL-C as much as 16:O (16% for TC and 19% for LDL-C). Lauric acid was estimated to increase LDL-C two thirds as much as 16:O. HDL-C concentrations were also increased by 14:O compared with 16:O (by 7%) and 18:l (by 15.7%), but overall, HDL-C and TAG did not differ among treatments. However, these results are difficult to extrapolate given that the research design used liquid diets, 12:O was a synthetic fat, and that the natural occurrence of 12:O is together with 14:0, which is regarded as more cholesterolemic than 16:O. Schwab et al. (15) in feeding mixed diets with 200 mg of cholesterol to 15 healthy women, did not find any differences between 12:O and 16:O exchanged at 4% of energy, with controlled amounts of 18:1 and 18:2. Myristic Acid (14:O). It is difficult to evaluate the specific action of 14:O because it commonly occurs together with 12:O in natural fats. Major sources are butter, coconut, and PKO. Nutmeg fat is also high in 14:0, but is rarely used as a dietary source. Only one experimental trial was used in the meta-analysis of Katan et al. (53) to compare the potency of 14:O; this was a human study (n = 59, 36 women and 23 men) by Zock et al. (57) in which 14:O was exchanged at 10% isoenergetic substitutions with 18:l and 16:O. All test fats were supplied as margarines, and the high content of the specific FA tested was obtained through blending of natural oils, or modified natural oils. The high 14:O test fat was obtained as a structured fat, synthesized from 14:0, glycerol, 18:0, and 18:2. The ratio of specific FA, as a percentage of total dietary energy, was in the order of myristic:palmitic:oleic acids, 11:5:11 in the 14:O-rich diet, 1:15:12 in the 16:O-rich diet and 1:5:21 in the 18:lrich diet, respectively. The diets provided cholesterol between 340 and 360 mg/d. Compared with 16:0, myristic acid significantly increased TC and LDL-C (>1.5 times) when substituted for 18:1. HDL-C concentrations increased with consumption of the 14:O diet compared with the 16:O (by 9%) and 18:l (by 10%) diets. In contrast to this finding is a study by Tholstrup et al. (58), who did not find significant differences in TC and LDL-C between 14:O and 16:O diets when exchanged with 18:1, but did find a decrease in HDL-C (by 8%) when 14:O was substituted for 16:O. That study used natural fats, and the researchers speculated that the presence of 12:0, 14:0, or 16:O in natural fats would have been additive according to the hypercholesterolemic effect. One limitation associated with the study of indi-
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vidual FA is that the test fat in question is often substituted at nonphysiologic doses. In relevance to 14:0, test fats have been used at 11-16% of dietary energy in human studies (57,58). Loison et al. (59) hypothesized that providing 14:O at the maximum physiologic dose of 2.4% of dietary energy would have no undesirable effects on cholesterol metabolism. In their study using hamsters, the 14:O content in test diets was at four different doses (0.5, 1.2, 1.8, and 2.4%), obtained by varying the natural fat permutations of lard and milk fat. The standard diets were semipurified, almost cholesterolfree (0.05%),and contained 12.5% total fats. The study design allowed for a baseline feeding (0.5% 14:0), treatment with the four doses of 14:0, and a post-treatment period in which half the hamsters in each group (1.2-2.4% of 14:O) were switched to the baseline feeding. Plasma TC, LDL-C, and HDL-C levels increased with increasing permutations of 14:O but did not differ between doses. Linear regression was significant ( r = 0.49, P = 0.01) only for HDL-C. Hepatic scavenger receptor class B type 1 protein (SR-B1) levels were lower, but CE concentrations were higher when 14:O was increased beyond 0.5% energy. A negative correlation was found between HDL-C and SR-B1 mass ( r = -0.69; P < O.OOOl), and an inverse linear regression between 14:O content and SR-B1 mass ( r = -0.75; P < 0.0001). Interestingly, there was a regain in SR-B1 mass when the animals originally fed 2.4% 14:O returned to the lowest 14:O dose. The conclusion from this study was that 14:O modulated HDL-C via a regulation in SR-B 1 expression. Increased amounts of 14:O in the diet were the most important factor in the increase of HDL-C concentrations, and this effect was linked to a decrease in the amount of SR-B 1 in the liver. Palmitic Acid (1 6:O). This is the most abundant SFA in the human diet; its prevalence in the food chain comes from meat and meat products, dairy products, and vegetable oils. The high concentration of 16:0, in palm oil for instance (-45%), makes it much easier to incorporate as a natural fat into research diets than 12:O and 14:O. The cholesterolemic effects of 16:O were studied extensively in comparison to the individual SFA 129-18:0, 18:1, 18:2, and trans FA. Results were variable depending on whether 16:O was compared with FA occurring in natural fats or synthetic forms. In isoenergetic exchanges (10-1 1%) with 18:1 in the presence of dietary cholesterol in human studies, 16:O generally caused a rise in TC and LDL-C concentrations (56,57). In contrast, in studies with a 5-6% isoenergetic exchange with 18:1, there was no difference in lipoprotein parameters (60-62). A shortcoming of the studies reporting the hypercholesterolemic effect of 16:O was that subjects were not normocholesterolemic, unlike in the studies reporting a neutral effect; in addition, the 18:l fats incorporated were modified TAG (63). With 18:l as a control in human studies, 16:O vs. 12:O elevated TC (by 5 % ) and LDL-C (by 7%) (56); compared with 14:0, it decreased TC (by 5 % ) and LDLC (by 4%) (57) or was similar (58,64); compared with 18:0, it either increased TC by 14% and LDL-C by 21% (65), or was similar (66). Both the 18:l and 16:O used
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in these studies were natural fats, but the 12:0, 14:0, and 18:O were all synthetic fats, making these results difficult to extrapolate. Because 12:O or 14:O occur together in natural fats such as dairy fat, PKO, and coconut oil, some investigators contend that comparisons should include natural combinations of these FA (12:O + 14:O) and should distinguish the lipoprotein setpoint of subjects (23,63). Studies with normocholesterolemic human subjects that compared 16:O with 12:O + 14:O and 18:2-rich diets used palm oil, coconut oil, and corn oil as fat sources exchanged at 70% of total fat content or alternately as a 5% isoenergetic exchange of a 12:O + 14:O-rich diet with palm oil and PKO (60). It was reported that 12:O + 14:O was more potent than 16:O in raising blood cholesterol levels. In the earlier discussion on dietary cholesterol, it was suggested that the cholesterol-response threshold depended on rLDL activity and fractional clearance rate of LDL (37), and individual lipoprotein set-points perhaps explain hypo- and hyper-responders to 18:1, 18:2, or 16:O (38,39). Further, the absence or presence of dietary cholesterol in reported studies is an important factor in 16:0, 18:1, and 18:2 affecting the response to blood cholesterol. When identical diets were fed to normocholesterolemic cebus and rhesus monkeys, there were identical effects on LDL-C irrespective of 16:0, 18:1, and 18:2 in both species, but only cebus monkeys were sensitive to the cholesterol-lowering properties of 18:2, and this was attributed to lowered HDL-C concentrations (39). In contrast, with cholesterol-sensitive monkeys (African green) that were fed cholesterol, 16:O elicited higher TC and LDL-C concentrations compared with either 18:l or 18:2 (67). This effect was also shown in a hamster study (36). Generally, human studies indicate that HDL-C concentrations are increased by 16:O compared with 18:0, 18:1, and 18:2 (58,60, 61,65).
Stearic Acid (78:O). This is the second most abundant SFA in the human food supply. Animal sources such as beef tallow, lard, and mutton fat, and vegetable fats such as cocoa butter and shea butter contain considerable amounts of 18:O. The neutrality of 18:0, with regard to LDL-C metabolism, was well-established by early predictive equations (9,20) and proven in human studies (56,65). Most animal studies are also consistent concerning the neutrality of 18:O. Evidence on its neutrality was based on the limited use of natural fats such as cocoa butter and shea butter, and a common problem in using a natural fat is the presence of significant amounts of 12:O + 14:O or 16:O or both. More often the norm has been to use synthetically manufactured fats, such as that obtained by Bonanome and Grundy (65). Their process required complete hydrogenation of soybean oil to obtain >90% 18:0, chemical hydrolysis of the hydrogenated soybean oil and high-oleic safflower oil, followed by random reesterification. Tholstrup et al. (68) compared 16:O with natural fats using shea butter as a source for 18:O (42%), PKO mixed with sunflower oil as a source of 14:O (lo%), and 12:O (30%). Shea butter has desirable characteristics in that the contents of
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12:O + 14:O and 16:O are low compared with cocoa butter. Data from this study indicated that 18:Oreduced TC and LDL-C compared with the more hypercholesterolemic 12:O + 14:O and 16:O.Another study, also using shea butter, was reported by Dougherty et al. (69),but the design of the study allowed for a comparison between 18:Oand 16:0,and had an extended feeding period of 40 d compared with the norm of 3 wk. The investigators reported a similar hypocholesterolemic effect associated with the 18:0diet by d 20 (31% drop in LDL-C levels); surprisingly, however, this effect rebounded (-55% of the decrease) by d 40. HDL-C concentrations tended to be lowered by a high 18:O diet when exchanged for 18:l or 18:2 in most reported studies (65,69).They were shown to remain lowered even with an extended period of feeding in which 18:l and 18:2 were controlled (69).Lipoprotein (a) [Lp(a)] is reported to remain unchanged with changes in dietary fat saturation or cholesterol (70). The mechanism by which 18:O is nonhypercholesterolemic compared with other long-chain SFA has been discussed at length but remains to be elucidated. Poor digestion and absorption of 18:O in the gut were suggested because 18:O by nature is solid at room temperature and difficult to dissolve (71).Experimental evidence from balance studies in humans and animals indicated that >90% of 18:O is readily absorbed (64,65,72).One study indicated that this was <90%, and there were greater fecal losses of 18:O (69).The position of 18:O on the TAG molecule, as well as the mixture of glycerides in the molecule, may explain the conflicting results. The use of interesterified fats with 18:O randomized to all three positions on the TAG molecule, or even tristearin (64,65,72), may render these fats less digestible compared with a mixture of glycerides in a natural fat (69).SFA, occupying the sn-1 and sn-3,are more easily hydrolyzed and absorbed than the more inaccessible sn-2position, whereas an unsaturated FA such as 18:l or 18:2in the sn-2position favors more rapid digestion and absorption. Another explanation is that 18:Ois rapidly converted into 18:l by desaturation at the n-9 position (65,73).Indeed, the FA composition in human plasma triacylglycerols in response to fat feeding is always at a higher ratio of 16:O to 18:O. Despite higher 18:O consumption, this status was maintained in the study of Bonanome and Grundy (65),but instead there was an increase in 18:l in both the plasma TAG and CE. An isotope-labeling experiment in mice by Bonanome et al. (73)found 18:O being rapidly converted into 18:l compared with 16:O.Further, a higher content of labeled 18:O was found in tissue phospholipids compared with 16:0,regardless of diet (rich in 18:l or 16:O or 18:O);in tissue TAG, however, the reverse was m e . The duration of feeding may make a difference as shown in a study by Dougherty et al. (69).The authors observed that with prolonged feeding (40 d) of 18:0,plasma FA composition, including that of red blood cells and platelets, showed increased incorporation of 18:O compared with a shorter period of feeding (20 d). This suggested that 18:O was metabolized similarly to 16:O albeit at a slower rate, and tissue incorporation took place only with prolonged feeding. One parameter of concern with SFA is the associated thrombogenicity. Miller et al. (74) established that activation of factor VII (FVII) occurs in both healthy
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adults and those with congenital deficiency of either factor XI or XII, after a highfat meal. Early studies by Connor (75) established through in vitro experiments that all SFA (129-1 8:O) were prothrombotic, and thrombogenicity decreased with decreasing chain length, implicating 18:O as the most thrombogenic of the FA. Time to thrombus formation was much shorter with 18:O than with 18:l or 18:2, which were neutral. In contrast, Tholstrup et al. (68) observed through in vivo human studies that 18:0, in addition to favorably affecting blood lipids, also favorably raised FVII coagulant activity compared with 12:0, 14:0, and 16:0, although other fibrinolytic variables did not differ among FA. Sanders et al. (76), using stearic acid-rich fats in the postprandial model, found that symmetrical TAG were absorbed more efficiently than asymmetrical TAG, and longer-chain FA in the sn-2 position led to activation of FVII. Animal Studies, Individual FA and LDL-C Metabolism
Direct evidence from animal studies was provided on increased rLDL activity by feeding unsaturated instead of SFA (22,77). These studies examined individual FA and LDL metabolism in relation to the regulatory mechanisms governing LDL concentrations, i.e., hepatic LDL fractional clearance rate, LDL production rate, rLDL activity, and the hepatic free cholesterollcholesteryl ester pool. Daumerie et al. (77) fed hamsters 8:0, 14:0, and 18:l; relative to 8:0, 14:O increased LDL-C and reduced hepatic CE. There was an associated decrease in rLDL activity and increased LDL-C production rate but switching to 18:1 had the opposite effect. Woollett et al. (12), also using the hamster model, substantiated that relative to a control, 6:0-10:0 were neutral, whereas 12:O-16:0 were negative regarding LDL-C concentration, rLDL activity, LDL-C production rate, or the hepatic cholesterol pool. Only 18:0, although reducing hepatic cholesterol to the same extent as 16:0, continued to upregulate rLDL activity and therefore had no effect on LDL-C concentrations. Ohtani et al. (35) in exchanging 18:2 and 16:O at 5% by weight, with and without dietary cholesterol in a hamster study, showed that 16:O augmented the effect of dietary cholesterol in elevating LDLC through suppressing rLDL receptor activity. However 18:2 diminished both the effects of 16:O as well as the addition of dietary cholesterol. Other study approaches have used single vegetable oils and fats, with diverse FA profiles or oil blends with a dominant FA profile, to evaluate LDL metabolism. It is difficult to attribute the true outcome of individual FA in these studies because they were less well-controlled for 18:l and 18:2 content. Observations in these categories found that 12:O + 14:O had the most negative effect on LDL metabolism, compared with 16:O and 18:O via reduced LDL fractional clearance rate and rLDL number (78). Plasma LDL-C concentrations were shown to be reduced in hamsters fed either a 12:O + 14:O-rich hydrogenated coconut oil diet or a 18:2-rich safflower oil diet at 0, 0.06, and 0.12% dietary cholesterol (79). Changes in LDL-C were associated with the 0.12% cholesterol diet, with the hepatic rLDL activity at 30% in the SFA diet and 77% in the PUFA diet.
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The different pathways of FA assimilation from the gastrointestinal system are hypothesized to explain the differential effects of FA chain length on LDL-C metabolism. These include the following: (i) A rapid absorption of 6:0, 8:0, and 1O:O and entry into portal circulation, conversion into acetyl CoA, and entry into oxidative and synthetic pathways (12); (ii) a sequence of changes in TAG structure for 12:0, 14:0, and 16:O during micellar solubilization in the gut, reesterification and incorporation into chylomicrons leading to accumulation of these FA in the liver, and subsequent downregulation of rLDL activity (12); (iii) the reason 18:0 does not enter either pathway is unclear, but it is further hypothesized that signal transduction, alternate metabolic pathways, or processes of membrane fusion are implicated (12); and (iv) the fast rate of conversion of 18:O into 18:l (73). It was also suggested that the regulatory effect of 12:0, 14:0, and 16:O may be exerted intracellularly through alterations in the hepatic cholesterol pool as follows: (i) Inhibition of ACAT by 12:0, 14:0, and 16:O prevents the conversion of free cholesterol (FC) coming in from dietary cholesterol into inert CE. The increased cholesterol flux across the liver coupled with “downregulation” of rLDL by the SFA will cause reduced LDL clearance and increased LDL-C levels (77) and (ii) ACAT favors 18:l or 1 8 2 as a substrate in the esterification of FC, shifting the equilibrium in favor of the CE pool, thus leading to the upregulation of rLDL and reduced LDL-C levels (77,79). Animal Studies, Individual FA and HDL-C Metabolism
Observations in human studies note that replacement of SFA (12:O-16:O) with 18:0 or an unsaturated FA not only has a cholesterol-lowering effect, but is also linked to a decrease in HDL-C concentrations (80). Numerous animal studies, all using the hamster model, generally agree with data from human studies, but in addition, have provided only limited information on HDL-C metabolism. Terpstra et al. (81) observed that decreasing dietary fat saturation did indeed lower HDL-C in hamsters, and this was associated with increased HDL binding in the liver. The hamsters were fed cholesterol-enriched diets with corn oil (predominantly 18:2, P/S = 4.77), olive oil (predominantly 18:1, P/S = 0.84), and palm oil (predominantly 16:0, P/S = 0.26) exchanged at -19% dietary energy, but this study reported only PIS ratios without individual FA values. Substituting 18:2 content for 16:O or 18:l lowered (P c 0.05) plasma TC, TAG, HDL-C, and non-HDL-C fractions. Although the 18:l diet tended to lower values compared with 16:0, the difference was not significant. The same study examined the mechanism by which increasing fat saturation lowered plasma cholesterol concentrations, particularly HDL-C through measured hepatic lipase, CETP, phospholipid transfer protein (PLTP), and 1ecithin:cholesterol acyltransferase (LCAT) activities. Dietary fat saturation did not significantly affect plasma LCAT and CETP activities, but PLTP activity increased with increasing saturation. There was a significant correlation between PLTP activity and the non-HDL-C fraction, as well as with the HDL-C fraction. Hepatic lipase activ-
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ity was significantly higher when 16:O was fed, which coincided with higher HDL-C concentration compared with 18:2 diets, whereas 18:1 diet had intermediate activities. Maximum HDL binding (Bma) was significantly higher with the 18:2 diet compared with 16:0, but 18:l was intermediate and not significant. The increased HDL binding to liver membranes associated with 18:2 may explain the decreased HDL-C concentrations. The positive correlation between hepatic lipase activity and HDL-C fractions is expected in hamster studies but is contrary to observations in human subjects in whom hepatic lipase and HDL-C concentrations are inversely related. The effect of a single FA on HDL metabolism was also evaluated in hamsters by Loison et al. (59). By feeding hamsters 14:O in varying concentrations, it was shown that 14:O modulated HDL-C via a regulation in SR-B1 expression. Increasing the amount of 14:O in the diet was the most important factor causing increased HDL-C concentrations, and this effect was linked to a decrease in the amount of SR-B 1 in the liver. Trans FA (TFA) Effects on Health
Within-population studies that link TFA intake from hydrogenated vegetable fats to CHD risk include the Nurses’ Health Study (82) and a case-control study on men and women with coronary artery disease (83). The Nurses’ Study, a prospective cohort study, reported positive relations with relative risk ( P c 0.001) at 1.5 times in the highest quintile group of TFA intake (5.7 g/d), compared with those in the lowest quintile of intake (2.4 g/d). The positive association was attributed to an intake of partially hydrogenated vegetable fats rather than isomers from ruminant sources. After adjusting for total energy consumption, the relative risk for developing CHD was 50% greater in the highest quintile ( P = 0.001), compared with those in the lowest quintile of TFA consumers. Ascherio et al. (83) established the relative risk of myocardial infarction at 2.4 times ( P < 0.001) for the highest quintile of TFA intake (6.5 g/d), compared with the lowest quintile of TFA intake (1.7 g/d). In both studies, dose-response relations were difficult to establish for the intermediate quintiles. Methodological limitations in the studies such as the use of semiquantitative food-frequency questionnaires, as well as food databases without comprehensive TFA content, led to difficulties in establishing a dose-response relation. In contrast, Kromhout et al. (84) reported for the Seven Countries Study, a cross-population study involving 12,763 men 40-59 yr old, a significant correlation ( r = 0.78; P < 0.001) between TFA intake, TC levels, and 25-yr coronary artery disease mortality. Food data were collected by the weighed food method, and the records were used to recreate foods that were subsequently analyzed for their individual FA composition. A confounding effect was the presence of SFA, which prevented establishing an independent effect for TFA. A prospective casecontrol study in a subgroup of the EURAMIC study established that those in the highest quartile of TFA intake from partially hydrogenated fish oil (PHFO) experi-
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enced five times the risk of myocardial infarction compared with those in the lowest quartile of intake (85). This study linked deleterious alterations in the LDL:HDL ratio to thrombogenesis. Another case-control study established a link between high TFA content in human erythrocyte membranes to cardiac arrhythmias and sudden death (86). Follow-up cohort studies in subjects with coronary disease indicate associations between TFA intake and the recurrence of cardiac events as well as mortality. Data collection depended again on food-frequency questionnaires or 24-h recalls. Hu et al. (87) reported a relative risk of 1.93 for myocardial infarction, with a 2% increment in energy from TFA intake in 939 women aged 34-59 yr. Gillman et al. (88), who followed up 267 men aged 45-64 yr, reported a relative risk of 0.99 for CHD for each increment of 1 tsp/d of margarine intake for the first 10 yr, whereas the risk increased to 1.12 during the follow-up period (1 1-21 yr) Tavani et al. (89) found that margarine could explain -6% of myocardial infarction in 429 women with relative risk varying between 1.0 for a low or no intake and 1.5 for a medium or high intake. A positive association between trans 18:2 content of partially hydrogenated-soybean oil (PHSO) and of adipose tissue to increased risk of nonfatal myocardial infarction ( P < 0.001) was reported recently in adult Costa Ricans (90).
Clinical Trials Effect on LDL-Cand HDL-C
A number of human clinical studies implicated trans-18:l in raising plasma cholesterol, primarily by raising LDL-C and reducing HDL-C levels. Mensink and Katan (91) were the first to observe that trans-18:ln-9 raised LDL-C and reduced HDL-C when exchanged at 11% energy with cis-18:l. However, the TFA dosage used in that study was suggested to be nonphysiologic given that its intake in freeliving populations was ~ 5 % of total dietary energy (92). In addition to the issue of the appropriate TFA dose for experimental diets, there was poor control for confounding variables. These are largely the unaccounted effect of the background trans content in the normal or control diet on blood lipids (93,94) and inadequate control for SFA, cis-18:1, and cis-18:2 content in the test and control diets (95). The IOM Report on Dietary Reference Intakes for Trans Fatty Acids (96) found that deleterious effects associated with higher amounts of TFA do not occur when sufficient amounts of 18:2 are consumed in animal studies. Findings from human studies by Sundram et al. (97,98) and French et al. (99) disagree with the IOM report. Those studies found that TFA diets adversely affected serum lipoprotein profiles, despite the fact that the diets provided more than sufficient amounts of 18:2 compared with 16:O (5.8 vs. 3.5% energy). A dose-response relation between the intake of TFA and human lipoprotein levels was demonstrated by Judd et al. (100). By feeding trans-l8:ln-9 at 3.8 and
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6.6% of total dietary energy exchanged with cis-unsaturated FA (18:l) and cis-saturated FA (12:O + 14:O + 16:0), LDL-C levels were significantly raised by 6 and 7.8%, respectively. However significant lowering of HDL-C levels by 2.8% occurred only with the higher dose. In this trial, SFA (at 6% isoenergetic exchange) raised LDL-C by 9% and HDL-C by 3.5% relative to cis-18:l. In reviewing the effect of dietary TFA on serum lipoproteins in humans, Zock and Mensink (101) observed a need to define the shape of the dose-response curve. Further, there was no evidence for a threshold of TFA intake below which there was no effect on lipoprotein levels (97,101). The cholesterol-raising potential of TFA was compared with nonhydrogenated vegetable oils rich in the hypercholesterolemic SFA (12:O + 14:O and 16:O) because, from the structural viewpoint, both appear similar. Some comparisons suggest that TFA have a similar or lesser effect on LDL-C than SFA (99,102). The focal point of these studies was to group SFA as a class rather than differentiate between individual FA classes. For instance, Judd et al. (100) ranked the effect of FA classes on TC and LDL-C as cis- 18:1 < moderate trans < high trans < SFA. In a review of the individual FA effects on plasma lipids and lipoproteins reported for human studies, TFA are ranked intermediate to the hypercholesterolemic SFA (12:O-16:O) and the neutral or mildly hypocholesterolemic cis-MUFA and the potently hypocholesterolemic cis-PUFA (4 1). The suggestion is that trans- 18:1 behaves similar to 18:0, which compared with unsaturated FA, significantly increases TC and LDL-C but appreciably lowers HDL-C. Khosla and Sundram (103) dispute comparisons of TFA with cis-18:1 because TFA came into the picture as a replacement for SFA. They advocated that in reality, TFA should be compared with SFA. Direct comparisons of TFA to individual SFA relative to cis-18:1, exchanged at 5.5% dietary energy were made by Sundram et al. (97). The ranking order of the ability of these FA to raise LDL-C was TFA > 12:O + 14:O > 16:O = 18:1.Further, differences between TFA and 12:O + 14:O were not significant, and the response of 16:O was identical to that of 18:1. Additionally, Sundram et al. (98) and French et al. (99), using similar designs, kept 18:2 constant at 5.8% of energy and found that although 16:O did not raise LDL-C concentrations, truns-18:l did. Their findings contradict the findings of Judd et ul. (100) and Kris-Etherton and Yu (41). HDL-C concentrations are unequivocally reduced by TFA as reported in all of the studies mentioned. Unlike SFA, which raise both LDL-C and HDL-C levels, the opposite effects seen with TFA adversely affect the TC:HDL-C and LDL-C:HDL-C ratios. Ascherio et al. (104), in combining a number of studies comparing TFA and SFA, found the magnitude of the dose-dependent effect on LDL: HDL-C ratio to be greater for trans fats compared with SFA; this difference was significantly different at <5% of energy intake ( P < 0.001) and at 7% ( P < 0.05). The LDL-C:HDL-C ratio ranking attributed in the study of Sundram et al. (97) was in the order of TFA > 12:O + 14:O > 18:1 > 16:0, which suggested that decreasing saturation adversely reduced HDL-C levels and that hydrogenation of unsaturated FA exacerbated the problem.
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The relative effect of margarines based on vegetable oils or fish oils was assessed for their effect on lipoprotein levels. When comparing margarines based on palm, soybean, and high-PUFA oils, high-PUFA margarines were most favorable to LDL-C concentrations, but also reduced HDL-C concentrations the most (102). The replacement of TFA in fish oil margarine (lower SFA but high trans) with palm oil-based margarine (higher SFA but less trans) improved plasma lipid levels by reducing LDL-C levels but did not significantly affect HDL-C levels (105). Increasing amounts of TFA in hydrogenated soybean oil products compared with the oil at the expense of PUFA increasingly raised LDL-C concentrations in the order of oil < semiliquid margarine < soft margarine < shortening < stick margarine and increasingly decreased HDL-C in the same order (106). Effect on TC:HDL-C Ratio
Another appropriate basis suggested for a comparison between SFA and TFA is the use of TC:HDL-C ratios, given that TFA depress, whereas SFA elevate HDL-C levels. Early predictive equations did not include TFA because they were recognized as a potential CHD risk factor only in the mid-1990s. A recent meta-analysis by Mensink et al. (43) included TFA, specifically truns-18:1, for the very fist time. Their analysis included eight studies for which the TFA intake, as a percentage of dietary energy, ranged between 0.0 and 10.9%. Isoenergetic replacement of trans-18:1 at 1% with SFA decreased TC:HDL-C by 0.019, with cis-18:1 by 0.048, and with cis-18:2 consumption by 0.054. A 1% isoenergetic replacement of trans-18: 1 with carbohydrates was beneficial on the TC:HDL-C ratio by 0.04 and was equivalent to a 7.3% replacement with SFA. Furthermore, trans-18:1 did not increase HDL-C or apo A1 concentration relative to carbohydrates. Overall, trans-18:1 was found to be the most harmful macronutrient with regard to the TC:HDL-C ratio. In comparing the margarines obtained from palm, soybean, and high-PUFA oils discussed in the previous section, stick margarine gave rise to the least favorable TC:HDL-C ratio, whereas the soft margarine was the most favorable (102). Increased Lipoprotein (a) Concentration
Lipoprotein (a) or Lp(a) is a subclass of LDL containing apo (a) in addition to apo B. Human Lp(a) is under genetic control; normal levels are <150 mgL, whereas CHD risk increases with Lp(a) concentrations A00 mg/L (107). Lp(a) is reported to remain unaffected by diet (108) or even by changes in dietary fat saturation or cholesterol (69). However, both cis-18:0 (5,109) and TFA are reported to raise Lp(a) concentrations (106,110-1 13). Hornstra et al. (109) found that replacing fat in the habitual Dutch diet with cis-16:0 decreased Lp(a) concentrations, and this was attributed to a >50% decrease in TFA. In a recent study using the postprandial model, Lp(a) concentrations increased in the triglyceride-rich lipoprotein (TRL) fraction after a trans-18: 1rich meal compared with fasting levels but not after a cis-18: 1-rich meal (1 14). In particular, the Lp(a) content of TRL peaked at 4 h with the trans-meal.
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It is clear that the magnitude of Lp(a) response is relative to a time x dose effect of feeding TFA compared with the various classes of FA (Tables 9.5 and 9.6). Increased Lp(a) concentrations are reported from 5.5% isoenergetic exchange of TFA relative to all classes of SFA, 18:1, and 18:2 (97,110,111,115), and even up to a maximum of 11% TFA energy (110). However the duration of feeding affects Lp(a) concentrations when diets containing 4%TFA isoenergetic exchanges are consumed (1 12,115). Taken together, the available data indicate that SFA (12:O-16:O) significantly decrease Lp(a) to a greater extent than cis-18:1, compared with trans- 18:1 or TFA from partially hydrogenated PUFA such as soybean oil or fish oil (97,110,115).
Summary Among nutritional factors, dietary fats per se have a profound effect on plasma lipoproteins. Fat composition, more than its quantity, dictates the greatest change in TC and LDL-C. The quantity of SFA determines the atherogenicity of the diet, and the reduction of SFA content by isoenergetic substitution with PUFA and MUFA reduces TC and LDL-C through an upregulation of rLDL and promotion of the fractional clearance rate of LDL. This fat composition model forms the basis of the Step 1 and Step 2 diets, otherwise referred to as LF-HC diets. Such diets cause a net reduction in TC and LDL-C but in the longer term, there is increased risk of inducing hypertriacylglycerolemia as well as reducing HDL-C, issues that remain to be resolved. The causal role of diet-induced decreases in HDL-C has yet to be shown in metabolic studies. However, isoenergetic substitution may institute proatherogenic TABLE 9.5 Percentage Change in Lp(a) Levels in Response to trans Fatty Acids (tl8:l n-9) vs. Dietary FA
cis-Fatty acid 12:O + 14:O + 16:O
12:O + 14:O 16:O 18:l n-9
18:O 18:2n-6
Feeding period
Lp(a) change
(wk)
(%)
(YO)
6 6 3 4 4 4 6 6 4 3 4 3 3
No change
3.8 6.6 11 6.9 6.9 5.7 3.8 6.6 5.7 11 6.9 8
ti3 t 73 ti4 126 ti9 No change t 4 t25 t41 ti9 t23 t23
lsoenergetic exchange
8
Reference 115 115 110 97 97 111 115 115 111 110 97 110 110
Dietary Fatty Acid Influences on Blood Lipids
195
TABLE 9.6 Percentage Change in Lp(a) Levels in Response to trans Fatty Acids (TFA) vs. Dietary Fatsa Feeding period (wk)
Dietary fat
TFA source
Corn oil
PHCO TFA-Marg PHSO PHFO PH-Soy PH-Soy
4.6
Palm olein Corn oil + palm kernel oil + coconut oil Palm olein + rapeseed oil + sunflower oil Vegetable oil
Lp(a) change
lsoenergetic exchange (Oh
1
Reference
4 3.9
4 4
Nochange t g t13 t33 t26 ~ 1 4
6.9 6.9
112 113 116 116 97 97
PH-Soy
4
t19
6.9
97
PHFO
2
t 6
6.6
105
5 3
3
a 8.5
aPH, partially hydrogenated; CO, corn oil; SO, safflower oil; Can, canola oil; Marg, margarine; Soy, soybean oil; FO,fish oil.
processes through a reduction in plasma HDL-C. Increased carbohydrate feeding is associated with elevations in both the TAG content of VLDL per particle as well as increased plasma VLDL particle number. The manipulation of dietary cholesterol in the management of elevated TC and LDL-C levels appears to mediate a very small effect compared with decreasing the saturation of the total diet. This is largely affected by the baseline dietary cholesterol intake and individual response. An important consideration is that not all SFA are equal in their effects on lipoprotein metabolism. Less certain also is the comparative hypercholesterolemic behavior of the different oils and fats available for human consumption. The environment of individual FA in the diet (intercorrelations with other SFA, PUFA, and MUFA), their predominance in various oils and fats, the position occupied by individual FA on the TAG molecule, and the percentage of SFA in the diet are all important variables modulating lipoprotein metabolism. The use of hydrogenated fats and oils has increased greatly in commercial food products because of economic advantage, as well as an appropriate replacement for SFA. Among trans-isomers, trans-18:1 as elaidic acid is most prevalent in the human diet. Epidemiologically, TFA has been associated with risk factors for CHD. The positive linear trend between TFA and lipoprotein levels was highlighted and concern voiced over the related risk to CHD. There is no evidence for a threshold of TFA intake below which there is no effect on lipoprotein levels. Studies implicate trans-18: 1 in raising LDL-C and reducing HDL-C concentrations. Unlike SFA which raise both LDL-C and HDL-C levels, the opposite effects are seen, with TFA adversely affecting the TC:HDL-C and LDL-C:HDL-C ratios. There is current debate on the potent effects of TFA compared with SFA, relative to cis-18:1, based on studies without adequate control for cis-18:2. Studies that did provide for sufficient cis-18:2 between test diets found that TFA are more deleteri-
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ous to lipoprotein levels than SFA. With TFA, there is an additional negative effect on Lp(a) concentrations compared with SFA, and the effect is dose-dependent. Clearly, dietary fats promote or prevent CHD through alternate pathways in lipoprotein metabolism, and a favorable action in one pathway may be detrimental to another pathway. The wider matrix of clinical management in CHD today recognizes that plasma HDL-Cand TAG also constitute risks for CHD,especially where there is a constellation of risk factors for “low metabolic capacity.” Testing individual FA using the postprandial mechanism will therefore provide definitive information on the relevance of dietary fat modulation to CHD. Acknowledgment The financial support and encouragement toward the preparation of this manuscript from Tan Sri Datuk Dr. Yusof Basiron, Director-General, Malaysian Palm Oil Board (MPOB) are gratefully acknowledged.
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87. Hu, F.B., M.J. Stampfer, J.E. Manson, E. Rimm, A. Wolk, G.A. Colditz, C.H. Hennekens, and W.C. Willett, Dietary Fat Intake and the Risk of Coronary Heart Disease in Women, N. Engl. J. Med. 337: 1491-1499 (1997). 88. Gillman, M.W., L.A. Cupples, D. Gagnon, B.E. Millen, R.C. Ellison, and W.P. Castelli, Margarine Intake and Subsequent Coronary Heart Disease in Men, Epidemiology 8: 144-149 (1997). 89. Tavani, A., E. Negri, B. D’Avanzo, and C. La Vecchia, Margarine Intake and Risk of Nonfatal Acute Myocardial Infarction in Italian Women, Eur. J. Clin. Nutr. 51: 30-32 (1997). 90. Baylin, A., E.K. Kabagambe, A. Ascherio, D. Spiegelman, and H. Campos, High 18:2 trans-Fatty Acids in Adipose Tissue Are Associated with Increased Risk of Nonfatal Acute Myocardial Infarction in Costa Rican Adults, J. Nutr. 133: 1186-1191 (2003). 91. Mensink, R.P., and M.B. Katan, Effect of Dietary trans Fatty Acids on High Density and Low Density Lipoprotein Cholesterol Levels in Healthy Subjects, N. Engl. J. Med 323: 439445 (1990). 92. Lemaitre, R.N., I.B. King, R.E. Patterson, B.M. Psaty, M. Kestin, and S.R. Heckbert, Assessment of trans Fatty Acid Intake with a Food Frequency Questionnaire and Validation with Adipose Tissue Levels of trans-Fatty Acids, Am. J. Epidemiol. 148: 1085-1093 (1998). 93. Wood, R., K. Kubena, B. O’Brien, S . Tseng, and G. Martin, Effect of Butter, Monoand Polyunsaturated Fatty Acid-Enriched Butter, trans Fatty Acid Margarine, and Zero trans Fatty Acid Margarine on Serum Lipids and Lipoproteins in Healthy Men, J. Lipid Res. 34: 1-11 (1993). 94. Noakes, M., and P.M. Clifton, Oil Blends Containing Partially Hydrogenated or Interesterified Fats: Differential Effects on Plasma Lipids, Am. J. Clin.Nutr. 68: 242-247 (1998). 95. Nicolosi, R.J., and J.M. Dietschy, Dietary trans Fatty Acids and Lipoprotein Cholesterol [letter], Am. J. Clin. Nutr. 61: 400401 (1995). 96. Institute of Medicine (IOM), Letter Report on Dietary Reference Intakes for Trans Fatty Acids, in Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids, Institute of MedicinelFood and Nutrition Board, National Academy Press, Washington, 2002. 97. Sundram, K., A. Ismail, K.C. Hayes, R. Jeyamalar, and R. Pathmanathan, Trans (elaidic) Fatty Acids Adversely Affect the Lipoprotein Profile Relative to Specific Saturated Fatty Acids in Humans, J. Nutr. 127: 5 14s-520s (1997). 98. Sundram, K., M.A. French, and M.T. Clandinin, Exchanging Partially Hydrogenated Fat for Palmitic Acid in the Diet Increases LDL-Cholesterol and Endogenous Cholesterol Synthesis in Normocholesterolemic Women, Eur. J. Nutr. 42: 188-194 (2003). 99. French, M.A., K. Sundram, and M.T. Clandinin, Changing Partially Hydrogenated Fat for Palmitic Acid in the Diet Increases LDL-Cholesterol and Endogenous Cholesterol Synthesis in Normocholesterolemic Women, in Frontiers in Cardiovascular Health, edited by N.S. Dhalla, A. Chockalingam, H.I. Berkowitz, and P.K. Singal, Kluwer Academic Publishers, Boston, 2003, pp. 353-366. 100. Judd, J.T., B.A. Clevidence, R.A. Muesing, J. Wittes, M.E. Sunkin, and J.J. Podczasy, Dietary trans Fatty Acids: Effects on Plasma Lipids and Lipoproteins of Healthy Men and Women, Am. J, Clin. Nutr. 59: 861-868 (1994). 101. Zock, P.L., and R.P. Mensink, Dietary transPatty Acids and Serum Lipoproteins in Humans, Curr. Opin. Lipidol. 7: 34-37 (1996).
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102. Muller, H., 0. Jordal, P. Kierulf, B. Kirkhus, and J.I. Pedersen, Replacement of Partially Hydrogenated Soybean Oil by Palm Oil in Margarine Without Unfavorable Effects on Serum Lipoproteins, Lipids 33: 879-887 (1998). 103. Khosla, P., and K. Sundram, Effects of Dietary Fatty Acid Composition on Plasma Cholesterol, Prog. Lipid Res. 35: 93-132 (1996). 104. Ascherio, A,, M.B. Katan, P. Zock, M.J. Stampfer, and W.C. Willett, Trans Fatty Acids and Coronary Heart Disease, N. Engl. J. Med. 340: 1994-1998 (1999). 105. Muller, H., 0. Jordal, I. Seljeflot, P. Kierulf, B. Kirkhus, 0. Ledsaak, and J.I. Pedersen, Effect on Plasma Lipids and Lipoproteins of Replacing Partially Hydrogenated Fish Oil with Vegetable Fat in Margarine, Br. I. Nutr. 80: 243-251 (1998). 106. Lichtenstein, A.H., L.A. Ausman, S . Jalbert, and E.J. Schaefer, Comparison of Different Forms of Hydrogenated Fats on Serum Lipid Levels in Moderately Hypercholesterolemic Female and Male Subjects, N. Engl. J. Med. 340: 1933-1940 (1999). 107. Bostom, A.G., A. Cupples, J.L. Jenner, J.M. Ordovas, L.J. Seman, P.W.F. Wilson, E.J. Schaefer, and W.P. Castelli, Elevated Plasma Lipoprotein (a) and Coronary Heart Disease in Men Aged 55 Years and Younger, J. Am. Med. Assoc. 276: 544-548 (1996). 108. Dahlen, G.H., Lp(a) Lipoprotein in Cardiovascular Disease, Atherosclerosis 108: 111-126 (1994). 109. Hornstra, G., A.C. van Houwelingen, A.D.M. Kester, and K. Sundram, A Palm-Oil Enriched Diet Lowers Serum Lipoprotein (a) in Normocholesterolemic Volunteers, Arteriosclerosis 90: 91-93 (1991). 110. Mensink, E.P., P.L. Zock, M.B. Katan, and G. Hornstra, Effect of Dietary cis- and trans-Fatty Acids on Serum Lipoprotein (a) Levels in Humans, J. Lipid Res. 33: 1493-1501 (1992). 111. Nestel, P., M. Noakes, B. Belling, R. McArthur, P. Clifton, E. Janus, and M. Abbey, Plasma Lipoprotein Lipid and Lp(a) Changes with Substitution of Elaidic Acid for Oleic Acid in the Diet, J. Lipid Res. 33: 1029-1036 (1992). 112. Lichtenstein, A.H., L.M. Ausman, W. Carrasco, J.L. Jenner, J.M. Ordovas, and E.J. Schaefer, Hydrogenation Impairs the Hypolipidemic Effect of Corn Oil in Humans: Hydrogenation, trans-Fatty Acids, and Plasma Lipids, Arterioscler. Thromb. 13: 154-161 (1993). 113. Judd, J.T., D.J. Baer, B.A. Clevidence, R.A. Muesing, S.C. Chen, J.A. Weststrate, G.W. Meijer, J. Wittes, A.H. Lichtenstein, M. Vilella-Bach, and E.J. Schaefer, Effects of Margarine Compared with Those of Butter on Blood Lipid Profiles Related to Cardiovascular Risk Factors in Normolipidemic Adults Fed Controlled Diets, Am. J. Clin. Nutr. 68: 768-777 (1998). 114. Gatto, L.M., D.R. Sullivan, and S. Samman, Postprandial Effects of Dietary trans Fatty Acids on Apolipoprotein (a) and Cholesteryl Ester Transfer, Am. J. Clin. Nutr. 77: 1119-1 124 (2003). 115. Clevidence, B.A., J.T. Judd, E.J. Schaefer, J.L. Jenner, A.H. Lichtenstein, R.A. Muesing, J. Wittes, and M.E. Sunkin, Plasma Lipoprotein (a) Levels in Men and Women Consuming Diets Enriched in Saturated, cis-, or trans-Monounsaturated Fatty Acids, Arterioscler. Thromb. Vasc. Biol. 17: 1657-1661 (1997). 116. Almendingen, K., 0. Jordal, P. Kierulf, B. Sandstad, and J.I. Pedersen, Effects of Partially Hydrogenated Fish Oil, Partially Hydrogenated Soybean Oil and Butter on Serum Lipoproteins and Lp(a) in Men, J. Lipid Res. 36: 1370-1384 (1995).
Chapter 10
Essential Fatty Acid Metabolism to Self-Healing Agents William E.M. Lands College Park, MD 20740
Introduction Healthful lipids include two different types-those we eat and those the body makes. Both types require wise management to maintain good health. The enzymes in our body readily make new lipids from the excess food we eat, and an important health principle for all foods is “eat no more than needed.” Each meal we eat usually has more food energy than is needed at that precise moment, and the “excess” food energy is converted into the hydrocarbon chains of fatty acids (FA) that are stored primarily as triacylglycerol esters (fat), which might be needed to supply energy at a later time. The time scale within which food is needed and when it gives benefits or harm is defined and interpreted by the dynamics of many different steps in lipid metabolism. Unfortunately, those metabolic steps are not tightly linked to the neural sensors regulating appetite, satiety, or food intake. Once food enters the body under the motivation of appetite, metabolic transformations proceed from the molecular collisions of the digested nutrients with multiple enzymes and cofactors, which give either needed energy or stored products (mainly fat). Fat provides 9 kcal/g or 4000 kcal/lb when metabolized to carbon dioxide and water. Many Americans carry 2540% of their body mass in the form of stored fat, which has large proportions of oleic (18:ln-9) and palmitic (16:O) acid. These nonessential FA are abundant in most diets, and they also are made by metabolism of the carbohydrate and protein in foods. Such nonessential FA compete in metabolic processes with the essential FA (EFA) that vertebrates cannot make from carbohydrate and protein. Competitive interactions of the mixture of FA with the many indiscriminate or promiscuous enzymes of lipid metabolism allow the relative supply of n-3 and n-6 EFA in the diet to have a large influence on the proportions of EFA that are maintained in tissues. Those food-induced proportions strongly influence several aspects of human health. Choosing food fats wisely remains an important way of preventing disease. The current epidemic of cardiovascular disease among Americans (along with its associated disorder, obesity) comes from a public health environment that has failed to educate people adequately about when food gives benefits or harm. Good
health requires preventing excessive ingested food being converted into unneeded structures, uncontrolled energy, or imbalanced signals. This chapter notes how 2 04
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imbalances in the intake and expenditure of energy and in the intakes of n-3 and n-6 EFA are causing a tragic epidemic of cardiovascular deaths among Americans (1). These two readily prevented imbalances cause vascular damage that begins in childhood (2) and slowly progresses toward the near-fatal onset of cardiovascular signs and symptoms that causes people to seek expensive treatments to extend their lives. Diseases caused by such imprudence can be prevented with better lifelong advice to the public about foods and lipid metabolism. Current treatments that fail to correct the dietary imbalances that cause disease leave patients vulnerable to a continued lifelong dependence on expensive medications and treatments. Essential polyunsaturated FA are like vitamins that must be taken regularly in small amounts relative to the larger amounts of food eaten to provide energy. Like all FA, EFA are metabolized and stored in tissue lipids and oxidized to CO, and H,O. The importance of EFA becomes evident after they are converted into important tissue structures and tissue signals. The time scale within which EFA give benefit or harm depends on the dynamics of the specific action involved. Tissue structures needed in the body often develop over hours, days, and weeks with long-lasting effects, whereas tissue signaling actions occur transiently within seconds and minutes. The relative proportions of n-6 (0-6) and n-3 ( 0 - 3 ) types of EFA derivatives stored in tissue lipids come from foods eaten at an earlier time. Rapid conversion of the stored EFA to hormone-like autacoids (auto = self, akos = healing) is part of normal tissue adjustment to changing conditions (3,4), but the amplifying dynamic actions sometimes go beyond desired healthy intensities. Although both n3 and n-6 types participate in the transient tissue signaling that regulates body responses to stress, signaling by the n-6 derivatives can sometimes be more intense than that by n-3 derivatives. Excessive n-6 eicosanoid signaling occurs in inflammatory/immune vascular disorders, thrombotic heart attacks, and cardiac arrhythmic events, as well as arthritis, asthma, cancer proliferation, and other chronic disorders of serious concern to aging adults (5). Billions of dollars are spent every year to develop and market pharmaceuticals that moderate imbalanced actions of these potent hormone-like autacoids (1,6), but little publicity is given to the fact that the sole source of the precursors for these autacoids is the food that people eat. Altering the foods eaten can alter the intensity of the signaling in tissues. This chapter briefly notes how the fats and FA that we eat and make are transported, stored in tissues, and made into hormone-like agents by metabolic processes that include the following: (i) remodeling of glycerolipid molecules, (ii) converting food energy to other forms in tissues, (iii) rearranging the EFA into signaling lipids during self-healing events, and (iv) preventing imbalances when choosing foods. Digestion and Transport of Fats and Phospholipid Metabolism
Lipids in the food we eat are digested by a relatively efficient and indiscriminate hydrolysis of the ester bonds in the intestine. Triglyceride lipase and phospholipase
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are capable of eventually hydrolyzing all of the lipid ester bonds consumed. However, esters of docosahexaenoic acid (DHA; 22:6n-3) are cleaved a bit more slowly, presumably due to steric hindrance by the presence of the A4 double bond too close to the carboxyl ester, In addition, faster lipase hydrolysis of the 1- and 3-ester bonds (compared with the more hindered 2-ester) yields many FA soaps and 2-monoglycerides, which are efficiently absorbed by intestinal epithelial cells. There, long-chain soaps are converted into coenzyme A thiol esters (acyl-CoA), esterified mainly into triglycerides (fats), secreted into the lymph, and eventually moved through the thoracic duct into the bloodstream as fat-rich chylomicron lipoprotein particles. For hours after every meal, the blood plasma is "milky" as the plasma carries elevated amounts of fat on lipoproteins being distributed to various tissues. In the bloodstream flowing through tissue capillaries, the triglycerides on plasma lipoprotein are hydrolyzed by lipoprotein lipase, and the NEFA released enter the tissue where they again form acyl-CoA esters that transfer acyl chains onto glycerolipid acceptors, forming diglycerides, triglycerides, and phospholipids in the tissues (Fig. 10.1). A continual formation and cleavage of esters in tissues rearrange the different FA in accord with their relative abundance and their efficacy in competing for the small amounts of the vitamin-based cofactor, CoA, in tissues. Each molecule of this cofactor is used and reused billions of times as various acyl chains esterify to it briefly during metabolism. Figure 10.1 notes that acyl-CoA
Fig. 10.1. Metabolism of FA. FA from circulating lipoproteins enter cells and form acylCoA esters that can be elongated, desaturated, transferred to glycerolipids, or oxidized to CO,. NEFA, nonesterified FA; HUFA, highly unsaturated FA; LPS, lipopolysaccharide.
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esters can be made longer and more unsaturated as the 18-carbon n-3 and n-6 acids form 20- and 22-carbon highly unsaturated FA (HUFA). When FA are in the liver in large amounts, their acyl-CoA esters form triglycerides (fat), which are packaged into fat-rich very low density lipoproteins (VLDL) and secreted into the circulating blood plasma. The process parallels the intestinal cell secretion of fat-rich chylomicron lipoproteins into lymph and plasma after every meal. In the blood, lipoprotein lipase hydrolyzes the fat in VLDL, releasing NEFA and forming smaller, more dense low density lipoproteins (LDL). Unfortunately, high levels of NEFA and LDL can increase oxidative stress to vascular endothelial tissue, especially when lipopolysaccharide (LPS) enters from the gut and triggers inflammatory responses (Fig. 10.1). Such conditions with overabundant NEFA and LDL occur transiently after every meal. Eating less food energy per meal may help prevent cumulative vascular endothelial injury from such repetitive transient postprandial stress. The enzymes transferring the acyl chain from acyl-CoA to form phospholipid and triglyceride esters have general selectivities (Fig. 10.2) that tend to put saturated acids (16:0, 18:O) at the l-position and unsaturated acids (18:ln-9, 18:2n-6)at the 2-position (7,8). The 3-position of triglycerides also tends to have unsaturated FA (9), giving typical fats in humans comprised of 33% saturated acids and 66% unsaturated acids, whereas phospholipids tend to have 50% saturated acids and 50% unsaturated acids (Fig. 10.2). These trends are less evident when a great abundance of substrate is present to “push” enzymes into faster reactions with whatever acids are available (as occurs after every large meal). However, during periods between meals, these general selectivities eventually influence the net composition of tissue lipids. High-carbohydrate, low-fat meals induce high rates of FA synthesis from excess acetyl-CoA, and the Ag desaturase forms large amounts of n-7 and n-9 nonessential FA (16:ln-7, 18:ln-7, 18:ln-9) via palmitate and stearate (16:O and 18:0, respectively). In this condition, the acyl-CoA pool has much more abundant nonessential FA than essential n-3 and n-6 acids (11). When the percentage of food energy (en%) as fat is c20%, the proportion of n-7 FA in tissue lipids is inversely related to the en% of fat
G-3-P
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Fig. 10.2. General selectivity in forming tissue lipids. In general, the I-position has much palmitic acid (16:O) and the 2-position has much oleic acid (1 8:l n-9). G-3-P, glycerol-3-phosphate; SFA, saturated fatty acids; UFA, unsaturated FA; HUFA, highly unsaturated FA. Reprinted from Lands (10) with author’s permission.
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intake (12). As a result, the elongation and desaturation enzymes form n-7 and n-9 HUFA (rather than n-3 and n-6 HUFA from dietary 18-carbon EFA). Alternatively, the very low levels of the n-9 HUFA, 203n-9, typical of many human studies, indicate a strong competition from n-3 or n-6 fats (or both). In fact, the almost undetectable level of 20:3n-9 in Americans can be regarded as preliminary evidence of excessive dietary supplies of either n-3 or n-6 EFA (12). Inattention to h s important metabolic marker leads to another serious flaw in current dietary advice from the many committees attempting to advise Americans about “healthy eating” (13). The HUFA differ from other unsaturated FA in being particularly enriched at the 2-position of phospholipids, where acyltransferases and phospholipases react with them more rapidly (14) than with other acyl chains during the retailoring processes indicated in Figure 10.2. As a result, the molecular species of lecithins in liver (and therefore in secreted plasma lipoproteins) of laboratory rats fed customary rat chow have a large proportion containing HUFA (especially 20:4n-6) that the body makes from linoleate, 18:2n-6 (Fig. 10.3). Fasting followed by eating fat-free sugar-rich diets shifts the supply of acyl-CoA substrates, and the composition of phospholipid species shifts dramatically from the initial “normal” pattern of 04 and 02 dominance among the molecular species (1 1). The results in Figure 10.3 illustrate a “normal” pattern of FA in tissue phospholipids from the wide range of tissue compositions that are possible from eating different foods. The “normal” status of this composition is correct in the statistical sense of representing a mean value obtained under common dietary inputs that typically occur with current feeding conditions. However, the promiscuous activities of the normal lipidLecithin species mol (%) Female
00 01 11 02 12 03 13 04 14+24 06
1.6 10.7 .7 24.5 2.7 2.9 1.o 47.2 .5 7.7
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00 01
04
1.8 11.4 2.0 28.0 6.5 2.1 2.0 34.9 4.5 5.1
00 01 11 02 12 03 13 04 14+24 06
4+24 0~ 06
Fig. 10.3. The bar graph patterns and adjacent percentage values result from standard laboratory chow containing relatively more n-6 than n-3 type of essential fatty acids. Species noted as 01 have one saturated and one monounsaturated acid, whereas 04 species have one saturated and one arachidonic acid, a HUFA. Reprinted from Lands & Hart (1 1) with authors’ permission.
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metabolizing enzymes permit greatly different compositions of tissue phospholipid molecular species to occur from these otherwise normal processes. Unfortunately, researchers have often failed to explore the range of compositions that normal tissues can make when mixing the acids we eat with those we make. Failure to explore the diversity attainable leaves investigators uninformed about their own narrow range of experience and about the diverse effects of dietary unsaturated FA on tissue composition and on health and disease. The amounts of n-3 and n-6 EFA used in typical feeding studies cannot moderate the n-6 eicosanoid excesses that occur in experimental tissue responses. Thus, typical (“normal”) conditions in most studies of laboratory animals and humans are not desirable, and desired healthy conditions are not typical (“normal”). The relatively slow actions of the elongation and desaturation enzymes and the small supply of available 2-ester positions in the limited phospholipids of tissue membranes create intensely competitive conditions for entry of HUFA into tissue esters. These constraints cause the HUFA made in tissues from dietary 18-carbon EFA to appear in tissue phospholipids in a hyperbolic response to the en% in the dietary supply (15-17). In contrast, dietary acids appear readily in tissue triglycerides in linear proportion to their en% abundance in the diet (12). A strong enzyme preference for the n-3 and n-6 EFA by elongating and desaturating enzymes allows very small amounts of 18-carbon EFA to be efficiently converted to HUFA and placed into phospholipids in preference to more abundant n-7 and n-9 FA (15). Unfortunately, the small amounts of n-3 or n-6 vitamin-like EFA shown to be needed for normal growth and health of laboratory animals and humans (0.3 en%) are now seldom discussed by nutritionists. Instead, foods with much more than needed n-6 fat and little n-3 fat are typically provided and often recommended as desirable. An empirical quantitative hyperbolic equation describing the relationship of dietary EFA to the proportions of HUFA that are maintained in tissues of laboratory rats (16,18) was also able to estimate diet-tissue patterns in women eating typical American foods (17). The successful fit illustrates the great similarity in enzyme specificities between humans and the laboratory animals used to model human processes. It also reflects similar relative EFA abundances in rat chow and typical average American food. Subsequent constants developed to fit results from a small clinical study with diets supplemented with n-3 HUFA (17) were recently revised using more data from nearly 200 healthy men and women eating voluntarily chosen diverse diets (6,19). The equation and constants used now closely predict the observed HUFA proportions in plasma phospholipids from dietary assessments (6). Figure 10.4 shows how competition between dietary 18-carbon n-6 EFA, linoleate (18:2n-6), and dietary n-3 HUFA affects the proportion of tissue HUFA that is n-6 HUFA. When diets have very low en% of n-3 HUFA, the n-6 HUFA dominate the tissue HUFA. However, the effect of increasing intakes of dietary n-3 HUFA is indicated by the family of curves with lower proportions of n-6 HUFA (Fig. 10.4). The four ovals in the figure represent voluntary dietary mixtures typically seen in average daily foods of four different ethnic populations. The resultant propor-
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Fig. 10.4. Influence of dietary essential FA on proportions of n-6 highly unsaturated fatty acids (HUFA). Higher intakes of linoleate (1tk2n-6) give a higher percentage of n-6 HUFA, whereas a higher intake of n-3 HUFA gives lower percentage of n-6 HUFA. TG, triglyceride. Source: http://efaeducation.nih.gov/sig/food2.html.
tions of n-6 HUFA in total HUFA (noted on the vertical axis) closely correlate with observed cardiovascular heart disease mortality rates (deaths per 100,000) noted in the ovals. The relationship is likely due to different intensities of n-6 eicosanoid responses when the mixture of tissue HUFA is mobilized (as discussed later in this chapter). Diverse ethnic food habits among many hundreds of people in the Canadian province of Qutbec created proportions of plasma phospholipid HUFA that ranged from 13% n-6 HUFA to 91% n-6 HUFA. The average HUFA proportions for Qukbec Inuits (20), Qutbec Cree (21), and more urban Qutbecers (22) correlated with cardiovascular mortality rates from these three groups in the province. The correlation agrees with that seen for nearly 6000 Americans in the Multiple Risk Factor Intervention Trial study (19) and likely involves excessive n-6 eicosanoid signaling from the high proportion of n-6 HUFA stored in tissue HUFA of the people studied. Although this chapter focuses on the metabolic and physiologic consequences of eating the precursors of self-healing autacoids, another vital aspect of EFA biology is in forming important membrane phospholipids needed for healthy brain and retina function (23). The recent production of infant formulas containing the 22-carbon HUFA, DHA (22:6n-3), reflects a recent awareness of the importance of this n-3 HUFA for healthy development of humans. Unfortunately, entry and exit of FA across the blood-brain “barrier” involve transport selectivities and dynamics that are still not well defined or understood. In addition, uncertainty about the different relative rates of elongation and desaturation in brain compared with other tissues
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remains a serious limitation in understanding what quantity of the different EFA should be present in healthy diets. Clearly, essential n-3 acids cannot be in brain membranes if they are not in an infant’s food supply. Conversion of Food Energy into Stored and Expended Energy
Protein, carbohydrates, and lipids all eventually break down to the two-carbon metabolite, acetyl-CoA (see Fig. 10.5). This metabolite is a ready substrate for energy release by the mitochondrial enzymes that convert acetyl-CoA to carbon dioxide via the metabolic steps of the citric acid cycle. The conversion gives large amounts of reduced cofactors that carry away the electrons removed, eventually reducing oxygen to water. Useful work is done as the electron-carrying cofactors are regenerated by transferring the electrons through the cytochrome electron transport chain to form water from oxygen. The work energy released during electron transport to oxygen is transferred into forming ATP from ADP in a tightly coupled process. As a result, the overall coordinated process of forming CO, and H,O depends on ATP being used and ADP being regenerated. Without expending energy and regenerating ADP, the electron transfer cofactors are not regenerated, citrate can not be oxidized to CO,, and metabolites carrying the excess food energy “spill over” into storage forms (especially fat that readily accumulates in adipose tissues; see Fig. 10.1). The standard measure of energy being expended by a person at a given time is the overall volume of oxygen consumed (VO,) as mitochondrial enzymes convert it to water. When resting, consumption is typically 3.5 mL O,/(kg body weightemin), and it might increase up to 15-fold depending on the intensity of exercise (24). Without energy expenditure, unoxidized mitochondrial citrate accumulates and “spills out” to the cytosol, where it tends to increase acetyl-CoA levels and promote the synthesis of FA (Fig. 10.5). This “spilling” occurs continually, but it “pushes” fat formation more vigorously after every meal when ingested protein, carbohydrates, and lipids all transiently add to the two-carbon metabolite pool. In healthy adults maintaining a constant body weight, all ingested essential and nonessential proteins, carbohydrates, and lipids are eventually converted to CO, and water with no net increase in stored metabolites. However, Figure 10.5 shows that accumulating fats, isoprenoids, and cholesterol (as well as stored energy as body mass) are inescapable results whenever food energy intake exceeds expenditure. Dietary EFA (when converted to HUFA; 25) suppress lipid synthesis in the liver, upregulate FA oxidation, increase glycogen storage, and increase insulin sensitivity (26). For these effects, n-3 EFA are more potent than n-6 EFA on a carbonfor-carbon basis. This “repartitioning” of food energy likely involves n-3 HUFA activating peroxisome proliferator-actived receptor alpha (PPARa) (27). Another result of excess food energy associated with a large throughput of food and its electrons is an uncoupling or “leaking” of electrons from reduced cofactors to form reactive oxygen species (ROS; superoxide, hydrogen peroxide,
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+ Food Energy Low n-3 Fats
High Cytosollc AcetylCoA + NAD(P)H
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Mortality
Plasma Cholesterol Suppresses Mevalonate Formation
Arrhythmia
The Chain of Events Linking Diet and Disease Fig. 10.5. Two dietary imbalances cause inflammation, thrombosis, ischemia, morbidity, and mortality. Preventing inadequate ratios of energy expenditure to energy intake and n-3 to n-6 dietary intake can prevent morbidity and mortality. Nitric oxide decreases ischemia and thrombosis, reactive oxygen species; some prenylated proteins decrease nitric oxide synthesis, and statins decrease mevalonate synthesis.
and lipid peroxides) rather than water and ATP. The resulting oxidant stress sets in motion several inflammatory signaling events that are amplified and made worse by some prenylated proteins and by n-6 eicosanoids (1,28) as noted in Figure 10.5. Unfortunately, inflammatory processes in vascular plaques induce the generation of still more ROS that oxidize the HUFA at the 2-position of nearby lipoprotein phospholipids. The resulting fragmented phospholipid formed by the ROS mimics platelet activating factor (PAF), a potent inflammatory signaling agent formed by phospholipase removing the HUFA from the 2-position and acetyl transferase adding an acetyl group from acetyl-CoA (29). The actions of PAF and PAF-mimics can amplify small inflammatory events into more serious events, and their chronic presence promotes vascular disease. Oxidized phospholipids may explain much of the harmful consequence of LDL being present at inflammatory sites in vascular walls. The presence of increased plasma LDL (from VLDL hydrolysis) may be a marker of increased cardiovascular risk because of accompanying high levels of NEFA and PAF-mimics rather than
because of any cholesterol being carried on the LDL. In a related way, the presence of anti-inflammatory enzymes in high density lipoprotein (HDL) aggregates, which
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inactivate PAF and PAF-mimics, may better explain the benefits of HDL, irrespective of any cholesterol molecules present in the aggregates (30). Preventing repeated chronic postprandial exposures to high levels of NEFA and PAF-mimics seems more likely when less food energy per meal is ingested. The higher blood levels of glucose and nonesterified FA that follow large meals create transient oxidative/inflammatory conditions (3 1,32) that impair endothelial function and cause leukocyte adhesion and stress-related responses (33). Even apparently harmless transient postprandial inflammatory events that are 99.9% reversible can eventually accumulate inflammatory plaques over 3-4 decades of 1000 mealdyr. Inflammatory vascular disease starts in childhood and develops progressively for decades (34) before clinical signs and symptoms make older people seek a doctor’s advice and become a patient who needs treatment. Fatty streaks were seen in abdominal aortae of -20% of 15- to 19-yr-old Americans, and this percentage increased to -40% for 30- to 34-yr-olds (2,34). In the right coronary arteries, raised fatty streaks were present in -10% of 15- to 19-yr-old people, and 30% for 30- to 34-yr-olds (34). Less arterial disease exists in people whose tissues have relatively more n-3 EFA and less aggressive inflammatory responses. Two nutritional imbalances make vascular inflammatory damage worse, i.e., a relatively greater ratio of intake to expenditure of energy and a relatively greater ratio of n-6 to n-3 EFA (Fig. 10.5). Without awareness and education about ways to prevent the poor food and health habits that cause the disease, people face continued lifelong disability with expensive treatments and medications.
Signaling Actions by Tissue Eicosanoids: Physiology and Pathology Earlier parts of this chapter discussed how voluntary food choices alter the dynamics of maintaining essential and nonessential FA in body tissues. Once the choice to eat has been made, the eventual speed and intensity of self-healing responses are regulated by enzymes and autacoid receptors that are beyond our voluntary control. Understanding these dynamics and the diversity of autacoids and their receptor actions can help readers better appreciate how their food choices can have such widespread effects on their health (35). Transient postprandial inflammation from NEFA, oxidative stress, ROS, and prenylated proteins (Fig. 10.5)can be amplified out of control by excessive activation by n-6 tissue eicosanoids that are formed after tissue phospholipases release stored HUFA. Oxidant hydroperoxides activate the FA oxygenases (lipoxygenase and cyclooxygenase; Fig. 10.6) that convert 20-carbon HUFA into potent hormone-like self-healing autacoids called eicosanoids (4). Both types of oxygenase have an absolute requirement for hydroperoxide activators (36), and they are inhibited by a normal continuous removal of such activators (3,37). As a result, conditions of oxidant stress allow both pathways of eicosanoid formation to more readily produce the self-healing agents that normally are not present. In fact, the first products of oxygenase action are hydroperoxide intermediates [5-hydroperoxy-eicosatetraenoic acid and prostaglandin (PG)G; Fig. 10.61 that give positive feedback and accelerate
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-v-vvwac 12-HPETE
nrc I c
COOH
Arachidonic Acid
Fig. 10.6. Many leukotrienes and prostaglandins are formed from 20:4n-6 and 20:5n3 . The n-6 structures in this figure have similar corresponding n-3 structures with the additional n-3 double bond. Each prostanoid autacoid at the right of the figure and many leukotrienes at the left bind a specific receptor that transmits autacoid signals during tissue adaptations, Source: http:Nefaeducation.nih.gov/sig/icosa.html. HPETE, hydroperoxy-5,8,10,14-eicosatetraenoic acid; LT, leukotriene; TX, thromboxane; PG,
prostaglandin; HHT, 5,8,1O-heptadecatrienoic acid; HMG, 3-hydroxy-3-methylglutaryl. Source: http:Nefaeducation.nih.gov/sig/dietdisease.html.
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explosively the oxygenase reaction from its normally suppressed state. The leukotriene (LT)A and PGH formed by the oxidative reactions eventually are rearranged to form leukotrienes and prostaglandins, respectively. Fortunately, a self-catalyzed inactivation eventually stops oxygenase action (3,38) and halts the explosive eicosanoid production that had escaped from its normal basal suppression. The acceleration of synthesis by hydroperoxides has important consequences because nearly all active eicosanoids are rapidly inactivated by either spontaneous decomposition or by ubiquitous catabolic enzymes (3). The fast inactivation allows only small transient amounts of active autacoid to bind to nearby tissue receptors and provide transient regulatory self-healing events during normal tissue responses. Too slow a rate of synthesis that does not exceed the inactivation rate cannot give adequate signaling actions, even when considerable autacoid is produced and inactivated over time. However, too intense a rate of forming active autacoid can activate more receptors than is compatible with the needed balanced self-healing action. Conversion of the n-6 HUFA, 20:4n-6, to PGG is several-fold faster than conversion of the n-3 HUFA, 20511-3 (39,40). This kinetic difference creates an important consequence of eating more n-3 EFA, which competitively moderate excessive n-6 eicosanoid actions. The faster synthesis of the various n-6 PG-based autacoids shown at the right side of Figure 10.6 makes their access to receptors more intense than would occur if signaling were coming from comparable amounts of n3 precursors. Such a kinetic difference does not appear to occur with the 5-lipoxygenase that supports leukotriene signaling events, and some n-3 and n-6 leukotrienes may give similar signal intensities. One pathophysiologic process in which eicosanoid kinetics plays an important role is aggregation of blood platelets, which has multiple amplified explosive positive feedback features. A key step is the explosive formation of n-6 thromboxane (TXA,) from arachidonate released from platelet membranes. Thromboxane is unstable, and it rapidly decomposes with a half-life of seconds (4 1). This instability provides reversible, transient platelet aggregation. Nevertheless, the explosive formation often goes beyond the minimum needed to aggregate platelets (42), and consolidation of the thrombus can cause irreversible loss of blood flow-as occurs in ischemic heart attacks. The slower formation of the n-3 thromboxane (TXA,) makes less active autacoid available at the thromboxane receptor (TP). The lower aggregation when platelets have high proportions of competing n-3 HUFA (42) seems due to combined effects of the lower proportion of n-6 HUFA substrate released, slower formation of TXA, from the n-3 HUFA, and less signal transmitted by selective TP action when competing TXA, is the bound ligand (43,44). The strong linear correlation between the proportion of n-6 HUFA in tissue HUFA and the observed mortality rates for coronary heart disease (6,13,19) may have the intensity of TXA, formation and action as the probable causal mechanism. The eight known human prostanoid receptors (45) are still not well characterized in terms of their selectivity for n-3 and n-6 prostanoids. Although selective responses seem apparent for the TP, the prostacyclin receptor (IP) acts equally
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with n-3 and n-6 PGI forms. Thus, differences in dietary EFA have a greater effect on TP signaling than IP signaling. Some of the four receptors for PGE (EP-1, EP-2, EP-3, and EP-4) and the receptors for PGD (DP) and PGF (FP) likely have different signaling responses with n-3 and n-6 ligands that allow dietary EFA to alter immunehnflammatory signals. The leukotriene receptor for LTB (BLT) gives very much stronger signaling with the n-6 LTB, than with the n-3 LTB,. As a result, diets with higher proportions of competing n-3 EFA create less neutrophil chemotaxis and less inflammatory tissue responses. Preventing Imbalances When Choosing Food
Although repeatedly recommending “good nutrition,” the broader health network often waits until disease occurs before applying specific treatments to individuals who are in trouble rather than preventing the disease from developing in the population. For each expensive treatment that decreases clinical signs and symptoms, we should consider, “Does removal of the clinical signs remove the original underlying nutritional imbalances that cause people to develop the disease?’ An important ethical issue is in the decision to withhold community-wide primary preventive nutrition efforts and wait until disease signs appear in individuals who are then selectively treated with secondary prevention to slow further signs. Such a strategy ensures a continuing supply of new patients for the expensive treatments rather than preventing their entry into disease states with primary prevention efforts. Among the causes of cardiovascular disease and many chronic diseases of the elderly, much evidence points to nutritional imbalances in expenditure/intake of energy and in n-3/n-6 EFA. The epidemic of early onset and life-long progression of vascular inflammatory disease in Americans (2,34) begs for a well-designed nutritional education program of primary prevention for the two readily corrected imbalances noted in this chapter. Although people are not free to choose their genes or the metabolic enzymes made from them, they can choose what they put into their mouths. Nutrition education can help them choose wisely. To facilitate the education effort among health professionals and the public, interactive computer-aided access to needed information is available at distance learning sites for essential FA education, http://efaeducation.nih.gov/,and dietary supplements, http://ods.od.nih.gov/eicosanoidd. The wide range (13-91% n-6) of tissue proportions of HUFA in response to different diets that are voluntarily eaten by people is illustrated at http://efaeducation.nih.gov/sig/foodl.html. A convenient calculator for general planning of daily diets that balance tissue HUFA at whatever proportion desired appears at a related site, http://efaeducation.nih.gov/sig/dietbalancel.html. The simple calculator estimates the likely outcome from four measures of EFA in the diet: the en% of the n-3 and n-6 18-carbon EFA, and the en% of the n-3 and n-6 HUFA. The empirical equation and fitted constants that relate dietary intakes to tissue HUFA proportions (17) in this calculation are given at http://efaeducation.nih.gov/ sighufacaIc.htm1.
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To help people plan a widely diverse menu of nutrient-rich foods that are pleasing to their own personal taste and lifestyle, the distance learning sites provide a free interactive personalized computer program to inform people as they choose daily food items from the nearly 12,000 servings in the U.S. Department of Agriculture nutrient database that also can be searched at http://www.nal.usda.gov/fnickgi-birdnut-search.pl. The software, KIM-:! (Keep It Managed-2nd version), shows the milligrams of n-3 and n-6 EFA in each serving selected and provides estimates of the daily total and the likely surrogate clinical outcome for each day’s food choices. By seeing the variety of nutrient intakes that can be selected along with the likely outcome of their voluntary food choices, people can plan daily menus that meet their personal goals for preventing imbalanced expenditure and intake of energy and imbalanced n-3 and n-6 EFA. Experience with the nutrient information in KIM-2 rapidly alerts users to the high levels of linoleic acid, 18:2n-6, in foods that are currently common in daily menus in the USA. For example, replacing household oils with either olive or canola oil is one simple step toward diminishing the current imbalance in dietary EFA. Another valuable step is to include more seafood in daily menus as advised by the American Heart Association (46,47). To make educated choices among the possible food servings, the food choice software quickly sorts selected food lists in rank order to put at the top foods for which the amounts of n-3 EFA are relatively greater than those of n-6 EFA. As food producers and marketers consider ways for the public to diminish excessive n-6 intakes and to eat fortified foods that increase n-3 intakes, consumers will face a continually changing set of new food products to better maintain health and prevent disease.
Overview Healthful lipids that we eat mix with those the body makes and affect overall health. Food energy not needed at any given moment is converted into hydrocarbon chains of FA and stored until it is eventually burned to carbon dioxide when energy is expended. The n-3 and n-6 EFA that cannot be made by vertebrates can be made longer and more unsaturated and stored (primarily at the 2-position of phospholipids) until released when environmental stimuli activate cellular phospholipase A,. The released 20-carbon HUFA form hormone-like eicosanoids that activate specific tissue receptors in self-healing responses. Eicosanoids made from n-6 precursors often have excessively vigorous actions that can become pathological when not moderated by sufficient competing n-3 precursors in tissues. Healthful diets ensure a balance between expenditure and intake of food energy and a balance between the n-3 and n-6 essential precursors of eicosanoids. References 1. Lands, W.E.M., Primary Prevention in Cardiovascular Disease: Moving Out of the Shadows of the Truth About Death, Nutr. Metub. Curdiovusc. Dis. 13: 154-164 (2003).
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2. Zieske, A.W., G.T. Malcom, and J.P. Strong, Natural History and Risk Factors of Atherosclerosis in Children and Youth: The PDAY Study, Pediatr. Pathol. Mol. Med. 21: 213-237 (2002). 3. Lands, W.E.M., The Biosynthesis and Metabolism of Prostaglandins, Annu. Rev. Physiol. 41: 633-652 (1979). 4. Samuelsson, B ,, Prostaglandins, Thromboxanes, and Leukotrienes: Formation and Biological Roles, Harvey Lect. 75: 1 4 0 (1979). 5. Lands, W.E.M., Fish and Human Health, Academic Press, Orlando, FL,1986. 6. Lands, W.E.M., Functional Foods in Primary Prevention or Nutraceuticals in Secondary Prevention? Curr. Top. Nutraceut. Res. I : 113-120 (2003). 7. Lands, W.E.M., Lipid Metabolism, Annu. Rev. Biochem. 34: 313-346 (1965). 8. Hill, E.E., and W.E.M. Lands, Phospholipid Metabolism, in Metabolism and Function of Lipids, edited by S.J. Wakil, Academic Press, Orlando, FL,1970,Vol. 1, pp. 185-277. 9. Slakey, P.M., Sr., and W.E.M Lands, The Structure of Rat Liver Triglycerides, Lipids 3: 30-36 (1968). 10. Lands, W.E.M., Selective Recognition of Geometric and Positional Isomers of Fatty Acids In Vitro and In Vivo, in Geometrical and Positional Fatty Acid Isomers, edited by E.A. Emken and H.J. Dutton, American Oil Chemists’ Society, Champaign, IL, 1979, pp. 18 1-2 12. 11. Lands, W.E.M., and P. Hart, The Control of Fatty Acid Composition in Glycerolipids, J. Am. Oil Chem. SOC.43: 290-295 (1966). 12. Lands, W.E.M., Long-Term Fat Intake and Biomarkers, Am. J. Clin. Nutr. 61 (Suppl.): 721s-725s (1995). 13. Lands, W.E.M., Please Don’t Tell Me to Die Faster, INFORM 13: 896-897 (2002). 14. Lands, W.E.M., M. Inoue, Y. Sugiura, and H. Okuyama, Selective Incorporation of Polyunsaturated Fatty Acids into Phosphatidylcholine by Rat Liver Microsomes, J. Biol. Chem. 257: 14968-14972 (1982). 15. Mohrhauer, H., and R.T. Holman, The Effect of Dose Level of Essential Fatty Acids Upon Fatty Acid Composition of the Rat Liver, J. Lipid Res. 4: 151-159 (1963). 16. Lands, W.E.M., A.J. Morris, and B. Libelt, Quantitative Effects of Dietary Polyunsaturated Fats on the Composition of Fatty Acids in Rat Tissues, Lipids 25: 505-516 (1990). 17. Lands, W.E.M., B. Libelt, A. Morris, N.C. Kramer, T.E. Prewitt, P. Bowen, D. Schmeisser, M.H. Davidson, and J.H. Burns, Maintenance of Lower Proportions of n-6 Eicosanoid Precursors in Phospholipids of Human Plasma in Response to Added Dietary n-3 Fatty Acids, Biochim. Biophys. Acta 1180:147-162 (1992). 18. Mohrhauer, H., and R.T. Holman, Effect of Linolenic Acid upon the Metabolism of Linoleic Acid, J. Nutr. 81: 67-74 (1963). 19. Lands, W.E.M., Diets Could Prevent Many Diseases, Lipids 18: 317-321 (2003). 20. Dewailly, E., C. Blanchet, S. Lemieux, L. Sauve, S. Gingras, P. Ayotte, and B.J. Holub, n3 Fatty Acids and Cardiovascular Disease Risk Factors Among the Inuit of Nunavik, Am. J. Clin. Nutr. 74: 464-473 (2001). 21. Dewailly, E.E., C. Blanchet, S. Gingras, S. Lemieux, and B.J. Holub, Cardiovascular Disease Risk Factors and n-3 Fatty Acid Status in the Adult Population of James Bay Cree, Am. J. Clin. Nutr. 76: 85-92 (2002). 22. Dewailly, E.E., C. Blanchet, S . Gingras, S. Lemieux, L. Sauve, J. Bergeron, and B.J.
Holub, Relations Between n-3 Fatty Acid Status and Cardiovascular Disease Rsk Factors Among QuCbecers, Am. J. Clin. Nutr. 74: 603-61 1 (2001).
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23. Salem, N., Jr., B. Litman, H.Y. Kim, and K. Gawrisch, Mechanisms of Action of Docosahexaenoic Acid in the Nervous System, Lipids 36: 945-959 (2001). 24. National Research Council Recommended Dietary Allowances, 10th ed., National Academy Press, Washington, 1989. 25. Nakamura, M.T., H.P. Cho, and S.D. Clarke, Regulation of Hepatic A-6 Desaturase Expression and Its Role in the Polyunsaturated Fatty Acid Inhibition of Fatty Acid Synthase Gene Expression in Mice, J. Nutr. 130: 1561-1565 (2000). 26. Clarke, S.D., Polyunsaturated Fatty Acid Regulation of Gene Transcription: A Molecular Mechanism to Improve the Metabolic Syndrome, J. Biol. Chem. 131: 1129-1132 (2001). 27. Pawar, A,, and D.B. Jump, Unsaturated Fatty Acid Regulation of Peroxisome ProliferatorActivated Receptor Alpha Activity in Rat Primary Hepatocytes, J. Biol. Chem. 278: 35931-35939 (2003). 28. Lands, W.E.M., Eicosanoids and Health, in The Third International Conference on Nutrition in Cardio-Cerebrovascular Diseases, edited by K.T. Lee, Y. Oike, and T. Kanazawa, The New York Academy of Sciences, New York, 1993,pp. 46-59. 29. Marathe, G.K., G.A. Zimmerman, S.M. Prescott, and T.M. McIntyre, Activation of Vascular Cells by PAF-Like Lipids in Oxidized LDL, Vasc. Pharmacol. 38: 193-200 (2002). 30. Marathe, G.K., G.A. Zimmerman, and T.M. McIntyre, Platelet-Activating Factor Acetylhydrolase, and Not Paraoxonase-1, Is the Oxidized Phospholipid Hydrolase of High Density Lipoprotein Particles, J. Biol. Chem. 278: 3937-3947 (2003). 31. Bae, J.H., E. Bassenge, K.B. Kim, Y.N. Kim, K.S. Kim, H.J. Lee, K.C. Moon, M.S. Lee, K.Y. Park, and M. Schwemmer, Postprandial Hypertriglyceridemia Impairs Endothelial Function by Enhanced Oxidant Stress, Atherosclerosis 155: 5 17-523 (2001). 32. Hsieh, T.J., S.L. Zhang, J.G. Filep, S.S. Tang, J.R. Ingelfinger, and J.S. Chan, High Glucose Stimulates Angiotensinogen Gene Expression Via Reactive Oxygen Species Generation in Rat Kidney Proximal Tubular Cells, Endocrinology 143: 2975-2985 (2002). 33. Griendling, K.K., D. Sorescu, B. Lassegue, and M. Ushio-Fukai, Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species and Their Role in Vascular Physiology and Pathophysiology, Arterioscler. Thromb. Vasc. Biol. 20: 2175-21 83 (2000). 34. McGill, H.C., Jr., C.A. McMahan, A.W. Zieske, G.D. Sloop, J.V. Walcott, D.A. Troxclair, G.T. Malcom, R.E. Tracy, M.C. Oalmann, and J.P. Strong, Associations of Coronary Heart Disease Risk Factors with the Intermediate Lesion of Atherosclerosis in Youth. The Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group, Arterioscler. Thromb. Vasc. Biol. 20: 1998-2004 (2000). 35. Lands, W.E.M., Biosynthesis of Prostaglandins, Annu. Rev. Nutr. 11: 41-60 (1991). 36. Kulmacz, R.J., and W.E.M. Lands, Peroxide Tone in Eicosanoid Signaling, in Oxidative Stress and Signal Transduction, edited by H.J. Forman and E. Cadenas, Chapman & Hall, New York, 1991,pp. 134-156. 37. Kulmacz, R.J., and L.H. Wang, Comparison of Hydroperoxide Initiator Requirements for the Cyclooxygenase Activities of Prostaglandin H Synthase-1 and -2, J. Biol. Chem. 270: 24019-24023 (1995). 38. Smith, W.L., and W.E.M. Lands, Stimulation and Blockade of Prostaglandin Synthesis, J. Biol. Chem. 246; 6700-6704 (1971)
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39. Kulmacz, R.J., R.B. Pendleton, and W.E.M. Lands, Interaction Between Peroxidase and Cyclooxygenase Activities in Prostaglandin-Endoperoxide Synthase, J. Biol. Chem. 269: 5527-5536 (1994). 40. Malkowski, M.G., E.D. Thuresson, K.M. Lakkides, C.J. Rieke, R. Micielli, W.L. Smith, and R.M. Garavito, Structure of Eicosapentaenoic and Linoleic Acids in the Cyclooxygenase Site of Prostaglandin Endoperoxide H Synthase- 1, J. Biol. Chem. 276: 37547-37555 (2001). 41. Hamberg, M., J. Svensson, and B. Samuelsson, Thromboxanes: A New Group of Biologically Active Compounds Derived from Prostaglandin Endoperoxides, Proc. Natl. Acad. Sci. 72: 2994-2998 (1975). 42. Lands, W.E.M., B.R. Culp, A. Hirai, and R. Gorman, Relationship of Thromboxane Generation to the Aggregation of Platelets from Humans: Effects of Eicosapentaenoic Acid, Prostaglandins 30: 819-825 (1985). 43. Needleman, P., A. Raz, M.S. Minkas, J.A. Ferrendelli, and H. Sprecher, Triene Prostaglandins: Prostacyclin and Thromboxane Biosynthesis and Unique Biological Properties, Proc. Natl. Acad. Sci. 76: 944-948 (1979). 44. Needleman, P., H. Sprecher, M.O. Whitaker, and A.Wyche, Mechanism Underlying the Inhibition of Platelet Aggregation by Eicosapentaenoic Acid and Its Metabolites, Adv. Prostaglandin Thromboxane Res. 6: 61-68 (1980). 45. Abramovitz, M., M. Adam, Y. Boie, M. Caniere, D. Denis, C. Godbout, S. Lamontagne, C. Rochette, N. Sawyer, N.M. Tremblay, M. Belley, M. Gallant, C. Dufresne, Y. Gareau, R. Ruel, H. Juteau, M. Labelle, N. Ouimet, and K.M. Metters, The Utilization of Recombinant Prostanoid Receptors to Determine the Affinities and Selectivities of Prostaglandins and Related Analogs, Biochim. Biophys. Acta 1483: 285-293 (2000). 46. Krauss, R.M., R.H. Eckel, B. Howard, L.J. Appel, S.R. Daniels, R.J. Deckelbaum, J.W. Erdman, Jr., P. Kris-Etherton, I. J. Goldberg, T.A. Kotchen, A.H. Lichtenstein, W.E. Mitch, R. Mullis, K. Robinson, J. Wylie-Rosett, S. St. Jeor, J. Suttie, D.L. Tribble, and T.L. Bazzarre, AHA Dietary Guidelines: Revision 2000: A Statement for Healthcare Professionals from the Nutrition Committee of the American Heart Association, Circulation 102: 2284-2299 (2000). 47. Kris-Etherton, P.M., W.S. Harris, and L.J. Appel, for the Nutrition Committee Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease, Circulation 106: 2747-2757 (2002).
Chapter 11
Dietary n-6:n-3 Fatty Acid Ratio and Health Sarah Gebauera, William S. Harrisb, Penny M. Kris-Ethertona, and Terry D. EthertonC Departments of aNutritional Sciences and CDairy and Animal Science, The Pennsylvania State University, University Park, PA 16802 and bLipid and Diabetes Research Center, Saint Luke’s Hospital and the University of Missouri-Kansas City School of Medicine, Kansas City, MO 641 11
Introduction Polyunsaturated fatty acids (PUFA) are remarkably diverse molecules, both structurally and functionally. The two predominant PUFA classes are the n-6 and n-3 fatty acids; they are distinguished by the position of the first double bond proximate to the methyl terminus. Within each PUFA class, there are many different fatty acids. The predominant dietary n-6 PUFA is linoleic acid (18:2; LA, representing -84-89% of total PUFA), and the primary n-3 PUFA is a-linolenic acid (18:3; ALA, representing -9-11% of total PUFA). Both LA and ALA are derived primarily from plant sources. Marine sources of the n-3 fatty acids, eicosapentaenoic acid (20:5; EPA) and docosahexaenoic acid (22:6; DHA), are a quantitatively minor component of total PUFA (contributing <0.1-0.2% of energy). LA and ALA are not synthesized in the body; therefore, they are required nutrients. They serve as a substrate for the synthesis of numerous longer-chain fatty acidderived bioactive eicosanoids. The pathways involved in the metabolism of n-6 and n-3 fatty acids are well characterized. Both pathways result in the synthesis of related but unique bioactive compounds that regulate many important biological processes. A key precept in the metabolism of LA and ALA is that both absolute amounts and relative proportions (i.e., their ratio) are integrally involved in modulating flux through the n-6 and n-3 metabolic pathways. Thus, the absolute amounts and relative proportions of LA and ALA affect the respective metabolism of each fatty acid, as well as the metabolism of the other fatty acid. This is manifested in a way in which changes in LA can affect the flux of ALA and vice versa. Consequently, changes in the quantity and ratio of n-6 and n-3 fatty acids orchestrate flow through the respective PUFA metabolic pathways. The n-6:n-3 ratio has attracted attention because changes in one have consequent effects on metabolism of the other fatty acid. The purpose of this chapter is to review our current understanding of the n-6: n-3 ratio and how changes in the ratio and, importantly, the absolute amounts of LA and ALA affect numerous conditions including cardiovascular disease (CVD), cancer, diabetes, bone health, mental status and cognitive function, rheumatoid 221
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arthritis, asthma, immune function, and inflammatory status. Emerging evidence suggests that the diet strategies that have the greatest health benefits ensure adequate intake of LA and increased n-3 intake beyond that currently consumed. PUFA Structure
PUFA are long-chain fatty acids that contain 18 or more carbons with two or more double bonds. PUFA are described using a structural designation that identifies the fatty acid by the number of carbon atoms, and the number and position of the unsaturated double bonds. Depending upon the position of the first double bond proximate to the methyl end of the fatty acid, these fatty acids are categorized as n-9, n-6, or n-3 (also called w-9, 0-6, w-3) series, with the first double bonds being between carbons 9 and 10, carbons 6 and 7, and carbons 3 and 4, respectively. Using this nomenclature, LA and ALA are designated as 18:2n-6 and 18:3n-3, respectively. The three classes of unsaturated fatty acids are not interconvertible; however, they are metabolized by a common series of elongases and desaturases (see Fig. 11.1). Humans are capable of desaturating stearic acid to form oleic acid (18: ln-9) by means of a A9 desaturase enzyme (1) (Fig. 11.1). However, humans lack the ability to insert a double bond at the n-6 or n-3 position of a fatty acid carbon chain because of the absence of the A1* and A15 desaturases. The presence of these two enzymes is required for the production of LA and ALA (18:3n-3), which is why these fatty acids are essential and must be derived from the diet. PUFA Metabolism
The parent fatty acids of the n-6 and n-3 fatty acid families contain 18 carbon atoms and are LA and ALA, respectively. LA and ALA are metabolized by the same microsomal enzyme system, by alternating desaturation and elongation, generating two different pathways of subsequent metabolic products of up to 22 or more carbons (2) (Fig. 11.1). Marcel et al. (3) were the first to identify the preferred pathway for LA metabolism as the following: 18:2n-6 + 18:3n-6 + 20:3n-6
-
20:4n-6
+
22:4n-6
+.22511-6
The pathways for LA metabolism discovered by Klenk and Mohrhauer (4) are: 18:3n-3 + 18:4n-6 + 20:4n-3 + 20511-3
--+
22511-3
+
22:6n-3
It is important to note that although LA and ALA are further metabolized by desaturation and elongation, they cannot be interconverted (5). The major site of desaturation and elongation is the liver. The desaturase enzymes necessary for synthesis of arachidonic acid (AA; 20:4n-6) and DHA (22:6n-3) are present in animal, but not plant cells. Synthesis of DHA involves two successive elongations of 20511-3
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18:3
4
a-linolenic A6 dessturase
18:3n-6 Series-1Prostaglandins: TXA, PGE, PGFI,, PGDi
-
18:4n-3
y-linolenic
1
stearidonic
eloqase
20:4n-3
20:3n-6
Dihomo-y-linolenic
S e h . 2 Prostaglandins:
TXAz, PGEi, PGFz,, PGDz, PGHz, PGLi
eicosatetraenoic
y 1
-
1 1
AS desaturase
20:4n-6
1
20:5n-3
arachidonic
eicrtaenoic
Series4 Leukothnes r
Seties-3 Prostaglandins: PGE,, PGHr, PGlr, TXPU
22:4n-6
22511-3
adrenic
m3docosaDentaenolc
1
ua Seriesd Leukothnes
f
elorgase
24:4n-6
24511-3
d-tetracosatetraenoic
Eicosanoids
03-tetracosapentaenoic
f
A6 de urase
24:5n-6
24:6n-3
o6-tenacosapentaenoic
1 1
w3-tetracosahexaenoic
p-oxidation
22:5n-6
I
22:6n-3
w6-docosapentaenoic p-oxidation
docosahexaenoic
1
energy metabolic pathway
Fig. 11.1. Metabolic pathways for n-6 and n-3 fatty acids. Source: References 21, 23.
to form 24:5n-3, which is then desaturated at position 6 to form tetracosahexaenoic acid (24:6n-3) and translocated to the peroxisomes where one cycle of P-oxidation leads to the formation of 22:6n-3 (DHA) (6-8). The limited data available from stable isotope studies indicate that ~ 8 % of ALA is converted to EPA in adults, whereas conversion to DHA is marginal (<0.02-4%) (9-14). There is evidence that dietary EPA andor DHA can decrease the conversion of labeled ALA to both EPA and DHA (12,15,16). This suggests
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that EPA + DHA availability can inhibit conversion of ALA to the longer-chain n-3 fatty acids by feedback inhibition. Moreover, Burdge et al. (15) showed using stable isotopes that increasing dietary ALA does not increase (upregulate) conversion of ALA to the longer-chain n-3 fatty acids. Collectively, these findings indicate that the pathways responsible for converting ALA to longer-chain n-3 fatty acids and n-3 eicosanoids are complex and subject to regulation at multiple points, and that EPA and/or DHA plays a key role in modulating flow of n-3 fatty acids through these pathways. The ability of humans to convert the essential fatty acids to long-chain PUFA at different stages of the lifespan and in different disease states is important because many of the essential roles of n-6 and n-3 fatty acids are fulfilled by AA, EPA, and DHA, rather than their precursors 18:2n-6 and 18:3n-3, respectively. Desaturation of 18:2n-6 and 18:3n-3 is believed to involve the same enzymes. In vitro, the A6 desaturase enzyme shows clear preference in the order 18:3n-3 > 18:2n-6 > 18:ln-9 (5). In the absence of a dietary supply of n-6 and n-3 fatty acids, oleic acid (18:ln-9), derived from the diet or synthesized de novo from acetyl CoA, undergoes A6 and A5 desaturation and elongation to form eicosaetrienoic acid (20:3n-9). Concentrations of 20:4n-6 decrease due to the absence of 18:2n-6 (5,17). Mohrhauer and Holman (18) first reported that increasing the level of dietary ALA with LA held constant suppressed the level of n-6 products. Similarly, when ALA was held constant, increasing quantities of LA suppressed the production of subsequent n-3 products. This led to the recognition that n-3 and n-6 PUFA compete at certain enzymatic sites in these two pathways, particularly those involved in desaturation and elongation, and thereby inversely affect each other’s tissue levels [reviewed in (19)]. PUFA are important structural membrane components that confer membrane fluidity and selective permeability. Moreover, PUFA serve as precursors for eicosanoids, growth regulators and hormones, and are constituents of membrane phospholipids involved in signal transduction [reviewed in (20,2 l)]. They have important biological effects on cognitive function and immune function. One of the more important functions of PUFA (both n-3 and n-6 series) relates to their enzymatic conversion to eicosanoids, which are a class of short-lived hormones synthesized from 20-carbon fatty acids [reviewed in (22)]. Eicosanoids are biologically potent and have a broad array of functions: they play a key role in modulating inflammation and immune responses and are critically involved in platelet aggregation, cell growth, and cell differentiation. The precursor 20-carbon fatty acids for the formation of eicosanoids are dihomo-y-linolenic acid (DGLA; 20:3n-6), AA, and EPA. These PUFA serve as substrates for cyclooxygenases (COX), lipoxygenases (LOX), or cytochrome P450monoxygenases. COX give rise to prostaglandins and thromboxanes, whereas LOX catalyze the synthesis of leukotrienes, hydroxyl fatty acids, and lipoxins [reviewed in (22)]. The n-6 and n-3 20-carbon fatty acids share the same enzymes (LOX and COX) for the synthesis of prostaglandins and leukotrienes. The type and amount of these different PUFA in
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cell membranes play an important role in regulating which eicosanoids are produced. Because the major PUFA in cell membranes is AA, most eicosanoids produced are the 2-series prostanoids and the 4-series leukotrienes (see Fig. 11.1). EPA is the substrate for the 3-series prostanoids and the 5-series leukotrienes. The 2-series prostanoids and 4-series leukotrienes are involved in biological actions such as platelet aggregation, vasoconstriction, and the production of inflammatory mediators in response to physiologic stressors. The EPA-derived prostanoids and leukotrienes are less potent than those derived from AA, and act to attenuate excessive 2-series prostanoids. Thus, adequate production of the 3-series prostanoids appears to be important for protection against heart attack, stroke, and various inflammatory diseases (23). In general, AA-derived eicosanoids have proinflammatory effects, whereas EPA-derived eicosanoids have anti-inflammatory effects. The latter occurs in part because EPA (as the n-3 homologue of AA) can decrease production of AAderived eicosanoid inflammatory mediators by competition for COX and LOX enzymes and consequently suppress synthesis of products produced by these enzymes (24). In addition, n-3 fatty acids inhibit eicosanoids synthesis and signaling and nuclear factor (NF)KB activation, which accounts for the anti-inflammatory and antithrombotic effects observed (20). Collectively, increasing dietary n-3 fatty acids can shift the balance of eicosanoids to a less inflammatory mixture (25). Limitations of the n-6:n-3 Ratio
The early recognition that LA and ALA competitively inhibited the metabolism of the counterpart n-6 or n-3 fatty acid metabolic pathway led to the belief that the ratio of these fatty acids regulated the quantity of metabolites produced in these pathways downstream from LA and ALA. Evolution of the LA:ALA concept led to a very simplistic translation that frequently is referred to as the n-6:n-3 ratio. Appreciating that the n-3 fatty acid family is comprised of different fatty acids that have varying metabolic effects was an important indicator that the ratio, per se, provides very little information about which fatty acids are included in the ratio. This is also the case for the n-6 fatty acids. Furthermore the ratio does not provide information about the mass of constituent fatty acids in each of the PUFA classes. Because competitive inhibition by LA and ALA is based on reaction kinetics, changes in the mass of ALA and LA (and not n-3 or n-6 fatty acids collectively) are key in determining the flux of fatty acid metabolites through the n-6 and n-3 pathways. The n-6:n-3 ratio (as a composite of all n-6 and all n-3 fatty acids) masks the quantity of individual n-6 or n-3 fatty acids presented to cells for metabolism. For example, data presented in Table 11.1 (upper panel) demonstrate that the same n-6:n-3 ratio is associated with very different quantities of LA and ALA. At a ratio of 5 : 1, a 1600 kcal diet provides 8.9 g of LA and 1.8 g of ALA, whereas a 3200 kcal diet with the same ratio provides twice the amount of each fatty acid. Similarly, the
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TABLE 11.I
Relation Between n-6:n-3 Ratio and Fatty Acid Mass at Two Levels of Energya Energy intake (kcal) 1600
YO Fatty acid LA
n-6:n-3
5
3200
%en
(g)
%en
(g)
5
8.9
5
17.8
1
1.8
1
3.6
5
8.9 1.8 1 .o 2 .a
5 1 0.3 1.3
5:l; 5 : l ALA
1
LA ALA EPA + DHA
5 1 1g
3.3:l; 3.8:l
1
0.5 1.5
X n-3
17.8 3.6 1 .o
4.6
aAbbreviations: LA, linoleic acid; ALA, a-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
same ratio at the same energy intake can result in dramatic differences in the quantity of fatty acids consumed (see Table 11.2). For example, given an n-6:n-3 ratio of 20:l (based on LA and ALA intake) there is a twofold greater quantity of LA and ALA in a diet that provides 10% of energy from LA and 0.5% of energy from ALA vs. a diet that provides 5% of energy from LA and 0.25% of energy from ALA (Table 11-2). Thus, it is evident that multiple strategies can be implemented to achieve the same ratio of LA and ALA, yet the quantity of these fatty acids in the diet is vastly different. Of note, also, is that adding other fatty acids in the respective fatty acid classes can alter the ratio. One example is presented in the bottom panel of Table 11.1. The relative contribution of a fairly sizeable intake (1 g) of the n-3 fatty acids, EPA + DHA, at 1600 kcal changes the ratio from 5: 1 to 3.3: 1 and at 3200 kcal, the ratio changes from 5:1 to 3.8: 1. The variables that can affect the n-6:n-3 ratio are presented in Table 11.3. As shown, the greatest effect is associated with changes in both LA and ALA. Specifically, increasing LA and decreasing TABLE 11.2
Relation Between n-6:n-3 Ratio and Fatty Acid Mass at the Same Level of Energya Energy intake (kcal) 2000 n-6:n-3 LA
2000
%en
(g)
%en
(g)
5
11.0
10
22.2
0.25 4
0.6 8.9
0.5 8
1.2 17.8
1
2.2
2
4,4
20:l ALA LA 4:l ALA 5 e e Table 11.1 for abbreviations.
227
Dietary n-6:n-3 Fatty A c i d Ratio
TABLE 11.3
Multiple Strategies for Changing the n-6:n-3 Ratioa Strategy
Effect on ratiob ~~
~
Increase the ratio Increase linoleic acid Increase arachidonic acid Increase both linoleic acid and arachidonic acid Decrease a-linolenic acid Decrease EPA + DHA Decrease a-linolenic acid and EPA + D H A Increase linoleic acid and decrease a-linolenic acid
++
+ +++ + + + ++++
Decrease the ratio Decrease linoleic acid Decrease arachidonic acid Decrease both linoleic acid and arachidonic acid Increase a-linolenic acid Increase EPA + D H A Increase a-linolenic acid and EPA
+ DHA
Decrease linoleic acid and increase a-linolenic acid aSee Table 11.1 for abbreviations. bMagnitudeof effect: + = least effect;
++ + +++ + + + ++++
+ + + + = greatest effect on ratio.
ALA increase the ratio the most, whereas decreasing LA and increasing ALA reduce it the most. In addition to changing only the n-3 fatty acid, ALA, a sizeable change in EPA + DHA (either with or without changing ALA) in conjunction with alterations in LA also can modify the ratio appreciably. Given the many health benefits associated with n-3 fatty acids, many believe that decreasing the n-6:n-3 ratio is best done by increasing n-3 fatty acid levels (26). In fact, decreasing the ratio without deliberating increasing n-3 fatty acids is not considered acceptable. Based on the previous discussion, it is apparent that the ratio of n-6:n-3 fatty acids alone does not provide sufficient evidence to understand the biological consequences of diets that vary in PUFA content. As will be discussed, it is important to know absolute amounts of all dietary n-6 and n-3 fatty acids for sufficient evaluation of study outcomes that involve these fatty acids. Information about the LA:ALA ratio would provide additional insight. Also, information about the content of EPA + DHA would provide further valuable information. The overarching goal is to collect as much information as possible about the amount of all constituent n-6 and n-3 PUFA so that the ratio can be calculated and put in an appropriate context. This is consonant with a recent extensive and systematic review of the literature on n-3 fatty acids and disease endpoints published by the Agency for Healthcare Research and Quality (23), which recommended that it is still advisable
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to estimate and report the total dietary n-6:n-3 fatty acid ratio in diet studies that focus on the biology of dietary n-3 fatty acids. As discussed herein, this should be done with an appreciation of the limitations inherent in that ratio. Further Insights About the n-6:n-3 Ratio
There is an extensive literature on the n-6:n-3 ratio (as well as the n-3:n-6 ratio) and how this relates to many different disease states and risk of these diseases, including CVD, cancer, diabetes, bone health, mental status and cognitive function, arthritis, asthma, immune function, and inflammatory status (26). The basis for the interest in the n-6:n-3 ratio is multifaceted and linked to how our diet has changed over time, the interrelated metabolism of these fatty acid classes, and how changes in the quantity of each class (and specific fatty acids within a class) contribute to health outcomes. With respect to the contemporary diet, the n-6:n-3 fatty acid ratio has changed significantly as humans evolved from consuming a diet that consisted primarily of fruits, berries, nuts, lean meat, fish, and green leafy vegetables, to a typical Western diet characterized by foods high in refined carbohydrates, saturated and trans fatty acids, and vegetable oils high in n-6 fatty acids that contain little n-3 fatty acids (27). Because of these changes, the present Western diet has an n-6:n-3 ratio of -1O:l to 20-25:l compared with the diet our early ancestors likely consumed, which was estimated to provide a ratio of 1:l. As reviewed by Simopoulous (27,28), a growing number of scientists believe the present n-6:n-3 ratio is involved in the development of many chronic diseases. Dietary n-6 and n-3 fatty acids have both beneficial and potentially adverse effects on health outcomes. Consequently, a better understanding is needed about the optimal quantity of each fatty acid class and the constituent fatty acids that comprise each class. There are differences in opinion about the usefulness of the n-6:n-3 ratio. Nonetheless, there is general consensus that n-3 fatty acids have many health benefits and, therefore, should be increased beyond levels currently consumed. This alone would decrease the n-6:n-3 ratio. Other scientists believe that LA should be markedly decreased because of some adverse health effects. This, too, would decrease the n-6:n-3 ratio. However, some ascribe to the idea that both n-6 and n-3 are beneficial, and at a minimum n-6 should be maintained and n-3 be increased (29). To provide the appropriate context for future dietary recommendations independent of the n-6:n-3 ratio, it is important that the health effects of n-6 and n-3 fatty acids, both beneficial and adverse, be reviewed. The Effects of n-6 and n-3 Fatty Acids on Health
Beneficial Effects of n-6 Fatty Acids. Both within- and cross-population studies have reported either a beneficial or no association between dietary PUFA and CVD morbidity and mortality. Two population studies (30,31) reported an inverse association between PUFA intake and CVD mortality after adjusting for dietary saturated fat. Moreover, a number of prospective studies (32-36), two longtudinal studies (37,38),
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229
and one cross-sectional study (39) reported a beneficial association between dietary PUFA intake and CVD morbidity and mortality. The Seven Countries Study, however, reported no significant association between dietary PUFA and CVD (40,41). Consistent with this, other epidemiologic studies also have not found any association between PUFA intake and CVD (42,43). In a study with a Jewish population in Israel that consumed a diet high (10% of energy) in n-6 PUFA (44), high intakes of LA were not associated with increased risk of acute myocardial infarction (MI). However, adipose tissue arachidonic acid levels were positively associated with acute MI. Several clinical trials published between 1968 and 1983 reported that high levels of dietary PUFA (ranging from 13 to 21% of energy) decreased total cholesterol (TC) levels -13-15% and coronary event rates by -2540% over a period of 4-8 yr of follow-up (4549). In a comparable study (50) that reported a 14.5% reduction in TC levels, CVD events and/or deaths did not differ between the treatment and control groups, likely because of the short study duration (I yr). Numerous clinical studies demonstrated the cholesterol-lowering effects of PUFA. The results of these studies were used to develop blood cholesterol predictive equations that demonstrate cholesterol-lowering effects (both TC and LDL cholesterol) of PUFA. These equations predict that a 1% increase in PUFA will decrease TC by 0.024 mmol/L. The cholesterol-lowering response is approximately half the cholesterol-raising effect of saturated fatty acid (SFA) (5132). More recent predictive equations that evaluated the effect of specific fatty acids on blood cholesterol levels indicate that LA is the most potent TC and LDL cholesterol-lowering fatty acid. Beneficial Effects of n-3 Fatty Acids. Three epidemiologic studies conducted among U S . populations found that an ALA intake of 0.53-2.8 g/d was associated with a reduced risk of CVD events (39), fatal ischemic heart disease (53), and allcause mortality (54). In addition, the Health Professionals Follow-Up Study reported that a 1%increase in ALA intake was associated with a 40% lower risk of MI ( P c 0.01) after adjustment for total fat intake (32). Two secondary prevention clinical trials (55,56) reported a beneficial effect of ALA on CVD in post-MI patients. In these studies an ALA intake of 2.0 and 2.9 g/d, -0.8 and 1.2-1.3% of energy, respectively, reduced the risk of recurrent coronary events. In a recent prospective study, Albert et al. (56a) reported that increasing ALA (1.5 g/d) decreased risk of sudden cardiac death by 46% and fatal coronary heart disease (CHD) by 2 1%. There is convincing epidemiologic evidence from U.S. studies of a cardioprotective effect of fish consumption (54,57-60). The range of EPA and DHA intake in the studies that conferred the lowest risk was 246-919 mg/d. In the Zutphen Study, an increase in fish consumption from 0 to 45 g/d was associated with a progressive decrease in the risk of CHD after 20 yr ( P < 0.05) (61). Two recent metaanalyses reported that consuming fish 2 5 times/wk was associated with lower CHD mortality (62) and a lower incidence of stroke (63). Three secondary prevention clinical trials demonstrated beneficial effects of EPA and DHA and fish consumption on recurrent coronary events. In the DART
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study (64), male MI survivors who received fish oil capsules containing 900 mg/d of EPA and DHA or 200-400 g/wk of fatty fish, which provided an additional 500-800 mg/d of n-3 fatty acids, had a 29% reduction in all-cause mortality over a two-year period. The greatest benefit was seen for fatal MI. In the Gruppo Italian0 per lo Studio della Sopravvivenza nell’Infarto Miocardico (GISSI) Prevention Study (65), subjects with preexisting CHD randomly assigned to the EPA + DHA supplement group (850 mg/d of n-3 fatty acid ethyl esters) with and without 300 mgjd vitamin E experienced a 15% reduction in the primary endpoint of death, nonfatal MI, and nonfatal stroke (P < 0.02). In addition, all-cause mortality was reduced by 20% (P = 0.01) and sudden death was reduced by 45% (P < 0.001) compared with the control group (vitamin E provided no benefit). Other evidence demonstrating the efficacy of EPA and DHA was reported in the Indian Experiment of Infarct Survival study (56). MI survivors who were treated with either fish oil capsules (1.8 g/d EPA + DHA) or mustard oil (2.9 g/d ALA) for one year had fewer total cardiac events, nonfatal infarctions, arrhythmias, and less left ventricular enlargement and angina pectoris than the control group. A decrease in cardiac deaths was observed only in the group treated with fish oil (56). The cardioprotective effects of EPA and DHA may be due to multiple mechanisms, including antiarrhythmic effects, decreased platelet aggregation, and decreased triglyceride (TG) levels (66,67). Dietary EPA and DHA decrease the production of prostaglandin E, (PGE,) metabolites, thromboxane A,, a vasoconstrictor and platelet aggregator, and leukotriene B,, an inflammatory and adherence agent. In addition, there is an increase in the production of thromboxane A,, a weak vasoconstrictor and platelet aggregator, prostacyclin PGI,, and leukotriene B,, a weak inducer of inflammation (28). EPA and DHA were shown to reduce arrhythmias and ventricular fibrillation, as well as inflammatory markers including C-reactive protein, interleukin-6, and serum amyloid A (68-70). Adverse Effects of n-6 Fatty Acids. Because LA is converted to AA, increased levels of LA will result in elevated concentrations of AA and ultimately increased production of proinflammatory and prothrombotic factors, which contribute to increased blood viscosity, vasoconstriction, decreased bleeding time, and plaque development (71). In addition, LA increases the susceptibility of LDL to oxidative modification, which is problematic because oxidized LDL are atherogenic (72-74). A high intake of n-6 PUFA may result in hyperinsulinemia and subsequent insulin resistance (75). Dubnov and Berry (26) suggested that an increase in insulin levels activates phospholipase A,, resulting in the release of more PUFA from cell membranes and a resultant increase in eicosanoid synthesis. Adverse Effects of n-3 Fatty Acids. There is some epidemiologic evidence of a positive association between diet and blood levels of ALA and increased risk of prostate cancedadvanced prostate cancer (76,77), In a meta-analysis of nine observational studies (four prospective studies and five nonprospective studies), the risk
Dietary n-6:n-3 Fatty Acid Ratio
23 1
estimate was 1.70 [95% confidence interval (CI), 1.12-2.581 (76). Leitzmann et al. (77) reported that the multivariate relative risk of advanced prostate cancer from comparisons of extreme quintiles of ALA intake from non-animal sources was 2.02 (95% CI, 1.35,3.03). In the latter study, there was no association of ALA from meat and dairy sources with prostate cancer. Some studies showed that very high intakes (via supplementation) of n-3 fatty acids, especially EPA and DHA, at levels 7-15 times higher than typical U.S. intakes diminish the potential of the immune system to attack pathogens (78-80). However, this immune response also was associated with the suppression of inflammatory responses (81). Very high intakes of EPA and DHA (average of 6.5 g/d) were linked to excessive cutaneous bleeding time (82). Diets high in n-3 fatty acids may cause prolonged bleeding due to the reduction in platelet aggregation. However, moderate n3 fatty acid supplementation, between 2 and 5 g/d, was not found to increase bleeding tendency (28). Similar to n-6 fatty acids, n-3 fatty acids are also susceptible to lipid oxidation, leading to oxidative damage. Adequate intake of antioxidants may counteract the oxidative effects of n-3 fatty acid supplementation (83,84). The Role of AA Effects of LA on Tissue AA. In discussions about the n-6:n-3 or LA:ALA ratio, AA is a focal point because it is the 20-carbon n-6 precursor to inflammatory eicosanoids. Consequently, an obvious question is the following: what is the relation between tissue levels of AA and disease? Cleland and colleagues conducted a series of informative studies to address this question. In one, they fed healthy subjects LA intakes of between 2.5 and 17.5% (85), and in another, 1.8 and 12.8% of dietary energy (86). There was no correlation between LA intake and tissue AA in either study. Although altering dietary LA intake does not modify tissue n-6 PUFA content, relatively small increases in n-3 PUFA intake can. For example, providing 1, 2, or 3 g/d (0.5, 1, or 1.4% of energy) of EPA and DHA (from fish oil) in a typical Western diet containing 6 5 % LA decreased red blood cell (RBC) phospholipid n-6 PUFA (expressed as a percentage of total PUFA) from 62% at baseline to 46, 38, and 32%, respectively (87). Thus, a relatively small increase (in terms of mass) in n-3 PUFA is more efficient in lowering n-6 PUFA tissue proportions than markedly decreasing LA intake. Tissue AA Levels and the Risk of CHD. If elevated tissue AA content contributes to an increased risk for CHD events, it stands to reason that epidemiologic studies would demonstrate an association between tissue AA and risk. This is not what the literature indicates. Miettinen et al. (88) followed a cohort of men free of CHD for five year and correlated risk for developing MI as a function of the plasma phospholipid fatty acid composition. In this study, men who later developed MI had lower AA levels. Their n-3 levels were higher, consistent with a cardioprotective effect. The AA/EPA ratio was 3.2 in the infarct group and 3.6 in the healthy controls. A study from Finland also supported this finding (89). An increased
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amount of cholesteryl ester AA predicted reduced risk of death from heart disease in subjects followed for five year. In other studies, the AA levels did not differ between those with and without CHD. A Swedish group studied the fatty acid composition of coronary arteries taken from individuals who had died from either sudden cardiac death or noncardiac causes (90). They found that AA (as well as n-3 fatty acids) levels were lower in the sudden death cases. The AA/EPA ratio was 48 in the controls and 32 in those with sudden cardiac death. In 1987, Wood et al. (91) compared platelet fatty acid composition between patients with acute MI, angina pectoris, and controls. In that study, the patient group did not have a high AA level but did have a low level of n3 fatty acids. In contrast, the healthy controls had higher AA levels in platelet membranes. In the case-controls study nested in the Physicians’ Health Study, blood fatty acid levels were measured and correlated with future risk for sudden cardiac death (SCD) (92). Although EPA and DHA levels were lower in the SCD group, the AA levels did not differ, i.e., they were not associated with future risk. Measurement of EPA, DHA, and AA in the nonesterified fatty acid (NEFA) fraction (the fraction that may be a critical determinant of risk for MI or thrombosis) shows the same pattern. Comparing 103 patients with MI to 104 age-, sex-, and body weight-matched controls, the EPA in the NEFA fraction in the latter group was twice as high, DHA was 55% higher, but AA was virtually identical (93). Similar results were reported in five other studies examining the fatty acid composition of RBC membranes (94-97), plasma (96,98), or platelets (95). Thus, tissue AA levels are not associated with risk for CHD, whereas EPA (and DHA) levels are. Although the ratio may be higher in cases than controls, it is only because the denominator has fallen with no change in the numerator. Consequently, with respect to the ratio, and specifically AA:EPA, inclusion of AA in the ratio serves only to diminish and obscure the clear relation between n-3 fatty acids and risk; it does not add, but subtracts, discriminatory power. Effects of the n-6:n-3 Ratio
There is a rapidly growing literature about the relationships between the ratio of n-6 to n-3 fatty acids and many diseases and conditions including CVD (both coronary heart disease and stroke), cancer, diabetes, bone health, conditions associated with mental health (Alzheimer’s disease and cognitive function, depression, aggressive behavior), arthritis, and asthma. The basis for this relation is often linked to changes in the diet over the years from one that was high in n-3 fatty acids and low in n-6 fatty acids to a contemporary diet in which these proportions have been inverted. The increase in n-6:n-3 ratio frequently has been associated with an increase in these diseases and conditions. Although there is some biological basis for the “ratio change” and increased prevalence of many diseases, there are other factors that could contribute to the initiation and progression of the many diseaseskonditions that currently confront virtually all populations.
Dietary n-6:n-3 Fatty Acid Ratio
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CVD. With respect to CVD, there is epidemiologic evidence that a higher intake of ALA is protective against fatal ischemic heart disease (53). Importantly, a higher intake of either LA or ALA was inversely related to coronary artery disease in the NHLBI Family Heart Study (39). In addition, a higher habitual intake of both n-6 and n-3 fatty acids is associated with the lowest levels of inflammation (99). Collectively, it is not unreasonable to conclude that health benefits are conferred when both n-6 and n-3 fatty acids are increased in the diet. However, there is evidence that increasing LA can induce an inflammatory environment (100). The lack of an adverse effect of LA in the epidemiologic studies suggests that ALA may blunt the inflammatory effects of LA. Support for the cardioprotective benefit of a low n-6:n-3 ratio was put forth on the basis of the results of the Lyon Diet Heart Study (101) and the Indo-Mediterranean Diet Heart Study (102), two secondary prevention studies that implemented a high ALA diet in coronary patients. The n-6:n-3 ratios of the treatment diets were 4:l and 9:1, respectively. However, the treatment diets also were lower in saturated fat and higher in fruits and vegetables, making it difficult to frame the ratio alone as being the important factor accounting for the significant treatment effects observed in these studies. In the studies conducted in the 1960s and 1970s with very high intakes of LA (45-49), there were very significant decreases in TC levels (see above). Soybean oil was used as a source of PUFA. Of note is that soybean oil also is a source of ALA: hence, the effects observed could have reflected the increase of ALA in these studies, irrespective of the n-6:n-3 ratio (which was not reported in these studies-although it had to be very high). Clearly, the fish and fish oil studies indicate that inclusion of EPA and DHA, even in small amounts, has a marked cardioprotective effect. In a recent study, Mozaffarian et d.(102a) reported that both plant and marine derived n-3 fatty acids reduced CHD risk when LA intake is either high (711.2 g/d) or low (<11.2 g/d) suggesting that LA does not blunt the beneficial effects of n-3 fatty acids. Cancer. Recent epidemiologic and experimental data indicated that high intakes of n-6 PUFA accompanied by low intakes of n-3 PUFA were linked to increased risks for breast, colorectal, and prostate cancers. The Lyon Diet Heart Study (102) compared newly diagnosed cancer rate and overall survival among patients following either a Mediterranean-type diet or controls following a Step 1 diet [30% energy as total fat, 10% SFA, 10% monounsaturated fatty acid (MUFA), 10% PUFA, dietary cholesterol <300 mg/d]. The Mediterranean diet was high in fruits and vegetables, breads, cereals, legumes, and fish: intake of meat, butter, and cream was decreased. A specially formulated canola oil-based margarine rich in oleic acid and ALA was provided. After adjustment for variables such as age, sex, smoking, leukocyte count, and cholesterol, individuals following the Mediterranean diet vs. the control diet experienced a 56% ( P = 0.03) reduction in risk for total deaths and 61% ( P = 0.05) reduction in cancer risk. Although the reduction in cancer risk and total deaths cannot be attributed solely to n-3 or n-6 fatty acids, this study suggests that individuals
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consuming a Mediterranean diet, which is characterized by a low ratio of n-6:n-3 fatty acids, have a reduced cancer risk. Although the exact mechanisms by which fatty acids affect the development of cancer are unclear, it may be due to multiple effects including the following: alteration of cell signaling leading to changes in gene expression; induction of DNA damage due to peroxidation of conjugated double bonds in PUFA; alteration in eicosanoid production; and structural and functional changes in cell membrane composition and membrane-bound enzymes leading to changes in hormone synthesis (103). The European Community Multicenter Study on Antioxidants, Myocardial Infarction and Cancer of the breast (EURAMIC) was a case-control study conducted in five European countries that examined whether n-3 fatty acid levels were inversely associated with breast cancer risk when also considering n-6 fatty acid levels. There was no consistent association with either n-6 or n-3 fatty acid levels; however, the ratio of total n-3:n-6 PUFA, as well as the ratio of long-chain n-3 fatty acids to total n-6 fatty acids, was inversely correlated with breast cancer risk in four of the five countries. The strongest inverse association was observed with the ratio of long-chain n-3 fatty acids to n-6 fatty acids, for which a dose-response was reported (104). Colorectal cancer mortality also was correlated with the n-6:n-3 fatty acid ratio (105-108). An epidemiologic study involving 24 European countries found an inverse correlation in men between fish intake and colorectal cancer mortality. Analysis of the n-6:n-3 fatty acid ratio revealed there was a significant inverse correlation with consumption of fish and fish oil and colorectal mortality in men and women with high fish intakes relative to intake of n-6 fatty acids. This correlation was also significant for breast cancer mortality in women (109). Two human studies conducted by Bartram et al. (1 10,111) investigated the amount of fish oil supplementation necessary to suppress rectal epithelial cell proliferation and PGE, biosynthesis. Cell proliferation was suppressed with a n-6:n-3 ratio of 2.5, but this effect was not seen with the same absolute level of fish oil intake (4.4 g/d n-3 fatty acids) and an n-6:n-3 fatty acid ratio of 4 (1 10,111). These two studies demonstrate the importance of considering both n-6 and n-3 fatty acid levels in the development of colorectal cancer. In a prospective cohort study in Norway conducted by Harvei et al. (112), there was no significant association between total n-6 and n-3 fatty acids and prostate cancer risk. However there was an increased risk of prostate cancer with increasing ALA ( P = 0.03). In addition, the n-6:n-3 fatty acid and AA:EPA ratios were negatively correlated with prostate cancer risk. In contrast, a cohort study in the Netherlands reported a negative correlation with ALA intake and prostate cancer risk (1 13). In that study, no associations for AA, EPA, and DHA existed. Two studies specifically evaluating the effect of the n-6:n-3 fatty acid ratio on the risk of prostate cancer found higher n-6:n-3 fatty acid ratios in cancer patients vs. control subjects as well as higher ratios in prostate tissue from prostate cancer patients compared with tissue from patients with benign hyperplasia (1 14,115).
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Collectively, on the basis of the research to date, some scientists believe that a ratio of -1:l-2:l can provide a protective effect against the development and growth of breast and colon cancers (27). Due to conflicting results, further research is warranted to evaluate the effects of PUFA on prostate cancer and determine whether the n-6:n-3 ratio is involved in determining cancer risk. Non-Insulin Dependent Diabetes Mellitus. In NIDDM, a loss of first-phase insulin release in response to glucose occurs followed by sustained insulin secretion during the second phase (1 16). PGE,, a metabolite of AA, is an inhibitor of first-phase insulin release in response to glucose, whereas a product of an AA lipoxygenase causes sustained increased second-phase insulin release (1 17). Evidence suggests that the prevalence of NIDDM may be correlated with the n-6:n-3 fatty acid ratio (117). In that study, there was a significant increase in the prevalence of NIDDM and coronary disease in urban and upper socioeconomic classes, which was associated with a change in the predominant sources of fat used in Indian cooking. In the past, the three traditional fats comprising the typical Indian diet were ghee and coconut oil, both sources of SFA, and rapeseed mustard oil, a MUFA with high n-3 fatty acid content (117). Each of these fats contains a low amount of n-6 PUFA with a very low n-6:n-3 ratio. Studies evaluating the association of n-3 fatty acids and the incidence of diabetes have been inconclusive. Fish oil intake was associated with a low incidence of diabetes (118). In a 20-yr follow-up of the Finnish and Dutch cohorts of the Seven Countries Study, Feskens et al. (119) reported that high fat intake, specifically high intake of saturated fat, was positively associated with the development of glucose intolerance and NIDDM; however, high fish intake was protective. Another study demonstrated that hyperinsulinemia and insulin resistance were inversely associated with the amount of 20- and 22-carbon fatty acids in muscle cell membrane phospholipids (120). In a clinical trial conducted by Conner et al. (121), 6 g/d of n-3 fatty acids consisting of EPA and DHA, administered for 6 mon in addition to medication for individuals with diabetes, resulted in a nonsignificant increase in fasting serum glucose concentrations during the n-3 fatty acid phase vs. the control phase. There was also no significant change in glycated hemoglobin concentrations; however, TG levels decreased 43%. Bone Health. PGE, is the predominant bone cell-derived prostaglandin and is regarded as a potent regulator of bone modeling and remodeling (122). An increase in AA could lead to an overproduction of PGEz in bone, resulting in reduced bone formation. Dietary n-3 PUFA were reported to lower the concentration of AA in bone (123) and cartilage (124), and to have anti-inflammatory effects (125). Decreasing the n-6:n-3 fatty acid ratio in the diet was shown to increase bone marrow cellularity (126), bone strength (127), and bone growth (128) in animals, and reduce bone PGE, production (129). Tumor necrosis factor (TNF)-a-induced osteoclast recruitment is believed to play a key role in the pathogenesis of postmenopausal
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osteoporosis (130,13 1). Studies showed that TNF-a promotes osteoclastic bone resorption and inhibits bone collagen synthesis in vitro (132). Moreover, AA enhances and EPA attenuates TNF-a expression in human osteoblast cells (133). Mental Health. There is evidence that individuals with depressive disorders have a higher ratio of n:6 to n:3 fatty acids in plasma phospholipids (134). The authors suggested that fatty acid composition of plasma phospholipids affects mood. In addition, another study showed that higher proportions of both stearic acid and n-6 PUFA in RBC membranes were associated with a greater risk of cognitive decline in 246 men and women ages 63-74 yr (135). Moreover, in that study, a higher proportion of n-3 fatty acids was associated with a lower risk of cognitive decline. There is evidence that the n-6:n-3 ratio may affect neuronal membranes, and a decrease in the ratio may improve the membrane fluidity index, which is a key factor in neuronal activity (136). As a result, an increase in membrane fluidity may affect learning and memory, cognition, Alzheimer’s disease, seizures, among other neurodegenerative diseases. Arthritis and Asthma. The fact that n-3 fatty acids act to decrease inflammation by reducing inflammatory cytokines has been the basis for studies to evaluate the effects of n-3 fatty acid supplementation on inflammatory diseases such as arthritis (137). There is evidence that n-3 fatty acids may reduce risk from certain types of arthritis and help provide relief when symptoms occur. There is some evidence that n-3 fatty acids may modify leukotriene biosynthesis, thereby favorably affecting bronchial smooth muscle contraction. Thus, these fatty acids may be of some benefit in established allergic diseases (such as asthma and atopic dermatitis), although these effects are not strong (138). PUFA Intake. PUFA contribute -7% of total energy intake and -19-22% of energy intake from fat (139). The predominant long-chain dietary polyunsaturated n-6 fatty acid is LA, comprising 84-89% of total PUFA energy, whereas ALA contributes 9-11% of the total PUFA energy. The major long-chain n-3 fatty acid in the diet is the plant-derived ALA; the marine-derived EPA (20:5n-3) and DHA (22:6n-3) represent -10% of total n-3 intake.
Dietary Recommendations for n-6 A N D n-3 Fatty Acids
The Institute of Medicine (IOM) of the National Academies recently released recommendations for macronutrients and energy (8 1). The Dietary Reference Intakes (DRI) that were made for n-6 and n-3 fatty acids are based on a nutrient requirement model, which has as its basis the strategy of preventing a fatty acid deficiency (defined by classical fatty acid deficiency symptoms). The recommendation is based on median intakes in the United States where deficiencies in n-6 fatty or n-3 fatty acid are nonexistent. The Adequate Intake (AI) for LA is 17 g/d for men (19-50 yr) and 12 g/d for women (19-50 yr). An A1 was set for ALA also. The A1 for men 19 to >
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70 yr old is 1.6 g/d and for women 19 to >70 yr old, it is 1.1 g/d. In terms of an Acceptable Macronutrient Distribution Range (AMDR), 5-10% of energy is recommended for LA and 0.6-1.2% of energy is recommended for ALA. Because the biological potencies of EPA and DHA are much greater than that of ALA, the IOM did not recommend one AMDR for the entire n-3 fatty acid class. The IOM recommended that up to 10% of the AMDR for ALA can be consumed as EPA and/or DHA (which corresponds to 0.06-0.12% of energy). Although an n-6:n-3 ratio was not specifically recommended, the recommendations for the individual n-6 and n-3 fatty acids (i.e., LA and ALA) correspond to a ratio that ranges from 4.2 to 16.7:1. Other groups have made recommendations for n-6 and n-3 fatty acid intake that translate into a lower n-6:n-3 ratio than those of the IOM. For example, the International Society for the Study of Fatty Acids and Lipids (ISSFAL) recommends an A1 for LA of 2% of energy, a healthy intake of ALA of 0.7% of energy, and a minimum intake of EPA and DHA of 500 mg/d (140). The ISSFAL recommendations translate into an n-6:n-3 ratio of -2: 1. The Japanese Society for Lipid Nutrition also recommends a ratio of 2: 1 (141). The recognition that fish, and specifically EPA and DHA, confers many health benefits has prompted numerous organizations and government agencies worldwide to make dietary recommendations for fish consumption. These are summarized as follows: American Heart Association: two servings of fish (preferably fatty) per week (142); National Cholesterol Education Program: recommends fish as a food item for people to choose more often (143; Table V.2-6); World Health Organization: regular fish consumption (1-2 servings per week; each serving should provide the equivalent of 200-500 mg of EPA + DHA) (144); European Society for Cardiology: oil fish and n-3 fatty acids have particular protective properties for primary CVD prevention (145-147); United Kingdom Scientific Advisory Committee on Nutrition: consume at least two portions of fish per week, of which one should be oily, and provide 450 mg/d of EPA + DHA (148); American Diabetes Association: 2-3 servings of fish per week provide dietary n-3 polyunsaturated fats and can be recommended (149). Collectively, the guidance about n-6 and n-3 fatty acid intake is evolving in a way that is very specific with respect to individual fatty acids. Figure 11.2 presents the spectrum of “recommendation scenarios” ranging from nonspecific to very specific guidance. Clearly, very specific recommendations guided by well-defined public policy with accompanying strategies for effective implementation will provide the necessary guidance to minimize ambiguities in the translation of the nutrient recommendations (150). This is far preferable to making nonspecific recommendations using the n6:n-3 ratio because the ratio alone ignores individual fatty acid recommendations. The recognition that n-3 fatty acids have many health benefits has prompted the development of new food products that are enriched in these fatty acids. In addition, genetic modification has been used to develop a new generation of plants (Lee,corn and soybeans) that produce seeds that have a modified fatty acid profile. Strategies are available to increase (151) or decrease (152,153) target fatty acids in the
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Specificity of Dietary Advice -specific Eat a balanced
Eat X% n-6 and Y% n-3
Eat an n-6/n-3 of X%
Eat X% n-6, Y% ALA, and Z% EPA+DHA
Very specific Eat X% n-6, Y% ALA, Z% EPA, and A% DHA
Fig. 11.2. Specificity of dietary advice for n-6 and n-3 fatty acids. Abbreviations: ALA, a-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
target plant (or animal). Table 11.4 presents the fatty acid profile, including the n-6:n-3 ratio of common liquid vegetable oils. With respect to the ratio, those with a relatively low ratio include flaxseed oil, canola oil, walnut oil, soybean oil, and even olive oil. In addition to considering the ratio of the oil, it also is important to be mindful of the amount of ALA in each respective oil. As noted previously, a low ratio could reflect small quantities of both LA and ALA such as in olive oil. It is likely this list will expand remarkably with the approval of genetically modified plants. Consequently, the evolving marketplace will help individuals meet fatty acid recommendations in a way that could not be envisioned 25 years ago. Thus, it will be far easier for individuals to meet the contemporary recommendations for n-6 and n-3 fatty acids.
Summary PUFA have an impressive array of biological effects. There has been, and currently is, great interest in gaining a better understanding of their biological effects and how these can be exploited to develop innovative strategies to improve health. Historically, the n-6:n-3 ratio has been used as a metric to provide insight about TABLE 11.4 n-6:n-3 Ratio of Common, Liquid Vegetable Oilsa Polyunsaturatedfat Oil Flaxseed Canola Walnut Soybean Olive Corn Sunflower Cottonseed Peanut Safflower
Saturated fat
Linoleic acid
a-Linolenic acid
Monounsaturated fat
10 7 10 15 15 13 12 27 19 10
17 22 54 54 9 57 71 54 33 76
55 10 11 8 1 1 1 0.2
18 61 25 23 75 29 16 19 48 14
0 0
aListed according to n-6:n-3 ratio, lowest to highest. NA, not available
n-6:n-3 ratio
0.3 2.2 4.9 6.7 9 57 71 270 NA NA
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how relative changes in one PUFA class affect the metabolism of the other PUFA class, and what might be the consequent biological effects and health outcomes. It is clear that the n-6:n-3 ratio alone provides very limited information about the individual PUFA effects and the interactive effects of the n-6 and n-3 fatty acid classes. Nonetheless, there is an “explosion” in the literature of studies assessing the effects of the ratio, and changes in the ratio, on various health outcomes, and the underlying mechanisms of action. Despite this, it must be appreciated that changes in the ratio reflect changes in the mass of the constituent fatty acids, and this likely accounts for the observed end-point response. As research brings clarity to what the previous and present scientific findings mean, we will better understand the biology of PUFA and the diseases/conditions they affect. These advances will improve our ability to make optimal dietary recommendations for PUFA to prevent and treat many diseases/conditions. References 1. Pereira, S.L., A.E. Leonard, and P. Mukerji, Recent Advances in the Study of Fatty Acid Desaturases from Animals and Lower Eukaryotes, Prostaglandins Leukot. Essent. Fatty Acids 68: 97-106 (2003). 2. Holman, R.T., The Slow Discovery of the Importance of Omega 3 Essential Fatty Acids in Human Health, J. Nutr. 128: 427S433S (1998). 3. Marcel, Y.L., K. Christiansen, and R.T. Holman, The Preferred Metabolic Pathway from Linoleic Acid to Arachidonic Acid In Vitro, Biochim. Biophys. Acta 164: 25-34 (1968). 4. Klenk, E., and H. Mohrhauer, Metabolism of Polyene Fatty Acids in the Rat, Z. Physiol. Chern. 320: 2 18-232 (1960). 5. Innis, S.M., Essential Fatty Acids in Growth and Development, Prog. Lipid Res. 30: 39-103 (1991). 6. Ferdinandusse, S., S. Dennis, P.A.W. Mooijer, Z. Zhang, J.K. Reddy, A.A. Spector, and R.J. Wanders, Identification of the Peroxisomal P-Oxidation Enzymes Involved in the Biosynthesis of Docosahexaenoic Acid, J. Lipid Res. 42: 1987-1995 (2001). 7. Sprecher, H., D.L. Luthria, B.S. Mohammed, and S.P. Baykousheva, Reevaluation of the Pathways for the Biosynthesis of Polyunsaturated Fatty Acids, J. Lipid Res. 36: 24712477 (1995). 8. Sprecher, H., Q. Chen, and F.Q. Yin, Regulation of the Biosynthesis of 22:5n-6 and 22:6n-3: A Complex Intracellular Process, Lipids 34: S153-Sl56 (1999). 9. Emken, E.A., R.O. Adolf, and R.M. Gully, Dietary Linoleic Acid Influences Desaturation and Acylation of Deuterium-Labeled Linoleic and Linolenic Acids in Young Adult Males, Biochem. Biophys. Acta 1213: 277-288 (1994). 10. Salem, N., R. Pawlosky, B. Wegher, and J. Hibbeln, In Vivo Conversion of Linoleic Acid to Arachidonic Acid in Human Adults, Prostaglandins Leukot. Essent. Fatty Acids 60: 407410 (1999). 11. Vermunt, S.H.F., R.P. Mensink, A.M.G. Simonis, and G. Hornstra, Effects of Age and Dietary n-3 Fatty Acids on the Metabolism of [13C]-a-Linolenic Acid, Lipids 34: S127 (1999). 12. Vermunt, S.H.F., R.P. Mensink, A.M.G. Simonis, and G. Homstra, Effects of Dietary a-
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Chapter 12
CLA Sources and Human Studies Marianne O’Sheaa, Margriet Van Der Zeeb, and lnge Mohedeb aLoders Croklaan, Lipid Nutrition, Channahon, IL 60410 and bLoders Croklaan, Lipid Nutrition, Worrnerveer, The Netherlands
Introduction Conjugated linoleic acid (CLA) refers to a class of positional and geometric conjugated dienoic isomers of linoleic acid; cis-9, trans- 11 (c9,tll) CLA and trans-10, cis12 (tlO412) CLA are the main isomers. CLA is naturally present in the milk and meat of ruminants due to its production by anaerobic bacteria in the rumen of these animals; it can also be produced industrially by isomerization of linoleic acid (LA) (12).CLA concentrations in ruminant-derived products range from 3 to 7 mg CLNg fat depending on source and processing of products (1,3-5). The estimated average daily intake of CLA from these dietary sources ranges from 0.19 to 1.0 g CLA (6,7) and varies for different countries (2). In Germany the average daily intake of CLA is estimated at 350 mg for women and 430 mg for men (8). Various positive health effects are ascribed to CLA consumption, including anticarcinogenic (9-1 l), antiatherogenic (12-14), and antidiabetic activity (15,16). In addition, animal and human studies suggested that CLA has favorable effects on certain aspects of immune function (17-24). CLA was also reported to decrease body fat while increasing lean body mass (LBM) (25-27). Many human intervention studies with a mixture of both (c9,tll CLA and tlO,cl2 CLA) and 4-isomer preparations from industrial sources were published recently. Furthermore two human intervention studies with single-isomer material were published (282.9). Intervention with mixed isomers in doses ranging from 0.7 to 6 g/d, for periods of 8 wk to 1 yr in various populations [healthy, overweight, obese, noninsulin-dependent diabetes mellitus (NIDDM), syndrome XI investigated clinical outcomes in relation to body composition, immune function, insulin sensitivity, lipid metabolism, and safety (18,27,28,3041). There is a difference in the outcome of these human intervention trials based on the isomer composition of the CLA used, including many other variables such as diet, exercise, and population. However in this chapter, the focus will be on the sources and composition of the material used. The purpose then will be to discuss the sources of CLA, both natural and industrial, produced from vegetable oil. The levels of CLA that may be obtained from both sources will be compared with the levels demonstrated to have beneficial effects in
the intervention studies in humans. In addition, the differences in the isomer composition in relation to biological effects will be discussed. 249
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Sources of CLA Natural Origins of CfA
CLA is an intermediate in the biohydrogenation of linoleic acid (LA); until recently, it was generally accepted that CLA in ruminants originated from the incomplete biohydrogenation of LA by rumen bacteria ( 6 ) .Complete biohydrogenation of LA in the rumen is a three-step process, leading to the production of C,,:, (42) (Fig. 12.1). CLA is formed as the first intermediate of this pathway by the action of LA isomerase, an enzyme of the anaerobic rumen bacteria Butyrivibrio fibrisolvens (43); it is unusual in the respect that the reaction occurs in the middle of a hydrocarbon chain away from any activating functional groups. In addition, it has an absolute substrate requirement for a c9,c12 diene structure and a free carboxyl group. The enzyme was shown to exhibit maximum activity with the substrates LA and C,,:, (44). Lipid biohydrogenation in the rumen is affected by the type and amount of fatty acid substrate (45), the forage to grain ratio (46), and the nitrogen content of the diet fed to ruminants (47). In accepting this explanation for the presence of CLA in ruminant lipids, it was assumed that the amount of CLA escaping rumen biohydrogenation and being absorbed was adequate to account for CLA levels in milk and body fat. However, the absence of any reported measurements of the amount of CLA escape and absorption, together with in vitro studies showing rapid conversion of CLA, formed by LA isomerase, to trans vaccenic acid (tl l-C18:l)has cast considerable doubt on the rumen as the sole source of CLA in tissues and milk fat. Work by Griinari et al. (48) showed that CLA could be produced endogenously from tll-C,,:, in tissues by A9 desaturase. Figures 12.1 and 12.2 illustrate the two pathways of CLA biosynthesis, which together may account for the high CLA concentrations observed in milk fat even when cows are fed diets that are low in LA, e.g., pasture feeding or fish oil supplements. It is proposed that tll-C1,:, accumulates in the rumen and that a portion escapes further biohydrogenation (49). After absorpc18:2 c9,
c12 (Linoleic Acid)
Linoleate c12, tl 1 isomerase Butyrivibriojbriosolvens C18:~ c9,
tl 1 conjugated octadecadienoic acid
cl8:ltl 1 octadecamonoenoic acid
Cl8:O stearic acid
Fig. 12.1. Biohydrogenation of linoleic acid in the rumen.
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Rumen
Tissues v
C 9 ) c12
c9, c12
(c18:2)
(c18:2)
C9) t l l
c9, t l 1 (conjugated, Cl8:Z)
(conjugated, c18:2)
1 b
tll
f tll
(cI8:l)
(Cl8.1)
c1s:o
c18:O
b
___,
cl8:l
c9
Fig. 12.2. Pathways of conjugated linoleic acid biosynthesis.
tion from the digestive tract, tl l-C18:lis utilized by different tissues in which a portion is desaturated to CLA and incorporated into tissue and milk lipids. This “desaturase hypothesis” was proposed to explain the relatively constant ratio of t l l-Cls:l and CLA in bovine milk fat across a range of diets. The presence of t7,c9 CLA and c9,t13 C18:2 supports the role of an active A9 desaturase, an enzyme that introduces a cis double bond between carbons 9 and 10 (50,51). Evidence suggests that As desaturation of t l l C,,:, may be more relevant in the production of CLA than previously thought. Several studies reported a strong correlation coefficient between t l l-C18:1and CLA concentrations in milk fat (52-54). A close linear relationship between milk fat tll-C18:land CLA was observed, suggesting a precursor-product association in which tl l-Cls:l is the precursor and CLA is the product (48). A slope of 0.5 suggests that approximately one third of circulating tl l-C18:lwas desaturated by A9 desaturase (49). A study in which t11-C18:1and CLA were fed in equal quantities to mice reported that 12% of the tll-C18:l consumed during a 2-wk feeding period was recovered in the carcass as CLA ( 5 5 ) . Of the proportion of tll-C18:lin the tissues that was available for bioconversion, 48.8% was desaturated. CLA was found in the carcass only when tl l-Cls:l or CLA was fed. CLA was found in both triglyceride and phospholipids when CLA was fed, but only in triglyceride when tllc18:1 was fed, suggesting that bioconversion occurred in the adipose tissue ( 5 5 ) .
M. O'Shea e t a /
252
CLA in human serum was shown to be derived in part from the diet and in part by conversion of dietary trans fatty acids (TFA) (56). Serum levels of CLA varied between 0.17 and 0.43%, when healthy subjects were fed a dairy fat diet for 5 wk, followed by either a TFA diet or a C18:odiet for 5 wk (56). The proportions of CLA in the dairy fat, TFA, and C,,:, diets were 0.37, 0.04, and 0.10% of total fatty acid methyl esters (FAME).The corresponding proportions in serum were 0.32,0.43, and 0.17% (Table 12.1). The difference in the CLA content of serum between subjects fed the dairy fat and C,,:, diets was explained by the different dietary intakes of CLA. Intake of CLA from Natural Sources
Levels of total CLA in various foods range from as low as 0.2 mg/g fat in corn and peanut oil (57) to as high as 17 mg/g in beef (4) and 30 mg/g in milk fat (2); c9,tll is the predominant isomer in milk comprising 90% of the total CLA (Table 12.2). Negligible amounts of c9,tll and t10,c12 CLA occur in seafood and vegetable oils. Estimates of CLA intakes for humans were based on CLA databases in conjunction with 3-d or 5-wk dietary records, national food intake surveys, and data from biochemical analyses of food portions. Using the last-mentioned approach, in conjunction with 5-wk dietary records, it was estimated that CLA intake by Finnish men and women ranged between -40 and 310 mg/d (56). Such variation was attributed to marked differences in the type of fat (dairy, trans, and stearic acid) in the diet. Herbel et al. (58) reported that young men and women living in the United States consumed -127 mg CLA/d. Another US.study estimated that the dietary intake of CLA was lower in women (52 44 mg/d) than in men (137 & 84 mg/d) (59). Dietary intake of CLA in Germany was also estimated to be lower in women than in men (350 vs. 430 mg CLA/d) (8). The differences between these studies can be accounted for by the fact that fat intake is higher in Germany than in the United States (60). The German study was not age-specific, whereas the U S . studies were limited to young men and women, suggesting that age may influence CLA intake in humans.
*
TABLE 12.1 Mean Percentage Fatty Acid Composition of Total Serum from Healthy Subjects Fed a Dairy Fat Diet Followed by a trans Fatty Acid Diet or a Stearic Acid Dieta Fatty acid
C, 6:O Cl8:O C,,:, trans fatty acid
c,,:, Cl,:, LA c18:2 CLA
Trans fatty acid diet
Stearic acid diet
Dairy fat diet
(n = 40)
(n = 40)
23.74 7.74 0.59 23.39 30.03
20.31 6.68 4.47 24.39 29.73
21.42 10.30 0.49 23.1 1 29.78
OS32
0,43
0.1 7
aSource: Reference 56. LA, linoleic acid; CLA, conjugated linoleic acid.
CLA Sources and Human Studies
253
TABLE 12.2 Total CLA Content a n d Percentage of c9,tll CLA in Food Products Food
Total CLA (mp/p fat)
Butter Nonfat frozen dairy dessert Condensed milk Natural cheeses Processed cheeses Cheddar cheese Ice cream Sour cream Yogurt T-bone (cooked) T-bone (raw) Round beef Milk fat Seafood Vegetable oils
9.4-1 1.9 0.6 7 0.6-7.1 3.2-8.9 5.1-5.4 3.8-4.9 7.5 5.1-9.0 4.7-9.9 4.4-6.6 2.9 2-30 0.5 0.2
c9,tl 1 CLA (O/O
of total CLA)
91 90 90 17-90 17-90 82-88 73-76 78 82 65 59 79 90 NDa 45
Reference
(4) (57) (57) (5736)
(57,86,87) (88) (8) (8) ( 8)
(89) (89) (90) (21 (57) (57)
aND, not detected. See Table 12.1 for other abbreviations.
The highest level indicated (i.e., 1000 mg/d) was found in a Hare Krishna community in Australia. The relatively high level of ghee and butter consumption appeared to be the reason of this high intake. Also, measurements of the CLA content in the breast milk of women in this population resulted in relatively high values, and was clearly related to the high intake of CLA-containing products (61). Specific intervention studies showed that increasing the CLA content of the diet increased the CLA content in human milk (62), plasma (63), and adipose tissue (64). Feeding a high dairy fat diet containing 29 1 i 75 mg CLA/d led to a 1.6fold increase (13.5 i 0.1 pmol/g fat) in CLA content of human milk (62). Plasma CLA increased 19-27% to 9.6 1.1 pmol/L when men were fed cheddar cheese containing 178.5 mg CLA daily for 4 wk (63).
*
CLA Produced from Vegetable Oils
Alkali isomerization of LA is the common method used in the synthesis of mixtures of CLA isomers resulting in commercial CLA products. The amount of LA in the starting material relates to the amount of conjugated isomers formed. Typically, safflower and sunflower oils are used as starting materials in the production of CLA isomers due to their high LA content. The two most common processes for the production of CLA both use an alkali-catalyzed conjugation step starting from safflower oil containing -80% LA as raw material (65). During the process, the oil or its fatty acid esters are either saponified or interesterified with an alcohol, and one of the double
bonds in the LA is “flipped’ in the direction of the second double bond. Depending on the process conditions used, many different isomers can be produced; consequently,
254
M. O'Shea et al.
products initially contained a minimum of four isomers (t8,clO; c9,tll; t10,c12; ~ 1 1 ~ 1 3In) recent . years manufacturers have refined the process in such a way that the conjugation step leads to a CLA product in free fatty acid (FFA) or ester form, consisting only of c9,tll and t10,c12 CLA isomers in an -5050 ratio (66-68). The ester product consists of either methyl- or ethylesters. The esters can be hydrolyzed to give FFA, or, if desired, FFA and esters can be esterified with glycerol, and bleached and deodorized to form triglyceride oil. Refining the process to result in a product consisting of two main isomers led to the development of a standardized product that was demonstrated to be both efficacious and safe in comparison with the four-isomer mixtures, which generally have led to inconsistent results in human intervention trials (65). The standardized two-isomer mixture is produced in both FFA and triglyceride forms, and the efficacy of each was demonstrated and compared in many studies. These products have paved the way for the introduction of dietary supplements, bars, drinks, and soon many more food products enriched in CLA isomers. CLA has been on the market as a dietary supplement in many countries all over the world for -7-8 yr. Both CLA FA and CLA glyceride are sold for that purpose, although the labeling may not always clearly distinguish between them (65).
Human Intervention Studies Several studies examined the effects of CLA in humans. A review of these studies was reported by Gaullier et al. (65). The daily doses of CLA ranged from 3 to 7 g and the treatment periods ranged from 4 wk to 1 yr. The trials were carried out in a range of population groups, e.g., body builders, healthy individuals, overweight and obese individuals, individuals with metabolic syndrome, and populations with NIDDM. The studies discussed below are categorized according to the isomeric composition of the CLA preparation, in view of the fact that this is a major influence on the effects of CLA. As stated before, the CLA preparations containing mainly the ~ 9 ~ isomer 1 1 and the tlO412 isomer in approximately equal proportions were demonstrated to have beneficial effects in human intervention trials (65). Human Studies with Two-Isomer Preparations
Most studies on CLA were performed with preparations such as ClarinoP, which consist mainly of two isomers, the c9,tll isomer and the t10,c12 isomer, in approximately equal amounts (5050) (Table 12.3). These studies focused on body composition, insulin glucose sensitivity, immunomodulatory properties, milk fat synthesis, and lipid metabolism as the main parameters. The composition of the material, dose, population, and primary outcomes will be discussed here. The studies on body composition focused on changes in body fat mass (BFM) and lean mass as primary outcome variables, with insights into lipid metabolism and safety. A pilot study (33) investigated the efficacy and tolerability of a daily intake of
CLA Sources and Human Studies
255
1.8 g/d CLA 5050 for 12 wk in 20 healthy exercising humans with body mass index (BMI) <25 kg/m2. It was a randomized, double-blind, placebo-controlled study. Body fat was significantly reduced in the CLA group after 4, 8, and 12 wk but not in the placebo group. The strenuous exercise performed by the participants may have had a positive influence on the effect of CLA. None of the participants withdrew from the study due to adverse events, and no serious adverse events were reported. Mougios et al. (34) conducted a study with 24 healthy subjects (BMI <30 kg/ m2), receiving 0.7 g/d of a two-isomer CLA or placebo for 4 wk and 1.4 g/d two-isomer CLA or placebo for the next 4 wk. The CLA preparation consisted of 69% CLA containing a 5050 ratio of the c9,tll CLA and t10,c12 CLA. BFM was significantly reduced in the CLA group during the second period, but not overall during the study. A trend toward lower triglycerides, as well as a significant decrease in HDL cholesterol, was observed during the low CLA intake period, but the lower HDL cholesterol was no longer observed during the high CLA intake period. Total cholesterol levels were not reported, and no adverse effects were reported. A randomized, double-blind study by Blankson et al. (27) and Berven et al. (32) included 60 overweight and obese men and women (BMI 27.5-39.0 kg/m2).The subjects were divided into 5 groups administered placebo (9 g olive oil), 1.7, 3.4,5 .I, or 6.8 g (blended with olive oil for a total intake of 9 g/d) CLA/d for 12 wk, respectively. The active capsules (12/d) contained 75% CLA. Eight subjects withdrew from the study due to adverse events; however, no differences among treatment groups were found regarding adverse events. Numerous parameters were analyzed, including blood lipids, hematology, and chemistry at baseline, and wk 6 and 12. Vital signs (blood pressure and pulse) and liver and kidney safety parameters (aspartate amino transferase, amino alanine transferase, bilirubin, y-glutamyltransferase) were measured at baseline and at wk 12. A significant reduction of body fat was found in the 3.4 and 6.8 g/d CLA groups only (5.7 and 3.7%, respectively). No significant changes were found in blood glucose or insulin levels. Some changes were observed within each group in hematological parameters, but none of the changes was considered clinically important. No clinically relevant changes in blood parameters, liver and kidney safety parameters, or vital signs occurred. A series of trials conducted at the University of Uppsala investigated and reported the effects of CLA (given as the commercially available mixture and as the purified trans- lO,cis-12 isomer) on anthropometry, lipid and glucose metabolism, and markers of lipid peroxidation (69). None of the studies showed any effects by CLA on body weight or BMI. There were, however, indications of a certain reduction of the proportion of body fat (especially abdominal fat) by CLA and t10,c12 CLA. In the first study, body fat decreased by 3.8% ( P < 0.001, paired ttest) after 3 mon in a CLA-treated group of healthy men and women (30). In that study examining the effect of CLA on body composition, 52 healthy men and women (BMI 19.1-34.5 kg/m2) received either 4.2 g/d CLA or olive oil for 12 wk. The CLA capsules contained 75.9% CLA consisting mainly of the c9,tll-isomer and the tlO,cl2-isomer, which were present in equal amounts. There were no sig-
TABLE 12.3 Overview of Results of Studies G-80 on ClarinolTMG-80 and CLA Preparations Similar to ClarinolTMin Hurnansarb
Intake (g/d)
Duration
Metabolic hormones
Liver parameters
Serum lipid profile
Other effects
Healthy a;' vaccinated for hepatitis B
1.7 (FFA)
12 wk
Glucose:
u
Total cholesterol: LDL cholesterol: HDL cholesterol: Triglycerides:
Treatment-related adverse events:
Obese, d a n d 9
2.7 (FFA)
26 wk
NA
NA
NA
Treatment-related side or adverse events: ++
Reference Subjects
-
---
-
Healthy, d a n d 9
4.2 (FFA)
12 wk
NA
NA
NA
Lipid peroxidation:
Subjects with type 2 diabetes mellitus
6 (FFA)
8 wk
Glucose: .1 Leptin: 1
NA
NA
NA
Healthy overweight and obese, d and 0
1.7, 3.4, 5.1, 6.8 (FW
12 wk
Glucose: Insulin:
AST, ALT, Bilirubin, y-Glutamyltransferase:
Healthy overweight, d and 9
3.4 (FFA and TG)
52 wk
Glucose:
NA
NA
Healthy overweight, d and 9
1 4 3 . 6 (TC)
13 wk
Glucose: Insulin:
NA
c,
--
--
-
?
-
Blood safety parameters: Vital signs: Treatment-related adverse events:
-
-
-
Treatment-related adverse events:
NA
1.2 (?)
2x5d
NA
NA
NA
0.7-1.4 (TG)
8 wk
NA
NA
HDL cholesterol: Triglycerides: 1
Healthy, dand9
3 (FFA)
8 wk
Glucose: Insulin:
Obese middleaged, d
4.2 (FFA)
4 wk
Glucose: Insulin:
Healthy lactating 9 Healthy, dandQ
Obese middleaged, d
Healthy, d a n d 9
3.4 (FFA)
4.2 (FFA)
12 wk
12 wk
--- --
Triglycerides:
A-
1
Infant milk consumption: c-, Total milk fat: -1 Creatine kinase: Treatment-related adverse events:
-
NA
-
- -
Treatment-related adverse events:
NA
c,
Glucose: Insulin: Leptin:
NA
HDL cholesterol: -1 Other cholesterol: Triglycerides: Free fatty acids: c-,
Insulin sensitivity: Lipid peroxidation: 7 a- and y-Tocopherol: IL6: CRP TNFa:
Glucose: (NS) Insulin:
AST and ALT
ApoB: f
NA
NA
NA
-
--
Healthy exercising, dand0
1.8 (FFA)
12 wk
NA
Healthy overweight, d and 9
6 (TG)
52 wk
Glucose: Insulin:
--
-
-
AST, ALT, AIkP
- - --
? 3
ii
2 nl
4
s3 nJ
--
Cholesterol: Triglycerides:
- -
Treatment-related adverse events:
39 2
Adverse events: 4 T4, TSH: Blood parameters:
“These preparations were composedof equal amounts of the L9,tll CLA isomer and the tlO,c12 CLA isomer. bAbbreviations: AST, aspartate amino transferase; ALT, amino alanine transferase; IL, interleukin; CRP, C-reactive protein; TNF, tumor necrosis factor; Apo, apolipoprotein; alkP, alkaline phosphatase; -, unchanged; J, decreased; 7, increased; FFA, free fatty acids; TG, triglycerides; LDL, lowdensity lypoprotein; HDL, high-densitylipoprotein. See Table 12.1 for other abbreviations.
N VI U
258
M. O’Shea et al.
nificant differences between the two groups with regard to serum lipid concentrations. No clinically relevant effects on insulin and glucose levels or liver enzymes were found. A second study with the same CLA preparation was performed in 25 abdominally obese men (BMI 27-39 kg/m2) with signs of the metabolic syndrome (31). Subjects received 4.2 g/d of the same two-isomer CLA or olive oil for a period of 4 wk. As in the previous study, no adverse events were clinically detected or reported. Sagittal abdominal diameter (SAD) is suggested to be the best simple anthropometric measurement of visceral fat. After 4 wk of supplementation of abdominally obese men, there was a significant decrease in SAD in the CLA group (-2%; P = 0.003, paired t-test) compared with placebo ( P = 0.04, unpaired t-test). BMI did not differ between the groups. Plasma glucose, insulin, and leptin levels and insulin sensitivity were not affected, nor were C-reactive protein (CRP) levels. The formation in the urine of 15-keto-dihydro-prostaglandin(PG)F,, and of 8-isoPGF,,, measures of lipid peroxidation and possibly inflammation, increased. Other inflammatory parameters such as tumor necrosis factor (TNF)-a, a-tocopherol, ytocopherol, and interleukin (1L)-6 were not affected. HDL cholesterol decreased, but total cholesterol, LDL cholesterol, triglycerides, and FFA levels were not affected. The same study included the effects of CLA preparations enriched in the t10,c12 CLA isomers. The results of that part of the study will be discussed in the section on pure isomer preparations. In a study by Basu et al. (70), 53 healthy men and women (average BMI = 25.1 kg/m2) received 4.2 g of olive oil or a 2-isomer CLA mix for 3 mon. No participant experienced any side effects during the study period. Significant increases in both 8iso-PGF2, and 15-keto-dihydro-PGF2, in urine were observed after 3 mon of daily CLA intake (4.2 g/d) compared with the control group ( P c 0.0001). However, the magnitude of the increase of 8-iso-PGF2, and 15-keto-dihydro-PGF2, in that study was not comparable to the earlier described inflammatory response-related (7 1,72) or oxidative injury-induced (73) formation of these compounds. The compounds were measured by new highly sensitive radioimmunoassays. To investigate whether CLA might reduce regain of BFM and enhance regain of fat-free mass, a trial with 54 overweight men and women (BMI = 25-30 kg/m2) was conducted (39,74). Before the intervention, subjects consumed a very-low-energy diet for 3 wk. After the 3-wk period, subjects received daily 1.8 g CLA (low dose, LD) or placebo (oleic acid) or 3.6 g CLA (high dose, HD) or 3.6 g/d CLA (HD) of a two-isomer preparation for 13 wk. Multiple regression analysis showed that at the end of the 13-wk intervention, CLA did not have an effect on body weight regain. Feelings of fullness and satiety were increased and feelings of hunger were decreased after the 13-wk CLA intervention compared with placebo, independent of the percentage of body weight regain (39). The regain of fat-free mass was increased by CLA (LD 6.2 f 3.9, HD 4.6 f 2.4%) compared with placebo (LD 2.8 f 3.2%, HD 3.4 f 3.6%), independent of the percentage of body weight regain and physical activity. As a consequence of an increased regain of fat-free mass from CLA consumption, resting metabolic rate was increased by CLA (LD 12.0 11.4%, HD 13.7 f 14.4%) com-
*
CLA Sources and Human Studies
259
pared with placebo (LD 9.1 f 11.O%, HD 8.6 f 8.5%). Substrate oxidation and blood plasma parameters were not affected by CLA. In conclusion, the regain of fat-free mass was favorably, dose-independently affected by 13 wk of consumption of 1.8 or 3.6 g CLA/d, leading to an increased resting metabolic rate (74). CLA did not affect plasma glucose, insulin, triglycerides, or FFA concentrations. Thus, body composition rather than body weight was affected by CLA. There were no differences between the HD and LD CLA groups. Most recently, long-term intervention studies were conducted with the two-isomer mixture of CLA (37,40). Men and women (n = 180) with BMI of 25-30 kg/m2 were included in a double-blind, placebo-controlled study for 1 yr (40).Subjects were randomly assigned to three groups: 3.6 g/d CLA-FFA, 3.6 g/d CLA-triacylglycerol (TAG), or placebo (olive oil). Change in BFM, as measured by dual-energy X-ray absorptiometry (DEXA), was the primary outcome. Secondary outcomes included the effects of CLA on LBM, adverse events, and safety variables. Mean (* SD) BFM in the CLA-TAG and CLA-FFA groups was 8.7 f 9.1 and 6.9 f 9.1%, respectively, lower than that in the placebo group ( P < 0.001). Subjects administered CLA-FFA had 1.8 f 4.3% greater LBM than those receiving placebo ( P = 0.002). These changes were not associated with diet or exercise. LDL cholesterol increased in the CLA-FFA group ( P = 0.008), whereas HDL cholesterol decreased in the CLA-TAG group ( P = 0.003), and lipoprotein(a) increased in both CLA groups ( P < 0.001) compared with mon 0. Fasting blood glucose concentrations remained unchanged in all three groups. Glycated hemoglobin increased in all groups from mon 0 concentrations, but there was no significant difference between groups. Adverse events did not differ significantly between groups. A further study has now confirmed the finding of Gaullier et al. (40) that supplementation with CLA mixture of isomers for 1 yr is safe in healthy overweight and obese humans at doses higher than the previous study (37). This was a randomized, double-blind study consisting of three phases in which subjects were given 6 g/d of CLA mixed isomer (c9,tll and t10412 in a 5050 ratio) or placebo. Phase 1 was a low-energy diet (13 kcalkg desirable weight) for 12 wk or until 10-20% of initial body weight was lost. In Phase 2, from wk 12 to 28, subjects were refed a diet providing 25-30 kcal/kg of desirable body weight. Phase 3 was open label, with subjects from both groups taking CLA from wk 28 to 52. At biweekly visits, subjects completed a questionnaire evaluating side effects and adverse events. Blood was taken for assay of liver function, glucose, insulin, serum lipids, blood counts, and general chemistry. Overall, body composition did not differ between the groups. Laboratory tests showed no adverse effects of CLA. There were significantly fewer ( P < 0.05) adverse events and side effects in the CLA group compared with placebo. There were no significant differences between groups in insulin levels at any of the time points. CLA subjects had a significantly higher serum glucose level compared with placebo subjects at wk 2 (93.1 f 1.5 vs. 87.2 f 1.6 mg/dL, P < 0.007), but differences were not significant at any other time points. The difference at wk 2 resulted from a decrease in mean glucose levels in
2 60
M. O’Shea et a/.
the control group of 1 mg/dL, coupled with an increase of 2 mg/dL in the CLA group. Using homeostasis model assessment (HOMA) as a measure of insulin resistance (75), there were no differences between groups at any time point throughout the study. These studies confirm that CLA supplementation in healthy overweight and obese humans is safe for up to 1 yr at doses of up to 6 g/d active c9,tll and t10,c12 in a 5050 ratio. In line with effects on BFM, one would expect changes in metabolic parameters, particularly related to glucose and insulin homeostasis. In a study of Belury et al. (36), subjects with type 2 diabetes mellitus were randomized into one of two groups receiving either a supplement containing 6 g/d of CLA 5050 or safflower oil for 8 wk. Although this study was not performed with preparations enriched in one particular CLA isomer, the effects were correlated with plasma levels of the individual isomers. There was a stronger correlative of the t10,c12 CLA isomer with decreased leptin levels and associated decreased glucose levels than the c9,tlI CLA isomer. Although insulin levels were not affected, the decreased glucose levels indicate a potential beneficial effect of CLA on subjects with type 2 diabetes mellitus that can be attributed to the t10,c12 CLA isomer (36). Another recent study examined whether CLA supplementation can improve insulin sensitivity in sedentary humans (41). Young sedentary individuals [n = 16; age, 21.5 f 0.4 y (mean f SEM); body mass, 77.6 f 3.4 kg] participated in this study. Ten subjects received 4 g/d of mixed CLA isomers (35.5% c9,tll; 36.8% t10,c12 CLA) for 8 wk, whereas six subjects received placebo (safflower oil). Oral glucose tolerance tests were performed at baseline (0), 4, and 8 wk of supplementation. After 8 wk of CLA supplementation, the insulin sensitivity index (ISI) increased (14.4 f 1.O, 8 wk vs 11.3 f 1.3,O wk; P < 0.05), which corresponded to a decrease in fasting insulin concentrations. Six of the 10 subjects had large increases in their IS1 (range, 4-27 to 90%), whereas two demonstrated essential no change (3-5%), and two had a decrease in insulin sensitivity (12-13%). IS1 was unchanged over 8 wk in the placebo group. These results indicate that a common dosage of a commercially available CLA supplement can improve IS1 in young, sedentary individuals. However, there is considerable individual variability in the response. Additional studies are required to identify underlying metabolic changes in human skeletal muscle. In addition to effects on body composition, there are now intervention trials focused on immunomodulatory effects. Several animal and recently human studies indicated that CLA can positively affect aspects of the immune response in addition to basal immune parameters, while negating the adverse effects of inflammation (17). In a study by Albers et al. (18), a promising infection model was used to measure the effects of CLA on the immune system. In that model, the main dynamic immunologic parameters of the humoral and cellular immune response after hepatitis B (HB) vaccination were investigated. In a 12-wk, randomized, placebo-controlled, double-blind trial, the immunomodulating effect of CLA in 7 1
subjects (healthy men, aged 30-70 yr) was studied. Subjects were randomly assigned to CLA supplements consisting of either 50% c9,tll and 50% t10,c12, or
CLA Sources and Human Studies
261
CLA supplements consisting of 80% c9,tll and 20% t10,c12, or sunflower oil fatty acids (placebo). A dosage amount of 3 g/d in soft-gel capsules provided 1.7 g CLA/d in the treatment groups. At baseline and after 12 wk in vivo, cell-mediated immune responsiveness was measured. The results of that study indicated an increased response after HB vaccination that was reflected in HB-specific antibody titers and seroprotection rate. That study is the first in which CLA was shown to stimulate the humoral immune response in humans as reflected in an increased seroprotection rate after vaccination. Furthermore, the effects of milk fat synthesis investigated in animal models have now been explored in humans. A study by Masters et al. (76) was designed to investigate whether maternal consumption of a CLA 5050 mixture decreased milk fat in humans. Healthy lactating women (n = 9) took part in a 17-d study consisting of three periods: intervention I (5 d), washout (7 d), and intervention I1 (5 d). During the intervention period, subjects consumed supplements containing 1.2 g CLA 50:50 and 0.3 g other fatty acids or 1.5 g olive oil daily. There was no effect of treatment on mean infant milk consumption. The concentration of both c9,tll and t10,c12 CLA isomers was increased in maternal plasma as well as in maternal milk. Total milk fat was lower during CLA consumption compared with the placebo consumption, but not compared with the washout period. There was no significant change in fatty acid composition of the milk fat, as measured by gas chromatography. No significant effects on infant weights were found in this study. The milk fat content remained within normal ranges. The control used in this study was olive oil, which increased the milk fat content. In fact, both the increase of fat by olive oil and the decrease of fat by CLA were not significant compared with the washout period, in which no supplements were taken. The effect of CLA consumption on milk fat was investigated also in rats (1) and cows (77). Although the milk fat content was found to decrease (77), supplementation with CLA did not affect litter size or induce apparent abnormalities in rats (1). On the contrary, feeding CLA to the rat dams during gestation and lactation improved the postnatal body weight gain of pups. Pups that continued to receive the CLA-supplemented diet after weaning had significantly greater body weight gain and improved feed efficiency relative to control rats. Finally the effect of CLA on cardiovascular risk factors and lipid metabolism was monitored in many studies focused on body composition parameters. However, a study designed specifically to investigate these effects in humans was also conducted (35). Noone et al. (35)examined the effects of dietary supplementation using two isomeric blends of CLA on TAG-rich lipoprotein metabolism and reverse cholesterol transport in human subjects and evaluated whether CLA modulated cardiovascular disease risk factors. Normolipidemic subjects (n = 51) participated in this randomized, double-blind, placebo-controlled, intervention trial. Subjects were randomly assigned to receive daily 3 g c9,tll and t10,c12 CLA isomeric blend (5050) or a c9,tll and t10,clZ CLA isomeric blend (8020) CLA or LA (control) for 8 wk. The 5050 CLA isomer blend significantly reduced ( P s 0.005) fasting plasma TAG con-
2 62
M. O’Shea e t a / .
centrations. The 80:20 CLA isomer blend significantly reduced ( P 5 0.05) very low density lipoprotein (VLDL) cholesterol concentrations. CLA supplementation had no effect on LDL cholesterol, HDL lipid-protein composition, or reverse cholesterol transport. CLA supplementation had no effect on body weight, plasma glucose, or insulin concentrations. Fatty acid analysis revealed that the c9,tll CLA isomer was incorporated into total plasma lipids after supplementation with both isomeric blends of CLA. The present study demonstrates that CLA supplementation significantly improves plasma TAG and VLDL metabolism in human subjects. The study confirms that some of the cardioprotective effects of CLA that were shown in animal studies are relevant to humans. Human Studies with Four-Isomer CLA Preparations
Numerous studies were conducted with a four-isomer preparation of CLA, containing approximately equal amounts of the t8,c10-, c9,tl1, and t10,c12 CLA, and cll,t13 CLA isomers and high levels of trans, trans isomers (Table 12.4).Studies on CLA preparation with four different isomers were published by Benito et al. (78), Kelley et al. (79), Medina et al. (80), and Zambell et al. (81). Healthy female volunteers (n = 17; BMI -23 kg/m2) received 3.9 g/d CLA or placebo (sunflower oil) for 63 d. A supplement of d-a-tocopherol was included in the diet to ensure adequate antioxidant levels in the volunteers during the study. There were no changes in BFM. The authors claim to have found a nonsignificant trend toward an increase in insulin levels at the end of the study, although this was not indicated by the published data. No significant effect on glucose levels was observed. CLA did not alter the plasma LDL cholesterol and HDL cholesterol levels in this study, and there were no adverse effects on the health of the volunteers. Body builders who consumed 7.2 g/d CLA for a period of 6 wk showed no effects on serum glucose levels. In addition, liver enzymes and serum lipid profiles revealed no significant differences (82). A similarly high level of 7 g/d CLA was tested in resistance-trained athletes in an open, single-center, nonplacebo-controlled study for 6 mon (83). Two groups of seven athletes were included in this study, one group with (experienced) and one group without (novice) experience in weight training, with BMI of 27.1 and 26.4 kg/m2,respectively. Both groups experienced a loss of body fat relative to body weight compared with baseline values. Plasma LDL and total cholesterol levels were increased in the novice group, but not in the experienced group compared with baseline levels. HDL and triglyceride levels were not affected in either group. Significant changes in leptin, soluble leptin receptor, or insulin-like growth factor-I levels were not detected. Without a control group, the influence of CLA on any of these parameters cannot be deduced. However, no adverse effects were reported in this 6-mon study. In a study by Kreider et al. (84), resistance-trained men (n = 23) were supplemented daily with 9 g olive oil or 6 g CLA + 3 g unspecified fatty acids for 4 wk. The CLA preparation consisted of 65% CLA isomers, with t10,c12 CLA isomer (22.6%), cll,t13 isomer (23.6%), c9,t11, (17.6%) and t8,clO isomer (16.6%) as the
TABLE 12.4 Overview of Results of Published Studies on CLA Preparations Composed of Four Different Isomersa ~
Dose Reference
Subjects
(78-81)
Healthy,
9
(g/d)
Duration
Adipose tissue
3.9
9 wk
u
(84)
Resistancetrained, d
6
(83)
Novice and experienced athletes, dand 9 Healthy body builders, d
7.2
6 rno
7.2
6 wk
(82)
aSee Tables 12.1 and 12.3 for abbreviations.
4 wk
u
J
-
Metabolic hormones
Liver parameters
Glucose: Insulin: t(?) Leptin: J Glucose:
NA
-
--
Leptin:
--
Glucose: Insulin:
Liver enzymes:
-
Serum lipid profile
-
NA
AST, ALT, Bilirubin, y-Glutamyl transferase:
TNFa:
-
Cholesterol: ?(novice) Triglycerides:
-
-
--
No placebo group!
NA
?
0 rn
Side effects: Bone density:
+3
t ,
Other effects
C
ii
IE! 3 LI,
4 >
-L
2 ? 3 a
2 64
M. O’Shea e t a / .
major constituents. The CLA preparation contained a relatively high amount of trans, trans CLA isomers (7.7%) and unknown isomers (11.9%). There were no effects on BFM and no clinically significant changes in general markers of health, such as blood glucose levels and liver enzymes. In conclusion, the effects seen in the previous section with the 5050 mixture of isomers are not substantiated when they are present with other isomers (65). Furthermore physiologic effects in animal and human trials were associated only with the c9,tll and t10,c12 CLA isomers to date; the effects of the other isomers remain largely unknown and warrant further investigation. Human Studies with Purified CLA Isomer Preparations
The effects of the t10,c12 CLA isomer were studied in overweight men (BMI = 27-39 kg/m2) with signs of the metabolic syndrome (29,85). Three groups received either 3.4 g control oil (olive oil), 3.4 g CLA (50:50),or 3.4 g purified (76.5%) t10,c12 CLA isomer for a period of 12 wk. The major isomer content of the CLA mix preparation (80% FFA) was 35.9% t10412 isomer and 35.4% t9,cll-isomer. There was a decrease in SAD in the CLA group of men treated for 12 wk with a reduction of SAD within both the CLA group (-3%) and the group given the purified tlO,cl2-CLA isomer (-3%). Compared with the control group (-1.5%), the difference between the groups tended to be significant ( P = 0.07, ANOVA). No effects on insulin and glucose levels were found in subjects receiving the 5050 CLA. The HDL cholesterol concentration decreased significantly (4%; P < 0.01, unpaired t-test) with a concomitant (nonsignificant) tendency to increased VLDL TAG concentrations. Blood glucose concentrations increased (4%; P < 0.001, unpaired t-test), and there was a significant reduction in insulin sensitivity (-19%; P < 0.01, unpaired t-test), measured as the IS1 with the euglycemic clamp, among the men already experiencing insulin resistance in the group receiving the t10,c12 isomer, but not in those receiving the 5050 mix. The results indicate isomer-specific metabolic actions of CLA, at least in abdominally obese humans. When evaluated separately in the obese men, it was clearly shown that the increase of lipid peroxidation was significantly more pronounced after the t10,c12 CLA isomer than after the CLA mixture, again indicating that the lipid peroxidation might, at least to some extent, be an isomer-specific effect of CLA (71). In that study, there was also a significant increase of CRP by 110% compared with placebo ( P < 0.01, unpaired ttest) after supplementation with truns-lO,cis-12 CLA. The changes in CRP were significantly associated with the changes in 8-iso-PGF2, (72). No effects were found on TNFa, a-tocopherol, y-tocopherol, or IL-6. HDL cholesterol levels were lowered by treatment with both the tlO412 CLA isomer and the CLA mix. A further trial using the same purified isomer material was completed recently (28). Middle-aged, overweight, healthy men and women (n = 81) participated in this bicentric, placebo-controlled, double-blind, randomized study. For 6 wk (runin period), all subjects consumed daily a drinkable dairy product containing 3 g of high-oleic acid sunflower oil. Volunteers were then randomly assigned to five
CLA Sources and Human Studies
265
groups receiving daily 3 g of high-oleic acid sunflower oil, 1.5 g of c9,tll CLA, 3 g of c9,tll CLA, 1.5 g of t10,c12 CLA, or 3 g of t10,c12 CLA administered as TAG in a drinkable dairy product for 18 wk. The percentages of BFM and fat and lean body mass were assessed at the end of the run-in and experimental periods by DEXA. Dietary intake was also recorded. Body fat mass changes averaged 0.1 f 0.9 kg (mean f SD) in the placebo group and -0.3 f 1 . 4 , a . g f 2.1,O.O & 2.3, and -0.9 f 1.7 kg in the 1.5-g c9,tl1,3-g c9,tl1,1.5-g t10,c12, and 3-g t10,c12 groups, respectively. Changes among the groups were not different ( P = 0.444). Monthly monitoring of biological side effects, by assessment of hematological and clinicalchemical parameters, including plasma insulin and glucose concentrations, revealed no systematic adverse effects. In conclusion, the effects of the individual isomers do not reflect the findings for the 5050 mixture, suggesting that there may be a synergistic effect of combining the two main isomers c9,tll and t10,c12 CLA. In addition, the possible negative effects of the pure t10,c12 CLA in men with the metabolic syndrome were not present in the group receiving the 50:50 mixture, again suggesting a synergy between these bioactive isomers of CLA (29,85).
Conclusions The predominant natural sources of CLA are dairy products, with 90% of CLA present as the c9,tll isomer. Vegetable oil sources consist of both the c9,tll and t10,c12 CLA isomers but at much lower levels compared with dairy products. Furthermore, the daily intake of CLA isomers from these sources is quite low at -300 mg/d, varying with sex and dietary choice. The production of CLA isomers from vegetable oil led to an oil that consisted of an equal mixture of the two main isomers in a high concentration (80%); this paved the way for intervention trials investigating the physiologic effects of these isomers, in addition to the safety of effective dose levels. In addition, these products have been available on the consumer market in Europe and the United States since 1996 in the form of soft gel capsules and as ingredients in various meal replacers, drinks, and dietary supplement bars for application in improving body composition by decreasing fat mass and increasing LBM. Many human intervention studies were performed with the CLA produced from vegetable oil sources. The 50:50 mixture of isomers has beneficial effects in reducing BFM while increasing lean mass in humans. This was demonstrated clearly in healthy overweight subjects in many studies (27,30,33,34) and most recently in the longest intervention (1 yr) with the largest population (n = 180) (40). The doses varied considerably in the trials with beneficial effects on body composition being observed at doses from 1.7 (34) to 3.6 g/d (40). The effects on obese populations are not as evident (37,38) and warrant further studies. Obese populations are more heterogeneous than overweight populations and therefore may require stricter inclusion and exclusion criteria and higher numbers to demonstrate the effects seen in overweight populations. It is possible there
266
M . O’Shea eta/.
may be specifically good and bad responders to CLA intervention and these have to be identified in further trials, ideally multicenter studies that can compare the effects across a range of populations using a similar trial design. Furthermore, the effects on lipid metabolism are not clear, Gaullier et al. (40) demonstrated a significant decrease in HDL cholesterol in the CLA-TAG group but not compared with placebo. When comparing all human trials, there was no consistency on effects of CLA on HDL cholesterol levels. Levels of HDL cholesterol can vary greatly with diet and exercise. Furthermore, it is recommended that levels of HDL cholesterol be evaluated in combination with other blood lipid levels. For example, a decrease in HDL cholesterol would not be harmful if found in combination with a decrease in total cholesterol. However, human data on levels of all blood lipids are equally as variable as data on HDL cholesterol. This is likely due to the fact that most human studies were designed to measure effects of CLA on body composition. Only the study by Noone et al. (35) was designed specifically to address blood lipids, and this study showed beneficial effects of CLA. In addition, animal data repeatedly showed beneficial effects of CLA on cardiovascular health. These data were more consistent because diet and exercise are more easily controlled in animal studies than in free-living subjects. Certainly the composition of the material used in the intervention trials affected the outcome whereby the products containing four isomers did not show the same efficacy as the two-isomer preparations (64). Furthermore, studies investigating pure isomer material did not demonstrate the physiologic effects that occurred when the mixture was given. The same two-isomer preparations used in the studies by Riserus et al. (29,85) were used in a study by Malpuech-Brugere et al. (28). The populations used in both studies were very different, as were the observed physiologic effects. Riserus et al. (29) demonstrated an increase in insulin resistance and inflammatory markers in metabolically obese men with 3 g/d of the t10,c12 CLA isomer; such effects were not evident in the same population receiving the 5050 mixture of both isomers. Human supplementation with high doses of the t10,c12 CLA isomer without the presence of c9,tll CLA should be avoided until further information on possible effects and side effects becomes available. However, it cannot be excluded that future studies could point to clinical applications, eg., as a result of antiturnongenic properties or as a tool to prevent weight gain with this isomer. This possibility certainly requires more research to increase our understanding of the mechanisms behind the effects of CLA and specific CLA isomers on a molecular level. However, the mixture of isomers when given at doses of up to 6 g/d for a period of 1 yr did not have any adverse effects in obese humans (37). Evidently the dose here contained the same level of t10,c12 CLA as that used by Riserus et al. (31) without any significant differences on HOMA-calculated insulin sensitivity. Furthermore, this was confiied in healthy overweight subjects in many studies (27,30,33,34,40).In addition, the data from Eyjolfson et al. (41) in sedentary subjects demonstrated a positive effect on insulin sensitivity using the 5050 mixture,
This would suggest that the two isomers have synergistic effects and that certain populations, in this case syndrome X, may be sensitive to the pure isomer form.
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Multicenter studies in defined populations are warranted, as are controlled studies for comparisons of the effects of different and well-defined (mixtures of) isomers and to completely understand populations who are good responders to the beneficial effects of CLA. Undoubtedly, the long-term safety of two independent trials for up to 1 yr with doses of 3.6 g/d in overweight subjects and 6 g/d in obese subjects demonstrated that the 5050 mixture is safe in these populations, which will encourage the production of CLA-enriched food products to help improve body composition. References 1. Chin, S.F., J.M. Storkson, W. Liu, K.J. Albright, and M.W. Pariza, Conjugated Linoleic - 10,12-0ctadecadienoic Acid) Is Produced in Conventional but Not Acid ( 9 ~ 1 and Germ-Free Rats Fed Linoleic Acid, J . Nutr. 124: 694-701 (1994). 2. Parodi, P.W., Conjugated Linoleic Acid: An Anticarcinogenic Fatty Acid Present in Milk Fat, Austr. J . Daily Technol. 49: 93-97 (1994). 3. Shantha, N.C., and E.A. Decker, Conjugated Linoleic Acid Concentrations in Processed Cheese Containing Hydrogen Donors, Iron and Dairy-Based Additives, Food Chem. 47: 257-261 (1993). 4. Shantha, N.C., L.N. Ram, J. O’Leary, C.L. Hicks, and E.A. Decker, Conjugated Linoleic-Acid Concentrations in Dairy-Products as Affected by Processing and Storage, J . Food Sci. 60: 695-697 (1995). 5 . Lin, H., T.D. Boylston, M.J. Chang, L.O. Luedecke, and T.D. Shultz, Survey of the Conjugated Linoleic Acid Contents of Dairy Products, J . Dairy Sci. 78: 2358-2365 (1995). 6. Fritsche, J., and H. Steinhart, Analysis, Occurrence, and Physiological Properties of trans Fatty Acids (TFA) with Particular Emphasis on Conjugated Linoleic Acid Isomers (CLA)-A Review, Fett-Lipid 100: 190-210 (1998). 7. Fritsche, J., H. Steinhart, V. Kardalinos, and G. Klose, Contents of trans-Fatty Acids in Human Substernal Adipose Tissue and Plasma Lipids: Relation to Angiographically Documented Coronary Heart Disease, Eur. J . Med. Res. 3: 401-406 (1998). 8. Fritsche, J., and H. Steinhart, Amounts of Conjugated Linoleic Acid (CLA) in German Foods and Evaluation of Daily Intake, Z. Lebensm-Unters. -Forsch A 206: 77-82 (1998). 9. Ip, C., M. Singh, H.J. Thompson, and J.A. Scimeca, Conjugated Linoleic Acid Suppresses Mammary Carcinogenesis and Proliferative Activity of the Mammary Gland in the Rat, Cancer Res. 54: 1212-1215 (1994). 10. Ip, C., C. Jiang, H.J. Thompson, and J.A. Scimeca, Retention of Conjugated Linoleic Acid in the Mammary Gland Is Associated with Tumor Inhibition During the PostInitiation Phase of Carcinogenesis, Carcinogenesis 18: 755-759 (1997). 11. Thompson, H., Z. Zhu, S. Banni, K. Darcy, T. Loftus, and C. Ip, Morphological and Biochemical Status of the Mammary Gland as Influenced by Conjugated Linoleic Acid: Implication for a Reduction in Mammary Cancer Risk, Cancer Res. 57: 5067-5072 (1997). 12. Lee, K.N., D . Kritchevsky, and M.W. Pariza, Conjugated Linoleic Acid and Atherosclerosis in Rabbits, Atherosclerosis 108: 19-25 (1994). 13. Nicolosi, R.J., E.J. Rogers, D. Kritchevsky, J.A. Scimeca, and P.J. Huth, Dietary
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14. Kritchevsky, D., S.A. Tepper, S. Wright, and S.K. Czarnecki, Influence of Graded Levels of Conjugated Linoleic Acid (CLA) on Experimental Atherosclerosis in Rabbits, Nutr. Res. 22: 1275-1279 (2002). 15. Houseknecht, K.L., J.P. Vanden Heuvel, S.Y. Moya-Camarena, C.P. Portocmero, L.W. Peck, K.P. Nickel and M.A. Belury, Dietary Conjugated Linoleic Acid Normalizes Impaired Glucose Tolerance in the Zucker Diabetic Fatty fa@ Rat, Biochem. Biophys. Res. Commun. 244: 678-682 (1998). 16. Ryder, J.W., C.P. Portocarrero, X.M. Song, L. Cui, M. Yu, T. Combatsiaris, D. Galuska, D.E. Bauman, D.M. Barbano, M.J. Charron, J.R. Zierath, and K.L. Houseknecht, Isomer-Specific Antidiabetic Properties of Conjugated Linoleic AcidImproved Glucose Tolerance, Skeletal Muscle Insulin Action, and UCP-2 Gene Expression, Diabetes 50: 1149-1 157 (2001). 17. O’Shea, M., J. Bassaganya-Riera, and I.C.M. Mohede, Immunomodulatory Properties of Conjugated Linoleic Acid, Am. J . Clin. Nutr. 79 (Suppl.): 1199s-1206s (2004). 18. Albers, R., R.P.J. van der Wielen, E J . Brink, H.FJ. Hendriks, V.N. Dorovska-Taran, and I.C.M. Mohede, Effects of cis-9, trans-11 and trans-10, cis-12 Conjugated Linoleic Acid (CLA) Isomers on Immune Function in Healthy Men, Eur. J . Clin. Nub. 57: 595-603 (2003). 19. Turek, J.J., Y. Li, I.A. Schoenlein, K.G.D. Allen, and B.A. Watkins, Modulation of Macrophage Cytokine Production by Conjugated Linoleic Acids Is Influenced by the Dietary n-6:n-3 Fatty Acid Ratio, J. Nutr. Biochem. 9: 258-266 (1998). 20. Hayek, M.G., S.N. Han, D. Wu, B.A. Watkins, M. Meydani, J.L. Dorsey, D.E. Smith, and S.N. Meydani, Dietary Conjugated Linoleic Acid Influences the Immune Response of Young and Old C57BL/6NCrlBR Mice, J . Nutr. 129: 32-38 (1999). 21. Wong, M.W., B.P. Chew, T.S. Wong, H.L. Hosick, T.D. Boylston, and T.D. Shultz, Effects of Dietary Conjugated Linoleic Acid on Lymphocyte Function and Growth of Mammary Tumors in Mice, Anticancer Res. 17: 987-993 (1997). 22. Yang, M., M.W. Pariza, and M.E. Cook, Dietary Conjugated Linoleic Acid Protects Against End Stage Disease of Systemic Lupus Erythematosus in the NZB/W F1 Mouse, Immunopharmacol. Immunotoxicol. 22: 433-449 (2000). 23. Cook, M.E., C.C. Miller, Y. Park, and M.W. Pariza, Immune Modulation by Altered Nutrient Metabolism: Nutritional Control of Immune-Induced Growth Depression, Poult. Sci. 72: 1301-1305 (1993). 24. Miller, C.C., Y. Park, M.W. Pariza, and M.E. Cook, Feeding Conjugated Linoleic Acid to Animals Partially Overcomes Catabolic Responses Due to Endotoxin Injection, Biochem. Biophys. Res. Commun. 198: 1107-1 112 (1994). 25. Delany, J.P., F. Blohm, A.A. Tmett, J.A. Scimeca, and D.B. West, Conjugated Linoleic Acid Rapidly Reduces Body Fat Content in Mice Without Affecting Energy Intake, Am. J . Physiol. 276: R1172-R1179 (1999). 26. West, D.B., J.P. Delany, P.M. Camet, F. Blohm, A.A. Truett, and J. Scimeca, Effects of Conjugated Linoleic Acid on Body Fat and Energy Metabolism in the Mouse, Am. J . Physiol. 275: R667-R672 (1998). 27. Blankson, H., J.A. Stakkestad, H. Fagertun, E. Thom, J. Wadstein, and 0. Gudmundsen, Conjugated Linoleic Acid Reduces Body Fat Mass in Overweight and Obese Humans, J.Nutr. 130: 2943-2948 (2000). 28. Malpuech-Brugere, C., W.P.H.G. Verboeket-van de Venne, R.P. Mensink, M.A. Arnal, B. Morio, M. Brandolini , A. Saebo, T.S. Lassel, J.M. Chardigny, J.L. SCbtdio, and B.
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Acids. IV. Substrate Specificity and Inhibition of Linoleate 12 cis, 11 m n s Isomerase from Butyrivibriofibrisolvens,J . Biol. Chem. 245: 3612-3620 (1970).
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44. Kepler, C.R., K.P. Hirons, J.J. McNeill, and S.B. Tove, Intermediates and Products of the Biohydrogenation of Linoleic Acid by Butyrivibrio fibrisolvens, J. Biol. Chem. 241: 1350-1354 (1966). 45. Nobel, R.C., J.H. Moore, and C.G. Harfoot, Observations on the Pattern on Biohydrogenation of Esterified and Unesterified Linoleic Acid in the Rumen, Br. J. Nutr. 31: 99-108 (1974). 46. Gerson, T.A., R. John, and A.S.D. King, The Effects of Dietary Starch and Fibre on the In Vitro Rates of Lipolysis and Hydrogenation in Sheep Rumen Digesta, J. Agric. Sci. 105: 27-30 (1985). 47. Gerson, T.A., R. John, and B.R. Sinclair, The Effect of Dietary N on In Vitro Lipolysis and Fatty Acid Hydrogenation in Rumen Digesta from Sheep Fed Diets High in Starch, J. Agric. Sci. 101: 97-101 (1983). 48. Griinari, J.M., P.Y. Chouinard, and D.E. Bauman, Trans Fatty Acid Hypothesis of Milk Fat Depression Revised. Proc. Cornell Nutr. Conf. Feed Manuf. Ithaca, NY, 1997, pp. 208-216. 49. Griinari, J.M., and D.E. Bauman, Biosynthesis of Conjugated Linoleic Acid and Its Incorporation into Meat and Milk in Ruminants, in Advances in Conjugated Linoleic Acid Research, Volume I , edited by M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, and G.J. Nelson, AOCS Press, Champaign, IL, 1999, pp. 180-200. 50. Ulberth, F., and M. Henninger, Quantitation of trans Fatty Acid in Milk Fat Using Spectroscopic and Chromatographic Methods, J. Dairy Res. 61: 517-527 (1994). 51. Yurawecz, M.P., J.A.G. Roach, N. Sehat, M.M. Mossoba, J.K.G. Kramer, J. Fritsche, H. Steinhart, and Y. Ku, A New Conjugated Linoleic Acid Isomer, 7 trans, 9 cisOctadecadienoic Acid, in Cow Milk, Cheese, Beef and Human Milk and Adipose Tissue, Lipids 33: 803-809 (1998). 52. Jahreis, G., J. Fritsche, and H. Steinhart, Conjugated Linoleic Acid in Milk Fat-High Variation Depending on Production System, Nutr. Res. 17: 1479-1484 (1997). 53. Jiang, J., L. Bjorck, and R. Fonden, Occurrence of Conjugated 9,ll-Octadecadienoic Acid (CLA) in Bovine Milk: Effects of Diet and Feeding Regime, J. Dairy Sci. 79: 4 3 8 4 4 5 (1996). 54. Precht, D., and J. Molkentin, Effect of Feeding on Conjugated cis Delta-9, trans Delta11-Octadecadienoic Acid and Other Isomers of Linoleic Acid in Bovine Milk Fats, Nahrung Food 41: 330-335 (1997). 55. Santora, J.E., D.L. Palmquist, and K.L. Roehrig, trans-Vaccenic Acid Is Desaturated to Conjugated Linoleic Acid in Mice, J. Nutr. 130: 208-215 (2000). 56. Salminen, I., M. Mutanen, M. Jauhiainen, and A. Aro, Dietary trans Fatty Acids Increase Conjugated Linoleic Acid Levels in Human Serum, Nutr. Biochem. 9: 93-98 (1998). 57. Chin, S.F., W. Liu, J.M. Storkson, Y.L. Ha, and M.W. Pariza, Dietary Sources of Conjugated Dienoic Isomers of Linoleic Acid, a Newly Recognized Class of Anticarcinogens, J. Food Compos. Anal. 5: 185-197 (1992). 58. Herbel, B.K., M.K. McGuire, M.A. McGuire, and T.D. Schultz, Safflower Oil Consumption Does Not Increase Plasma Conjugated Linoleic Acid Concentrations in Humans, Am. J. Clin. Nutr. 67: 332-337 (1998). 59. Ritzenthaler, K., M.K. McGuire, R. Falen, T.D. Schultz, and M.A. McGuire, Estimation of Conjugated Linoleic Acid (CLA) Intake, FASEB J. 12: A527 (Abstr.) (1998). 60. Adlof, R., W. Eberhardt, H. Heseker, S. Hartmann, A. Herwig, B. Matiaske, K J . Moche. R. Schneider, and W. Kuebler, kbensmittel und NaehrstofSaufnahmeinder Bundesrepublic
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Deutschland, edited by W. Kuebler, H J . Anders, and W. Heeschen, VERA Schriftenreihe, Band XII, WissenschaftlicherFachverlag Dr. Fleck, 1994, Niderkleen. 61. McGuire, M. K., and M.A. McGuire, Dietary CLA Intake in Humans: What We Know and What We Should Know, in Perspectives on Conjugated Linoleic Acid Research, Current Status and Future Directions, Bethesda, 2002. http://ods.od.nih.gov/News/ CLA-Conference .aspx 62. Park, Y., M.K. McGuire, R. Behr, M.A. McGuire, M.A. Evans, and T.D. Schultz, HighFat Dairy Product Consumption Increases Delta 9c ,11t- 18:2 (Rumenic Acid) and Total Lipid Concentrations of Human Milk, Lipids 34: 543-549 (1999). 63. Huang, Y.C., L.O. Ludecke, and T.D.Schultz, Effect of Cheddar Cheese Consumption on Plasma Conjugated Linoleic Acid Concentrations in Men, Nutr. Res. 14: 373-386 ( 1994). 64. Jiang, J., A. Wolk, and B. Vessby, Relation Between the Intake of Milk Fat and the Occurrence of Conjugated Linoleic Acid in Human Adipose Tissue, Am. SOC.Clin. Nutr. 70: 21-27 (1999). 65. Gaullier, J.M., G. Berven, H. Blankson, and 0. Gudmundsen, Clinical Trial Results Support a Preference for Using CLA Preparations Enriched with Two Isomers Rather Than Four Isomers in Human Studies, Lipids 37: 1019-1025 (2002). 66. Loders Croklaan BV, U S . Patent 6160140 B1 (2000). 67. Loders Croklaan BV, U.S. Patent 6271404 B1 (2001). 68. Natural ASA, US.Patent Application 20040058998 A1 (2004). 69. Riserus, U., A. Smedman, S. Basu, and B. Vessby, Metabolic Effects of Conjugated Linoleic Acid in Humans: The Swedish Experience, Am. J . Clin Nutr. 79: 1146s-1148s (2004). 70. Basu, S., U. Riserus, A. Turpeinen, and B. Vessby, Conjugated Linoleic Acid Induces Lipid Peroxidation in Men with Abdominal Obesity, Clin. Sci. 99: 51 1-516 (2000). 71. Basu, S., and M. Eriksson, Conjugated Linoleic Acid Induces Lipid Peroxidation in Humans, FEBS Lett. 438: 159-160 (1998). 72. Basu, S., and M. Eriksson, Lipid Peroxidation Induced by an Early Inflammatory Response in Endotoxaemia, Acta Anaesthesiol. Scand. 44: 17-23 (2000). 73. Basu, S., Oxidative Injury Induced Cyclooxygenase Activation in Experimental Hepatotoxicity, Biochem. Biophys. Res. Commun. 254: 764-767 (1999). 74. Kamphuis, M.M., M.P. Lejeune, W.H. Saris, and M.S. Westerterp-Plantenga, Effect of Conjugated Linoleic Acid Supplementation After Weight Loss on Appetite and Food Intake in Overweight Subjects, Eur. J. Clin. Nutr. 57: 1268-1274 (2003). 75. Matthews, D.R., J.P. Hosker, A.S. Rudenski, B.A. Naylor, D.F. Treacher, and R.C. Turner, Homeostatis Model Assessment: Insulin Resistance and P-Cell Function from Fasting Plasma Glucose and Insulin Concentrations in Man, Diabetologia 28: 412-419 (1985). 76. Masters, N., M.A. McGuire, K.A. Beerman, N. Dasgupta, and M.K. McGuire, Maternal Supplementation with CLA Decreases Milk Fat in Humans, Lipids 37: 133-138 (2002). 77. Baumgard, L.H., E. Matitashvili, B.A. Corl, D.A. Dwyer, and D.E. Bauman, Trans-10, cis- 12 Conjugated Linoleic Acid Decreases Lipogenic Rates and Expression of Genes Involved in Milk Lipid Synthesis in Dairy Cows, J. Dairy Sci. 85: 2155-2163 (2002). 78. Benito, P., G.J.Nelson, D.S. Kelley, G. Bartolini, P.C. Schmidt, and V. Simon, The
Effect of Conjugated Linoleic Acid on Plasma Lipoproteins and Tissue Fatty Acid Composition in Humans, Lipids 36: 229-236, correction Lipids 36: 857 (2001).
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79. Kelley, D.S., P.C. Taylor, I.L. Rudolph, P. Benito, G.J. Nelson, B.E. Mackey, and K.L. Erickson, Dietary Conjugated Linoleic Acid Did Not Alter Immune Status in Young Healthy Women, Lipids 35: 1065-1071 (2000). 80. Medina, E.A., W.F. Horn, N.L. Keim , P.J. Havel, P. Benito, D.S. Kelley, G.J. Nelson, and K.L. Erickson, Conjugated Linoleic Acid Supplementation in Humans: Effects on Circulating Leptin Concentrations and Appetite, Lipids 35: 783-788 (2000). 81. Zambell, K.L., N.L. Keim, M.D. Van Loan, B. Gale, P. Benito, D.S. Kelley, and G.J. Nelson, Conjugated Linoleic Acid Supplementation in Humans: Effects on Body Composition and Energy Expenditure, Lipids 35: 777-782 (2000). 82. Lowery, L.M., P.A. Appicelli, and P.W.R. Lemon, Conjugated Linoleic Acid Enhances Muscle Size and Strength Gains in Novice Body Builders, Med. Sci. Sports Exerc. 30: S182 (Abstr.) (1998). 83. Von Loeffelholz, C., J. Kratzsch, and G. Jahreis, Influence of Conjugated Linoleic Acid on Body Composition and Selected Serum and Endocrine Parameters in ResistanceTrained Athletes, Eur. J . Lipid Sci. Technol. 105: 251-259 (2003). 84. Kreider, R.B., M.P. Ferreira, M. Greenwood, M. Wilson, and A.L. Almada, Effects of Conjugated Linoleic Acid Supplementation During Resistance-Training on Body Composition, Bone Density, Strength, and Selected Hematological Markers, J . Strength Cond. Res 3: 325-334 (2002). 85. Riserus, U., S . Basu, S . Jovinge, G.N. Fredrikson, J. Arnlov, and B. Vessby, Supplementation with Conjugated Linoleic Acid Causes Isomer-Dependent Oxidative Stress and Elevated C-Reactive Protein-A Potential Link to Fatty Acid-Induced Insulin Resistance, Circulation 106: 1925-1929 (2002). 85. Ha, Y.L., N.K. Grimm, and M.W. Pariza, Newly Recognised Anticarcinogenic Fatty Acids: Identification and Quantification in Natural and Processed Cheeses, J . Agric. Food Chem. 37: 75-81 (1989). 86. Garcia-Lopez, S . , E. Echeverria, I. Tsui, and B. Balch, Changes in the Content of Conjugated Linoleic Acid (CLA) in Processed Cheese During Processing, Food Res. Zntl. 27: 61-64 (1994). 87. Werner, S.A., L.O. Luedecke, and T.D. Shultz, Determination of Conjugated Linoleic Acid Content and Isomer Distribution in Three Cheddar-Type Cheeses: Effects of Cheese Cultures, Processing and Aging, J . Agric. Food Chem. 40: 1817-1821 (1992). 89. Shantha, N.C., A.D. Crum, and E.A. Decker, Evaluation of Conjugated Linoleic Acid Concentrations in Cooked Beef, J . Agric. Food Chem. 42: 1757-1760 (1994). 90. Ip, C., S.F. Chin, J.A. Scimeca, and M.W. Pariza, Mammary Cancer Prevention by Conjugated Dienoic Derivative of Linoleic Acid, Cancer Res. 51: 61 18-6124 (1991).
Chapter 13
Lipids with Antioxidant Properties Jan Pokorn): and Jana Parkdnyiovd Department of Food Chemistry and Analysis, Institute of Chemical Technology (Technical University), CZ-166 28 Prague, Czech Republic
Introduction Lipids contain unsaturated fatty acids (FA), which are oxidized by oxygen in the air into hydroperoxides. Dietary oils, recommended for healthful nutrition, often contain high amounts of polyunsaturated fatty acids (PUFA). These FA, such as linoleic or linolenic acid, are particularly sensitive to oxidation (1). Lipid hydroperoxides are very unstable, especially at higher temperatures. They are dissociated into free radicals and decomposed by several consecutive reactions, forming volatile and nonvolatile aldehydes, alcohols, and ketones. Another possibility is the transformation of free radicals into oxypolymers. The oxidation products are toxic when ingested in large amounts. Essential PUFA bound as esters in lipid triacylglycerols (TAG) lose their physiologic activity. Minor lipids, such as sterols or lipophilic vitamins, are also oxidized into inactive or even toxic products. If foods containing PUFA and their oxidation products are ingested, the oxidation may proceed in vivo. Free radicals and their reaction products participate in the development of atherosclerosis, cancer, and other diseases. Therefore, it is very important to protect edible lipids from oxidative changes, especially by antioxidants.
Protection of Lipids by Antioxidants Antioxidants inhibit lipid oxidation via several mechanisms (2). Polyphenolic substances react with free radicals produced by the decomposition of lipid hydroperoxides, reducing them to nonradical products (Fig. 13.1A). During the reaction, antioxidants are converted to free radicals as shown in Figure 13.1. The antioxidant free radicals are much less reactive, however, than lipid free radicals; thus, they cannot produce appreciable amounts of free lipid radicals. They are likely to react with another antioxidant free radical, forming dimers (Fig. 13.1B), or with a lipidic free radical, forming a copolymer (Fig. 13.1C) . Another mechanism of antioxidant activity is the protection of lipid hydroperoxides against their decomposition to free radicals. The free-radical formation is catalyzed by metal ions with transient valency, such as copper, iron, or manganese.
Therefore, the metal-chelating substances, such as phosphoric or citric acid, also protect lipids against oxidation. 2 73
1. Pokornyand 1. Parka’nyiovd
2 74
R-OOH
(A)
R-0.
H0.
%*
R-OH
A.
(6) 2Ao
(C) A.
___)
A-A
R-O.
A-OR
Fig. 13.1. Mechanism of reactions between lipid hydroperoxides (R-OOH)and antioxidants (AH).
Antioxidants do not stop the oxidation completely; rather, they reduce the rate of oxidation. In the beginning of storage or heating, small amounts of free radicals are produced, but they are rapidly neutralized by antioxidants present in the system. Antioxidants are slowly consumed during the reaction. This period of slow oxidation is called the induction period. The activity of antioxidants may be measured by determination of the length of the induction period. When antioxidants are completely destroyed, free radicals are no longer eliminated from the system. They are accumulating and autocatalytically increase the reaction rate. This period is called the stage of rapid oxidation. The induction period is not a linear function of the concentration of an antioxidant. At low concentrations, the induction period increases with rising concentration. At high concentrations of antioxidants, however, the activity becomes lower. At very high concentrations, the activity may even decrease in some cases, and the antioxidant becomes a prooxidant. Therefore, it is not reasonable to add large amounts of antioxidants. If lipids are stabilized with a mixture of antioxidants, the resulting activity is not always the sum of contributions of individual components. Either they do not interact with one another, or more often, the resulting antioxidant activity is greater than expected (synergism of the antioxidants) or it is lower (antagonism of the antioxidants). Apart from the chemical structure and concentration of individual antioxidants in a system, the antioxidant activity depends on several other factors, such as the degree of lipid unsaturation, temperature, partial oxygen pressure, the presence of prooxidants and metal chelators, the presence of proteins and other food components, and the method of assessing the antioxidant activity. Some fats, oils, and foods already contain natural antioxidants in sufficient amounts to guarantee the expected shelf-life; in other cases, however, e.g., in systems high in PUFA and low in natural antioxidants, the application of additional antioxidants is advisable. Until the last two or three decades, synthetic antioxidants were preferred because they are inexpensive, pure, and their safety has been tested by sophisticated methods. They have been approved by authorities, and are still widely used for the stabilization of many industrial products (3). The use of synthetic antioxidants in foods depends on the legislation of the particular country. Some
Lipids with Antioxidant Properties
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antioxidants previously used are no longer allowed because of negative results from more modern safety tests. The amount of antioxidants, added alone or in a mixture, should not exceed 0.02%,based on the fat content. The majority of consumers now prefer natural antioxidants, mainly for ideological reasons; therefore, we shall discuss the stabilization of food lipids with natural antioxidants. Mixtures of synthetic and natural antioxidants are sometimes used; identical substances of synthetic nature or antioxidants synthesized by biochemical processes may be applied.
Occurrence of Native Antioxidants in Edible lipids Natural Content of Antioxidants in Edible Fats and Oils
Lipidic products produced and recommended for human nutrition should be sufficiently stable against oxidation. High stability is particularly expected in the case of healthful lipids. The resistance against oxidation depends on the FA composition (4). Animal fats, such as lard or butter, have a high saturated FA content and low unsaturated FA content, but they contain only trace amounts of antioxidants. They are efficiently stabilized against oxidation by the addition of either synthetic or natural antioxidants. On the contrary, vegetable oils are more unsaturated, but they generally contain natural antioxidants, most often tocopherols. Some vegetable oils are more stable than would be expected given the unsaturation and tocopherol content because they contain specific antioxidants in addition to tocopherols. The natural content of antioxidants is important for the choice of healthful lipids. Tocopherols and Tocotrienols as Antioxidants
All vegetable oils contain tocopherols as antioxidants, at least in small amounts. Tocopherols are chroman derivatives (Fig. 13.2), substituted with a hydroxyl group and with one, two, or three methyl groups in the phenolic cycle. They also contain a long terpenic side-chain. The side chain is either saturated (in tocopherols), diunsaturated and monounsaturated (present in rare cases in food materials), or triunsaturated (tocotrienols). From the standpoint of substitution with methyl groups, several tocopherols are possible, but only four (Fig. 13.2) exist in edible oils. The role of tocopherols with respect to the antioxidant activity was discussed by Kamal-Eldin and Appelqvist ( 5 ) . The real antioxidant activity of tocopherols in edible oils is evident when tocopherols are added to vegetable oil TAG, prepared by stripping the respective oil with water vapor to remove natural tocopherols (6).The activity is more pronounced than would be expected. The tocopherol antioxidant activity is due not only to the deactivation of free radicals produced by the decomposition of lipid hydroperoxides, but also to the inhibition of lipid hydroperoxide decomposition (7). The antioxidant activity in a vegetable oil increases as follows: a-tocopherol < P-tocopherol = y-tocopherol < &tocopherol
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CH,
R = tocopherol
tocotrienol
Fig. 13.2. Chemical structures of tocopherols and tocotrienols.
Trolox, a synthetic compound, has a basic structure similar to that of tocopherols, but it is not substituted with a terpenic side-chain, making it much more polar than the tocopherols. All tocopherols are viscous liquids, well miscible with edible oils. During the extraction of oils from oilseeds, tocopherols are coextracted, even when they are not dissolved in oil droplets in the original material. The content and composition of tocopherols depend on the source of the oils. They often occur in smaller amounts in more saturated oils, such as coconut oil, and in higher amounts in highly polyunsaturated oils (Table 13.1). Corn germ oil and soybean oil are particularly rich in y-tocopherol. Tocotrienols are present in palm oil (4-36 mg of a-tocotrienol, but 14-710 mg of y-tocotrienol, and up to 377 mg of 8-tocotrienol in 1 kg), in cereals, and other sources. The natural tocopherol content in oils is near the optimum from the standpoint of antioxidant activity; therefore, further addition of tocopherols does not substantially increase the oxidative stability of oil. An extremely high tocopherol content may even decrease the stability against oxidation under storage conditions (8).Prooxidant activity of tocopherols in extreme concentrations was observed in lipid emulsions (9). During crude oil refining, vegetable oil is treated with superheated steam under low pressure in the deodorization step. Tocopherols are partially lost under these conditions, e.g., in the case of deodorization of zero-erucic rapeseed oil at 230°C, the losses were as follows: 36.3% a-tocopherol, 41.8% y-tocopherol, and 48.7% 8-tocopherol, 39.9% total tocopherols (10). There was no significant difference in losses during alkali refining and physical refining. In a similar plant-scale experiment in Poland, losses of a-tocopherol and y-tocopherol were the same (30.2%), but losses of 8-tocopherol were significantly higher (11). Because tocopherol losses depend on the technological conditions, it is desirable to verify the actual tocopherol content in edible oils recommended as healthful lipids or in other cases, when the oxidative stability is important. Among other factors, tocopherol losses contribute to the lower oxidative stability of refined oils compared with crude oils. In the deodorization process, volatiles are collected, and tocopherols may be recovered from deodorization sludges. They are used as natural antioxidants in foods, especially in animal products, which contain practically no natural antioxidants.
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TABLE 13.1 Tocopherol Concentration in Vegetable Oils (rng/kgla ~~
Oil Corn Cottonseed Linseed Olive Peanut Rapeseed Safflower Sesame Soybean Sunflower
a
13
23-573 136-674 5-1 0 85-1 00 403-935 105-1 58 234-660 0-3 9-352 403-935
0-359 0-2 9
0-41 34 0-1 7 0
0-3 6 045
Y
268-2468 138-746 430-575 10 88-389 652-788 0-1 2 52 1-983 89-2 3 07 0-34
6 23-75 0-2 1 4-8
0-7 222-31 5 0
4-21 1 0 0-7
Total
331-3716 389-1 185 440-588 70-1 50 176-1291 1165 2 4 6-6 64 531-1 003 601-3363 447-1 5 14
aSources:Reference 126 and unpublished results.
Tocopherols also have vitamin E activity, with a-tocopherol possessing the highest value. Therefore, they are often added to refined vegetable oils to improve their vitamin value. Synthetic a-tocopherol, which is identical in nature, is used for this purpose, usually as an acetate. Tocopherol acetate is more stable against oxidative deterioration during storage of vegetable oils than free tocopherol. It possesses no antioxidant activity in foods, but when such fortified oil has been ingested, tocopherol acetate is cleaved by digestive enzymes into acetic acid and free a-tocopherol. It is then absorbed and can act as both a vitamin and an antioxidant in blood plasma. Unlike tocopherols in deodorization sludges, synthetic a-tocopherol has no optical activity. The antioxidant efficiency of the nature-identical synthetic tocopherols is the same as that of natural, optically active tocopherols, but the vitamin activity is lower. During storage of vegetable oils, tocopherols inactivate free radicals. They are converted to free radicals, like other antioxidants, Free radicals then polymerize to a mixture of dimers and higher oligomers. These oligomeric compounds also possess moderate antioxidant activity. Copolymers of tocopherol free radicals with free radicals generated by decomposition of lipid hydroperoxides are also formed (12). If oil or foods contain iron or copper ions, they oxidize tocopherols with the formation of very reactive quinones, which easily polymerize or copolymerize. The antioxidant activity of tocopherols is substantially increased by the addition of ascorbyl palmitate. During the process of deodorization, citric acid is often added to hot oil, which also improves the antioxidant activity of the tocopherols left in oil after deodorization. Antioxidants of Sesame Oil and linseed Oil
Sesame (Sesamum indicum L.) seeds are traditional food components in Asian countries; for -100 years, they have also been consumed in the United States and Europe. Sesame oil is also used as an edible oil. It offers the advantage of good
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resistance against oxidative deterioration, caused by the presence of lignans and their degradation products. Lignans present in sesame seeds, mainly sesaminol, are derivatives of ferulic acid. They are decomposed during the roasting process into sesamin, samin, sesamolin, sesaminol, and sesamol (Fig. 13.3), which is the best known compound of this group. Sesamol and sesaminol are active free radical quenchers (13). Both compounds are formed from sesamolin. Sesamol was more active in sunflower oil than sesaminol, or sesamine (14). Another decomposition product is trans-ferulic acid (15). Sesame oil is sometimes mixed with other edible oils to improve their stability at higher temperature, e.g., GoodFry oil is a mixture of 94% low-linoleichigh-oleic sunflower oil, sesame oil, and rice bran oil (16). The methanol extract of defatted sesame flour contains 41 mgkg free phenolic acids, 324 mgkg esterified phenolic acids, and 14 mgkg insoluble phenolic acids (17). Cultivated flax (Linum usitutissimum L.) seeds have been used as a source of edible oil for several thousand years. The oil contains a high level of linolenic acid; thus, it is now used only very rarely as an edible oil because of rapid rancidification. Linseeds are a rich source of lignans that are related in their chemical structure to sesame seed lignans. The most important derivatives are secoisolariciresinol and matairesinol, esters of ferulic acid with sterol glycosides. The secoisolariciresinol diglucoside content varied between 11.7 and 24.1 g k g in defatted linseed flour, and between 6.1 and 13.3 g k g in whole seeds (18). Among free phenolic acids, sinapic, p-hydroxybenzoic, coumaric, and ferulic acids dominated (17). Antioxidants in Olive Oil
Olive (Oleu europuea L.) fruits have always been consumed in the Mediterranean area and have become a common food in most countries around the world. For the produc-
0P L
O
H
O
sesamol
Fig. 13.3. Sesame oil antioxidants.
Lipids with Antioxidant Properties
279
R’ OH H O C H r CHz6
\ /O
0 H3CO-i&
CH,C-0-CH,-CH, 0 II &OH H-CH,
H
0-x R’ = H; OH X = Glc: ligstroside; oleoeuropein X = H: ligstroside aglycone;
hydroxytyrosol
oleoeuropein aglycone Fig. 13.4. Olive oil antioxidants.
tion of olive oil, the pericarp is cold-pressed. The crude, unrefined product is called virgin olive oil, and the highest quality olive oil is qualified as extra-virgin oil. The tocopherol content of olive oil is low, but both olive fruits and virgin olive oil contain a mixture of other antioxidants, which are derivatives of hydroxytyrosol (Fig. 13.4). The most important representatives are secoiridoids, especially oleoeuropein (Fig. 13.4), the aglycone of ligstroside, and their respective dialdehyde derivatives. Flavonoids, such as rutin and luteoline-7-glucoside, and lignans, such as acetoxypinoresinol and pinoresinol, and phenolic acids are also present. The polyphenol content in extra-virgin olive oils was reported to be as high as 48 mgkg, compared with sesame (6 mgkg) or soybean (4 mgkg) oils (19). In another experiment (20), the content of phenolics in virgin olive oil was 4-6 times higher than in refined olive oils. All of these compounds are efficient antioxidants in edible oils (21,22). The antioxidant activity in emulsions of tocopherol-free olive oil ranks as follows: 3,4-dihydroxyelenolicacid > its dialdehyde > oleuropein = a-tocopherol >> control (23). Olive oil antioxidants are also active in vivo as free radical scavengers (24). Virgin (unrefined) olive oils have high stability against oxidation, yet they should be protected against sunlight because they contain chlorophylls, which catalyze photooxidation of unsaturated FA. In addition, chlorophylls impart the greenish color tint to virgin oils. Antioxidants of Rice Bran Oil
Rice (Oryza sutivu L.) bran, obtained during the rice dehulling, is a material less rich in oil than oilseeds, but it is still economically advantageous to process the bran to produce oil, which has good flavor, a high content of unsaturated FA, and high stability against oxidation,The stability is due in part to tocopherols, but also to y-oryzanol. It is a mixture of several closely related substances, consisting of steryl ferulates (Fig.
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13S),i.e., cycloartenyl ferulate, 24-methylenecycloartenyl ferulate, and campesteryl ferulate (25). The first-mentioned compound is the major component. Oryzanol was found to be an efficient free radical scavenger, tested with 2,2'-azobis (2-methylpropionamide) dihydrochloride. Its concentration is particularly high in the outer layers of brans, more than 10 times higher than that of tocopherols. Traces of wax from the surface layer of brans are also present in the oil. Both rice wax residues and y-oryzanol were shown to be active antioxidants in peanut oil (26). Carotenes and Carotenoids as Antioxidants
Carotenes (hydrocarbons) and related xanthophylls (containing an additional oxygen atom) are components of many fruits, vegetables, and oilseeds. Because of their nonpolar character, they are co-extracted by hexane with TAG from oilseeds. Therefore, they are minor components of almost all crude vegetable oils. Palm oil is particularly rich in carotenes (-0.054.20%), which impart a specific red color to the oil. Other crude oils, such as rapeseed oil, are also rich in carotenes and xanthophylls. Consumers dislike colored edible oils; therefore, carotenes and xanthophylls are removed in the process of refining at the stage of bleaching. For specific dietetic oils, however, carotene pigments are desirable; therefore, carotenes removed in the bleaching process are restored by the addition of synthetic pcarotene or a mixture of carotenoid pigments. Several refined palm oil products with a reddish tint are available on the market. The role of carotenes and xanthophylls as antioxidants is important in photooxidation, especially in the presence of chlorophyll pigments (27). During photooxidation, catalyzed by chlorophylls, triplet oxygen commonly present in the air is converted into singlet oxygen, which is much more reactive. It has several hundred times greater ability to oxidize double bonds than triplet oxygen. Carotenoid pigments convert singlet oxygen back to the less active triplet stage. The activity of xanthophylls is similar to that of carotenes (28). The structurally similar vitamin A is also active, but it is present only in trace amounts in animal tissues, and it is absent in plant tissues. The quenching efficiency depends on the number of conjugated double bonds in the molecule. Carotenes are also able to inhibit free radical reactions (29). Phytosterols as Antioxidants
All vegetable oils contain up to 1% sterols; these sterols are called phytosterols. They are oxidized via free radicals similarly to unsaturated FA into ketones,
A4
HO
Fig. 13.5. Major component of y-oryzanol.
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A5-avenasterol
281
A7-avenasterol
Fig. 13.6. Sterols possessing antioxidant activity.
hydroxylic and epoxy pigments. Avenasterols possess antioxidant activity. Both A5- and A7-avenasterols (Fig. 13.6) contain a (24a-24-ethylidene group in the side chain, which forms a relatively stable free vinyl radical and can thus inhibit oxidation. Greek olive oils are particularly rich in As-avenasterol; values as high as 36% total sterols were reported (22). Avenasterol contributes to the stability of olive oil as a deep-frying oil (30).
Phenolic Substances Suitable as Natural Food Antioxidants Chemical Structures of Natural Antioxidants in Plants
The oxidative stability of vegetable oils or their mixtures is often not sufficient in spite of the presence of tocopherols and other natural antioxidants. The application of additional antioxidants is desirable in such cases. Synthetic antioxidants can be used, but natural antioxidants are preferred in dietary oils and other healthful lipid products (3 1). Nearly all plant materials contain phenolic substances possessing antioxidant activities. They most often contain two or three hydroxyl groups in a benzene ring, which are in an ortho- or a para-position, whereas meta-disubstituted derivatives are normally quite inefficient. A hydroxyl group may by replaced with a methoxy group or an alkyl group. The content of active phenolic compounds in plants may be enriched using enzymatic methods (32). The simplest natural antioxidants are phenolic acids, belonging either to the benzoic acid series or the cinnamic acid series. Vanillic (4-hydroxy-3-methoxybenzoic) acid, syringic (4-hydroxy-3,5-dimethoxybenzoic) acid, and gallic (3,4,5-trihydroxybenzoic) acid are the most important representatives of the benzoic acid series. Gallic acid is a rather polar compound; it is only sparingly soluble in fats and oils. Therefore, esters are often added to fats and oils instead of the free acid, e.g., propyl, tert-octyl or tert-dodecyl gallates. During digestion, they are cleaved into the respective alcohol and free gallic acid. Gallic acid esters are almost exclu-
sively synthetic compounds, but they are permitted for food use in most countries because gallic acid is a natural food component.
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The cinnamic acid series has an additional double bond in the side-chain. The simplest representative of the cinnamic acid series is p-coumaric (4-hydroxycinnamic) acid; however, it is almost inactive in fats and oils. Ferulic (4-hydroxy-3methoxycinnamic) or sinapic (4-hydroxy-3,5-dimethoxycinnamic) acids are more active. They often occur as esters or glycosides, e.g., in oat lipids, they are bound to a variety of lipidic and other components (33). Oat diferulates are bound mainly to long-chain diols; thus, the resulting esters are lipophilic (34). The most active and frequently encountered antioxidant in this group of compounds is caffeic (3,4dihydroxycinnamic) acid, often bound as esters to quinic acid. Several isomers are possible. These esters are called chlorogenic acid. They widely occur in edible plants and are the main precursors of enzymic browning of food products, e.g., in apples, potatoes, or bananas. Coumarins are lactones of cis-o-hydroxycinnamic acids. Furocoumarins (psoralens) and pyranocoumarins belong to the most important species in this class of compounds (35). They occur mamly in plants of the Umbelliferae and Rutaceae families and possess moderate antioxidant activities, e.g., in some spices. Another widely occurring large group of phenolic compounds is flavonoids. Several thousand flavonoids have been identified in plants. Their structures are discussed in any textbook of food chemistry; thus, only a few examples will be given here. They were reviewed from the standpoint of a lipid scientist (36). Flavonoids have a configuration fitting the schema: C6-C3-C6 (Fig. 13.7), in which the 3-carbon unit may have different structures, such as a pyrane or a pyrone ring. Flavones, flavonols, flavanones, flavanols, and flavans are the most important flavonoid classes. Flavones have a carbonyl group on the carbon atom 4; flavonols have an additional hydroxyl group on the carbon atom 3; flavanones have a carbonyl group on the carbon atom 4, but no double bond between the carbon atoms 2 and 3; flavanols have also no double bond between carbon atoms 2 and 3, but have a hydroxyl group on the 3-carbon atom in the 3-carbon ring (Fig. 13.7). The 3-carbon ring is open in chalcones and dihydrochalcones. Flavonoids may occur either free or bound as glycosides. Quercetin (Fig. 13.7) is the best known flavonol, often present in the form of its diglycoside rutin. The isomeric isoflavones, such as genistein (Fig. 13.7), are not only antioxidants, but also have hormone activity. The best known flavans are catechins (Fig. 13.7). Anthocyanins and anthocyanidins are related to flavanones, but the 4-carbonyl group is absent; therefore, their antioxidant activities are lower than those of the corresponding flavones. The oxygen atom on the 3-carbon ring is ionized (Fig. 13.7). Anthocyanins thus occur as salts, imparting red, violet, purple, or blue color notes to food products. Their condensation products with other flavonoids are proanthocyanidins or condensed tannins (36). They usually consist of dimers, oligorners or polymers, up to the molecular weight of 4OOO Da. In fruits they are classified into three groups, i.e., procyanidins, propelargonidins, and prodelphinidins. The terminal group of a proanthocyanidin is mainly a catechin or an epicatechin unit (37). In legumes, they are linear polymers of flavan-3-01s and flavan-3,4-diols, which are linked together by a C-C bond between
Lipids with Antioxidant Properties
283 OH
H
b) quercetln o
\ 5
I OH
4
a) basic C,-C,-C,
configurationof flavonoids
OH
/
HO
OH
I
/
OH
d
\
c) genistein OH
0
HO&
/
\
\
/ OH
OH d) catechin
OCH,
e) malvidin
OH
Fig. 13.7. Chemical structures of flavonoids.
carbon-4 of one unit and carbon-6 or carbon-8 of another unit (38). The antioxidant activity of both flavonoid aglycones and glycosides was found to be high in methyl linoleate (39). Such highly unsaturated materials as fish oils can be satisfactorily stabilized with flavonoids (40). Lignans are phenolic compounds consisting of two phenylpropyl units connected together by their propyl groups. Lignans are not sources of antioxidants, but sesame, flaxseed, and olive oils are sources of lignans, which act as antioxidants. A lignan from the creosote bush (Larrea tridentutu), nordihydroguaiaretic acid, was proposed and used as an antioxidant, but its use is no longer permitted because its toxicity was proven by modem, more sophisticated tests. The same is true for guaiac gum, which has a very similar composition. Copolymers of phenylpropyl units are the basis of lignin, found in wood, nutshells, and cereal brans, where it forms macromolecular tridimensional structures. Natural antioxidants belonging to diterpenes will be discussed below. Applications of Antioxidants from Oilseeds
In the last section, the contents and activities of different phenolic substances in plant food materials were discussed. The majority of plant antioxidants are very polar; thus, it would be hardly possible to protect food lipids, which are nonpolar, against autoxidation by these compounds alone. The easiest way would be to add the respective food raw materials directly to fats, oils, or oil emulsions. However, it is not possible because of the lack of solubility. It is necessary to fractionate them first by simple, inexpensive procedures to achieve successful results. The application of antioxidants from nuts and oilseeds seems to be the best way
to stabilize edible lipids. Of course, both oilseeds and nuts are rich in lipids, making the isolated lipid phase a good source of natural antioxidants. Several good examples
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were provided earlier. They could be mixed with less stable oils to improve the oxidative stability. However, the lipidic phase prepared in the conventional way does not supply antioxidants to the stabilized product other than those present in the respective edible oils. During oil extraction, the majority of phenolic antioxidants remain in the extracted meal because of their higher polarity. Therefore, extracted meals should be considered as suitable sources of additional antioxidants. For the separation of oil, oilseeds are extracted with hexane or similar hydrocarbon solvents. To separate the maximum phenolic antioxidants from meals, the oilseed meals already extracted with hexane should be reextracted with more polar organic solvents (41), such as acetone, ethyl acetate, diethyl ether, or methanol (42). Extraction with purified edible oil should also be considered (43). Liquid carbon dioxide is also a good solvent, but it is rather expensive. Rapeseed oil is among the most important oils in the world. Rapeseed is produced from winter rape (Brussicu mpus L.), and is very similar in its composition to summer rape (B.rupa L.), mainly called canola in the case of zero-erucic oil. Rapeseed-extracted meals contain 1-2% phenolics, i.e., -10 times more than soybean meal (44), but -80% are bound to choline as esters called sinapine. Unfortunately, sinapine is quite inefficient as an antioxidant. Nevertheless, rapeseed expeller cakes or extracted meals show some antioxidant activity in lard or in sunflower oil. Crude tannins extracted from rapeseed hulls with 70% aqueous acetone were active in a carotene-linoleic acid system, and in a free radical-scavenging test (45). The content and composition of antioxidants in mustard (Simpis albu L.) seed, used as a flavoring, are similar to those of rapeseed. In spite of that, defatted mustard flour and the respective methanol or ethanol extracts possess strong antioxidant properties (46). Soybean (Glycine mux Merr. or G. hispida Maxim.), the leading oilseed on the world market, was also tested. Soybean powder and soybean extracts are among the oldest natural antioxidants proposed for animal fats and meat products. Defatted soybean flour is rich in flavonoids, mainly isoflavones, present in amounts of up to 0.25% (47,48). Isoflavones are present mainly as glycosides, but also as aglycones, which are more active as antioxidants. Isoflavone aglycones prevail in fermented soybeans. They have a strong synergistic effect with tocopherols and their copolymers (49). Peanuts (Aruchis hypogaeu L.) are relatively rich in phenolic acids, -1.75 g k g defatted peanut flour (50). Caffeic acid, dihydroquercetin, and other compounds are also present (5 1). The content of phenolic compounds is still substantially greater in peanut skins than in deskinned peanuts. Methanolic extracts of peanut hulls were active as antioxidants in soybean and peanut oils (52). Sunflower (Helianthus annuus L.) seeds contain 30-35 g phenolicskg defatted flour, consisting mainly of chlorogenic and caffeic acids (53). Evening primrose (Oenotheru biennis L.) is not a typical oilseed, but it is processed for dietary oil so that the extracted meal is available. The methanol extract contained 862.2 g phenolics/kg, consisting mainly of flavanols, proanthocyanidins, and isoflavones (54). After a preliminary concentration of phenolics
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using a combined extraction with organic solvents, the extracts were efficient in rapeseed and sunflower oils (55).Palm (Elaeis guineensis L.) fruits contain mainly tocotrienols and carotenoids, which enhance the oxidative stability of palm oil, but little is known about the effect of the phenolic antioxidants present. Antioxidants derived from lignans and contained in olive, sesame, flaxseed, and rice bran oils were discussed above. Application of Antioxidants from Spices
Spices are natural food ingredients, but consumption of large quantities could be harmful. Nevertheless, they could be added to lipids without special problems with food regulation. The main disadvantages are the specific aroma and flavor of spices, which could be objectionable for some purposes. Deodorized extracts have a more neutral flavor, but the final product would have to be examined using sensory tests. Rosemary (Rosmarinus oficinalis L.) antioxidants are the best-known compounds because they have been available on an industrial scale for many years. Their primary application is as flavoring agents. Therefore, rosemary extracts and their fractions are not listed as natural preservatives and antioxidants, i.e., they could be added without restrictions. They were proposed as antioxidants even before World War 11, and later by Matijakvie. However, their industrial application became possible after the research of Chang and co-workers (56). Safe rosemary antioxidants were proposed, based on extraction of rosemary leaves and their purification (43). Extraction by carbon dioxide under pressure is particularly suitable (57).Rosemary antioxidants act not only as antioxidants, but also as metal chelators and scavengers of superoxide radicals (58). Rosemary antioxidants are mainly nonpolar or only somewhat polar; therefore, hexane or ethyl acetate extracts were found to be more efficient than methanol or ethanol extracts in emulsions (59-61). Rosemary extracts are suitable as additives to frying oils because they suppress polymer formation (62). Rosemary resins, obtained as the nonvolatile residue after distillation of volatiles from the essential oil from rosemary leaves, consist of a complicated mixture of structurally related diterpenic compounds. Cuvelier et al. (63) identified 24 active compounds in an extract from rosemary. Carnosol and carnosic acid (Fig. 13.8) are the main components (43). Rosmarinic acid, epirosmanol, isorosmanol, rosmaridiphenol, and rosmariquinone are also present. Carnosol is more active than other components in emulsions, but the more polar carnosic and rosmarinic acids are more active in bulk edible oils (64). Sage (Salvia oficinalis L.) is another plant from the same family. Its leaves are used for flavoring and seasoning foods. Active antioxidants in sage are similar to those present in rosemary (63), but their concentration is lower. Sage is a more efficient source of antioxidants than other Lamiaceae plants, but still less efficient than rosemary. Oregano (Origanum vulgare L.) is another related plant, used as spice. Rosmarinic and caffeic acids are the most active components. The methanol extract
J. Pokornfand J. Parka'nyiovd
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from oregano was compared with other spice extracts (65), and activity decreased in the following sequence: oregano > thyme > marjoram > lavender. The acetone extract was reported to be an active antioxidant both in sunflower oil and its 20% oil-in-water emulsions (66). Another related plant, thyme (Thymus vulgaris L.), is also used as a spice and has antioxidative activity. The most active flavonoids in thyme were eriodictyol-7(67). Extracts from summer savory rutinoside and luteolin-7-O-~-glucopyranoside (Satureja hortensis L.) have similar antioxidant activity. The ethanol extract was active in sunflower oil (68). Extracts from the leaf spices had only moderate activity in rapeseed oil, except for rosemary and sage (69). Rhizomes of ginger (Zingiber officinale Roscoe) and turmeric (Curcuma domestica L.) are widely used as spices. They contain a mixture of several derivatives of 3-hydroxy-4-methoxy-benzene, which are efficient antioxidants in lipids. Gingerol, tested by Jitoe et a / . (70), and curcumin, tested by Kikuzaki and Nakatani (7 I), are the best-known representatives of these spices. Juniper extracts were also tested as antioxidants and possessed medium activity (72). The major active component in clove (Eugenia caryophyllata Thunb.) is eugenol (73), but the isomeric isoeugenol is still more active in lard and sunflower oil. Licorice (Glycyrrhizza glabra L.) extract also possesses active flavonoids (74). Application of Antioxidants from Tea, Herbs, and Plant Materials Used for the Preparation of Beverages
The most important members of this group are the tea beverages. They are prepared from green (nonfermented) tea, oolong (semifermented) tea, or black (fermented) tea.
carnosic acid
carnosol
rosmarinic acid Fig. 13.8. Rosemary antioxidants
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Green tea is harvested, and the leaves are only dried; thus, their composition does not change appreciably. Green tea is now consumed traditionally mainly in China and Japan, and to a small extent also in Europe and in America, mainly for dietetic reasons. Green tea (Camellia sinensis Kuntze) leaves are cultivated in subtropical and tropical countries worldwide. Green tea is produced mainly in countries of drier climate, where the moisture does not start the fermentation. Leaves are collected, rapidly dried to eliminate enzymatic activities, and packed (75). Green tea leaves are rich in catechins and related phenolics (up to 40% in content). The antioxidants of green tea extracts were active in lard and other bulk lipids (76). The activity of green tea catechins increased in lard (77) as follows: epicatechin < epicatechin gallate < epigallocatechin < epigallocatechin gallate. The activity of green tea antioxidants depends on the lipid systems to be stabilized (78). They were active in corn oil, prooxidants in oil-in-water emulsions, but antioxidants in lecithin liposomes in the presence of copper ions. The antioxidant activity is due to the high content of catechins (flavan-3-01s) and related substances (mainly flavonols), which constitute -30% of the dry weight of tea leaves. Epigallocatechin gallate is the most important catechin in tea leaves, with -100 mg present in a cup of tea infusion. Green tea catechins are now considered to be an important source of antioxidants in the human diet as a protection against in vivo oxidative changes in blood lipids. Tea catechins, especially epigallocatechin gallate, are important free radical scavengers (79). They were found to be active in emulsions using the 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) procedure (80). Green tea extracts are a commercially available product, suitable for applications in edible oils and other lipid products. Fermented teas (black tea and oolong tea) are also important sources of antioxidants even when close to half of the native antioxidants are oxidized into tea pigments (theaflavins and thearubigins) in the course of the fermentation. Catechins are oxidized enzymatically by the action of polyphenol oxidases (81). Tea pigments ,especially theaflavin gallates and digallates (82), also have moderate antioxidants activities. The residual nonoxidized catechins impart most of the antioxidant activities to fermented tea extracts (83). They are also active as in vivo antioxidants, inhibiting the oxidation of erythrocyte membranes (84) and plasma lipoproteins. Wine (Vitis vinijera L.) grapes and wine, especially red wine, are an important source of natural antioxidants (85), particularly the fraction containing proanthocyanidins and resveratrol. Their consumption is correlated with protection against ischemic diseases. Resveratrol is highly appreciated, perhaps more than is warranted. However, these substances are not likely to be applied for the stabilization of anhydrous lipid foods against autoxidation. On the contrary, they efficiently stabilized liposomes (86). Only 6 1 3 % total antioxidants belonged to the lipophilic fraction (87). Common herbal plants used in Central Europe for the preparation of herbal tea, such as strawberry, blueberry, raspberry, cranberry, blackberry, and nettle
leaves, hips, or other sources are not sufficiently active as antioxidants in bulk lipids, according to our unpublished results, to be considered as potential sources
2 88
1. Pokornf a nd I. Parka’nyio vd
of dietary antioxidants in bulk vegetable oils. Hawthorn leaves are another example of potential antioxidants of moderate activity. Various herbal drugs have been used in the Far East, especially in China, for many centuries (88). Gingseng (Panax gingseng and P. quinquefolium) extracts contain a mixture of ginsenosides and other phenolics, which influence nitrosyl oxide metabolism. Extracts from roots are active as free radical scavengers (89). The extracts can be used as an ingredient for beverages, but also for applications in lipid foods and edible fats, oils, and emulsions. Extracts from Gingko biloba are the most frequently discussed antioxidants in this group of materials (90). They contain a mixture of gingkosides, whose benign effects have not been established by clinical tests necessary for use in foods. These extracts are efficient as food antioxidants (91), including clinical applications. However, they should be subjected to the same sophisticated tests of their safety as those required for food applications before they are used for human nutrition. The same is valid for extracts from pine bark (Pinuspinaster), which are commercially available (92) and may be used for both the preparation of beverages or for application in lipid foods. They contain flavonoids, primarily procyanidins, consisting of condensed tannins, monomeric catechins, and phenylcarboxylic acids. They can be used in medicine and for pharmaceutical preparations; however, for food applications, when larger amounts would be consumed, their use would require more research. Application of Antioxidants from Cereals and Pulses
Cereals and pulses contain liposoluble antioxidants, which could be used for the stabilization of lipids and lipid emulsions. They would be not likely used directly or as extracts, but they can increase the stability of lipid phase in food products and ready meals, which contain them. Oat (Avena sativa L.) antioxidants belong to the first antioxidants suggested by Musher (127) for the protection of lard and other lipids even before World War 11. Esters of caffeic and ferulic acids with higher alkanols or hydroxyacids, such as n-hexacosyl ferulate, 26-0-26-feruloyl-hydroxyhexacosanoic, or 26-0-caffeoyl26-hydroxyhexacosanoicacid, were isolated from oat bran (93). They have antioxidant properties, and are liposoluble. Phenolic acids are also bound to DAG (94). Another group of oat antioxidants contains anthranilic acid. Avenanthramides, amides of anthranilic acid and cinnamic acid, were detected in oat brans (95). They are extracted with methanol. Avenasterol extracted with oat lipids, contributes to the antioxidant activity of the oat nonpolar fraction. Torn (Zea mays L.) antioxidants are located mainly in the germ, which is used as an oilseed raw material. It is rich in tocopherols, especially y-tocopherol. Ferulic and sinapic acids are present free and bound as glycosides. Corn germ oil is also very rich in A7-avenasterol. Buckwheat (Fagopyrum esculentum L.) grains are now popular in dietetic food preparations. They contain several phenolic acids, especially vanillic and caf-
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feic acids (96), and a mixture of flavonoids, which, however, are less important from the standpoint of their antioxidant effect. Barley (Hordeum sativum L.) antioxidants are both nonpolar and polar. Tocopherols and tocotrienols belong to the former group, and isoflavone derivatives such as 2'-O-~-glucosylisovitexinbelong to the latter group (97). Another group of important food commodities are legumes or pulses. Peanut and soybean antioxidants were discussed above. Beans (Phaseolus vulgaris L.) contain cyanidin, pelargonidin, delphinidin glucosides, and other anthocyanins (98,99). Similar anthocyanins, proanthocyanins, and other related inhibitors were identified in other legumes (100). Lentil (Lens culinaris) seed extracts are rich in phenolics, and have high free radical-scavenging activity (101). Yellow pea (Pisum sativum L.) and pea flour contain phenolic antioxidants similar to those in other legumes (102). Their direct application to lipids is not likely, but they may improve the resistance of lipids to oxidation in lipid-rich food products. Application of Antioxidants from Vegetablesand Fruits
Vegetables and fruits contain various phenolic substances of higher or lower antioxidant activity, but it is unlikely that they would be used directly as sources of antioxidants for the stabilization of lipids. Nevertheless, if they are an integral part of the foods or ready meals, their natural antioxidants would positively affect the resistance of the product to oxidation. Vegetables of the Brassicaceue family, such as cabbage, cauliflower, broccoli, or Brussels sprouts, contain glucosinolates or their breakdown products, mainly isothiocyanates, which deactivate free radicals. Various phenolics are also present. Carrot (Daucus carota L.) extracts have only low antioxidant activity. Potato (Solanurn tuberosum L.) tubers and peels are a good source of antioxidants because of high content of polyphenolics, such as chlorogenic acids (103), which cause enzymic browning of potato tubers. Wastes from the production of potato starch also inhibit lipid oxidation. Onion (Allium cepa L.) and garlic (A. sativum L.) have antioxidant activity in liposomes , lipoproteins, and lipid emulsions. Alliin (S-allylcysteine sulfoxide), its degradation product allicin (allyl-2-propenylthiosulfinate), and similar sulfur compounds reduce lipid hydroperoxides in inactive products (104). Quercetin is present in the outer layers of onion tubers. The antioxidant effect of tomato (LycopersicumescuZentum Mill.) is attributed to the carotenoid lycopene. Bell peppers (Capsicum annuum L.) may be moderately antioxidative or prooxidative. The active substances in bell peppers are capsaicinoids, flavonoids, ascorbic acid, p-carotene, and tocopherols (105). Therefore, they suppress the photooxidation of linoleic acid by quenching singlet oxygen (106). The presence of antioxidant activities is often controversial because it depends greatly on the experimental conditions, The content of active substances depends very much on the cultivar, climate, harvesting, and storage conditions, food to be stabi-
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lized, and many other factors. It is necessary to test the material or extracts before the application. The antioxidant activities of fruits and berries are due mainly to flavonoids and anthocyanins (107) and to ascorbic acid. Prunes (Prunus domestica) contain high levels of chlorogenic acid and neochlorogenic acid. Fresh grapes (Vitis vinifera) and grape juices contain flavonoids, such as catechins, anthocyanins, and resveratrol (108). Wild berries possess higher antioxidant activity than cultivated berries. Cherries (Cerasus avium) and tart cherries (Cerasus vulgaris) contain high concentrations of phenolics; thus, their extracts are relatively active as oxidation inhibitors. The reported activities depend greatly on the lipid system studied. Application of Essential Oils as Antioxidants
Many sources of plant antioxidants, especially spices, herbs, and fruits, have specific aromas because they contain small amounts of essential oils. The main components of essential oils are monoterpenes and sesquiterpenes, which are volatile, but the nonvolatile fraction is in many cases also active, as in the case of orange (Citrus sinensis) essential oil (109). Rosemary resins were discussed above. Some terpenes are active as moderate antioxidants, e.g., essential oils from thyme and clove were active in the protection of cottonseed oil against oxidation (1 10). Extracts from such materials still contain small amounts of essential oils after evaporation of extraction solvents. To remove the specific odor, extracts may be deodorized. The deodorized samples often possess lower antioxidant activities than the original extracts (111). A few examples are shown in Table 13.2. Essentially, it would be possible to use nondeodorized extracts as antioxidants; their disadvantage is that they impart a specific aroma to lipids stabilized by their application. They can be thus used as inhibitors of oxidation only in those cases in which the resulting aroma is not objectionable.
Synergists and Metal Chelators Synergists are substances that have no antioxidant activity in the absence of antioxidants, but augment the activity of phenolic antioxidants. Their application is particularly useful in the case of vegetable oils that also contain tocopherols (112). As noted above, metals are efficient catalysts of lipid oxidation because they catalyze the decomposition of lipid hydroperoxides with the formation of two free radicals. Chelating agents bind heavy metals in inactive complexes, thus inhibiting indirectly the lipid oxidation. Synthetic chelators such as EDTA or similar substances are sometimes used for stabilization of lipids, but natural substances are now preferred. Some natural antioxidants, especially flavonoids and anthocyanins, have metalchelating activity, as well. Because most synergists are also metal-chelating agents, we will discuss both classes of inhibitors at the same time. Nonvolatile organic acids with 2-3 carboxyl groups in the molecule are the best-known synergists. Citric acid is the oldest synergist known and used. It is a
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TABLE 13.2 Effect of Essential Oils on the Antioxidant Activity of Spicesatb Methanol extract of spice
Sage (sample A) Sage (sample B) Savory Dragonhead Roman chamomile Sweetgrass (sample A) Sweetgrass (sample B)
Protection factor before deodorization 4.9 4.5 2.2 1.6 2.5 2.6 1.7
Protection factor after deodorization 3.3 2.5 1.7 1.4
1.6 2.8 1.6
aProtectionfactor = induction period (IP) of stabilized sample/lP of original sample. "Source: Pokornt eta/., unpublished results.
good synergist of tocopherols and an efficient metal chelator at the same time. Taussig proposed its use more than 100 years ago, as reviewed by Pardun (1 13). Citric acid was added to edible oils during refining, and this practice is continued in the superdegumming procedure. During deodorization, citric acid is partially pyrolyzed at high deodorization temperatures (>2OO0C).Both the residual citric acid and its degradation products increase the stability of refined oils against oxidation. Citric acid esters, such as isopropyl citrate, are more soluble in oils than free citric acid, and are more resistant to degradation on heating. Malic, succinic, or tartaric acids have similar activities. Ascorbic acid is still more efficient as a synergist than citric acid. Synthetic nature-identical ascorbic acid or its mixture with isoascorbic acid is used. To increase the solubility in oils, esters of ascorbic acid are preferred to the free acid. Ascorbyl palmitate is frequently used because it is highly soluble in oils and more resistant to oxidation than esters with an unsaturated acid. It is very active in edible oils rich in tocopherols, such as rapeseed, soybean, or corn oils, because it possesses strong synergistic activity. Ascorbyl palmitate is useful for the stabilization of frying oils as well because it is relatively thermostable (114). Ascorbyl palmitate was found to be efficient in quenching singlet oxygen during photooxidation of oils under sunlight, if they contained traces of chlorophylls (115). Ascorbic acid and ascorbyl palmitate are relatively polar; therefore, they are less active in emulsions than in bulk oils. They have only a small effect on the composition of off-flavor compounds (1 16). Phospholipids are important synergists, though of only moderate efficiency. However, phospholipids have generally regarded as safe (GRAS) status, which allows their use without restrictions. Their advantages include good solubility in oils and other lipids. When added to dietary oils, they increase not only the stability against oxidation (1 17,118), but also the nutritional value. Phosphatidic acids can also bind heavy metal ions in inactive complexes, giving them a metal-chelating activity (1 19). Trimethylamine oxide is cleaved from phosphatidylcholine oxidation products. It is an efficient synergist of &tocopherol (120).
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Amino acids are also active as synergists, several of them also as metal chelators. Amino acids are not soluble in oils, but can be used in lipid emulsions and in lipidcontaining foods. Proteins have an inhibitory effect in lipid emulsions, membranes, and lipoproteins. Amino acids were efficient as synergists of tocopherols in vegetable oils in spite of their low solubility (121). Lower peptides, such as carnosine, inhibited lipid oxidation catalyzed by iron salts and heme pigments (122). Pure amino acids are expensive, but protein hydrolysates are also active, eg., soy sauce, which contains a part of the original soybean natural antioxidants. By-products of animal production, especially from poultry processing, may be hydrolyzed, but their application in lard or in lipid emulsions showed only moderate activity (123). Protein hydrolysates from by-products of aquatic food materials were also active (124). Biogenic amines, occurring in food materials of animal origin and in food hydroly-sates, were found to be active, inhibiting the oxidation of PUFA and efficiently protecting fish oils (125). Phytates and phosphates represent another type of metal chelating agents, which may be added to the aqueous phase of lipid emulsions.
Summary Oxidation damages the nutritional value of healthful and dietary lipids such as PUFA, and lipophilic vitamins are destroyed. The sensory value deteriorates because of the development of rancid off-flavors. In larger amounts, lipid oxidation products are harmful to health. For these reasons, the protection of lipids against oxidation is desirable. Lipids of plant origin contain natural antioxidants, mainly tocopherols, but other antioxidants are also present in some oils, such as olive, sesame, rice, or linseed oils. Antioxidants are found in only trace amounts in animal fats and oils. The stability of lipids is improved by the addition of antioxidants. Synthetic antioxidants are active and readily available, but natural antioxidants are now preferred to synthetic antioxidants, especially in healthful oils. The most frequently used natural antioxidants are tocopherols, followed by rosemary resins and geen tea catechins, but the application of many other natural antioxidants is possible. Only antioxidants with GRAS status or antioxidants permitted by the latest government regulations should be used. Safe limits of such compounds should be observed. Antioxidant activity is further increased by the application of synergists and metal-chelating agents, most often ascorbic or citric acids and their esters. The efficiency of natural antioxidants depends on the FA composition of lipids and on the presence of polar substances, such as emulsifiers and water. Less-polar antioxidants are active in lipid emulsions, whereas more polar antioxidants are more active in bulk fats and oils. References
Frankel, E.N., Lipid Oxidation,The Oily Press, Bridgwater,UK, 1998. 2. Shahidi, F., Natural Antioxidants: Chemistry, Health Effects, and Applications, AOCS Press, Champaign, IL, 1997. 1.
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Chapter 14
y-Linolenic Acid: The Health Effects Rakesh Kapoor Bioriginal Food & Science Corporation, Saskatoon, Canada S7J OR1
Introduction y-Linolenic acid (GLA, cis 6, cis 9, cis 12-octadecatrienoic acid) can be called a functional essential fatty acid (EFA) because it can correct the symptoms of EFA deficiency, which is produced by the elimination of EFA from diet (1,2). It is produced in animals as the first product of the metabolism of linoleic acid (LA), an EFA of the omega-6 series. This reaction is catalyzed by the enzyme A6-desaturase (D-6-D), the slowest reaction in the metabolic pathway. Hence, this reaction is known as a rate-limiting reaction (3,4). Once formed endogenously from LA, or administered, GLA is rapidly elongated to dihomo-y-linolenicacid (DGLA) by the elongase enzyme (Fig. 14.1). Subsequently, DGLA is acetylated and incorporated into cell membrane phospholipids. A small amount can be converted into arachidonic acid (AA) and this reaction is catalyzed by the A5-desaturase (D-5-D) enzyme. Different animal species and different tissues vary in their capacity to convert DGLA to AA. Rats metabolize DGLA to AA in large amounts, whereas
Linoleic acid (18:Z)c A-6-desaturation
y-Linolenic acid (18:3) elongation
i Arachidonic acid (20:4)Fig. 14.1. Metabolic pathway for linoleic acid. 301
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humans and other species have limited capacity to form AA from DGLA. Cats are deficient in the D-6-D enzyme; hence, they cannot synthesize GLA and subsequent metabolites from LA (5). Therefore, cats must eat a meat-based diet to obtain longer-chain metabolites of LA (DGLA, AA). Activity of the D-6-D enzyme decreases with age and in people suffering from various diseases including arthritis, diabetes, hypertension, eczema, and psoriasis. Lifestyle factors such as stress, smoking, excessive consumption of alcohol, linoleic acid (6), saturated and trans fatty acids, and nutritional deficiencies of vitamin B,, zinc (7), and magnesium also inhibit this desaturase. As a result, the in vivo production of GLA and subsequently DGLA is compromised. Populations in industrialized countries (North America and Europe) consume meat-based diets supplying preformed AA. They also use vegetable oils rich in LA for cooking and as salad dressings. As a result, these populations consume excessive amounts of LA and AA in their diet. Excessive amounts of LA inhibit the D-6-D enzyme, thereby inhibiting production of GLA and hence DGLA. DGLA competes with AA for cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, and the metabolites of DGLA and AA from these enzymes [prostaglandins (PG) and leukotrienes (LT)] have opposing actions (discussed in detail below). This may result in a functional deficit of DGLA in these populations because of excessive consumption of LA and AA. As a result, there is an imbalance in PGLT production and excessive production of AA-derived inflammatory PG. This may be contributing to chronic diseases such as arthritis, cancer, or heart disease, which are more prevalent in industrialized countries. Supplementation with GLA balances inflammatory and anti-inflammatory cytokines, which may explain why supplementation with GLA exerts beneficial effects in these populations, even though their intake of total n-6 fatty acids is excessive.
Commercial Sources of GLA GLA is distributed in small amounts in many plants belonging to the families Aceraceae, Boraginaceae, Cannabinaceae, Liliaceae, Onagraceae, Ranunculaceae, Saxifragaceae, and Scrophulariaceae (8,9); however, only a limited number of these plants contain commercially relevant amounts. To date, only borage (Borugo officinalis), evening primrose (Oenotheru officinalis), and black currant (Ribes nigrurn) oils are exploited commercially for the production of oil rich in GLA. Of these sources, borage oil contains the highest amounts of GLA (18-26%) followed by black currant oil (12-18%) and evening primrose oil (8-12%). The GLA content in these oils varies due to geographic location, length of light period during the growing season, average temperature, and diurnal temperature variations. The average fatty acid composition of these oils as provided by Bioriginal Food & Science Corporation (unpublished data) and published information is reported in Table 14.1. Echium (Echium pluntugineum L.) seeds are another source of GLA that has not yet been commercialized.
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TABLE 11.1 Fatty Acid Profile of Borage, Evening Primrose, Black Currant, and Hemp Oilsa Borage oil
Evening primrose oil
Fatty acid
16:O 16:l 18:O 18:l 18:2 18:3(n-3) 18:3(n-6) 18:4 20:o 20:l 22:o 22:l 24:O 24:l
Black currant oil
Hemp oil
7.10 0.10 1.50 10.90 45.20 13.20 16.90 3.30 0.10 0.80 0.10 0.20
5.60 0.10 2.60 11.50 56.60 18.50 1.60 0.50 0.90 0.60 0.30
(YO)
9.90 0.30 4.1 0 17.70 36.70 0.30 22.40 0.20 0.30 4.1 0 0.20 2.60 0.10 1.70
5.98 0.05 1.84 7.20 73.87 0.28 9.74 0.07 0.30 0.1 9 0.1 0 0.04
0.10
Values are means.
Metabolism of GLA After oral intake of GLA-rich oils, GLA is rapidly absorbed. Part of the absorbed GLA is oxidized; the rest is taken up by various tissues/cells and is rapidly elongated to DGLA (Fig. 14.1). The oxidation rate of GLA was found to be 28% of that for LA (10). DGLA can be acetylated and incorporated into membrane phospholipids or it can be desaturated to AA by D-5-D. The distribution of GLA and its metabolites is tissue and species specific (1 1). Feeding a GLA-rich diet to rats caused an accumulation of DGLA in milk (12) and a rise in DGLA and AA in aorta and platelets (13), in immune cells, including macrophages, Kupffer cells, and endothelial cells (14,15), and in the brain and liver (16). In human volunteers, administration of GLA in varying doses (from 0.4 to 5.23 g) resulted in a rise in DGLA levels in neutrophils (17,18), mesenteric and lymph node spleen lymphocytes (1 l), and platelet phospholipids (19) without affecting AA levels. After daily administration of 5.23 g of GLA from borage oil for 42 d to human volunteers, a differential distribution of DGLA in various phospholipid fractions was observed (19). Platelet phosphatidylcholine (PC) had maximal (67.6%) DGLA followed by phosphatidylethanolamine (16.7%), phosphatidylserine (12.9%), and phosphatidylinositol(2.6%). There was no change in sphingomyelin. In all of the phospholipid fractions, the ratio of DGLA/AA decreased significantly. At the same oral dose, GLA caused a rise in DGLA levels in PC fraction of plasma high density lipoprotein (HDL) and cholesteryl esters (19). AA levels increased only in the PC fraction of HDL. In this study, the dose of GLA employed was much higher than the doses used
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in any of the clinical trials (usual range is from 240 mg to 2.1 g GLA). The difference in the observed rise in platelet AA levels after the feeding of GLA sources in the above studies is due to species difference. Rat platelets have the D-5-D enzyme required for the conversion of DGLA to AA, whereas human platelets lack this enzyme and obtain preformed AA from the circulation. DGLA and AA are substrates for COX and LOX enzymes. The metabolites of these enzymes, called eicosanoids, play an important role in the signal transduction process and act as second messengers. These are discussed below under inflammation.
Actions of GLA Anti-Inflammatoryand lmmunomodulatoryActions Immune and inflammatory reactions are complex and tightly regulated responses that involve the interplay of several cell types (phagocytic and immune cells) and humoral factors. Controlled response is essential for survival because it plays an important role in destroying pathogenic microorganisms entering the body (selfdefense) and the damaged cells. Uncontrolled inflammatory response causes damage to cells and has been implicated in many diseases including cancer, cardiovascular, diabetes, Alzheimer’s, cystic fibrosis, multiple sclerosis, ulcerative colitis, and inflammatory bowel disease, and autoimmune diseases including arthritis and psoriasis. GLA has been studied mainly for its anti-inflammatory and immunomodulatory properties. The immune cells (including polymorphonuclear leukocytes, monocytes, splenocytes, Kupffer cells, macrophages, natural killer cells) take up GLA and rapidly elongate it to DGLA. The DGLA is incorporated into cell membrane phospholipids. A small amount can be desaturated to AA in some species (rat) but not in humans because human inflammatory cells lack the D-5-D enzyme required for synthesis of AA. After an inflammatory stimulus, the enzyme phospholipase A, releases DGLA from the cell membranes. The released DGLA competes with AA for COX and LOX enzymes. DGLA produces PGE, and thromboxane A, (TXA1) by COX and 15-hydroxyeicosatrienoicacid (15-HETrE) by the action of 15-LOX (Fig. 14.2). The in vitro inhibitory concentration of 15-HETrE against 5-LOX was 5 pmol/L (18). PGE, exerts mainly anti-inflammatory and vasodilatory actions (20), whereas 15-HETrE inhibits the enzyme 5-LOX, thus inhibiting the production of LTB, from neutrophils (18,21). LTB, is a very potent chemotactic factor that attracts neutrophils at the site of inflammation. It also increases the adherence of leukocytes to endothelial cells, enhances the migration of T-lymphocytes in vitro, stimulates release of interferon-y and interleukin (1L)-2 production by T cells, and promotes the biosynthesis of IL-1 from monocytes. Hence, LTB, amplifies the inflammatory response; by inhibiting the production of LTB,, the inflammatory response is reduced. Several studies showed inhibition of the production of LTB, from stimulated neutrophils by GLA (17,18,22).
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I
**..*
%*
.*
DGLA (20:3)
AA (20:4)
LTB,
Anti-inflammatory
PGE,
PGI,,TXA,
Pro-inflammatory
Fig. 14.2. Production of cytokines from dihomo-y-Iinolenic acid (DGLA) and arachidonic acid (AA). Abbreviations: PL, phospholipase; COX, cyclooxygenase; LOX, Iipoxygenase; PG, prostaglandin; 15-HETrE, 15-hydroxyeicosatrienoicacid; LT, leukotriene; TX, thromboxane.
GLA also affects cytokine pathways, inhibiting mitogen-induced production of IL-2 by human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner (23). This effect appeared to be independent of the COX pathway because indomethacin, a COX inhibitor, did not suppress the IL-2 inhibitory effect of GLA, although it inhibited PGE release from fatty acid incubated PBMC. The inhibition of IL-2 release could be mediated by the effect of GLA and DGLA on early response genes because GLA was shown to reduce an increase in c-fos and a decrease in cmyc oncogenes in T cells (24). DGLA was shown to inhibit IL-2-dependent proliferation of T lymphocytes isolated from synovial tissue and synovial fluid from arthritic patients (25). GLA and DGLA inhibited IL-1-induced proliferation of thymic lymphocytes, and GLA was less potent than DGLA (26). This action was not mediated through the PG pathway because COX inhibitors had no effect on the actions of these fatty acids that might exert a direct effect on lymphocytes. DeLuca et al. (27) stimulated the PBMC for 30 min, followed by stimulation with LPS for 16 hr. They observed a dose-dependent decrease in LPS-induced release of IL-lP and tumor necrosis factor (TNF)-aby GLA and DGLA. They observed a similar reduction in the release of these mediators when 2.4 g of GLA was administered to humans as a single dose. The LPS-stimulated IL-1P release was further increased by IL-1, a process known as autoinduction. GLA was shown to inhibit IL-1P release from LPSstimulated monocytes mainly by inhibiting the autoinduction process (28). This
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information suggests that GLA may be inhibiting excessive release of IL-lP to prevent inflammation but may not interfere with basal release of IL-lp, which plays a role in host defense. Thus, dietary administration of GLA-rich oils may modulate the inflammatory response and immune function in the prevention and/or treatment of autoimmune diseases including arthritis or psoriasis. Rheumatoid Arthritis. Rheumatoid arthritis is an autoimmune disease, associated with the destruction of cartilage and the inflammation of joints. There is no cure for the disease, Treatment is only symptomatic and involves steroids, and nonsteroidal anti-inflammatory drugs (NSAID). Ultimately, joint replacement surgery is required. Oils containing GLA exert anti-inflammatory and immunomodulatory actions in laboratory animals challenged with inflammatory insults and humans suffering from inflammatory conditions. These studies are summarized below. Dietary administration of GLA to rats reduced inflammation induced by the injection of monosodium urate crystals (29) and Freund’s adjuvant-induced arthritis (30). These actions of GLA were associated with inhibition of polymorphonuclear leukocyte recruitment, phagocytosis and lysosomal enzyme release, and reduced proliferation of pouch-lining cells. In a randomized, placebo-controlled trial, patients suffering from rheumatoid arthritis were administered 1.4 g of GLA from borage oil for 6 mon. This resulted in a significant reduction in swollen joint count and score, tender joint count and score, and platelet counts (31). This trial observed a 33% reduction in duration of morning stiffness. The only side effects of GLA treatment were belching, flatulence, and soft stools. Zurier et al. (32) studied the effect of a higher dose (2.8 g/d) of GLA for 6 mon. The effects were compared with a placebo group. After 6 mon, all of the patients were switched to a GLA arm.At the end of 6 mon, patients in the GLA group had reductions in swollen joint count and score, morning stiffness, and tender joint count and score. At the end of 12 mon, all the patients administered GLA had improved, but the improvements were greater in patients who started with GLA from the beginning. No patients in the GLA group experienced deterioration of condition in first 6 mon but at the end of 12 mon, 2 patients (out of 21) reported deterioration in condition; 7 of these 21 patients required a reduction in the dosage of NSAID and/or prednisone. In most patients, the disease condition was exacerbated 3 mon after stopping the treatment with borage oil, suggesting that borage oil must be continued for relief of symptoms. Daily treatment of arthritic patients with 540 mg of GLA or 450 mg of GLA and 240 mg of EPA for 12 mon significantly reduced the requirement for NSAID (33). In that study, 3 mon after stopping the treatment with GLA, all of the patients required a full dose of NSAID, indicating that GLA and/or EPA had NSAID-sparing effects and were not disease-modifying agents. In an open-label clinical study, 1.1 g of GLA was given for 12 wk; it reduced inflammation in arthritic patients and also reduced the release of PGE,, LTB,, and LTC, (34). Hansen et al. (35) did not observe any benefit from the administration of 4 g
evening primrose oil supplying 360 mg GLAld along with zinc, ascorbic acid,
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niacin, and pyridoxine to a group of 20 arthritis patients for 12 wk. The failure of GLA treatment could be due to the low dosage and/or the short duration of treatment. A recent double-blind, placebo-controlled trial (36) of 4 mo duration, supplying nutrient supplement containing 0.5 g GLA, 1.4 g EPA, 0.21 1 g DHA, and other micronutrients including vitamin E, did not demonstrate any beneficial effects on rheumatoid arthritis. The studies that did not show any benefit of GLA either provided a lower dose or the treatment was for a shorter period than those showing benefits. A recent review of the literature (37) on alternative and complementary therapies found strong support for GLA in the treatment of rheumatoid arthritis. A meta-analysis of published studies on GLA concluded that GLA is beneficial in the reduction of morning stiffness by -73 min and exerts a NSAID-sparing effect (38). However, the dosage of GLA required for the treatment of arthritis is not well established because in various studies, dosages ranged from 340 mg to 2.8 g GLNd. Acute Respiratory Distress Syndrome (ARDS). ARDS is an acute, severe injury to lungs, associated with increased pulmonary capillary permeability, pulmonary edema, increased pulmonary vascular resistance, and progressive hypoxemia. ARDS can also lead to damage and failure of other organs. The exact cause of ARDS is not known but oxygen free radicals, cytokines, and prostaglandins have been implicated. Patients at risk of developing ARDS have significantly lower levels of GLA, DGLA, ALA, and EPA in plasma phospholipids, and patients with established ARDS have in addition lower amounts of AA (39), suggesting a potential for treatment with a combination of borage oil and fish oil (supplying GLA, EPA, and DHA). In a multicenter double-blind, placebo-controlled clinical trial, treatment for 4 d with a combination of borage oil, fish oil, and antioxidants reduced the number of total cells and neutrophils in bronchoalveolar fluid compared with the control group of patients (40). In that study, the treatment group (n = 51 patients) was administered daily a mixture of borage oil, fish oil (providing 5.8 f 0.3 g GLA, 6.9 k 0.3 g EPA, and 2.9 k 0.1 g DHA), and antioxidants via gastric or jejunal tube, whereas the control group was given an isoenergetic, isonitrogenous diet. Patients in the dietary intervention group had improved arterial oxygenation leading to lower requirements for ventilator support and supplemental oxygen, and a shorter stay in the ICU compared with patients in the control group. Significantly fewer patients in the treatment group developed new organ failure and there was an -17% reduction in the total number of infections in the treatment group. Because a combination of EPA and GLA with antioxidants was used, it is difficult to differentiate the effects of GLA alone. However, the study provides strong support for using a combination of EPA and GLA. Nelson et al. (41) reported an increased oxidative stress as indicated by lower total reactive antioxidant potential (TRAP), lower !evels of endogenous antioxidants (plasma @-carotene,atocopherol, and retinol) and increased lipid peroxidation levels (LPO) in ARDS
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patients compared with normal persons. After 4 d of feeding patients a diet enriched with GLA, EPA, and antioxidants, levels of a-tocopherol, @-carotene,and retinol were normalized compared with the control group, but there was no difference in TRAP and LPO values. ARDS patients can die of multiple organ failure, including cardiac depression. Murray et al. (42) studied the effect of fish oil alone or in combination with borage oil on cardiac function in pigs during acute lung injury induced by the infusion of Escherichia coli endotoxin. They reported a protection of cardiac function by fish oil or fish oil + borage oil. The combination of fish oil and borage oil acted synergistically compared with fish oil alone in attenuating the cardiac depression. Pigs administered the fish oil and borage oil combination had lower pulmonary vascular resistance during the 4-h experiment than either the control group or the group given fish oil alone. The fish oil and borage oil combination prevented the loss of platelets from circulation, whereas fish oil alone did not exert this effect. This observation indicates that GLA decreased the aggregatory and adhesive properties of platelets in vivo. A significant reduction in the amount of TXB, (fish oil, and fish oil + borage oil groups) and 6-keto prostaglandin F,, in the alveolar fluid in the fish oil + borage oil group was also observed, suggesting that the beneficial effects of the treatment may be mediated by a reduction in the levels of proinflammatory and vasoconstrictor metabolites of AA. In a subsequent study, the administration of EPA (fish oil) or EPA + GLA (fish oil + borage oil) to pigs altered the composition of pulmonary surfactant by reducing the concentration of oleic acid and increasing the concentration of DGLA, EPA, and DHA. However, there was no effect on pulmonary compliance or surfactant function (43). Mancuso et al. (44) observed attenuation of the endotoxin-induced rise in pulmonary microvascular protein permeability in rats administered a combination of fish oil and borage oil, and this was associated with a decrease in LTB,, TxA2, and PGE, production by pulmonary alveolar macrophages. Additionally, this treatment also attenuated endotoxin-induced early and late hypotension.
Cancer Cancer is a complex phenomenon whose etiology is not well understood. The risk of cancer increases with age and -77% of cancers are diagnosed in people [mt] 55 yr old. According to the American Cancer Society, -1.37 million new cases of cancer are expected to be diagnosed in 2004. This estimate excludes basal and squamous cell carcinoma of skin and carcinoma in situ of any site except urinary bladder; -563,700 people are expected to die from cancer in the United States in 2004. The role of the diet in cancer is gaining increasing understanding. Basic treatment for cancer includes chemotherapy, radiation, and surgery. These treatments have serious side effects. Strategies for prevention include modification of lifestyle factors and dietary interventions. The role of dietary fat in cancer is controversial.
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Many prospective studies found an increase in cancer risk (45-47), whereas others reported no association between fat intake and cancer (48-50). GLA was examined in several studies for its effects on various cancer cell lines in vitro. It exerted cytotoxic activities against several tumor cell lines in vitro and tumor implants in experimental animal models. Limited studies exist on the effect of GLA on tumors in humans. In Vitro Studies. GLA demonstrated growth inhibitory actions against human prostate, breast, and lung cancer cells with no effect on normal cells (51). The effects of GLA appear to depend on the cell line, dose, and incubation time. GLA inhibited the growth of mouse BL6 melanoma cells by 70% at a dose of 20 pg/mL (52). At that dose, GLA did not affect the growth of normal bovine kidney epithelial MDBK cells, suggesting that GLA acts as an anticancer agent and inhibits the growth of cancer cells without affecting normal cells. Human hepatoma cell lines differ in sensitivity to GLA because they require a continuous presence of GLA in culture media for 4 d before a growth-inhibitory effect is observed (52). Withdrawal of GLA from the growth media after 5 d of treatment suppressed the growth for an additional 5 d (53). It appears that the cancer cells may lack D-6-D and hence cannot make GLA and subsequently DGLA. Cancer cells incorporate GLA and DGLA into their cell membranes and DGLA may be acting via the COX pathway in inhibiting cancer cell growth because PGE, stimulates cyclic AMP formation and induces cell death in cancer cell lines (54). GLA inhibited 5a-reductase activity in androgen sensitive (LNCaP) and androgen insensitive (PC3) human prostate cancer cell lines (55). This observation may suggest that GLA could be acting as an anticancer agent against androgen-dependent prostate and skin cancers. Recently, it was demonstrated that 15-HETrE, a metabolite of DGLA, inhibited COX-2 in androgen-dependent prostatic carcinoma cells (56). In estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-23 1 and SK-Br3) human breast carcinoma cell lines, simultaneous treatment with GLA and docetaxel exhibited synergistic anticancer effects (57). The effect of GLA on docetaxel cytotoxicity was partially antagonized by vitamin E, suggesting that mechanisms other than oxidative stress mediate the effect of GLA. GLA markedly decreased the expression of pl85HER-2heu oncoprotein in MCF-7 breast cancer cells, suggesting that GLA enhanced the cytotoxicity of docetaxel in human breast cancer cells by mechanisms other than lipoperoxidation, and that GLA-induced transcriptional repression of HER-2/neu oncogene might be one component of the mechanisms of this interaction. In vivo Studies. Several in vivo studies were conducted to observe the effects of GLA treatment on carcinogen-induced cancers in animals. A study employing 20% evening primrose oil or corn oil in the diet after inducing mammary tumors in 50-dold female Sprague-Dawley rats with intragastric administration of 7,12-dimethylbenz(a)anthracene (DMBA) showed a significantly lower number of tumors in the
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evening primrose oil group (58). In that study, the two groups of rats differed only in GLA supplementation, suggesting that GLA must have played an anticancer role. In another study using 5% dietary evening primrose oil, there was no antitumor activity in pathogen-free female Sprague-Dawley rats in which the tumors were induced by intragastric administration of 10 mg of DMBA 1 wk after the rats consumed experimental diets containing 5% evening primrose oil, sunflower oil, or palm oil (59). The different results in these two studies (58,59) could be due either to differences in the dose of GLA given to rats or the timing of GLA treatment. The other difference could be the immune status of the rats because Lee and Sugano (59) conducted their studies on pathogen-free rats. Human Studies
Breast Cancer. Tamoxifen is the most widely used drug for the treatment of estrogen-responsive breast cancer patients. This drug has toxicity. Kenny et al. (60) compared the effects of co-administration of 2.8 g of GLA with 20 mg of tamoxifen to 38 breast cancer patients with a control group of 47 breast cancer patients administered 20 mg tamoxifen only. GLA acted synergistically with tamoxifen in reducing the expression of estrogen receptors (ER) in tumor cells and enhanced the efficacy of tamoxifen. The GLA + tamoxifen group of patients had an early response to therapy and a significantly better quality of life after 6 wk of therapy. The GLA treatment was well tolerated; 42% of the patients reported no side effects and a general feeling of well-being, and 34% of patients reported alterations in the bowel habits with a tendency toward loose stools (many elderly patients found this beneficial). In early responders, the GLA group had a much higher reduction in the expression of ER than those administered tamoxifen alone. In the GLA group, there was also a downregulation of expression of bcl-2 gene at 6 wk, compared with no effect or transient increase in the bcl-2 protein in the tamoxifen group. Because bcl-2 plays a role in the prevention of apoptotic cell death, this observation suggests that by reducing the expression of antiapoptotic protein, GLA stimulates apoptotic cell death in cancer cells, which may have contributed to the faster response at 6 wk.
Gliorna. Cerebral glioma requires aggressive treatment with radiation, surgery, and chemotherapy. The median survival time after aggressive treatment is -1 yr (61,62). Because GLA exerts cytotoxic effects against tumor cells without affecting the healthy cells, Naidu et al. (63) tested GLA for the treatment of malignant cerebral glioma. They treated six patients, suffering from histochemically c o n f i i e d malignant glioma. Of these patients, four patients received 1 mg of GLA daily for 10 d; the other two patients were treated only on alternate days. Treatment started 10 d after surgery; all of these patients demonstrated marked necrosis of their tumor immediately after the therapy, Of these six patients, three were alive after 2 yr, two were lost to followup, and one died. No side effect of therapy was observed during or after treatment.
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During subsequent follow-up, the investigators did not observe any increase in the size of the residual tumor or recurrence of the tumor. On the basis of the results of this study, the authors extended the treatment to 15 more patients and found increased survival by 1.5-2 yr. This study also confirmed necrosis of the tumor cells and the safety of GLA. They also injected GLA into normal dogs intracerebrally and found no cytotoxic effects (64). These studies demonstrated that GLA injected directly into a tumor mass may be a potentially useful treatment for malignant glioma. The lithium salt of GLA conjugated to iodized lymphographic oil caused complete occlusion of the tumor-feeding vessel when injected intra-arterially close to the origin of the vessel (65), leading to necrosis of tumor cells and ultimately a reduction in tumor size. Liver Cancer. GLA given at a dose of 1.44 g from evening primrose oil to patients with primary liver cancer was shown to significantly reduce y-glutamyl transaminase enzyme activity in seven patients, suggesting an effect on the tumor (66). In this double-blind, placebo-controlled trial, GLA increased mean survival time to 58 d compared with 42 d in the placebo group, although the difference was not significant. These patients were in an advanced stage of cancer with a tumor weight of up to 3 kg; the dose of GLA may not have been sufficient to obtain a significant effect on survival time. A major finding was that the quality of life was better for the evening primrose oil group as indicated by the patients self-assessment. Pancreatic Cancer. Patients with unresectable pancreatic cancer who have undergone either surgical bypass or have had the pancreas endoscopically stented normally survive for 3-6 mon. A group of 18 such patients was treated with the lithium salt of GLA intravenously for 10 d followed by oral administration (67). During the infusion period, the dose of GLA was gradually increased for the first 5 d and then continued at a maximal tolerated dose for five subsequent days. Patients received a mean dose of 5.7 g of lithium GLA for the last 5 d of the study and a mean oral dose of 3 g afterwards. The median survival rate was 8 mon, and 4 patients were still alive compared with normal life expectancy of 3-6 mon for these patients. GLA treatment increased T-cell function and reduced TNF production. In this report, the study design was not well defined; therefore, it was difficult to assess whether the protocol had any beneficial effect on patient survival, although the treatment was reported to be well tolerated. An in vitro study compared the cytotoxic effects of the lithium salt of GLA with the 1deoxy-1-methylamino-D-glucitol salt of GLA (MeGLA) on the growth of two human pancreatic cancer cell lines (Panc-1 and MIA PaCa-2) (68). It reported a similar inhibitory action of MeGLA, suggesting that this salt can also be investigated in in vivo human clinical studies. Gastric Cancer. Gonzalez et al. (69) performed a case-controlled study in four regions of Spain to investigate the association of dietary factors and the risk of gas-
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tric cancer. Zaragova is an area in Spain in which people eat borage leaves and stems, usually cooked by boiling in water. After adjusting for intake of fruits and vegetables and energy intake, a strong negative association was observed between the risk of gastric cancer and borage intake. The negative association showed a strong dose-response effect when the population was subdivided into quartiles. On analysis, the authors found that boiled borage leaves contained -4.4% GLA, whereas boiled stems contained 14.6% GLA. This is the first study of an association between dietary borage consumption and the risk of gastric cancer. Because very few populations are habitual borage eaters, it is difficult to repeat this study. In addition, this study could not definitively identify GLA as an anticancer agent in borage leaves and stems. Cancer Metastasis. Prevention of cancer metastasis is a huge challenge in the treatment of cancer patients. In experimental models, GLA inhibited the metastasis of cancer (70-74). The cellular mechanisms behind antimetastatic effects of GLA are not fully understood, although it appears to regulate cell-cell interactions by modulating the expression of various proteins, including integrins. In a study in colon cancer cell lines (HT115, HT29, and HRT18), GLA was reported to inhibit metastasis and the motility of cancer cells induced by a hepatocyte growth factor. This effect was mediated by the increased expression of cell surface E-cadherin receptors, whereby the adhesiveness of the cells to the matrix was increased (75). This study reported that GLA increased the expression of E-cadherin in lung, colon, breast, melanoma, and liver cancer cells, but no increase was reported in endothelial cells and fibroblasts (76). The effect of GLA on the E-cadherin pathway is not the only mechanism behind metastasis inhibitory actions because reduced metastasis and increased adhesion of tumor cells were observed in E-cadherin-negative HT115 and MDA-MB 231 cell lines. These effects were mediated by the increased expression of desmoglein, a desmosomal cadherin (77). Urokinase concentration is increased in malignant cancer cells, and it is reported to play a role in the invasiveness and metastasis of cancer. du Toit et al. (78) reported competitive inhibition of urokinase activity by GLA with a Kivalue of 120 ymoVL. In a subsequent study, they observed that GLA inhibited the production of urokinase activity in human prostate tumor (DU-145) cells (79). These observations suggest that GLA, by inhibiting urokinase activity, may be playing a role in preventing metastasis of cancers. GLA inhibited cell-matrix interaction at several stages by inhibiting focal adhesion kinase activation and paxilin activation. Both of these molecules are activated by tyrosine phosphorylation, which is inhibited by GLA in tumor cells. GLA also upregulates the expression of the metastasis suppressor nm-23 gene (74). A reduction in the level of nm-23 gene expression was reported in various cancers including colorectal, breast, liver, ovarian, and bladder. These studies indicate that GLA may act on different targets at the gene level to reduce the metastasis and invasiveness of cancers. Jiang et al. (73) demonstrated that GLA may be acting
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through activation of peroxisome proliferator-activated receptor-y (PPAR-y) via increased phosphorylation of these receptors. After phosphorylation, these receptors are translocated to the nuclear membranes and regulate the expression of various genes. The authors demonstrated that the removal of PPAR-y with antisense oligos abolished the effect of GLA on the expression of adhesion molecules and tumor suppressor genes.
Mechanism of Anticancer Effects of GLA. The anticancer effects of GLA may be mediated through a combination of mechanisms because no single mechanism appears to explain all of the observed anticancer activities. These mechanisms may be cancer specific. Many cancer cells lack D-6-D activity; hence, they do not produce GLA and subsequent metabolites. GLA, which is a polyunsaturated fatty acid, can increase oxidative stress by inducing lipid peroxidation in the cancer cells. Free radicals were implicated in the cytotoxic actions of several anticancer drugs. It is possible that GLA may be showing its anticancer effects through oxidative mechanisms (80). Leaver et al. (81) reported an increase in the production of free radicals by GLA and AA in normal and brain glioma cells; however, tumor cells responded with a much greater increase in the production of free radicals, and GLA was more potent than AA in increasing the free radical production in glioma cells. The anticancer effects of GLA could be mediated by its antiproliferative actions against cancer cells or by increasing apoptotic cell death. GLA inhibited cell proliferation in human osteogenic sarcoma cells (MG-63 cells) (82). In these cells, GLA caused an abnormal metaphase cell-spindle formation and the inhibition of protein synthesis in the G,- and S-phases. In human epithelial cervix carcinoma cells (HeLa cells), GLA increased hypercondensation of chromosomes, suggesting that increased apoptotic cell death was associated with increased protein synthesis for all GI proteins and selective S-phase proteins. In HeLa cells, GLA also inhibited the mitogen-activated protein (MAP)-kinase pathway and c-jun expression (83). Because c-jun is a transcription factor involved in cell proliferation and is activated by MAP-kinases, GLA is interfering with nuclear processes by inducing apoptosis in HeLa cells (83). GLA also reduced phosphorylation of ~ 2 7 ~ and P' which are inhibitors of cyclin-dependent kinases and play a role in the progression of mitotic growth (progression from GI to S-phase) (84). Decreased phosphorylation resulted in increased binding of these proteins to cyclin-dependent kinases including CDK4, cyclin E, and CDC2. GLA was also shown to increase the expression of p53, a proapoptotic protein, in the squamous esophageal carcinoma cell line WHC03 (85), suggesting a potential role of p53 modulation in anticancer effects of GLA. However, in another study, it was demonstrated that proapoptotic effects of GLA in cancer cells may not be mediated through p53 (86). This study employed skin fibroblasts and lymphoblast cells containing wild-type and mutant p53. The transformed cells containing wild-type and mutant p53 responded to the induction of apoptosis by GLA. The transformed cells
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in this study were more susceptible to apoptotic cell death induction by GLA, which did not have any appreciable growth inhibitory effect on normal cells. AA was more toxic to normal cells than GLA, because a much higher dose of GLA was required to induce apoptosis in normal cells.
Cardiovascular Effects Since the 1960s, CVD has ranked as the number one killer in industrialized countries, although considerable progress has been made in understanding the causes of the disease and new treatments have been developed. The role of diet has been recognized in modulating modifiable disease risk factors including hypertension, lipid abnormalities [high plasma cholesterol and triacylglycerol (TG) levels], atherosclerosis, obesity, and diabetes. Dietary GLA affects many of these variables and is discussed below. Effect on Blood Pressure. Rose et al. (87) administered DGLA intravenously to dogs and it produced a biphasic response on systemic arterial pressure that was characterized by an initial fall followed by a sustained fall in blood pressure, and an increase in myocardial contractility. Only the sustained fall in blood pressure was blocked by COX inhibition, whereas the early fall in blood pressure and positive inotropic effects were not affected. This observation suggested that the blood pressure-lowering action of DGLA could be mediated through its direct action and through PGE, pathways. In subsequent studies, borage (88) and evening primrose oils (89) reduced in vivo pressor responses to angiotensin-I1 and norepinephrine without affecting the in vitro contractile response of the aorta to potassium chloride and serotonin in rats. These observations suggested that GLA may be interfering with agonist-receptor interactions without affecting the contractility of vascular smooth muscles. In spontaneously hypertensive rats (SHR), borage oil reduced blood pressure without affecting the pressor response to angiotensin and norepinephrine, suggesting the action of other mechanisms (88). In SHR, GLA significantly reduced the ratio of plasma aldosterone to renin via a nonsignificant decrease in plasma aldosterone levels and a small increase in plasma renin activity (90). There was no effect of the borage oil treatment on plasma cortisol levels compared with rats fed a controlled diet, free of GLA. Borage oil treatment also reduced the angiotensin receptor number and affinity in SHR. This suggests that a reduction in the responsiveness of adrenal glomerulosa cells to angiotensin and interference with renin-angiotensin-aldosterone axis might contribute to the hypotensive effects. These studies cannot isolate the exact mechanism by which borage oil interferes with angiotensin receptors. GLA also inhibited an isolation (psychologic) stress-induced rise in blood pressure in rats (91). In the unstressed rats, there was no effect of GLA on blood pressure. No effects on heart rate, heart weight, or adrenal weight were observed in any of the rats. Male normotensive university students given 1.3 g GLNd for 28 d had a signifi-
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cantly smaller rise in stress-induced systolic blood pressure and heart rate compared with presupplementation testing (92). The borage oil treatment did not affect diastolic blood pressure or plasma norepinephrine levels. Borage oil treatment increased skin temperature and performance as determined by the number of correct responses after the Stroop color word conflict test. These data c o n f m the observations obtained earlier in rats and also indicate increased tissue perfusion by borage oil treatment. Leng et al. (93) also observed a blood pressure-lowering effect in patients with peripheral arterial diseases. In their study, a combination of GLA with EPA was used; thus, the probable contribution of EPA to the blood pressure-lowering effect cannot be entirely ruled out. Platelet Function and Plasma Lipids. The effects of GLA on blood lipids and platelet function are controversial. In hypertriglyceridemic patients, GLA had no effect on plasma TG levels or platelet function, although there was an increase in GLA and DGLA levels in plasma and platelet phospholipids (94,95). A fall in serum TG and cholesterol levels was observed in insulin-dependent diabetic patients who were administered 2 g GLNd, but not with a 500 mg daily dose for 6 wk (96,97). A negative correlation was observed in GLA levels and plasma TG levels as well as systolic and diastolic blood pressure (98). Guivernau et al. (99) fed GLA at a dose of 240 mg/d for 12 wk to 12 hypertriacylglycerolmic patients and 12 rats. They observed a significant decrease in plasma TG, total cholesterol, low density lipoprotein (LDL) cholesterol, and an increase in HDL cholesterol. The reactivity of platelets to low doses of adenosine diphosphate and epinephrine was significantly reduced. A reduction in plasma TXB, levels was also observed in humans. In rats, a rise in plasma 6-keto-PGF1, levels was observed, suggesting an increase in PGE, production by GLA administration. Changes in eicosanoids may contribute to the observed effects of GLA on platelet aggregation because TXB, is a potent platelet aggregator. GLA is rapidly metabolized to DGLA, which was shown to inhibit platelet aggregation in in vitro (100) and in vivo studies (101,102). A double-blind, crossover trial in hypercholesterolemic patients demonstrated that GLA lowered LDL cholesterol and apolipoprotein (apo) B in plasma, and increased HDL cholesterol levels, without affecting the levels of total cholesterol (103). In rheumatoid arthritic patients, GLA lowered plasma apo B concentrations without affecting plasma TG, total, or HDL cholesterol levels (104). In that study, evening primrose oil at a daily dose of 20 mL (-1.8 g GLA) was given for 12 wk. The effect of GLA on plasma cholesterol level depended on the pretreatment level. GLA treatment lowered plasma cholesterol levels in patients whose plasma cholesterol levels were >5 mmolL but had no effect in people whose plasma cholesterol levels were 4 mmol/L (105). GLA-rich diets lowered plasma, total cholesterol, and the sum of LDL, intermediate density lipoprotein (IDL), and very low density lipoprotein (VLDL) cholesterol levels in 8-wk-old rats fed a high-cholesterol diet (106). The cholesterol-lowering effects of a GLA-rich diet could be mediated by changes in the membrane lipid composition, affecting the absorption of choles-
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terol. This observation was confirmed by Koba et al. (107) in Caco-2 cells. When these cells were incubated with GLA, the absorption of cholesterol from the growth medium was attenuated and the cell membranes were enriched with GLA, DGLA, and AA. Atherosclerosis. Atherosclerosis is an inflammatory response to injured vascular smooth muscles. Because GLA exerts anti-inflammatory actions, it is expected to demonstrate antiatherosclerotic properties. Dietary GLA reduced the severity of atherosclerotic lesions in rabbits (108) and Japanese quail (109). Fan et al. (110) observed an inhibitory action of dietary evening primrose oil alone and in combination with fish oil on the aortic smooth muscle cell proliferative action of peritoneal macrophages from mice. The inhibitory action appeared to be mediated through the COX pathway because indomethacin (COX inhibitor) inhibited PGE, release and antiproliferative actions. The addition of the 5-LOX inhibitor to the culture medium had no effect on antiproliferative or DNA synthesis inhibitory actions of primrose oil. In apo E knockout mice, evening primrose oil inhibited aortic smooth muscle cell proliferation and reduced the aortic vessel wall medial layer thickness as well as the size of the atherosclerotic lesion (111). This study confirms the beneficial effects of GLA in lowering cardiovascular risks by inhibiting atherosclerotic plaque development.
Skin Conditions Skin is a metabolically active organ. It has the capability to elongate the fatty acids but lacks the capacity to desaturate, suggesting that dermal cells use preformed long-chain metabolites of LA (GLA, DGLA, and AA) and ALA (EPA, DPA, and DHA). EFA deficiency can cause dry, scaly skin (1 12). In studies with EFA-deficient rats, mice, and guinea pigs, it was demonstrated that skin undergoes hyperproliferation (acanthosis, hypergranulosis, and hyperkeratosis) with increased DNA synthesis. LA levels were significantly decreased with an increase in mead acid (20:3n-9, an abnormal fatty acid characteristic of EFA deficiency). Supplementing diets with a large dose of safflower oil (rich in LA) or a much smaller dose of evening primrose oil (rich in LA and GLA) reversed the signs of EFA deficiency on skin, whereas fish oil did not reverse these symptoms (1 13). In that study, a rise in EPA, DPA, and DHA levels in skin phospholipids was observed but the levels of LA did not increase. Employing labeled fatty acids, this study confirmed a lack of D-6- and D-5-D activities in skin, indicating that skin cannot metabolize LA or ALA. Atopic Dermatitis. Patients suffering from atopic dermatitis had higher concentrations of LA and a lower concentration of GLA, DGLA, and AA in plasma phospholipids (1 14), suggesting a defective D-6-D of LA. These patients also did not have a flushing response to topically applied niacin, suggesting that they have
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defects in prostaglandin pathways and do not produce vasodilatory prostaglandins. Subsequent studies showed lower levels of DGLA in the breast milk of atopic mothers than in the normal mothers (1 15,116). Because breast-fed infants receive their nutrient requirements from breast milk, they do not receive sufficient quantities of DGLA and may be prone to dermatitis. Infants suffering from atopic eczema were reported to have low levels of LA and AA (1 17) and responded well to supplemental lard containing LA and AA (1 17). On the basis of these observations, it appears logical that dietary GLA or DGLA should help prevendtreat atopic dermatitis. A double-blind, placebo-controlled clinical trial of evening primrose oil was conducted among 60 adults and 39 children suffering from moderate-to-severe atopic dermatitis (1 18). The adult patient groups received 4, 8, or 12 capsules daily, whereas children were given 2 or 4 capsules daily, with each capsule providing 45 mg GLA; the placebo group received a capsule containing liquid paraffin. Treatment for all groups continued for 12 wk. The lower dose of GLA provided relief only from itch, whereas patients administered higher doses of GLA had improvements in itch, scaling, and general impression of severity as assessed by a physician and the patient. Children in this study did not perform as well as the adults, possibly due to either an insufficient dose of GLA or to high placebo effects in children. Manku et al. (1 19) analyzed the blood samples of adult patients from the above study for plasma phospholipid fatty acids. They observed that LA levels were higher in the atopic patients, and the scatter of values for LA was also very high. Levels of DGLA and AA were lower in these patients. Treatment with 4 capsuledd did not affect blood GLA or DGLA or plasma PGE, levels, whereas 8 and 12 capsules/d significantly elevated the levels of DGLA and PGE,. Treatment of children suffering from atopic dermatitis with 3.0 g GLA for 28 d resulted in a significant reduction in itching and the use of antihistamines without any treatment-related side effects (120). None of the children in this study had a complete recovery, although gradual improvements in erythema, excoriations, and lichenification were reported. A double-blind crossover study employing treatment of atopic eczema in 3- to 17-yr-old patients with borage oil or corn oil reported no beneficial effects of borage oil treatment (121). In that study, 10 patients receiving borage oil treatment showed improvements, but they did not differ from nonresponders in any of the characteristics (e.g., age, sex, symptom severity). In that study, the dose may have been insufficient (360 mg for 10-14 wk) or the large placebo effect observed might have masked the effectiveness of borage oil due to the small number of subjects. Scarff and Lloyd (122) performed a crossover trial in dogs suffering from dermatitis to study the comparative effects of treatment with evening primrose oil and olive oil. The dogs were given an olive oil placebo for 3 wk followed by either olive oil or evening primrose oil for 9 wk. At the end of 9 wk, the treatments were switched over without any washout period. During the first 3 wk of olive oil treatment, all of the dogs deteriorated. During the first treatment period, all dogs
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showed improvement that could be ascribed to a placebo effect in the olive oil group. In the second treatment period, dogs administered olive oil worsened, whereas those administered evening primrose oil improved. The authors observed an interaction in the order of treatment with the evening primrose oil that could be due to the change in treatment between active and placebo without any washout period. Eczematous skin also has high transepidermal water loss compared with normal skin. Topical application of pure GLA to rats (123) with dry skin due to EFA deficiency or borage oil to infants (124) suffering from seborrhoeic dermatitis normalized the elevated transepidermal water loss. Topically applied borage oil also relieved the symptoms of dermatitis within 3 4 wk and caused a rise in serum LA content, suggesting transdermal absorption of LA from borage oil. The site of application of borage oil was not important because borage oil in the diaper area of the infants also relieved the symptoms at other sites. Henz et al. (125) reported no efficacy of borage oil in patients with atopic dermatitis in a double-blind, placebo-controlled, multicenter, clinical trial employing 160 patients with moderate eczema (Costa score between 20 and 36 points). In that study, the patients were divided into two groups. The active group received 3.0 g borage oil (690 mg GLA) daily for 24 wk and the placebo group received migliol, an oil containing no GLA. Patients were allowed to use a steroid cream during the trial. Some patients did not follow the guidelines and violated the conditions of protocol, e.g., poor compliance (<70% of dose consumed; 7 placebo, 6 borage oil), excessive use of steroid cream (3 times the median dose; 1 placebo and 4 borage oil), e l 1 wk of treatment (6 each treatment), and patients with unstable disease (Costa score <18 at week 2; 32 placebo and 21 borage). When all of the patients, including those who did not follow the protocol, were included in the data analysis, there were no significant differences in Costa scores between the two groups. Borage oil treatment did improve erythema, vesiculation, crusting, excoriation, lichenification, and insomnia scores over placebo, and a marked reduction in serum immunoglobulin E levels was observed but the difference was not significant due to the large inter-subject variations. Borage oil treatment also increased plasma and erythrocyte levels of GLA and DGLA in the majority of patients. When the subgroup of patients who did not follow the protocol was excluded from the analysis, the borage oil treatment showed significant improvement in reducing the use of steroid cream. Borage oil was well tolerated with minor side effects (headache, nausea, vomiting, and diarrhea). The frequency of these side effects was not different from that observed with the placebo treatment. Takwale et al. (126) also reported no beneficial effect of borage oil treatment in adults and children suffering from atopic eczema. In this single-center, doubleblind, clinical trial, adult patients were given 920 mg GLA from 8 capsules of borage oil, and children were given half of the adult dose daily for 12 wk. Patients were allowed to continue using a steroid ointment for symptomatic relief of symptoms. The efficacy of the treatment was evaluated from a change in total symptom score measured with the six-area, six-sign, atopic dermatitis (SASSAD) score as
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the primary endpoint. Secondary endpoints included symptom score assessment on visual analog scales, topical corticosteroid requirement, and global assessment of response by participants. This study did not report any effect of borage oil treatment on eczema, although the treatment was safe, well tolerated, and free from major side effects. The study discussed above (126) suffered from several major limitations. They recruited 151 patients; of these, 11 were lost at wk 2 of the 12-wk study. A further 16 participants withdrew during the trial, leaving only 124 subjects who completed the trial. However, they analyzed the data for 140 patients, including those who did not complete the protocol. A good clinical trial demands inclusion of data only from those patients that followed the protocol. Noncompliance with the treatment protocol is the singlemost important reason for failure of treatment in dermatological practice and was evident in the study by Henz et al. (125). They used two different placebo treatments: liquid paraffin for adults and olive oil for children. Liquid paraffin is an inert material for its effect on atopic dermatitis, whereas olive oil cannot be considered inert because it can modify cellular fatty acid profile. Olive oil was reported to increase tissue levels of DGLA (127,128) which may increase the dermal levels of LOX and COX metabolites of DGLA, which are reported to exert anti-inflammatory actions (129,130). Therefore, olive oil may have some beneficial effects due to the above-mentioned biochemical pathways; hence, this may not be a true placebo, thereby dampening the effect of treatment. Therefore, a separate analysis in adults and children was highly desirable to avoid the potential variations in outcome induced by different placebos. The scoring system (SASSAD) used in that study as a primary outcome parameter is reported to have a very high interobserver variation (7-30, median 15.5, out of a possible score of 108) (131). Diabetes
Diabetics are at high risk of complications including cardiovascular (atherosclerosis, heart attack, stroke, peripheral vascular disease), neurological (neuropathies), renal failure, skin diseases (dry, itchy skin, slun infections), impaired wound healing, retinopathy, and impotence. A combination of neuropathy and vascular disease in diabetics leads to amputations. The mechanism of complications of diabetes is not well understood. High blood glucose levels may contribute to various complications through several pathways; increased oxidative stress, modification of proteins by glucosylation, reduction in the production of vasodilator mediators including nitric oxide, prostacyclin, and altered cytokine production may be involved. Reduced tissue perfusion and the resulting cellular damage contribute to several pathophysiologic situations. Several studies confirmed that diabetes inhibits the activity of D-6-D (132-134), leading to a lower content of DGLA and AA in various tissues in diabetic patients (135). This may lead to an imbalance in different eicosanoids in diabetics and contribute to various complications that are
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common in diabetics. Supplementation with GLA-rich oils showed some benefits in protectingldelaying these complications associated with diabetes. Diabetic patients and animals have reduced nerve conduction velocity. Supplementation with GLA alone significantly attenuated a deficit in motor nerve fiber conduction velocity caused by streptozotocin-induced diabetes in rats (136). Combined treatment with GLA and EPA completely prevented the diabetesinduced reduction in motor nerve conduction velocity. In that study, GLA and/or EPA had no effect on diabetes-induced weight loss, increase in blood glucose, and glycosylated hemoglobin levels. Julu and Mutamba (137) compared the effects of GLA and insulin on sensory and motor nerve conduction velocity in streptozotocin-induced diabetes in rats. After induction of diabetes, a significant reduction in conduction velocity in myelinated sensory and motor nerve fibers was observed, whereas there was a trend for reduction in conduction velocity in unmyelinated sensory fiber. Due to high intersubject variability, the fall in conduction velocity in unmyelinated sensory fibers was not significant. Treatment with insulin for 3 d partially corrected the deficit in sensory nerve conduction velocity, and motor nerve conduction velocity was brought back to normal levels. GLA treatment for 3 d overcorrected the sensory nerve conduction velocity, whereas the motor nerve conduction velocity was brought back to normal levels. Treatment for 5 d with insulin returned the motor nerve conduction velocity to normal levels, and treatment with GLA for 5 d brought sensory nerve conduction velocity to normal levels. The overcorrection in sensory nerve conduction velocity experienced by the 3-d treatment with GLA cannot be explained. GLA treatment had no effect on blood glucose levels or diabetes-induced weight loss, whereas insulin corrected both of these parameters. Cameron et al. (138) studied the effect of GLA alone or in combination with fish oil on diabetes-induced reduction in nerve conduction velocity and resistance to conduction block and found that diabetes increased the resistance of nerves to hypoxic conduction block and reduced the nerve conduction velocity. Treatment with GLA prevented these changes, whereas a combination with fish oil was less effective. Their results on conduction velocity differed from those reported by Julu and Mutamba (137), who observed better efficacy of GLA and fish oil in combination. This difference is difficult to explain except for the sex differences in the rats in the two studies. Julu and Mutamba (137) used female rats, whereas Cameron’s (138) group used male rats. The role of female sex hormones in the differential observations of the two groups cannot be discounted because the polyol pathway is differentially affected in males and females. This pathway may be mediating a greater role in conduction velocity reduction in males than in females due to reduction in the perfusion of the vasa nervosum. GLA treatment also prevented a streptozotocin-induced fall in endoneural blood flow and oxygen tension (139). Diabetes-induced reduction in endoneural blood flow and oxygen tension may be causing hypoxic injury to the nerve cells, leading to reduced nerve conduction and neuropathy. By preventing the blood flow deficit, GLA may have prevented the
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reduction in nerve conduction velocity. GLA acts synergistically with antioxidants in the prevention of nerve conduction deficit (140). This synergistic effect on nerve conduction velocity was mediated by an improvement in the sciatic nerve capillary endoneural blood flow from the combined treatment. Other antioxidants given with GLA or as a conjugate with GLA led to similar improvements in diabetes-induced nerve conduction deficits. The antioxidants included ascorbic acid, a-lipoic acid (as conjugates), vitamin E, ascorbic acid, a-lipoic acid, n-acetylcystein, and butylated hydroxyltoluene. These observations confirm previously reported observations that diabetic complications may be mediated by a combination of increased oxidative stress and abnormalities in EFA metabolism. Treatment with GLA at a dose of 380 mg/d for 6 mon improved 9 of 12 neurological symptoms evaluated in patients with type 1 and 2 diabetes with established neuropathy (141). In that double-blind, placebo-controlled trial of evening primrose oil, treatment with GLA also increased the plasma phospholipid content of GLA, DGLA, and AA, which is reduced in diabetes. The GLA treatment had no effect on glycosylated hemoglobin levels. These observations indicate that GLAinduced improvements may be mediated by improved perfusion of nerves rather than a correction in metabolic derangements. By improving tissue perfusion, the treatment may have prevented hypoxic insult and related injury to the nerves, thereby improving nerve conduction and reducing the associated pain and numbness. Keen et aZ. (142) conducted a double-blind, placebo-controlled, multicenter clinical trial of GLA in 111 diabetic patients with mild neuropathy. They studied the effect of treatment over 1 yr on 13 symptoms including motor nerve conduction velocity, muscle strength, hot and cold thresholds, sensation, and tendon reflexes. GLA treatment at a dose of 480 mg/d had beneficial effects on functions, and these effects were more pronounced in well-controlled diabetics than in poorly controlled subjects lnfant Nutrition and Development
The role of GLA in fetal development and infant nutrition is not very clear. The body weight of infants at birth was positively associated with the proportions of AA and DGLA in plasma TG and choline phosphoacylglycerols in premature infants (143) and infants born at full term (143,144). The positive association of DGLA with birth weight is more consistent than that observed for AA or DHA (144). This information suggests that DGLA plays an important role in fetal development. The role of DHA in fetal development is well established, and strategies are being employed to increase the levels of DHA in infant formula and breast milk. Careful analyses of women’s breast milk composition from different geographical areas revealed that women who had a higher amount of DHA in their breast milk lipids also had a higher amounts of DGLA (145). This observation suggests that DGLA has a role in infant development. Earlier discussion in this chapter
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revealed that GLA and DGLA may be playing a role in atopy in infants because the breast milk of mothers of atopic children had lower GLA and DGLA levels. Fewtrell et al. (146) treated preterm infants with a combination of DHA and GLA (from borage oil) for 9 mon in a double-blind, randomized, placebo-controlled trial. They observed that the combination of GLA and DHA led to increased Bailey’s mental development index score at the age of 18 mon in supplemented boys. The supplemented infants had greater gains in body weight and length, and the effects were particularly greater in boys. Supplementation with GLA was without any side effects and supported growth and neural development. The recently popularized Barker hypothesis (147) (the fetal origins hypothesis) states that during fetal development, environmental exposures set the limit for metabolic capacity. When this capacity is exceeded later in life, overt disease develops. According to this theory, the effect of nutrient deficiency may be more marked than that during later life and may cause a predisposition to many chronic diseases such as heart disease, diabetes, or metabolic syndrome (148,149). In support of this hypothesis, it was observed that children who had higher levels of DGLA in umbilical cord plasma phospholipids at birth did not develop insulin resistance at the age of 7 yr, whereas those with lower levels of DGLA had increased insulin resistance, body fat, insulin, proinsulin, and leptin concentrations (150). The exact mechanism by which DGLA in infancy and fetal development can affect health conditions in later life is not clear. It may be possible that GLA, which is a ligand for PPAR, affects the transcriptional regulation of glucose and lipid homeostasis.
New Research Directions New research is focusing more on specific combinations of GLA with other drugshnterventions for the treatment of several disease conditions discussed above. GLA was reported to enhance the cytotoxicity of anticancer agents including vinca alkaloids (vincristine, vinblastin, and vindesine) (15 1). GLA may also increase the absorption of anticancer agents such as vinca alkaloids (15 1) and doxorubicin (152). It also increased the sensitivity of resistant cancer cells to doxorubicin toxicity, which was associated with increased superoxide dismutase activity with no effect on catalase activity and p-glycoprotein levels (152). This observation suggests that GLA has no effect on p-glycoprotein, which plays a role in the development of multidrug resistance. However, by increasing the levels only of superoxide dismutase and not catalase activity, GLA may stimulate the formation of hydrogen peroxide in the cancer cells, and this may contribute to the oxidative toxicity of doxorubicin. Hydrogen peroxide can form hydroxyl radicals, which are highly toxic to adjacent molecules. GLA also acted synergistically with soy protein in reducing the production of LTB4 (153) from peritoneal exudate cells, suggesting that soy protein may stimulate anti-inflammatory actions of GLA. Other research efforts are in the area of
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structured lipids containing GLA, EPA, and DHA on one TG molecule. Structured lipids can be produced by interesterifying a mixture of conventional fats and oils of interest using chemical or enzymatic methods. Chemical methods provide random distribution of different fatty acids on the glycerol backbone, whereas enzymatic reactions could be position specific, affording controlled production of TG with the desired configuration (154).
Safety of GLA-Containing Oils Safety h u e s
GLA-rich oils, such as borage and evening primrose, were used in several clinical trials in addition to laboratory animals as discussed in this chapter. None of these studies reported any serious side effects of GLA treatment. The most common side effects included gastric upsets such as soft stools, occasional diarrhea, and stomach cramps. Borage Oil Safety Issues. The borage plant contains pyrrolizidine alkaloids (PA). Seven pyrrolizidine alkaloids have been identified to date in borage leaves, flowers, and seeds. Thesinine, a saturated PA, is the major alkaloid in seeds, whereas the six unsaturated PA identified include amabiline, supinine, lycopsamine, interemedine, acetyllycopsamine, and acetylintermedine; they are minor constituents in the seed. The total alkaloid content of the plant was reported to be <0.001%, whereas mature seeds yield -0.03% crude alkaloids (155). Henman et al. (156) reported the presence of thesinine as a glycoside (thesinine-4’-O-P-D-glucoside) in seeds. Because borage oil is an item of commerce for its GLA content, there is a concern about the content of PA in the oil. Published research to date could not detect the presence of pyrrolizidine alkaloids in borage oil. Dodson and Stermitz (155) used a method with a detection limit of 5 ppm, whereas Parvais and Stricht (157) employed a method with a detection limit of 0.1 ppm. These authors reported the absence of PA in the oil samples tested. Mierendorff et al. (158) developed a method of detection of PA at a limit of 4 ppb. Employing Mierendorff‘s method at an independent testing laboratory in Germany, Bioriginal tested several lots of borage oil over several years and never detected any traces of PA even at such a low detection limit. The German health authority has limited the intake of unsaturated pyrrolizidine alkaloids to 1 pg/d. On the basis of the above results, and assuming that borage oil contains PA at a level of 4 ppb, one would have to consume >250 capsules (1000 mg each) every day to obtain a total of 1 pg PA. Based on this analysis, there is no likelihood of toxicity from PA with borage oil ingestion.
Evening Primrose Oil Safety Issues. Evening primrose oil was associated with reducing sensitivity to seizure threshold in patients suffering from temporal lobe epilepsy (159). At a recent FASEB meeting, Hubbard et al. (160) presented the
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findings of their clinical trial in epileptic children given 1.0 g of evening primrose oil. They observed a slight protective effect of evening primrose oil in reducing seizure activity. Borage or black currant oils were not reported to affect seizure activity. More research is required to c o n f m the effect of GLA-rich oils on seizure threshold. It is advisable to observe caution when giving these oils to epileptic patients.
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121. Borrek, S . , A. Hildebrandt, and J. Forster, [Gamma-Linolenic-Acid-Rich Borage Seed Oil Capsules in Children with Atopic Dermatitis. A Placebo-Controlled Double-Blind Study], Klin. Paediatr. 209: 100-104 (1997). 122. Scarff, D.H., and D.H. Lloyd, Double Blind, Placebo Controlled, Crossover Study of Evening Primrose Oil in the Treatment of Canine Atopy, Vet. Rec. 131: 97-99 (1992). 123. Hartop, P.J., and C. Prottey, Changes in Transepidermal Water Loss and the Composition of Epidermal Lecithin After Applications of Pure Fatty Acid Triglycerides to Skin of Essential Fatty Acid-Deficient Rats, Br. J. Dermatol. 95: 255-264 (1976). 124. Tollesson, A., and A. Frithz, Transepidermal Water Loss and Water Content in the Stratum Corneum in Infantile Seborrhoeic Dermatitis, Acta Dermato. -Venereal. 73: 18-20 (1993). 125. Henz, B.M., S . Jablonska, P.C. van de Kerkhof, G. Stingl, M. Blaszczyk, P.G. Vandervalk, R. Veenhuizen, R. Muggli, and D. Raederstorff, Double-Blind, Multicentre Analysis of the Efficacy of Borage Oil in Patients with Atopic Eczema, Br. J. Dermatol. 140: 685-688 (1999). 126. Takwale, A., E. Tan, S. Aganval, G. Barclay, I. Ahmed, K. Hotchkiss, J.R. Thompson, T. Chapman, and J. Berth-Jones, Efficacy and Tolerability of Borage Oil in Adults and Children with Atopic Eczema: Randomised, Double Blind, Placebo Controlled, Parallel Group Trial, Br. Med. J. 327: 1385 (2003). 127. Giron, M.D., F.J. Mataix, and M.D. Suarez, Changes in Lipid Composition and Desaturase Activities of Duodenal Mucosa Induced by Dietary Fat, Biochim. Biophys. Acta 1045: 69-73 (1990). 128. Campbell, K.L., and G.P. Dorn, Effects of Oral Sunflower Oil and Olive Oil on Serum and Cutaneous Fatty Acid Concentrations in Dogs, Res. Vet. Sci. 53: 172-178 (1992). 129. Miller, C.C., and V.A. Ziboh, Gammalinolenic Acid-Enriched Diet Alters Cutaneous Eicosanoids, Biochem. Biophys. Res. Cornmun. 154: 967-974 (1988). 130. Ziboh, V.A., C.C. Miller, and Y. Cho, Metabolism of Polyunsaturated Fatty Acids by Skin Epidermal Enzymes: Generation of Anti-Inflammatory and Antiproliferative Metabolites, Am. J. Clin. Nutr. 71: 361s-366s (2000). 131. Charman, C.R., A.J. Venn, and H.C. Williams, Reliability Testing of the Six Area, Six Sign Atopic Dermatitis Severity Score, Br. J. Dematol. 146: 1057-1060 (2002). 132. Ayala, S . , and R.R. Brenner, [Effect of Alloxan Diabetes on the Biosynthesis of Unsaturated Fatty Acids from Linoleic and Arachidonic Acids in Rat Liver and Testis (author’s translation)], Acta Physiol. Lat. Am. 25: 371-378 (1975). 133. Poisson, J.P., Comparative In Vivo and In Vitro Study of the Influence of Experimental Diabetes on Rat Liver Linoleic Acid Delta 6- and Delta 5-Desaturation, Enzyme 34: 1-14 (1985). 134. Mercuri, O., R.O. Peluffo, and R.R. Brenner, Depression of Microsomal Desaturation of Linoleic to Gamma-Linolenic Acid in the Alloxan-Diabetic Rat, Biochim. Biophys. Acta 116: 409-41 1 (1966). 135. Arisaka, M., 0. Arisaka, and Y. Yamashiro, Fatty Acid and Prostaglandin Metabolism in Children with Diabetes Mellitus. 11. The Effect of Evening Primrose Oil Supplementation on Serum Fatty Acid and Plasma Prostaglandin Levels, Prostaglandins Leukot. Essent. FattyAcids43: 197-201 (1991). 136. Julu, P.O., Essential Fatty Acids Prevent Slowed Nerve Conduction in Streptozotocin Diabetic Rats, J. Diabet. Complicat. 2: 185-188 (1988).
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137. Julu, P.O.O., and A. Mutamba, Comparison of Short-Term Effects of Insulin and Essential Fatty Acids on the Slowed Nerve Conduction of Streptozotocin Diabetes in Rats, J. Neurol. Sci. 106: 56-59 (1991). 138. Cameron, N.E., M.A. Cotter, and S. Robertson, Essential Fatty Acid Diet Supplementation. Effects on Peripheral Nerve and Skeletal Muscle Function and Capillarization in Streptozocin-Induced Diabetic Rats, Diabetes 40: 532-539 (1991). 139. Cameron, N.E., and M.A. Cotter, Effects of Evening Primrose Oil Treatment on Sciatic Nerve Blood Flow and Endoneurial Oxygen Tension in StreptozotocinDiabetic Rats, Acta Diabetologica 31: 220-225 (1994). 140. Cameron, N.E., and M.A. Cotter, Interaction Between Oxidative Stress and GammaLinolenic Acid in Impaired Neurovascular Function of Diabetic Rats, Am. J. Physiol. 271: E471-E476 (1996). 141. Jamal, G.A., and H. Carmichael, The Effect of y-Linolenic Acid on Human Diabetic Peripheral Neuropathy: A Double-Blind Placebo Controlled Trial, Diabet. Med. 7: 319-323 (1990). 142. Keen, H., J. Payan, J. Allawi, J. Walker, G.A. Jamal, A.I. Weir, L.M. Henderson, E.A. Bissessar, P.J. Watkins, M. Sampson, et al., Treatment of Diabetic Neuropathy with Gamma-Linolenic Acid. the Gamma-Linolenic Acid Multicenter Trial Group, Diabetes Care 16: 8-15 (1993). 143. Leaf, A.A., M.J. Leighfield, K.L. Costeloe, and M.A. Crawford, Long Chain Polyunsaturated Fatty Acids and Fetal Growth, Early Hum. Dev. 30: 183-191 (1992). 144. Rump, P., R.P. Mensink, A.D.M. Kester, and G. Hornstra, Essential Fatty Acid Composition of Plasma Phospholipids and Birth Weight: A Study in Term Neonates, Am. J. Clin. Nutr. 73: 797-806 (2001). 145. Wang, L., Y. Shimizu, S. Kaneko, S. Hanaka, T. Abe, H. Shimasaki, H. Hisaki, and H. Nakajima, Comparison of the Fatty Acid Composition of Total Lipid and Phospholipid in Breast Milk from Japanese Women, Pediatr. Znt. 42: 14-20 (2000). 146. Fewtrell, M.S., R.A. Abbott, K. Kennedy, A. Singhal, R. Morley, E. Caine, C. Jamieson, F. Cockburn, and A. Lucas, Randomized, Double-Blind Trial of LongChain Polyunsaturated Fatty Acid Supplementation with Fish Oil and Borage Oil in Preterm Infants, J. Pediatr. 144: 471-479 (2004). 147. Barker, D.J., P.D. Winter, C. Osmond, B. Margetts, and S.J. Simmonds, Weight in Infancy and Death from Ischaemic Heart Disease, Lancet 2: 577-580 (1989). 148. Lamont, D.W., L. Parker, M.A. Cohen, M. White, S.M. Bennett, N.C. Unwin, A.W. Craft, and K.G. Alberti, Early Life and Later Determinants of Adult Disease: A 50 Year Follow-Up Study of the Newcastle Thousand Families Cohort, Public Health 112: 85-93 (1998). 149. Jackson, A.A., Nutrients, Growth, and the Development of Programmed Metabolic Function, Adv. Exp. Med. Biol. 478: 41-55 (2000). 150. Rump, P., C. Popp-Snijders, R.J. Heine, and G. Hornstra, Components of the Insulin Resistance Syndrome in Seven-Year-Old Children: Relations with Birth Weight and the Polyunsaturated Fatty Acid Content of Umbilical Cord Plasma Phospholipids, Diabetologia 45: 349-355 (2002). 151. Ikushima, S., F. Fujiwara, S. Todo, and S. Imashuku, Gamma Linolenic Acid Alters the Cytotoxic Activity of Anticancer Drugs on Cultured Human Neuroblastoma Cells, Anticancer Res. 10: 1055-1059 (1990).
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152. Liu, Q.Y., and B.K. Tan, Effects of &-Unsaturated Fatty Acids on Doxorubicin Sensitivity in P388/Dox Resistant and P388 Parental Cell Lines, Life Sci. 67: 1207-12 18 (2000). 153. Kaku, S., S. Yunoki, K. Ohkura, M. Sugano, M. Nonaka, H. Tachibana, and K. Yamada, Interactions of Dietary Fats and Proteins on Fatty Acid Composition of Immune Cells and LTB, Production by Peritoneal Exudate Cells of Rats, Biosci. Biotechnol. Biochem. 65: 315-321 (2001). 154. Senanayake, S.P., and F. Shahidi, Structured Lipids via Lipase-Catalyzed Incorporation of Eicosapentaenoic Acid into Borage (Borago officinalis L.) and Evening Primrose (Oenotheru biennis L.) Oils, J. Agric. Food Chem. 50: 477-483 (2002). 155. Dodson, C., and F.R. Stermitz, Pyrrolizidine Alkaloids from Borage (Borago officinalis) Seeds and Flowers, J. Nat. Prod. 49: 727-728 (1986). the First 156. Herrmann, M., H. Joppe, and G. Schmaus, Thesinine-4'-O-P-~-glucoside Glycosylated Plant Pyrrolizidine Alkaloid from Borago officinalis, Phytochemistly 60: 399-402 (2002). 157. Parvais, O., and V. Stricht, TLC Detection of Pyrrolizidine Alkaloids in Oil Extracted from the Seeds of Borago oficinulis, J. Planar Chromutogr. 7: 80-82 (1994). 158. Mierendorff, H.J., Determination of Pyrrolizidine Alkaloids by Thin-Layer Chromatography in the Oil of Seeds of Borago officinalis, Fett Wiss. Technol. 97: 33-37 (1995). 159. Vaddadi, K.S., The Use of Gamma-Linolenic Acid and Linoleic Acid to Differentiate Between Temporal Lobe Epilepsy and Schizophrenia, Prostaglandins Med. 6: 375-379 (1981). 160. Hubbard, R.W., J. Westengard, D. Michelson, and S. Ashwal, Limiting trans-Fat Caused Mead Acid Production with Evening Primrose Oil (EPO), FASEB J. 18; 575.9 (abstr.) (2004).
Chapter 15
Phytosterols and Phytosterol Esters Robert A. Moreau Crop Conversion Science and Engineering Research Unit, Eastern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Wyndmoor, PA 19038
Introduction Phytosterols (plant sterols) are found in all plant cells and tissues. In the same way that cholesterol stabilizes animal organelle membranes, phytosterols (and mycosterols in fungi, mostly ergosterol, not discussed in this review) stabilize plant organelle membranes, Unlike the situation in animals in which cholesterol typically accounts for 99% of the sterols, most plant species contain at least three types of sterols; sometimes, there are as many as 20 different sterols in the same species. Within the various types of tissues in one plant species, there can be diversity in the phytosterol composition. During the last 10 yr, there has been a huge interest in phytosterols, prompted mainly by the commercialization of the first phytosterolenriched functional food in Finland in 1995 and in much of the rest of the world in the last five yr (1). There have been several excellent reviews on phytosterols, most notably: a very comprehensive review of biological, chemical, and nutrition aspects of phytosterols by Piironen and colleagues (2); a comprehensive review from our laboratory (3); and five reviews that focused on the clinical nutrition aspects of phytosterols (4-8). An excellent book by Dutta (9) devoted entirely to phytosterols was also published recently. The book contains 12 chapters that cover most of the major topics of phytosterol chemistry, biochemistry, safety, and nutrition. The purpose of this review is not to be a comprehensive treatise on all of phytosterol chemistry, structure, and function, but to focus on areas of phytosterol research that may be of interest to scientists not well acquainted with the field (mainly lipid chemists and biochemists). The Occurrence, Function, Biosynthesis, Biochemistry, and Molecular Engineering of Phytosterols in Plants
Occurrence. Phytosterols (plant sterols) are members of the “triterpene” family of natural products, which includes >lo0 different phytosterols and ~ 4 0 0 0other types of triterpenes (1,4). Cholesterol is the predominant sterol in animals; free cholesterol serves to stabilize cell membranes and, cholesteryl FA esters are a storagekransport form, usually found associated with triacylglycerols (4).Plant membranes contain little or no cholesterol; instead, they contain several types of phytosterols 335
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that are similar in structure to cholesterol but include a methyl or ethyl group at C-24. A convenient way to describe and catalog phytosterols is to divide them into three groups based on the number of methyl groups on carbon-4, i.e., two (Cdimethyl), one (4-monomethyl), or none (4-desmethyl). 4-Dimethylsterols and 4-monomethylsterols are metabolic intermediates in the biosynthetic pathway leading to the end-product, 4desmethyl phytosterols, but they are usually present at low levels in most plant tissues. Cycloartenol and cycloartanol are examples of 4-dimethylsterols, and gramisterol is an example of a 4-monomethylsterol (Fig. 15.1). 4-Desmethylsterols include the 27-carbon sterol cholesterol (Fig. 15,l) (ubiquitous and predominant in animals, but also generally present at low levels in plants) and all of the common 28-carbon and 29-carbon (Fig. 15.2) phytosterols, which are typically major membrane structural components in plant cells. Most 4-desmethyl phytosterols have a double bond between carbons 5 and 6 of the ring system and are thus called As-phytosterols. However, another group of common desmethylsterols that are abundant in plants of certain families have a double bond between carbons 7 and 8 instead of 5 and 6, and are hence referred to as A7-phytosterols. Both A5- and A7-desmethylsterols can include a second double bond in the alkyl side chain, most frequently between carbons 22 and 23. The cornrnon 29-carbon desmethylsterol stigmasterol (Fig. 15.2), which includes both C5, 6 and (trans) C22, 23 double bonds, is, for example, designated as As,22E. For the C28 and C29 phytosterols, the introduction of a methyl or ethyl group at C24 renders this position chiral and thus two epimers (aand p) are possible. In all plant tissues, phytosterols occur in five common lipid classes (Fig. 15.3), i.e., as the free alcohol (FS), as fatty-acyl esters (SE), as hydroxycinnamate steryl esters (HSE), as steryl glycosides (SG), and as acylated steryl glycosides (ASG). The last four forms (SE, HSE, SG, and ASG) are generically called “phytosterol conjugates.” In free phytosterols (FS), the 3P-OH group on the A-ring of the sterol nucleus is underivatized, whereas in the four conjugates, the OH is covalently bound with another constituent. The OH group is ester-linked with an FA in SE and HSE, and is linked by a 1-0-P-glycosidic bond with a hexose (most commonly glucose) in SG [first reported by Power and Salway in 1919 (lo)]. The last-mentioned group of phytosterol conjugates, ASG, differs from SG by the addition of an FA
Cholesterol
G;amisterol
Cycloartanol
(24-methylenelophenol) Fig. 15.1. Examples of 4-desmethy1, monomethyl, and dimethyl phytosterols.
Phytosterols and Phytosterol Esters
HO
Campesterol
HO
&
Ho HO
Brassicasterol %.
337
Sitosterol
Stigmasterol
i
HO
Ergosterol
& 28-lsofucosterol
HO A7-Stigrnasterol
HO
Carnpestanol
Spinasterol *,
Stigrnastanol Sitostanol
Fig. 15.2. Some common C28 and C29 desmethyl phytosterols.
esterified to the 6-OH of the hexose moiety [fist reported by Lepage in 1964 (Il)]. Seeds of corn and rice and other grains contain a fourth type of phytosterol conjugate, hydroxycinnamate steryl esters (HSE), in which the sterol 3P-OH group is esterified to ferulic or p-coumaric acid (Fig. 15.3). There is a widespread misconception that plant tissues are devoid of cholesterol. The fact is that this C27 sterol, which is predominant in animals and a contributing
factor in human cardiovascular disease, often accounts for 1-2% of the total plant sterols, and can comprise 5% or more in select plant families, species, organs, or
R.A. Moreau
338
Free Phytosterol (free OH at the 3 carbon)
(Sitosterol)
____& f0
0
Steryl Fatty Acid Ester (SE)
(Sitosteryl stearate) pI=CHCOO
Hydroxycinnamate Steryl Ester (HSE)
(Sitostanyl ferulate)
on Steryl Glycoside (SG)
yvc
(Sitosteryl P-D-glucoside)
Acylated Steryl Glycoside (ASG)
(Sitosteryl [6’-Ostearoyl] P-D-glucoside) Fig. 15.3. Common phytosterol lipid classes.
tissues. Despite the fact that the edible portion of some crop plants can include cholesterol as a significant portion of the total phytosterols, it should be noted that
this is inconsequential in the human diet relative to the amount of cholesterol in meat and dairy products. Many species of the Solanaceae (Nightshade family)
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339
contain relatively high levels of cholesterol (12). In total, sterols from the pericarp tissue of mature-green tomato (Lycopersicon esculentum) fruit, cholesterol constituted -6%, and in the SE fraction it ranged from 15 to 20% (13J4). A study of the sterol composition of seed oil from 13 species of Solanurn showed that in six species, the combined FS plus SE fractions contained >5% cholesterol, and the level in one species, S. pseudocapsicum, ranged from 10 to 22% (15). Several unique phytosterols and phytosterol conjugates were reported in cereal grains. Seitz (16) reported trans-hydroxycinnamate esters of phytosterols (HSE, including steryl ferulate and p-coumaroyl esters) in corn, wheat, rye, rice, and triticale. Norton ( 1 7 ~ 8extended ) these studies and separated several additional molecular species hydroxycinnamate esters from rice bran and corn bran. We reported that the levels of hydroxycinnamate esters in corn fiber were higher than in corn bran or any other grain (19). In a recent paper, we compared the levels of SE, FS, and HSE in 66 accessions of Zea, teoscinte, and Job’s tears, and identified several corn accessions with very high levels of HSE and total phytosterols (20). Both Seitz and Norton noted that sitostanol was the predominant phytosterol in corn HSE (16,18), whereas cycloartenol and 24-methylene cycloartenol were the predominant phytosterols in rice bran HSE (called “oryzanol”). Piironen and colleagues (21) recently compared the composition of total sterols (free + bound) in rye, oats, barley, wheat, corn, and other grains. They found that all grains contained significant levels of phytostanols (sitostanol and campestanol) in the total phytosterol fractions. Recent studies from our laboratory indicate that most of the phytostanols in corn are esterified in either SE or HSE, and all of the HSE is localized in the aleurone cells, which form a single layer in corn and fractionate into the corn fiber fraction during wet milling (22). Because commercial corn oil is obtained by extracting corn germ, the levels of HSE and phytostanols in corn germ oil are very low (23,24). Function. In general, phytosterols are thought to stabilize plant membranes, with an increase in the sterol/phospholipid ratio leading to membrane rigidification (25); however, individual phytosterols differ in their effect on membrane stability. Stigmasterol was reported to have a disordering effect on membranes (26), and the molar ratio of stigmasterol to other phytosterols in the plasma membrane increases during senescence (27). In plant as in animal cells, the plasma membrane is greatly enriched in sterols relative to other cell membranes (28,29). The profound effects of sterols on the physical properties of membranes are well-documented (30). Through interaction with phospholipids in a 1:2 stoichiometry, sterols condense the bilayer, reduce bulk fluidity and permeability, and broaden or eliminate phospholipid phase transitions (3 1,32), Sterol-phospholipid interactions influence membrane functions such as simple diffusion, carrier-mediated diffusion, and active transport, and also modulate the activities of membrane-bound enzymes or receptors (33). Although a wide
variation in sterol structure can be accommodated to fulfill the bulk membrane structural requirement (30), there appear to be other, more subtle functions or spe-
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cific situations, such as salt stress, for which sterol structure becomes more critical (34,35). Evidence from sterol biosynthesis inhibitor studies indicates that phytosterols also play an essential role in plant cell division (36-38). Compared with research on membrane phospholipids, the role of sterol lipids in pre- and postharvest plant physiology has received little attention (3,4,39,40). A sharp increase in the stero1:phospholipid ratio in microsomal membranes during plant senescence is associated with loss of membrane function (41,42). Changes in sterol composition that are likely to affect membrane function (28,43,44) can occur with greening, shading, maturation, aging, or ripening of plant tissues (13,4547). There is evidence that free sterols are tightly bound to the plasma membrane H+ATPase and may be essential for activity of this critical enzyme (48). Further work showed that H+-ATPase activity is dependent upon the kind and amount of sterol present in a reconstituted system (49,50). Zelazny and colleagues (51) showed that free sterols in the plasma membrane of the marine alga Dunuliellu are absolutely required for sensing osmotic changes. Sterol conjugation, the conversion of free sterols (FS) to steryl esters (SE), steryl glycosides (SG), or acylated steryl glycosides (ASG), is another potentially important aspect of membrane lipid metabolism. Like FS, SG and ASG are membrane structural components, whereas SE appear to be largely excluded from membranes (52,53), possibly because of their relatively low solubility in a phospholipid bilayer (54). Metabolic studies showed that the interconversion of sterols and sterol conjugates is quite rapid, suggesting a regulatory function (55). On the basis of reports that sterol interconversions are controlled by phytohormone levels and environmental factors such as light, temperature, and water stress, it was postulated that they are involved in the regulation of membrane properties in response to changing conditions (33). Kesselmeier and colleagues (56) reported an increase in the levels of SG and ASG during the enzymatic preparation of protoplasts from oat leaves. The extent of sterol glycosylation and esterification can be altered dramatically in response to ozone stress in snapbean leaves (57), growth conditions in bell pepper fruit (58), freezing stress in potato leaf plasma membrane (59), chilling stress in tomato fruit (60), and challenge by fungal elicitors, cellulase, xylanase, or copper ions in tobacco cells (61). In particular, it appears that different types of stress can promote conversion of FS to ASG (56,57,61). Recently, Peng and colleagues (62) reported that sitosterol-P-glucoside serves as a primer for cellulose synthase in plants. Because it is thought that most of the ASG and SG is localized in the plasma membrane (59,61), the involvement of SG in a biosynthetic process that occurs adjacent to the plasma membrane is a reasonable hypothesis. There is ample evidence that in plants, sterols serve as precursors in the synthesis of steroidal saponins and alkaloids, as well as ecdysteroids (insect molting hormones) and other pregnane- and androstane-type steroids (Fig. 15.4). In medicinal herbs and food plants, steroidal saponins and alkaloids are of interest because of their potential pharmacological activity and/or toxicity in animals.
Phytosterols and Phytosterol Esters
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Biosyntbesis and Biochemistry. The topic of plant sterol biosynthesis and metabolism was reviewed recently by Benveniste (63). The review describes the importance of recent studies with Arabidopsis mutants to the field of plant sterol metabolism (63). All triterpenes are synthesized via a pathway (Fig. 15.5) that starts with the reduction of HMG-CoA (six carbons) to mevalonate (five carbons) (2). Six mevalonate units are then assembled into two farnesyl diphosphate molecules, which are combined to make squalene (30 carbons or “three terpenes”). Enzymatic ring closure steps then form cycloartenol (also 30 carbons), and additional enzymatic reactions form common plant triterpenes such as phytosterols, triterpene alcohols, and brassinosteroids (Fig. 15.4). It was once thought that all
Phytosterol
/
.
(Campesterol)
Steroidal Glycoalkaloids
Phytoecdysteroids
P
H
20-H ydroxyecdysone Fig. 15.4. Phytosterols as precursors to other phytochernicals.
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HMG-COA
+MeValonate
--*Farnesyl d i ~ h o s ~ h a t e
-@ \
N
Squalene
d
*w Cycloartenol
24-methylene cycloartanol
d-24-ethylidene lophenol I f
w
24-methylene lophenol
lsofucosterol
/d&+ 24-methylene cholesterol
Sitosterol Campesterol
Campestanol
Stigmasterol
Sitostanol
Fig. 15.5. Biosynthesis of phytosterols.
isoprenoids in plants were synthesized via this mevalonate pathway. In the 1990s, Lichtenthaler et al. (64) discovered that sterols, sesquiterpenes, and polyterpenes were synthesized via the mevalonate pathway in the cytoplasm, whereas monoterpenes, diterpenes , and carotenoids were synthesized via a “non-mevalonate” (glyceraldehydes 3-phosphate/pyruvate) pathway in the plastids.
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A molecular-genetic study demonstrated that the activity of sterol methyltransferase 1 (SMT1) governs the level of cholesterol in plants (65). SMTl catalyzes the first step in the production of C28 and C29 phytosterols, methylation of cycloartenol to 24-methylene cycloartenol. In mature Arabidopsis plants bearing an smtl null mutation, cholesterol was the major sterol and comprised 26% of the total phytosterols, compared with 6% in wild-type plants. Because four of the five phytosterol classes are “conjugates” (meaning that the phytosterol moiety is covalently bound to another molecule), absorption and digestion of these conjugates probably require that they be hydrolyzed by digestive enzymes. These bonds are routinely cleaved via acids and bases during analysis, but very little is known about the hydrolysis of these conjugates by digestive enzymes (Fig. 15.6).Only for one of the four conjugates, phytosteryl fatty acyl esters (or phytostanyl fatty acyl esters), has it clearly been demonstrated that complete hydrolysis occurs in the upper portion of the small intestine (4,66). Further studies are required to determine whether and how the other phytosterol conjugates (HSE, SG, and ASG) are hydrolyzed and metabolized. Molecular Engineering, In the same way in which modem tools of genetic engineering have been applied to modify the FA composition of the vegetable oils from several oilseeds (66), several research papers (67,68) and patents (69-71) described various strategies to increase the levels of phytosterols in several plant species. A recent paper by Venkatramesh et al. (72) described the engineering of a Streptomyces gene into oilseeds (soybean and rapeseed) to modify their relative amounts of phytosterols and phytostanols. The enzyme that was inserted into the oilseeds catalyzed the reduction of the A5 double bond of phytosterols. Interestingly, in this study, the C5 double bond of brassicasterol and stigmasterol (which contain two double bonds) was reduced, but their C22 double bond was unaffected by the engineered reductase. Natural phytostanols occur at trace levels in many plants and at high levels in corn and some other grains (3). Phytostanols can also be produced by the chemical hydrogenation of phytosterols (3).
The Occurrence of Phytosterols in Foods All plant-derived foods contain phytosterols, with the levels in conventional foods (5,73-76) ranging from <1 mg to -100 mghtandard serving size (Table 15.1). The levels of phytosterols in functional food products (76) are typically -1 g/serving (Table 15.1). It has been estimated that the average daily Western diet contains -200-500 mg cholesterol and -200-300 mg phytosterols (4). Modem vegetarians and our human ancestors probably consumed higher levels of phytosterols (Fig. 15.7). The phytosterol drugs that were prescribed in the 1960s had recommended dosages of 15-20 g/d, and the ‘‘first generation” phytosterol functional foods were “high-fat’’ with recommended phytosterol dosages of 3-5 g/d (6). The “second generation” phytosterol functional foods are low-fat and nonfat, with new formulation strategies to reduce the amount of phytosterols required for cholesterol lowering (see formulations section below),
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ikr& go
0
Steryl Fatty Acid Ester (SE)
(Sitosteryl stearate)
Hydroxycinnarnate Steryl Ester (HSE) (Sitostanyl ferulate)
Steryl Glycoside (SG) (Sitosteryl P-D-glucoside)
Acylated Steryl Glycoside (ASG) (Sitosteryl [6’-Ostearoyl] P-D-glUCOSide) Fig. 15.6. Sites of hydrolysis of phytosterol lipid classes by acid hydrolysis (Ac), by alkaline hydrolysis (Alk), by the digestive enzymes in the digestive systems of colectcomized patients ( E l ) ( 7 8 ) , by the digestive enzymes in bovine pancreatin (E2) (Moreau et a/., unpublished), and enzymatic hydrolysis by digestive enzymes not yet demonstrated (E3).
The Effect of Dietary Phytosterols on Human Nutrition and Health
/nfesfina/Absopfion of Cho/esfero/and Phyfosferok Approximately 40-60% of dietary cholesterol is absorbed in the small intestines (3,4). Phytosterols are absorbed at a lower rate than cholesterol (Table 15.2), and phytostanols are absorbed at a lower rate than phytosterols (77). Low but detectable levels of phytos-
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TABLE 15.1 Phytosterol Concentrations in Some Conventional and Functional Foods Phytosterols (mgA 00 g)
Food Apple Orange Strawberry Avocado Potato BroccoI i Whole ground corn Whole wheat flour Toasted wheat germ Olive oil Soybean oil Sunflower oil Corn (germ) oil Corn fiber oil Benecol Take Control@(Pro-activTM)
12 34 10 83 5 40 120-1 80 40-80 41 0 221 250 100 968 -1 0,000 10,700 11,800
Ref. 73 73 74 73 73 74 74 74 75 73 73
73 73 76 76 76
terols can be measured in the serum (Table 15.3) ( 5 , 7 8 4 0 ) . Several studies demonstrated that ingestion of high levels of phytosterols (1-3 g/d) increases the levels of serum phytosterols, and ingestion of high levels of phytostanols (1-3 g/d) decreases the levels of serum phytosterols ( 6 ) .Cholesterol absorption and how it is influenced by phytosterols were studied by several methods (78-80). Wang et al. (8 1) recently reported a new single-isotope-labeled cholesterol-tracer approach for Vegetation diets New formulations 2002
W
Supplementation __ 1960s Medication
Fig. 15.7. Historical perspectives of phytosterol intakes and recommended supplementation levels (g/d) for cholesterol reduction relative to dietary form. Source: Reference 6, with permission.
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TABLE 15.2 Intestinal Absorption
of Cholesterol and Some Common Phytosterolsa
Absorption control (%)
Cholesterol Sitosterol Sitostanol Carnpesterol Carnpestanol
-50 (range 35-70) 4.5 0.512 i 0.038 2.6 0.0441 i 0.0041 14.8 1.89 0.27 2.3 0.155 0.01 7
* *
Absorption after consumption of stanol ester margarine ( O h ) .35 (range 10-55) L. I
0.1 6.3 0.2
Ref. 78 78 80
78 80 78 80 78 80
study measured sterol absorption in colectomized patients before and after consumption of stanol ester margarine (781, and the other measured sterol absorption by use of dual stable isotope (deuterated)tracers (80).
measuring human cholesterol absorption. This new method is less invasive than previous dual tracer methods. Phytosterolemia (sitosterolemia) is a rare human genetic disorder affecting phytosterol metabolism. Patients with this disorder absorb higher than normal amounts of phytosterols, and because their bodies are not able to catabolize phytosterols, their levels of serum phytosterols are extremely high (82). Phytosterolemia has been documented in -50 families since its discovery in 1973 (82). Cho/estero/-Loweringfropetfies. The mechanism of serum cholesterol lowering by phytosterols is thought to involve their ability to prevent dietary cholesterol from being absorbed in the small intestine (Fig. 15.8). In recent years, two transporter proteins (ABCG5 and ABCG8) were identified as part of this active transport process (6,83). Plant sterols are poorly esterified by intestinal acyl-CoA:cholesterol acyltransferase (ACAT) (82). After they are absorbed, only -12-25% of the sitosterol in the thoracic lymph is esterified, whereas 70-90% of the cholesterol is esterified. Because only esterified sterols are incorporated into chylomicrons, poor esterification of phytosterols may be the main reason for the poor absorption of plant sterols into the lymph (82). More than 60 clinical studies demonstrated the cholesterol-lowering efficacy of phytosterols. A recent meta-analysis involving 18 clinical trials with phytosterol and phytostanol margarines showed that consumption of phytosterols or phytostanols reduced LDL cholesterol (LDL-C) by 8-13%, which translates into an -25% reduction in the risk of cardiovascular disease (84). In a recent review by Normen et al. (85), the authors tabulated the results of 60 clinical studies and calculated an average LDL-C reduction of 10.9% (SD 4.6), with a mean dose of 2.8 g phytosteroldd (SD 1.7 g/d) (Fig. 15.9). Interestingly, when the data from these studies
were plotted (Fig. 15.9) free phytosterolslstanols had a slightly greater level of cholesterol-lowering ability than phytosterol/stanol esters (85).
TABLE 15.3 Serum Concentrations of Cholesterol and Phytosterols During Stanol/Sterol Ester Margarine Diets Concentration in serum Control diet
Phytostanol margarine diet
Phytosterol margarine diet
6.1 0 f 0.69 mrnol/L 7.20 f 1.04 rnrnol/L
5.52 f 0.75 mmol/L -
5.64 f 0.71 mrnol/L 6.84 i 1.1 2 mmol/L
94 95
Sitosterol
3.40 f 0.88 x 1C3mg/mg C 75.7 x mrnol/mol
2.21 f 0.73 88 x 10-3 mg /mg C -
4.23 f 1.02 88 x 1W3 mg/rng C 92.1 x 1C2mmol/mol
94 95
Campesterol
7.70 f 1.92 x 10-3 mg/rng C 115.7 x 1 o-2 mmol/mol
5.09 f 1.58 x 1@ mg/mg C -
10.46 2.44 x 1C3mg/mg C 197.9 x 1W2 rnrnol/rnol
94 95
Cholesterol
*
*
Reference
Sitostanol
0.08 0.04 x 1C3rng/mg C 117.7 x 1@ mmolhnol
0.27 f 0.07 x 10-3 mg/mg c -
0.06 f 0.04 x 10-3 rnglmg C 15.6 x 1W2mmol/mol
94 95
Carnpestanol
0.06 f 0.03 x 1C3mg/mg C
0.1 6 f 0.04 x 1C3mdmg C
0.05 f 0.02 x 1C3mg/mg C
94
W
P U
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Fig. 15.8. Proposed mechanism of action of phytosterols on cholesterol absorption.
0~ I
m'
2
3
4
I
6
7
8
9
A
-8
-5
-
-
v
e
I
8 s .-9
.lo.
;ij 2
-
43.
.-0 3
P u
-20.
m .25
a (g sterol equivalentdd)
J
10
Phytosterols and Phytosterol Esters
349
The Effect of Phytosterols on Various Types of Cancers and Prosfate Hea/fh. Several studies indicated that phytosterols may have health-promoting effects such as anticancer activity. The anticancer properties of phytosterols were reviewed recently by NormCn and Andersonn (86). Awad and colleagues (87-89) provided evidence that phytosterols were toxic to breast cancer cells. Other studies indicated that phytosterols were toxic to colon cancers (90,91). It has been suggested that phytosterols are one of the active ingredients in saw palmetto and contribute to its toxicity toward prostate cancer cells (92). If these results are confirmed, then the presence of some reasonable levels of serum phytosterols may be desired and conversely, consumption of any dietary component that would lower those levels could be detrimental to health. In this regard, a review of the literature would indicate that consumption of free sterols, free stanols, and their esterified forms all may differently affect serum phytosterol levels. As noted in clinical studies (93,94) and in a recent review ( 2 ) ,consumption of largely unabsorbable stanols and stanyl esters lowers all types of serum phytosterols. On the other hand, consumption of phytosteryl esters increases blood phytosterol levels (93). For example, administration of 2.5 g/d of plant steryl esters approximately doubled the subjects’ total serum phytosterol levels (95). Although steryl esters raise serum phytosterol levels and stanyl esters decrease them, two studies suggested that consumption of free phytosterols and mixtures of free phytosterols and phytostanols may result in no change (neither increase or decrease) in serum phytosterol levels (96,97). Additional research is warranted to clarify these results. There is evidence that some phytosterols may have antioxidant activity. White and Armstrong (98) demonstrated that A5-avenasterol,which occurs in high levels in oats, may have valuable antioxidant activity. Additional studies confirmed that A5-avenasterol and other phytosterols that contain an ethylidene group possess antioxidant and antipolymerization properties, especially valuable during frying (99-10 1). These antioxidant phytosterols probably improve the oxidative stability of oat oil and other oils that contain them, but whether these antioxidant properties have any significance to human health remains to be seen. The Effect of Various Food Matrices and Formulations on Phytosterol Efficacy
During the last five years much effort has been devoted to developing various types of formulations/dispersions for phytosterols and phytostanols (1,6),Although the “fist generation” phytosterol products focused on sterol/stanol esters formulated in high-fat foods, the “second generation” sterol/stanol products are mainly low-fat formulations. Early phytosterol products such as Cytellin contained free phytosterols such as sitosterol (102). Large dosages (>25 g/d) of Cytellin@were recommended, and the cholesterol-lowering efficacy was not reliable, probably because the free phytosterols were not adequately dispersed. Scientists at Procter & Gamble first suggested esterifying phytosterols to FA to make them soluble in fat matrices such as vegetable oils (4), but they failed to produce a commercial product.
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Raisio was the first company to develop and market a phytosterol ester product, Raisio focused on a phytostanol fatty acyl ester delivered in a fat matrix such as margarine or salad dressing. Raisio and McNeil (its U.S. licensee) sponsored many clinical studies of various forms of their products (see Chapter 29), and they hold many patents (1). In addition to its spreads and salad dressings, Raisio and partners now market (in Finland) phytostanol ester products dispersed in a variety of food matrices including cream cheese spreads, semi-hard ripened cheese, pasta, milk, mayonnaise, yogurt, meat products, and snack bars (see Chapter 29). Unilever’s approach (with its Take Control@,Becel Pro-ActivTM,Flora ProActivTM,and Rama Pro-ActivTMproducts) focused on phytosterol fatty acyl esters initially delivered in high-fat spreads and salad dressings but now also marketed in low-fat formulations. Unilever and Unilever Bestfoods North America (its U S . affiliate company) also sponsored many clinical studies of various forms of their products (1). Two recent phytosterol fatty acyl ester products were developed based on unique FA, one a phytostanol ester of CLA (103) and the other a phytosterol ester of DHA (104), but both concepts are in the developmental stage, and the types of formulations that will be used for each have not been announced. Corn fiber oil is a natural extract that contains phytosterol fatty acyl esters (5-9%), free phytosterols (1-2%), and phytostanol ferulate esters (4-6%) in a high-fat triacylglycerol matrix (102,105,106). Rice bran oil also contains 1-2% of a ferulate phytosterol ester called oryzanol (mainly cycloartenyl ferulate) in a triacylglycerol matrix. These natural ferulate phytosterol esters can be found in corn fiber oil and rice bran oil and in several other grains. Condo et al. (107) recently published a procedure to synthesize ferulate phytosterol esters at high yield. Much recent effort has been devoted to developing dispersion methods for free phytosterols. Forbes Medi-Tech’s approach for their Reducol@formulation involves a proprietary process to disperse free phytosterols (108). Forbes MediTech is actively developing many products (e.g., breakfast cereal, chocolate, beverages) with Reducol@formulated into various types of food matrices. Christiansen et al. (109-1 11) developed a process to prepare “microcrystalline” phytosterols that can be formulated into many types of low-fat foods. This process is being commercialized by Teriaka (Helsinki). Interestingly, a second Finnish company, Suomen Sokeri Oy, has a U.S. patent on a process to also make microcrystalline phytosterol for food use (112). A seemingly similar technology from scientists at Cognis involves a process to make “nanoscale” dispersions of phytosterols (nanoparticles of sterols and/or sterol esters with particle diameters of 10-300 nm) (113). In addition to dispersing phytosterols, lecithin also appears to play a valuable role in increasing the bioavailability of free phytosterols (1 14,115). A recent study (116) showed that sitostanol powder (1 g) reduced cholesterol absorption in humans by -1 1%. In contrast, only 300 mg of sitostanol administered in lecithin micelles reduced cholesterol absorption by 34%. The authors concluded that free sitostanol was less effective due to its slowness in dissolving in artificial bile.
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In Japan, the Kao Corporation pioneered a diacylglycerol oil, and it also developed a form of the oil that is enriched in phytosterols (117,118). A recent study (1 19) demonstrated that 500 mg/d of phytosterols in this product reduced serum LDL-C levels by -8% compared with the same amount of phytosterols in a triacylglycerol base, which caused no decrease in LDL-C levels. Kao and Archer Daniels Midland recently formed a partnership to launch a diacylglycerol oil in the United States under the name ENOVA@oil, but no plans have been revealed to also market the phytosterol-enriched diacylglycerol oil in the United States. Monsanto received a patent on a “phytosterol protein complex,” which is comprised of phytosterols, proteins, and edible oil (120). The complex is said to “increase the bioavailability of phytosterols” and “It is most preferred to extract the phytosterols from corn fiber oil.” Kraft has developed a phytosterol dispersion that involves emulsifiers and mesophase stabilized compositions (121,122). A recent clinical study indicated that phytosterols (two thirds esterified and one third in free form) could be successfully formulated into ground beef (123). Finnish scientists developed a product called Multi-BeneTM,a phytosterol formulation that combines phytosterols with calcium and other minerals (124). The ingredients in this product are intended to lower both serum cholesterol and blood pressure (125). Two clinical studies indicated that free phytosterols and stanol esters could be formulated into a low-fat yogurt (126,127). Another clinical study demonstrated that free phytostanols and phytosterol fatty acyl esters could be formulated in bread and breakfast cereals (128). It was suggested that the presence of A5-avenasterol in virgin olive oil may contribute to its high oxidative stability and possible health benefits (129). Some have suggested that “antioxidant phytosterols” (A5-avenasteroland other phytosterols that possess an ethylidene group) could be formulated into “antioxidant oils” (101). To prove that functional foods can have very appealing flavor, Forbes Medi-Tech recently reported promising cholesterol-lowering results with a phytosterol-enriched chocolate product. In December 2002, Forbes Medi-Tech announced the results of a clinical study with their functional chocolate: participants eating the phytosterol Reducol@-enrichedchocolate reduced their LDL cholesterol by 10.3% (130). Sterolins (sterol glycosides) were formulated with free phytosterols and sold as dietary supplements to enhance immune function (13 1-133). Recently two papers reported clinical nutrition studies with phytosterol beverages, Devaraj et aZ. (134) reported significant serum cholesterol reduction with a phytosterol-enriched orange juice. In another report, Jones et al. (135) studied phytosterol-enriched lowand nonfat beverages (1.8 g/d of tall oil phytosterols), and they concluded that this phytosterol formulation had no significant effect on LDL-C levels. Jenkins et al. (136) recently reported the results of a clinical study of a “dietary portfolio” of cholesterol-lowering foods, including phytosterols (1 g/1000 kcal), soy protein (21.4 g/1000 kcal), soluble fiber (9.8 g/1000 kcal), and almonds (14 g/1000 kcal). The level of LDL-C lowering by the portfolio diet (28.6%) was comparable to that achieved with 20 mg/d Lovastatin (30.9%).Although the efficacy
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of phytosterols alone was not measured in this study, it is interesting that a combination of several cholesterol-lowering functional foods lowered LDL-C by approximately the same degree as a statin drug. Recently, Ostlund et al. (75) compared the effect of wheat germ and phytosterol-stripped wheat germ (an 80-g dose of each) on the rates of cholesterol absorption. Cholesterol absorption was -40% lower in the wheat germ treatment than in the phytosterol-free wheat germ treatment. The dosage of wheat germ in this study contained 328 mg of phytosterols, thus demonstrating how low levels of dietary phytosterols, in certain food matrices, may effectively lower cholesterol. Although this wheat germ dosage had a dramatic effect on cholesterol absorption, the cholesterol-lowering efficacy of wheat germ has not yet been reported. The last studies described in the previous four paragraphs demonstrate the importance of food matrices and formulations for the efficacy of phytosterols. More work is required to fully understand these complex interactions between phytosterols and various food matrices. The Future of Phytosterols in Conventionaland Functional Foods
During the last 10 yr, tremendous research and development efforts have focused on understanding and harnessing the cholesterol-lowering properties of phytosterols. These research and development efforts have produced hundreds of research papers, numerous patents, and several successful phytosterol functional food products. In recent years, our understanding of the role of fat in phytosterol efficacy has changed. Some promising new phytosterol formulations have been identified (e.g., lecithin and diacylglycerol oil), but more will certainly be revealed in the coming years. In addition to natural phytosterols, there has also been an effort to design more potent artificial phytosterol analogs. Recently, Forbes Medi-Tech developed a new phytostanol-ascorbate drug, FM-VP4, which is reported to effectively lower LDL-C at much lower dosages than are required with natural phytosterols (137). It can be anticipated that the coming years will continue to see advances in this field with new foods with naturally and artificially elevated levels of phytosterols, new formulation chemistries, and new phytosterol analogs. Acknowledgments I would like to thank Kevin Hicks, Jari Toivo, and Bruce Whitaker for sharing many valuable concepts that were incorporated into this chapter.
References 1. Moreau, R.A., Plant Sterols in Functional Foods, in Phytosterols as Functional Foods. edited by P. Dutta, Marcel Dekker, New York, 2004, pp. 317-346. 2 . Piironen., V., D.G. Lindsay, T.A. Miettinen, J. Toivo, and A.-M. Lampi, Plant Sterols:
Biosynthesis, Biological Function, and Their Importance to Human Nutrition, J . Sci. Food Agric. 80: 939-966 (2000).
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3. Moreau, R.A., B.D. Whitaker, and K.B. Hicks, Phytosterols, Phytostanols, and Their Conjugates in Foods: Structural Diversity, Quantitative Analysis, and Health-Promoting Uses, Prog. Lipid Res. 41: 457-500 (2002). 4. de Jong, A., J. Plat, and R.P. Mensink, Metabolic Effects of Plant Sterols and Stanols (Review), J . Nutr. Biochem. 14: 262-269 (2003). 5. Ostlund, R.E., Phytosterols in Human Nutrition, Annu. Rev. Nub. 22: 533-549 (2002). 6. St-Onge, M.P., and PJ.H. Jones, Phytosterols and Human Lipid Metabolism: Efficacy, Safety, and Novel Foods, Lipids 38: 367-375 (2003). 7. Salo, P., I. Wester, and A. Hopia, Phytosterols, in Lipids for Functional Foods and Nutraceuticals, edited by F.D. Gunstone, The Oily Press, Bridgewater, UK, 2002, pp. 183-224. 8. Ostlund, R.E., Jr., Phytosterols and Cholesterol Metabolism, Curr. Opin. Lipidol. 15: 3 7 4 1 (2004). 9. Dutta, P., Phytosterols as Functional Foods, Marcel Dekker, New York, 2004. 10. Power, F.B., and A.H. Salway, The Identification of Ipuranol and Some Allied Compounds as Phytosterol Glucosides, J . Chem. SOC. 159: 3 9 9 4 6 (1913). 11. Lepage, M., Isolation and Characterization of an Esterified Form of Steryl Glucoside, J . LipidRes. 5: 587-592 (1964). 12. Heftmann E., Phytosterols, in Isopentenoids in Plants: Biochemistry and Function, edited by W.D. Nes, G. Fuller, and L.-S. Tsai, Marcel Dekker, New York, 1984,pp. 487-518. 13. Whitaker, B.D., Changes in the Steryl Lipid Content and Composition of Tomato Fruit During Ripening, Phytochemistry 27: 3411-3416 (1988). 14. Whitaker, B.D., Changes in Lipids of Tomato Fruit Stored at Chilling and Non-Chilling Temperatures, Phytochemistry 30: 757-761 (1991). 15. Zygadlo, J.A., A Comparative Study of Sterols in Oil Seeds of Solanurn Species, Phytochemistry 35: 163-167 (1994). 16. Seitz, L.M., Stanol and Sterol Esters of Ferulic and p-Coumaric Acids in Wheat, Corn, Rye, and Triticale, J . Agric. Food Chem. 37: 662-667 (1989). 17. Norton, R.A., Isolation and Identification of Steryl Cinnamic Acid Derivatives from Corn Bran,Cereal Chem. 71: 111-117 (1994). 18. Norton, R.A., Quantitation of Steryl Ferulate and p-Coumarate Esters from Corn and Rice, Lipids 30: 269-274 (1995). 19. Moreau, R.A., M.J. Powell, and K.B. Hicks, The Extraction and Quantitative Analysis of Oil from Commercial Corn Fiber, J . Agric. Food Chem. 44: 2149-2154 (1996). 20. Moreau, R.A., V. Singh, and K.B. Hicks, Comparison of Oil and Phytosterol Levels in Germplasm Accessions of Corn, Teosinte, and Job’s Tears, J . Agric. Food Chem. 49: 3793-3795 (2001). 21. Piironen, V., J. Toivo, and A.-M. Lampi, Plant Sterols in Cereals and Cereal Products, Cereal Chem 79: 148-154 (2002). 22. Moreau, R.A., V. Singh, A. Nuiiez, and K.B. Hicks, Phytosterols in the Aleurone Layer of Corn Kernels, Biochem. SOC.Trans. 28: 803-806 (2000). 23. Ham, B., B. Butler, and P. Thionville, Evaluating the Isolation and Quantification of Sterols in Seed Oils by Solid-Phase Extraction and Capillary Gas-Liquid Chromatography, LC-GC18: 1174-1181 (2000). 24. Moreau, R.A., V. Singh, S.R. Eckhoff, M.J. Powell, K.B. Hicks, and R.A. Norton,
Comparison of Yield and Composition of Oil Extracted from Corn Fiber and Corn Bran, Cereal Chem. 76: 449451 (1999).
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Chapter 16
The Effects of Eicosapentaenoic Acid in Various Clinical Conditions Andrew Sinclaird, JulieWallaceb, Marion Martind,Nadia Attar-Bashid, Richard Weisingerc, and Duo Lid aSchool of Applied Sciences (Food Science), RMlT University, Melbourne, Australia; k h o o l of Biomedical Science, University of Ulster, Coleraine, Northern Ireland, UK; =Howard Florey Institute, The University of Melbourne, Melbourne, Australia; and dDepartment of Food Science and Nutrition, Zhejiang University, Hangzhou, China
Introduction The n-3 polyunsaturated fatty acid (PUFA) have captured the imagination of scientists and the general public. The interest in these FA developed rapidly after two Nobel Prize-winning discoveries in Medicine and Chemistry in the mid- 1970s. The most common PUFA in our diet is linoleic acid, an n-6 PUFA. The n-3 family of PUFA is distinguishable biochemically and physiologically from the n-6 family of PUFA. There are two types of n-3 PUFA: plants are the major source of alinolenic acid (ALA), which is an 18-carbon n-3 FA, whereas fish and other marine products are the main source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are 20- and 22-carbon (long-chain) n-3 PUFA, respectively. Recent technological developments in genetic engineering have raised the possibility of plants being able to make EPA (1). Dominance of the n-6 PUFA in the Food Supply
The diets of most people in Australia and other Western countries are rich in the n-6 PUFA, linoleic acid, found in vegetable oils and derived products/foods such as margarines or salad dressings. The average linoleic acid intake in Australia is estimated to be -10.8 g/d (2). The content of n-3 PUFA in Western diets is low, especially in nations with a low reliance on fish consumption, such as Australia, the United Kingdom, the United States, and Canada. When recommendations are made for an adequate intake of long-chain n-3 PUFA (EPA + DHA), the figure is on the order of 20.2 g/d of these PUFA (3,4). In Australia, according to the 1995 Australian National Nutrition Survey, the average daily intake of EPA + DHA in adults was 0.189 g/d with the median intake being only 0.029 g/d (2). In other words, Australians are not meeting their recommended daily intake of EPA + DHA. 361
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Emerging lnterest in the n-3 PUFA: In the Brain
The marine food chain is dominated by the n-3 PUFA (3,and marine species have a requirement for these FA. Until the 1970s, the n-3 PUFA were not considered to be important for humans in physiologic or biochemical terms; however, at about that time, the n-3 PUFA became a focus of research in relation to the structure and function of the mammalian brain (6). The structural lipids (phospholipids) of brain grey matter of many different mammals, as widely divergent in size and habitat as mice and whales, were found to contain the same “fingerprint” pattern of PUFA. The fingerprint revealed two main n-6 PUFA, arachidonic acid (AA) and docosatetraenoic acid, and one n-3 PUFA (DHA) (7,8). The liver and muscle phospholipids from the same species showed that a wide range of n-6 and n-3 PUFA was present in varying amounts between species. The long-chain PUFA contribute 6% of the dry weight of the cortex in humans (9). Further research showed that DHA was located in specific membranes such as the cerebral cortex synaptosomes and synaptic vesicles, and the photoreceptor outer segments in the retina (10). Highly significant reductions in the level of DHA in the brain occur when animals are fed diets deficient in n-3 PUFA. This is associated with many dramatic changes in brain function, including a reduction in the size of neurons, changes in learning and memory, changes in the auditory and olfactory responses to stimuli, changes in nerve growth factor levels (1 1)(AQ2) and alterations in the level of 2-arachidonylglycerol (a putative endogenous ligand for cannabinoid receptors) (12). Additionally, n-3 deficiency is associated with a reduction in visual function as measured by the electrical response of the retina to light (13). Various mechanisms were suggested to account for these physiologic changes in the brain and retina in n-3 deficiency, as reviewed by Kurlack and Stephenson (14), Lauritzen et al. (15), and Salem et al. (16). These include effects on membrane receptors such as rhodopsin (17), effects on dopaminergic and serotoninergic neurotransmission (1 8), effects on the activity of membrane-bound enzymes (Na/K-dependent ATPase) (19), effects on signal transduction (20), and effects on ion flux through voltage-gated K+ and Na+ channels (21,22). Another mechanism of action of the n-3 PUFA could involve competition with AA for eicosanoid synthesis (14), as well as being precursors of docosatrienes and 17s resolvins (novel antiinflammatory mediators), which are derived from DHA (23). Finally, it is possible that n-3 PUFA exert their action in the brain through regulation of gene expression (24-26). Emerging lnterest in n-3 PUFA: The Cardiovascular System
At the same time that the studies on PUFA and the brain were being conducted, a considerable interest in the marine n-3 PUFA developed due largely to the obser-
vations among Greenland Eskimos by Dyerberg and Bang (27). The Greenland Eskimos had a low incidence of death from coronary heart disease (CHD) and
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other diseases of affluence (apart from stroke), even though their diet was rich in fat from seals and other marine species (4). Their plasma lipid levels were relatively low given their high-fat diet; their plasma FA patterns were dominated by the n-3 PUFA and they had a prolonged bleeding time compared with Danes consuming a Western diet. Although cholesterol levels were a focus of attention in CHD research for many years, the extent of thrombosis is also extremely important in terms of occlusion of blood vessels (28,29). The Japanese are another major group who have traditionally consumed high quantities of fish; however, their diet was low in total fat compared with the Eskimos. Epidemiologic studies revealed that there is also a low incidence of CHD in this population; however, they experience a high incidence of stroke (30).
Eicosapentaenoic Acid Metabolic Pathways A major catabolic pathway for EPA metabolism is P-oxidation, resulting in the production of ATP and carbon dioxide (31). Another route of metabolism of EPA is incorporation into tissue membrane lipids. In tissues, EPA can be metabolized to DHA via chain elongation, desaturation, and then chain shortening. After EPA is released from membrane phospholipids by the action of phospholipase A, (PLA,), the free EPA can then be metabolized by enzymes such as cyclooxygenase (COX), lipoxygenase, and cytochrome P450 (P4,0) to biologically active compounds. Lipoxygenases are cytosolic enzymes, whereas both COX and P,,, are membrane bound (32). Both EPA and its metabolites may act as competitive antagonists of AA and its products at different levels of metabolic pathways and receptor occupancy. COX- I Path way of €PA. Molecular oxygen is added to free EPA by COX to form prostaglandin G, (PGG,). PGG, is quickly converted to PGH, by the peroxidase. PGG, and PGH, are unstable, biologically active molecules, called endoperoxides; they are intermediates in the transformation of EPA to 3-series prostanoids such as prostaglandins (PGD,, PGE, and PGF,,), prostacyclin (PGI,), and thromboxane (TX) A,, which are antithrombotic agents (33). COX2 Pathway of EPA. Aspirin [acetylsalicylic acid; (ASA)] selectively acetylates the OH- group of a single serine residue within the polypeptide chain of the COX-1, resulting in a reduction in all COX metabolites such as prostaglandins, thromboxanes, and prostacyclins. However, the ASA-acetylated form of COX-2 is still active. After treatment with n-3 PUFA and ASA, human cells generate several novel 18R- and 15R-hydroxy series of compounds from EPA (34). Polymorphonuclear leukocytes (PMN) take up 18R-hydroxyeicosapentaenoic acid (HEPE) and convert it via 5-lipoxygenase (5-LOX) to an unstable 5,6-epoxide that gives rise to 5,12,18R-tri-HEPE. In an analogous biosynthetic pathway, 15R-HEPE released by endothelial cells is converted by activated PMN via 5-LOX to a 5-series lipoxin
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(LXA) analog that also retains its C15 R configuration, i.e., 15R-LXA,. Trout macrophages and human leukocytes can convert endogenous EPA to 15s-containing LX or 5-series LX, (35). It was suggested that stable synthetic analogs of lipoxins and the aspirin-triggered 15-epi-lipoxins can mimic many of the desirable anti-inflammatory, “pro-resolution’’ actions of native lipoxins (36). Lipoxygenase Pathway of EPA. The biosynthesis of leukotrienes (LT) involves the conversion of EPA to 5-HPEPE (5s-hydroperoxy-EPA) by 5-LOX. The 5 HPEPE is converted to the labile intermediate 5,6-epoxide LTA, by LTA synthetase. LTA, may then be converted to 5-series LT. LT are produced mainly by macrophages, monocytes, neutrophils, eosinophils, mast cells, and basophils. Additionally, transcellular synthesis of LTB, and LTC, from the 5,6-epoxide LTA, occurs in endothelial cells, platelets, mast cells, lymphocytes, and erythrocytes. The 5-series LT are less potent than the AA-derived 4-series LT (33). CytochromeP450 Pathway. P450is different from COX, because COX is a dioxygenase enzyme, whereas P4,0 enzymes are monooxygenase enzymes (37). Unlike COX and lipoxygenases, P4,0 enzymes require several cofactors to metabolize FA including P4,0 reductase and NADPH (38). P4,0, in the presence of these cofactors and molecular oxygen, can serve as the catalyst for the biotransformation of EPA to a variety of oxygenated metabolites (38), including epoxides and a series of FA alcohols such as epoxyeicosatetraenoic acids and hydroxyeicosapentaenoic acid(AQ3) (39,40).
Eicosapentaenoic Acid and Cardiovascular Disease Because of observations of the low prevalence of CHD in Eskimo populations in Greenland, the cardiovascular benefits of dietary EPA and DHA have been extensively studied. Results from an ecological study of 36 countries showed that fish consumption was associated with a reduced risk of ischemic heart disease, stroke, and all-cause mortality at the population level (41). There is also evidence from prospective, intervention, and case-control studies that the intake of EPA is protective against cardiovascular disease (CVD). Prospective Studies
Two recent prospective studies from the U.S. Nurses’ Health Study investigated the effect of n-3 PUFA intakes from fish, calculated from a food-frequency questionnaire, on CHD. The results from the first study showed that increased intakes of fish and long-chain n-3 PUFA were associated with a lower CHD incidence and total mortality among diabetic women (42). This study involved 5103 female nurses, 43-59 y old, with diagnosed type 2 diabetes but free of CVD or cancer at baseline
in 1980. There were 362 incident cases of CHD [141 CHD deaths and 221 nonfatal myocardial infarctions (MI)] and 468 deaths from all causes documented during 16 yr
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of follow-up. Compared with rare fish eaters ( e l serving/mo), the relative risks (RR) of CHD adjusted for age, smoking, and other established coronary risk factors were 0.36, 0.64, 0.60, and 0.70 for fish consumption 2 5 timedwk, 2-4 times/wk, 1 time/wk, and 1-3 timedmon, respectively ( P for trend = 0.002). In the second study, in 84,688 nurses, 34-59 y old, free from previously diagnosed CVD and cancer in 1980, there were 1513 incident cases of CHD (484 CHD deaths and 1029 nonfatal MI) during 16 y of follow-up (43). Compared with subjects who rarely ate fish (el serving/mo), after controlling for other CVD risk factors, the RR for CHD were 1.0, 0.93, 0.78, 0.68, and 0.67 ( P c 0.001 for trend) across quintiles of long-chain n-3 PUFA intake. RR of CHD were 0.66 for fish consumption 2 5 timedwk, 0.69 for 2 4 times/wk, 0.71 for 1 time/wk, and 0.79 for 1-3 times/mo, respectively ( P for trend = 0.001). For n-3 PUFA and fish intake, the negative relation appeared to be stronger for CHD deaths than for nonfatal MI; RR for fish intake 5 timedwk were 0.55 and 0.73, respectively (43). Results from the EUROASPIRE (European Action on Secondary Prevention through Intervention to Reduce Events) study showed that a high proportion of EPA in serum cholesteryl esters (CE) was associated with a low risk of death from coronary artery disease (CAD) (44). In that study, 285 men and 130 women aged 33-74 yr with CAD participated in the EUROASPIRE 5-yr follow-up study. During the follow-up, 36 patients died, 21 had MI, and 12 had strokes. Compared with the lowest tertile of EPA in CE, adjusted for CVD risk factors, the RR of death for subjects in the highest tertile was 0.33 (P for trend = 0.056). A large study in Japan is testing the hypothesis that the long-term use of highly purified EPA (1.8 g/d), in addition to a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, is effective in preventing CVD events in patients with hypercholesterolemia. The study is known as the Japanese EPA Lipid Intervention Study (JELIS); 15,000participants are enrolled in the primary prevention group; there are 3645 participants in the secondary prevention phase (45). Dietary Intervention Studies
The effect of long-chain n-3 PUFA from fish on fatal and non-fatal MI and overall mortality was evaluated in a meta-analysis of 11 randomized controlled trials, published between 1996 and 1999, in 7951 and 7855 patients in the intervention and control groups, respectively. The studies had at least 6 mo of follow-up and clinical end-point data (46). Compared with control diets or placebo, RR were 0.7 ( P c 0.01) and 0.8 ( P = 0.16) for fatal and nonfatal MI, respectively, for patients who were consuming n-3 PUFA-enriched diets. The authors concluded that n-3 PUFA reduced sudden death in patients with CHD and reduced overall mortality. Results from an earlier meta-analysis of four randomized controlled trials indicated that long-chain n-3 PUFA from fish oil reduced the percutaneous transluminal coronary angioplasty (PTCA) (47). The difference in restenosis rates between fish oilsupplemented and control groups was 13.9% (95% CI, 3.2-24.5). Results from regres-
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sion analysis indicated a positive linear relationship between the dose of n-3 PUFA intake and the absolute difference in restenosis rates ( r = 0.99, P c 0.03). A recent intervention study from Esapent for Prevention of Restenosis Italian Study (ESPRIT) showed that the restenosis rate was significantly decreased when CHD patients received long-term supplementation of n-3 PUFA before and after PTCA, compared with a placebo from olive oil (48). In that study, CHD patients were randomized in a double-blind, placebo-controlled study of n-3 PUFA (n = 123) vs. olive oil placebo (n = 132). Subjects in the n-3 PUFA group received 3 g/d EPA and 2.1 g/d DHA, starting 1 mo before PTCA, administered for 1 mo thereafter, and then continued at a half-dose for 6 mon. A large and long-term secondary intervention study from Italy, the GISSIPrevenzione study, found that up to 5.7 lives in every lo00 patients with previous MI could be saved by treatment with a l-g capsule/d containing 850-882 mg EPA + DHA (49). Patients suffering from MI within the previous 3 mon were randomized into n-3 PUFA alone (n = 2835), vitamin E alone (n = 2830), combined n-3 PUFA and vitamin E (n = 2830) and control with no supplement (n = 2828). During follow-up, clinical assessment and food-frequency questionnaires were collected from each subject at 6, 12, 18, 30, and 42 mon. Compared with the control group, the group administered n-3 PUFA alone had a significantly lower risk of total CHD events with an RR of 0.78 (0.65-0.94, P = 0.008). Case-Control Studies
A recent case-control study conducted by the Cardiovascular Health Study in the United States found that higher dietary intakes of EPA + DHA may lower the risk of fatal ischemic heart disease in older adults (50).In this study of subjects 265 yr old, 54 cases experienced incident fatal MI and other ischemic heart disease deaths, 125 suffered incident nonfatal MI, with 179 randomly selected matched controls. Plasma phospholipid concentrations of n-3 FA were used as biomarkers of intake. Blood samples were drawn -2 yr before the event. A higher plasma phospholipid concentration of EPA + DHA was negatively associated with risk of fatal ischemic heart disease with an odds ratio of 0.32 (0.13-0.78, P = 0.01); however, the EPA + DHA concentration was not associated with nonfatal MI. The nested case-control prospective Physicians’ Health Study from the United States reported that n-3 PUFA intake from fish was strongly associated with a reduced risk of sudden death among men without evidence of prior CVD who were followed up for 17 yr (51). In this study, blood levels of long-chain n-3 PUFA were used as biomarkers of intake. FA composition of previously collected blood was determined for 94 men in whom sudden death occurred as the first manifestation of CVD, and for 184 controls matched for age and smoking status. After adjustment for confounding factors, baseline blood levels of long-chain n-3 PUFA were inversely associated with the risk of sudden death ( P for trend = 0.007). Adjusted RR of sudden death was 0.28 and
0.19 in the 3rd and 4th quartiles of long-chain n-3 PUFA blood levels, respectively, compared with the lowest quartile ( P < 0.05).
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The mechanisms whereby EPA (and DHA) could prevent CHD include reduction of blood pressure (BP), reduction in the levels of plasmaherum TAG and lipoprotein lipids, antithrombotic and fibrinolytic activities, antiarrhythmia, anti-inflammatory and anti-immunity actions, and blood vessel functions.
Blood Pressure A recent cross-sectional study in 9758 healthy men, 50-59 yr old, from France and Northern Ireland showed that systolic and diastolic BP were significantly lower in those who consumed fish than in those who did not ( P c 0.006 and < 0.0001, respectively) (52). The effect of long-chain n-3 PUFA from fish on BP was evaluated in an earlier meta-analysis of 3 1 placebo-controlled trials in 1356 subjects. The results indicated that systolic BP fell by 3.4 mmHg and diastolic BP fell 2.0 mmHg after the ingestion of fish oil (5.6 g/d) in a group of hypertensive subjects (53). Two human intervention studies recently provided further evidence of the important effect of long-chain n-3 PUFA on BP. In the first study, the effect of medium-term supplementation with a moderate dose of n-3 PUFA from fish oil on BP was investigated in 16 subjects with mild essential hypertension (diastolic BP 95-104 mmHg) patients and 16 controls with normal BP. Both patients and control subjects were randomly assigned to either n-3 PUFA ethyl esters (2.04 g EPA + 1.4 g DHA) or olive oil (4 g/d) for a period of 4 mon. After 2 mo, both systolic (-6 mmHg, P c 0.05) and diastolic (-5 mmHg, P c 0.05) BP were significantly decreased in the n-3 PUFA supplementation group (54). In the second study, 69 overweight (BMI > 25 kg/m2) medication-treated hypertensive subjects were randomized to either a daily fish meal (-3.65 g/d of long-chain n-3 PUFA), weight reduction, the two regimens combined, or a control regimen for 16 wk; 63 subjects completed the study. Both systolic and diastolic BP, body weight, and heart rate were significantly decreased in the fish diet group compared with a control diet, even after adjustment for changes in urinary sodium, potassium, or the sodiudpotassium ratio, as well as dietary macronutrients (55). The effect of EPA on BP reduction was suggested to be due to changes in the activities of the membrane sodium transport systems (56). In that study, 17 men with essential hypertension were assigned to an 8-wk intervention with EPA (2.7 g/d) or placebo in a randomized, double-blind study with a crossover at wk 4. Compared with placebo, systolic BP and intraerythrocyte sodium content were significantly decreased in the EPA supplement group ( P < 0.05), and EPA levels were significantly increased in erythrocyte membranes ( P < 0.001). The increased EPA levels were negatively correlated with systolic BP (Y = -0.52, P c 0.05) and intraerythrocyte sodium content ( r = -0.57, P < 0.02). Decreased intraerythrocyte sodium content was positively correlated with the decrease in systolic BP ( r = 0.54, P < 0.05),and negatively correlated with the change in Na+-K+ATPase activity ( r = -0.59, P c 0.02) (56). In human endothelial cells, EPA was found to be more effective than DHA in reducing Na+-K+ATPase activity (57).
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Plasma/Serum and Lipoprotein Lipids
Consistent and extensive evidence from intervention, case-control, and prospective studies has shown that EPA lowers fasting or postprandial serudplasma TAG levels (58-62). Meta-analysis results from 26 trials with 425 diabetes patients showed that n-3 PUFA from fish oil decreased serum/plasma concentration of TAG by 0.60 mmol/L (95% CI, -0.84 to -0.33, P < 0.01) (63). A recent intervention study suggested that the reduction in serum TAG by EPA is due to accelerated chylomicron TAG clearance via increasing lipoprotein lipase (LPL) activity, and that EPA was as effective as DHA (64). In that study, 33 healthy subjects had a 4-wk placebo (4 g/d of olive oil) run-in period, followed by a 4-wk wash-out period. Subjects were then randomly assigned to 4 g/d of ethyl esters of safflower oil, EPA, or DHA for 4 wk. Data from the EPA and DHA groups were combined because EPA and DHA had similar effects on serum lipids and other variables. Postprandial concentrations of TAG, apolipoprotein B (apo B)-48 and apo B100 were reduced by 16% (P = 0.08), 28% (P < 0.001), and 24% (P < 0.01), respectively, and postheparin LPL increased by 50% ( P < 0.05) at the end of the n-3 PUFA supplementation. The n-3 PUFA supplementation decreased chylomicron particle size (P < 0.01) and reduced chylomicron TAG half-lives (P < 0.05). Chan et al. (65) suggested that n-3 PUFA from fish oil reduced serudplasma TAG mainly by decreasing VLDL apo B production, and not by altering the catabolism of apo B-containing lipoprotein or chylomicron remnants. They studied 24 dyslipidemic, viscerally obese men who were randomly assigned to receive either 4 g/d of fish oil capsules (45% EPA and 39% DHA as ethyl esters) or 4 g/d of corn oil for 6 wk. Compared with corn oil, fish oil supplementation significantly decreased plasma concentrations of TAG (-18%), VLDL apo B (-20%), and the hepatic secretion of VLDL apo B (-29%) (P < 0.05). There were also significant increases in the conversions of VLDL apo B to intermediate density lipoprotein (IDL) apo B (71%), VLDL apo B to LDL apo B (93%), and IDL apo B to LDL apo B (11%) (P < 0.05). Nestel et al. (61) found that 3 g/d of either EPA or DHA for 7 wk significantly lowered plasma total and VLDL TAG concentrations in dyslipidemic subjects, compared with a placebo group. Two recent studies reported that EPA supplementation significantly decreased serum/plasma concentration of HDL, cholesterol. In the first study, 39 men and 12 postmenopausal women aged 61.2 f 1.2 yr randomly consumed 4 g of purified EPA, DHA, or olive oiUd for 6 wk in a double-blind, placebo-controlled parallel study. Serum TAG decreased by 19% (P = 0.022) and 15% (P = 0.022) in the EPA and DHA groups, respectively. HDL, cholesterol increased by 16% ( P = 0.026) and 12% (P = 0.05) in the EPA and DHA groups, respectively. HDL, cholesterol decreased by 11% (P = 0.026) with EPA supplementation (66). The second study, which was similar in design to the first study, involved 56 overweight, nonsmoking, mildly hyperlipidemic men aged 48.8 ? 1.1 yr. HDL, cholesterol decreased significantly (6.7%; P = 0.032) in the EPA group. However, HDL, cholesterol increased by 29% (P= 0.004) with DHA, but not with EPA supplementation (60). Both studies
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found that purified EPA and DHA both significantly lowered serudplasma TAG concentrations. In a study of patients with familial combined hyperlipidemia, in whom there is a remarkable reduction in HDL, subfraction levels, it was shown that a dose of 1.88 g EPMd and 1.48 g DHA/d for 8 wk significantly increased the HDL2-cholesterol and mass by 40 and 26% respectively, compared with the placebo group (67). Higdon et al. (68) studied the influence of long-chain n-3 PUFA on plasma lipid peroxidation in postmenopausal women, as assessed by plasma F2-isoprostane and malondialdehyde levels. They found no evidence of increased lipid peroxidation compared with subjects consuming diets rich in oleic or linoleic acid. Thrombosis
Acute clinical disorders of the cardiovascular system are caused mainly by formation of thrombosis. Arterial thrombosis plays a major role in the transition from stable to acute ischemic heart and cerebral diseases, manifested by unstable angina, acute thrombotic infarction, and sudden death. In addition to local stimuli leading to the disruption of plaques, systemic thrombogenic factors, such as platelet hyperreactivity, increased concentrations of fibrinogen and factor VII, defective fibrinolysis, and abnormalities of blood flow may contribute to the occurrence, extent, and persistence of coronary thrombosis and its clinical sequelae. Platelet aggregation is an early event in the development of thrombosis. It is initiated by TXA,, discovered by Samuelson and colleagues in 1975; it is a potent platelet aggregation agent and vascular contractor, produced from AA in the platelet membrane (69,70). EPA competes with AA for access to COX to produce TXA,, an alternative form of TXA,, which is relatively inactive in promoting platelet aggregation and vasoconstriction (71). This situation can lead to a reduced TXA, production and thus a lower thrombosis tendency (27). In Eskimos, the main n-3 PUFA in the platelet membranes was EPA, which was hardly detectable in platelets from Danes in whom the main 20-carbon PUFA was AA (27). The AA to EPA ratio in the platelets from the Eskimos was approximately 1:l compared with 440:l for the Danes. Horrobin et al. (72) reported that a subject who consumed 12 g ethyl EPA/d for 16 mon had an AA:EPA ratio in platelets of 1.9:1, approximating the levels in Eskimos. A diet with a low ratio of n-3 to n-6 PUFA can cause a low tissue ratio of EPA to AA, which may promote production of TXA,, leading to an increased tendency toward thrombosis (73). Evidence from dietary intervention studies in humans indicated that the production of TXA, was decreased by EPA (74,75) and fish oil (76,77). EPA + DHA significantly decreased ex vivo platelet aggregability in healthy elderly subjects compared with ALA and oleic acid. In that study, 38 elderly (>60 yr) and 12 younger (<35 yr) subjects consumed an oleic acid-enriched diet for 3 wk. The elderly subjects were then randomly assigned to oleic acid (n = l l ) , ALA (n = 14) and EPA + DHA (EPA 1.05 g, DHA 0.55 g; n = 13) diet groups, whereas the younger subjects were assigned to an ALA-enriched diet for 6 wk. The
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ex vivo platelet aggregability was significantly decreased in the elderly group that received EPA + DHA as measured by filtragometry compared with the ALA (P = 0.006) and the oleic acid ( P = 0.005) diet groups (78). In another study, seal oil supplementation had a beneficial effect on some hemostatic variables. In that study, 19 healthy subjects consumed 20 g encapsulated seal oil/d for 42 d. Plasma fibrinogen decreased by 18%, whereas protein C, a coagulant inhibitor, increased by 7% (79). A recent prospective cohort study from U.S. Nurses’ Health study found that women with a higher intake of long-chain n-3 PUFA from fish had a lower risk of total stroke compared with women who ate fish
Vascular Function
Studies from humans, animals, and cell cultures showed that EPA improves vascular endothelial function through different mechanisms (73). EPA increases systemic arterial compliance in dyslipidemic subjects. In a recent parallel, double-blind trial, 38 dyslipidemic subjects were randomly assigned to three groups: 3 g EPA/d (n = 12), 3 g D H N d (n = 12) and a placebo (n = 14) for 7 wk. Arterial function was measured at the beginning and end of the interventions. Systemic arterial compliance was significantly increased in EPA (+36%) and DHA (+27%) groups compared with the placebo group (61).
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Results from a study of cultured rat aortic smooth muscle cells showed that EPA was incorporated into vascular smooth muscle cell phospholipids within 20 min, subsequently causing decreased intracellular calcium mobilization and protein kinase C activation, which later reduced vasoconstriction (83). Forearm blood-flow response to both acetylcholine and substance P was improved after treatment with EPA. In that study, forearm vascular responses to the endothelium-dependent vasodilator substance P and acetylcholine were examined before and after intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA, an inhibitor of NO synthesis). The same measurements were repeated after treatment with EPA (1800 mg/d) for 6 wk in 8 coronary artery disease (CAD) patients. The EPA-induced augmented response to acetylcholine was significantly inhibited by acute administration of I-NMMA, but not by substance P. These results indicate that long-term treatment with EPA increases both NO-dependent and non-NOdependent endothelium-dependent forearm vasodilatation in patients with CAD; therefore, the beneficial effects of EPA appear to extend to non-NO-dependent mechanism(s) (84). Another study from the same group found that long-term treatment with EPA improved both endothelium-dependent and exercise-induced forearm vasodilations in patients with CAD, and that NO was substantially involved in the EPA-induced improvement of the forearm blood-flow responses (85). In that study, 10 patients with stable CAD received treatment with EPA (1800 mg/d). EPA treatment significantly improved the forearm blood-flow responses to acetylcholine (P c 0.01), which was significantly reduced by acute administration of LNMMA (P c 0.01). EPA treatment significantly augmented the exercise-induced increases in forearm blood flow (P c 0.05) and L-NMMA acutely abolished this augmentation (P c 0.01) (85). In another study, it was shown that EPA improved endothelial function in hypertriglyceridemic subjects (62). In that study, the forearm blood-flow response to acetylcholine (but not to nitroprusside) was significantly lower in hypertriglyceridemic subjects (n = 8) before EPA than in control subjects (n = 8). Forearm blood-flow response to acetylcholine in hypertriglyceridemic subjects was normalized after 3 mo of EPA supplementation (1800 mg/d) despite the increase in VLDL oxidizability. Antiarrhythmic Effect
Cardiac arrhythmia is life-threatening and can cause sudden death. Recent evidence from animal models, cultured cardiac cells, and human intervention and epidemiologic studies suggested that EPA and other n-3 PUFA may prevent cardiac arrhythmia. This may be responsible in part for the protective effect of EPA and other n-3 PUFA on CHD mortality. Heart-rate variability is a predictor of arrhythmic events and sudden cardiac death in high-risk patients and in healthy subjects; it reflects cardiac autonomic regulation, with decreasing values indicating increasing arrhythmic events and mortality. A recent study showed that dietary fish consumption was associated with increased heart-rate variability. This observational study of 43 sub-
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jects, which investigated heart-rate variability as a function of fish intake, found those with the highest intakes of fish had the lowest blood pressure and highest heart-rate variability (86). The antiarrhythmic mechanisms of EPA and other n-3 PUFA have been addressed mainly in animal models and in cultured cardiac cells. Tachycardia, contracture, and fibrillation of the cultured rat myocytes occurred when arrhythmogenic toxins [e.g., P-adrenergic agonist, ouabain, high Ca(2+), lysophosphatidylcholine, acylcarnitine, and the Ca(2+) ionophore] were added to the myocyte perfusate. Adding EPA to the superfusate, before adding the toxins, prevented the expected tachyarrhythmias. If the arrhythmias were first induced, adding the EPA to the superfusate terminated the arrhythmias (87). The antiarrhythmic action of EPA and other n-3 PUFA is thought to be the result of the PUFA electrically stabilizing cardiac myocytes by modulating conductance of ion channels in the sarcolemma, particularly the fast, voltage-dependent sodium current and the L-type calcium currents, resulting in a prolonged relative refractory period (104). An increased ratio of EPA to AA within myocardial membranes increased the (Ca2+Mg2+)-ATPaseactivity of membranes (88).
Modulation of Inflammation and Immune Function by EPA As humans, we share our environment with potentially harmful agents; as a consequence, we have adapted a complex defense system, with the ability to distinguish self from non-self, for protection from such agents. The human immune system comprises a variety of organs, cells, and tissues located throughout the body. Immune cells produce and respond to a range of molecules including cytokines, the complement system, and antibodies, which have pleiotropic and synergistic effects and can act in an autocrine, paracrine, or endocrine manner. This interaction among molecules, cells, and tissues of the immune system is central to the protection of the host. The immune system is comprised of two components, the innate and the adaptive response. The innate immune response, which is the first line of defense, is a rapid response that is not antigen driven. The innate response is always present and does not change on exposure to the same pathogen. It includes anatomical barriers, mechanical removal, complement, phagocytosis, inflammation, and fever. The adaptive immune response, in contrast, is antigen dependent, taking longer to develop, improving with each infection, and memorizing the infectious agent to prevent reinfection. Adaptive immunity involves antigen-presenting cells, such as macrophages and dendritic cells; B- and T-lymphocyte activation and proliferation; the production of antibody molecules, cytotoxic T-lymphocytes; activated macrophages and natural killer (NK) cells; and cytokines. In vivo, there is considerable interaction and interplay between the innate and adaptive immune responses, particularly in relation to the inflammatory response.
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Inflammation is the tissue’s initial reaction to trauma or infection and is characterized by increased blood flow and movement of leukocytes into the tissues, which results in edema, redness, fever, and pain. Inflammation is mediated by molecules such as histamine and the cytokines, and involves neutrophils, which act in acute inflammation, and mononuclear cells, which act in chronic inflammation. Although inflammation is part of the normal immune response, it can result in tissue damage and disease when it is uncontrolled. Hypersensitivity is an overreactive immune response to a previously encountered antigen, which results in a deleterious outcome, such as tissue or organ damage, rather than a protective one. There are two categories of hypersensitivities, i.e., immediate hypersensitivity and delayed hypersensitivity. Immediate hypersensitivity involves complement and antibody proteins, whereas delayed hypersensitivity refers to cell-mediated immunity. Cytokines are small regulatory proteins that facilitate communications among immune cells and between immune system cells and the rest of the body. Proinflammatory cytokines, including tumor necrosis factor a (TNFa), interleukin (1L)-1, IL-6 and IL-8, together with IL-2 and interferon (1FN)-y derived from lymphocytes, enhance the immune response. Anti-inflammatory cytokines, including the IL-1 receptor agonist, IL-4, IL-10, and transforming growth factor p (TGFP), inhibit the synthesis of the major proinflammatory cytokines. Under physiologic conditions, the balance between these two classes of cytokines is tightly controlled; an imbalance, however, may lead to either inflammatory or allergic diseases or to an ineffective immune response. Dietary €PA and Immune Response
The recognition that nutritional deficiencies and excesses influence immune response has led to a large body of research investigating optimal nutrition for an optimal immune system. One class of nutrients studied extensively in this respect is the n-3 FA, in particular EPA. The majority of studies to date have provided EPA in the form of fish oil, thereby also supplying DHA; a few studies have supplied pure EPA. The purported immunological effects and modes of action of EPA were reviewed recently in detail (89,90). Therefore, this section will only briefly discuss the purported anti-inflammatory effect of EPA in vivo and will seek to highlight the most recent evidence from human studies. A large body of evidence has reported that EPA, given in the form of fish oil, has the ability to impinge on immune function in healthy humans as follows: lowering en vivo production of IL-1, IL-6, TNF-a,IFN-y, and IL-2 by peripheral blood mononuclear cells (9 1-96); lowering plasma IL-6 (97); decreasing lymphocyte proliferation (92,96,98); decreasing leukocyte chemotaxis (99); decreasing NK cell activity (100); lowering expression and plasma concentrations of adhesion molecules (101,102), and suppressing procoagulant activity (94,103). However, a significant number of supplementation studies have not observed an effect of supplementation on these indices of immune function (104-108). To explain the disagreement
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among studies, several factors must be considered. First, the supplementation dose varied widely among studies, with a range of 0.5 to 9.0 g n-3/d. This may be of critical importance because a recent paper reported that the effect of n-3 PUFA intake on cytokine production tends toward a “U”-shaped dose-response curve, with maximum effect apparent at an intake of 1.0 g EPA + DHA supplements/d (95). Second, several of the studies were poorly designed and lacked a placebo group which, given the reported seasonal effects on immune response (109), may have led to inappropriate interpretation of the results. Last, it is difficult to make comparisons between studies given the diversity of the types of samples analyzed, the intervention period, and the age and health status of the study population. It is encouraging to note, however, that recent results from well-designed, appropriately controlled interventions, providing intakes of EPA that could be achieved by dietary means, showed a reduction in proinflammatory cytokines, NK cell activity, and lymphocyte proliferation (88,95,96,100,107,110). Clinical Studies with EPA in Inflammatory Disorders
The immunoregulatory effects of EPA, together with the observation that autoimmune disorders are rare in the Greenland Inuit population (111) among whom fish intake is high, led researchers to investigate the clinical application of n-3 supplementation. Significant improvements in the clinical symptoms of rheumatoid arthritis (RA) and inflammatory status after fish oil supplementation generally occurred, with a reduction in the number of tender joints the most consistently reported benefit (112,113). Furthermore, in the studies in which drug use was examined, there was a reduction in the use of nonsteroidal anti-inflammatory drugs (1 14). Consequently, it was recommended that patients consume dietary supplements containing 3-6 g n-3 FA/d (1 15). The importance of the ratio of dietary n-3:n-6 was also highlighted, with the beneficial effect of fish oil supplementation in RA patients augmented by a diet low in the n-6 FA, AA (77,116). Indeed it was suggested that an n-3:n-6 ratio of 1 : 1 4 would be more physiologic than the ratio of 1:1620 provided by the current Western diet (90). Research also examined the effect of fish oil supplementation in atopic dermatitis and reported that lipid infusion with or topical application of n-3 may have the potential to improve symptoms (117,118). In contrast, there appears to be little evidence to suggest that fish oil improves asthma control (119,120). Studies also examined the effect of n-3 supplementation on Crohn’s disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, psoriasis (90), and in trauma patients (89). Although several of these trials suggest a significant benefit, there is a clear need for larger trials to confirm these findings. Studies reported altered FA levels in allergic mothers and their infants (121), and recent preliminary data indicate that maternal n-3 supplementation resulted in an altered neonatal cytokine profile, although no data are available as yet on the development of atopy in these children (122).
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Mechanisms of Action of €PA in Regulation of Immune Function
EPA affects immune function by a number of mechanisms; the immunomodulatory outcome of an intervention can, therefore, be the result of one or several of these mechanisms. Increased EPA intake results in an increased EPA content of immune cells at the expense of AA (93,100,106-108). The change in membrane lipid composition alters membrane fluidity, which can alter cellular responses. It also alters the substrate availability for eicosanoid formation and results in the synthesis of eicosanoids of a different type and potency, with those derived from EPA generally less potent than those derived from AA. Furthermore, EPA can inhibit AA metabolism. Eicosanoids are potent regulators of immune function, influencing cytokine production, lymphocyte proliferation, enhancing blood flow, inducing fever, edema, and pain, and stimulating the generation of reactive oxygen species. Indeed, increased production of AA-derived eicosanoids is implicated in a number of inflammatory diseases (33). The n-3 FA also affect immune response at the molecular level, regulating gene expression. Proinflammatory cytokine gene expression is initiated by the transcription factor nuclear factor (NF)-KB. Activation of NF-KB requires phosphorylation, degradation, and dissociation of the inhibitory KB (I-KB)from the NFKB-molecule. The n-3 FA significantly decrease NF-KB activity by reducing phosphorylation of I-KB (123). The effect of n-3 on NF-KB may also be mediated through another family of transcription factors, the peroxisome proliferator-activated receptors (PPAR). FA, including EPA, are ligands for PPAR, and oxidized EPA is a potent activator of PPARa (124). PPARa upregulates I-KB, thereby reducing NF-KB activation (125). Although the effect of EPA on PPAR or NF-KB expression in humans is unknown, it is likely that EPA exerts at least some of its antiinflammatory properties by regulating these and indeed other transcription factors (126). Inflammation is part of the normal immune response to infection or trauma. However, uncontrolled inflammation, which is typified by overproduction of inflammatory mediators including cytokines, eicosanoids, and reactive oxygen species, can result in tissue damage and disease. The n-3 FA, including EPA, which can act directly and indirectly to regulate immune function, are potent antiinflammatory agents. Their use as alternative and adjunctive therapies for inflammatory disease was investigated with significant success in some conditions, such as RA, but with limited benefit, albeit in a small number of studies, in others such as asthma. There is a clear need for further research into the therapeutic application of n-3 FA in autoimmune and inflammatory conditions.
EPA and NeuropsychologicalDisorders Manic-depressive illness (bipolar disorder), depression, and schizophrenia are common neuropsychiatric disorders. Results from case-control studies, clinical trials,
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and case studies showed that oils rich in n-3 PUFA play a beneficial role in these neuropsychiatric conditions. The incidence of depression has increased markedly in the past decades in Western countries (127). Epidemiologic evidence suggests that the condition has both genetic and environmental components. In 1995, it was hypothesized that a low n-3 PUFA status could predispose subjects to an increased risk of suicide and depression (128). Shortly after this, several studies reported that low n-3 PUFA levels in serum phospholipids, cholesteryl esters, and erythrocyte membranes were positively associated with depression (129,130). One of the first intervention studies was a 4-mon double-blind, placebo-controlled trial in 30 patients, aged 18-65 yr, with type I or I1 bipolar disorder (131). This study showed that episodes of severe mania and depression were significantly reduced in the n-3 PUFA (EPA + DHA) supplementation group (n = 14; 9.6 g/d) compared with the placebo group (n = 16). Peet and Horrobin (132) studied 70 patients with persistent depression, despite treatment with standard antidepressants. The patients were randomized to receive 1, 2, or 4 g ethyl-EPA/d or placebo for 12 wk. Interestingly, only those patients treated with 1 g/d (n = 17) showed a significant improvement (reduction in the depression rating); in this group, 69% of patients had a reduction of at least 50% of the symptoms rated on a standard depression rating scale compared with 25% of patients so improving in the placebo group. Another study with ethyl-EPA, in 20 patients with recurrent unipolar depression, showed that there were highly significant benefits from the treatment compared with placebo after 3 wk of treatment (2 g/d) (133). In a double-blind study, subjects with major depressive disorder were randomized to receive either 9.6 g EPA + DHA/d or placebo for 8 wk in addition to usual treatment (134). Patients in the n-3 group had a significantly decreased score on the Hamilton Rating Score for depression compared with the placebo group ( P < 0.0001). Noaghiul and Hibbeln (135) investigated cross-national prevalence rates of bipolar disorders and showed that there was a very strong relation between greater seafood consumption and lower prevalence rates. A single case report found that ethyl-EPA given to a treatment-resistant, severely depressed, and suicidal male patient resulted in dramatic and sustained clinical improvement in all symptoms of depression within 1 mon (136). The EPA treatment was accompanied by a reduction in the lateral ventricular volume in the brain. A double-blind study in 36 depressed patients who received 2 g DHMd for 6 wk showed no significant effect compared with the placebo treatment (137). It is of considerable interest that there is a relation between depression and heart disease. For example, patients with an episode of major depression have a threefold risk of cardiac mortality later in life (138). It is possible that the link between these conditions is that each of these is also associated with low intakes of n-3 PUFA. In a cross-national study of prevalence rates of postpartum depression and breast milk EPA, DHA, and AA levels and seafood consumption, Hibbeln (139) found that higher concentrations of DHA in milk and greater seafood consumption
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both predicted lower prevalence rates of postpartum depression. A small pilot open-label study among seven pregnant women with a past history of postpartum depression who were provided 2.96 g of EPA + DHA for 2 wk before giving birth did not have promising results (140). Unlike depression, schizophrenia does not differ in incidence among different races and cultures, suggesting that this disease has been with mankind for a very long time. Schizophrenia is one of the most severe mental illnesses. The dopamine system has been the focus of research into the pathogenesis of schizophrenia because most antipsychotic medications act as antagonists at the D2 dopamine receptor; however, because the main antidopaminergic treatments do not alleviate symptoms in many patients, many alternative hypotheses have been proposed (141). Several studies reported disturbances of phospholipid metabolism of the frontal and temporal lobes, including increased breakdown of the phospholipids (142,143). It was also reported that there is a significant correlation between abnormal phospholipid metabolism and schizophrenic symptoms (144). A case report showed that treatment of a drug-ndive patient with schizophrenia with ethyl-EPA (2 g/d) led to dramatic and sustained clinical improvement in both positive and negative symptoms and reversed the brain phospholipid abnormality and the cerebral atrophy (145). Several studies showed decreased n-3 and n-6 PUFA levels in erythrocyte membranes in schizophrenic patients (146-148), although the interpretation of this finding is likely to be confounded by the effects of medication and other factors such as diet and smoking (149). A recent review of environmental factors and membrane PUFA in schizophrenia indicated that there was conflicting information in the literature on the effect of smoking on PUFA levels in normal and schizophrenic subjects (150). Furthermore, these authors suggested that insufficient studies had been conducted to conclude that diet contributed to the membrane PUFA changes reported in schizophrenic subjects. Another study reported reduced levels of AA in brain tissue from schizophrenic subjects (151). A pilot study revealed that schizophrenic subjects had abnormalities in the electroretinogram (152), similar to that reported in animals that are deficient in n-3 PUFA (13). There have been several preliminary intervention studies with n-3 PUFA, and EPA in particular, in patients with schizophrenia with encouraging results (153). A study by Peet et al. (154) compared an EPA-enriched oil and a DHA-enriched oil with a corn oil placebo in 45 outpatients receiving existing antipsychotic medication in a 3-mon trial. The improvement in the EPA group was significantly greater than that in the DHA group. All patients receiving EPA improved, and half of them improved >25% on the rating scale score. There were three additional studies with ethyl-EPA, the first of which was a multicenter study in which 115 patients were given 1, 2, or 4 g ethyl-EPA or placebo, in addition to existing antipsychotic medication (155). In that study, the EPA treatment was effective only in patients receiving the background medication clozapine, but not in those receiving other antipsychotic agents. The results showed that the 2-g dose was the most effective and that the effect decreased with the 4-g dose. The second study was a placebo-
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controlled, double-blind study in 78 patients with schizophrenia or schizoaffective disorder to determine whether 3 g ethyl-EPA/d, in addition to neuroleptic drugs, could improve symptoms and cognition over a 4-mon period (156). The study found no differences between groups in positive or negative symptoms, mood, cognition, or global impression ratings. The final study was a randomized, parallelgroup, double-blind, placebo-controlled study in 40 patients, with persistent symptoms after at least 6 mon of stable antipsychotic treatment, who received 3 g ethylEPA/d or placebo for 12 wk (157). At 12 wk, the EPA group had a significantly greater reduction of positive and negative scores and dyskinesia scores than the placebo group. In depression, the epidemiologic and biochemical data suggest that there is a correlation between the prevalence of the disorder and the intake of dietary n-3 PUFA, whereas in schizophrenia it was suggested that the defect is either related to a more fundamental abnormality in dopamine activity or phospholipid metabolism. In both depression and schizophrenia, the studies to date indicate that EPA is more effective than DHA. This is surprising because the initial hypotheses assumed that DHA would be the more effective agent because neural membranes are rich in DHA and contain almost no EPA. Biophysical properties of synaptic membranes directly affect neurotransmitter biosynthesis, signal transduction, uptake of serotonin, binding of a-adrenergic and serotonergic receptors, and monoamine oxidase activity. The proposed mechanisms of action of the n-3 PUFA in neuropsychiatric disorders include effects on neurotransmitter receptors and G-proteins via effects on biophysical properties of the membrane, effects on secondary messengers and on protein kinases (128,158), and effects on the inflammatory response of eicosanoids derived from AA (159,160). Recent data showed that rats treated with lithium, a therapeutic used in treating bipolar disorder, demonstrated a reduced turnover of AA in brain phospholipids and decreased mRNA, protein levels, and enzyme activity of a cytosolic PLA,, which is AA specific (161). This treatment also reduced the brain concentration of PGE,, a product of the COX pathway. The same group showed that lithium reduced AA turnover in the brain, whereas the turnover of DHA and palmitic acid was unaffected (162). It was found in that study that lithium reduced the brain protein level and activity of COX-2. It is possible that EPA is effective in depression through its ability to inhibit COX activity, although the levels of EPA in the brain phospholipids are very low (7). It may also be possible that n-3 PUFA are operating through the neuromodulatory actions of the endocannabinoids [Nacylethanolamines (NAE) and 2-acylglycerols]. These lipids are endogenous ligands for the cannabinoid receptors found predominantly in the brain (163).(AQ10) In piglets, brain levels of NAE increased fourfold for AA, fivefold for EPA, ninefold for 22511-3 and ten-fold for DHA after being fed a diet with AA and DHA for 18 d compared with a diet without AA and DHA (164). More than 25 yr ago, two different investigators proposed an alternative hypothesis that involved abnormalities in brain phospholipid metabolism (165,166). Data
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gathered from a number of studies showed patterns of decreased PUFA and increased phospholipid turnover in schizophrenia. Skosnik and Yao (159) proposed that AA may play a crucial role in the pathophysiology of schizophrenia. It is not immediately apparent what mechanisms of action of n-3 PUFA and EPA, in particular, pertain in this disease; however, it was reported that EPA is able to reverse the phospholipid abnormalities in schizophrenia, perhaps via inhibition of PUFA-specific PLA,, an enzyme that removes PUFA from the sn-2 position of membrane phospholipids (147). It is becoming apparent that treatment with combinations of n-3 and n-6 PUFA and antioxidants is indicated and has yet to be fully explored in schizophrenia (159). Peet (153) recently suggested that there might be a common mechanism whereby diets rich in saturated fat, with a high glycemic load and also low in n-3 PUFA influence schizophrenia, namely, through a brain-derived neurotrophic factor (167). It was suggested that this factor might have a role in stabilizing the survival of brain circuitry in critical periods, by maintaining cortical neuron size and dendrite structure (168).
EPA and Cancer (Cancer Cachexia) Cachexia is a wasting syndrome experienced by cancer patients with progressive depletion of adipose tissue and skeletal muscle mass (169). The survival of cancer patients is related directly to the total weight loss and also the rate of weight loss (170). Up to half of all cancer patients experience cachexia, which can arise in patients with a tumor comprising less than 0.01% of the host’s weight (169). Patients with pancreatic and gastric cancer experience the highest frequency of weight loss (83-87%) (171) and patients with cancers of the pancreas, stomach, lung, esophagus, and colon experience a high incidence of cachexia (169). Various factors were investigated as mediators of tissue wasting in cachexia; however, the cause remains poorly understood. Although anorexia is common in cancer patients with reports of incidences between 15 and 40% at presentation (172), the decrease in food intake alone is insufficient to account for the metabolic changes that occur in cancer cachexia (173). In addition, cachexia can occur even in the absence of anorexia (174), suggesting the involvement of catabolic mediators produced by tumor or host cells. Nutritional supplementation and pharmacologic manipulation of appetite did not restore loss of lean body mass (170). Patients with cancer have highly variable changes in resting energy expenditure (173). It appears that tumor type plays an important role in determining energy expenditure; patients with lung (175) and pancreatic (176) cancer experience increased resting energy expenditure, whereas patients with gastric and colorectal cancer (175) had no elevation in resting energy expenditure. Increased energy expenditure might be related to the upregulation of uncoupling proteins, particularly uncoupling protein-3 in skeletal muscle (170). Several factors were proposed to influence fat and muscle metabolism in cachexia, and EPA was shown to attenuate some of these factors (170). Lean body
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mass and visceral protein depletion are characteristic of patients with cancer cachexia. A reduced rate of protein synthesis and an increased rate of degradation were reported in cancer patients with weight loss (177). The decreased protein synthesis could arise from the inactivity of the patient in addition to reduction in the supply or balance of amino acids due to an acute-phase protein production (170). The increased protein degradation seems to be due mainly to an increased expression of the components of the ubiquitin-proteasome proteolytic pathway in skeletal muscle, through proteolysisinducing factor (PIF) produced by tumor cells (169). EPA can attenuate the effect on protein degradation, but not protein synthesis. Pretreatment of mice with EPA (0.5 gkg body weight) was shown to abolish the cachectic effect of PIF in vivo (178), and it completely abolished weight loss in mice bearing the colon adenocarcinoma MAC16, which typically induces cachexia in the mice (179). In humans, there have been a few clinical studies with patients with unresectable pancreatic cancer. In one study, patients who had weight loss of 2.9 kg/mon received 12 g fish oil containing 18% EPA and 12% DHA/d by mouth (180). Within 3 mon of fish oil supplementation, the weight loss was transformed to a weight gain of 0.3 kg/mon (180). Another study was performed to c o n f m that the results were due to EPA and not DHA. All patients in the study were losing weight at a median of 2 kg/mon before supplementation with 6 g 95% pure EPNd (181). After 4 wk of supplementation, the body weight stabilized with a medium weight gain of 0.5 kg after 4 wk. Another study was performed in which patients consumed two cans of fish oil-enriched nutritional supplement/d (182). Each can contained 1.09 g EPA and 16.1 g protein; patients consumed a median of 1.9 cans/d. At the start of the study, patients had a weight loss of 2.9 kg/mon, but after 3 wk supplementation, they had a weight gain of 1 kg, which was increased to 2 kg after 7 wk supplementation (182). It was suggested that EPA exerts its effect by downregulation of the expression of the ubiquitin-proteasome proteolytic pathway in skeletal muscle (183). Another mediator that seems to affect muscle metabolism is protein mobilizing factor (PMF), which was isolated from mice bearing MAC16 tumors and from the urine of patients with cancer cachexia (184). EPA was shown to attenuate PMF (185) and preserve muscle mass in patients with cancer cachexia (180). Loss of adipose tissue occurs in these patients. Mechanisms that have been proposed to account for the decrease in body lipids in patients with cancer cachexia include inhibition of the enzyme LPL, which prevents adipocytes from extracting FA from plasma lipoproteins for storage and leads to a net flux of lipid into the circulation (173). The other mechanism is direct stimulation of triglyceride hydrolysis in adipocytes by activation of triglyceride lipase (173). Cytokines, such as TNF-y, IL-6, IFN-y, and leukemia inhibitory factor, are all suggested to decrease body lipids through inhibition of LPL (186). Loss of adipose tissue was suggested to arise predominantly via an increase in lipolysis, which is induced by the tumor product lipid-mobilizing factor acting through a P-3-adrenoreceptor (170). In vitro studies using LMF isolated from MAC16 mice showed that EPA was effective in inhibiting the cachexia induced by the MAC16 tumor (180). Studies to date suggest a potential role of EPA as an effective anticachex-
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ia agent. However, more studies are required to understand the mechanisms by which EPA acts to modulate cachexia before EPA is used as a therapeutic strategy.
Sources of EPA The sources of EPA for research can be found listed on the website of the Omega-3 Research Institute (www.omega3ri.com). These include products such as soft-gel capsules enriched in EPA, ethyl EPA, and microencapsulated powders enriched in n-3 PUFA. Health food shops also sell soft-gel capsules of various fish oils (salmon, menhaden, tuna) or PUFA-enriched fractions derived from fish oils. Finally, fish such as tuna, salmon, mackerel, herring, trout, sardines, and halibut are good sources of EPA and DHA (4,187); however, the amounts of EPA and DHA found in fish can be variable. For example, skinless fillets of whiting (Sillago ciliata) have 0.9% lipid with 0.1 g EPA and 0.2 g of DHN100 g flesh, whereas skinless fillets of salmon (Salmo salar) have 7.1% lipid with 0.5 g EPA and 1.1 g of DHN100 g flesh (187).
Conclusions The interest in the n-3 PUFA and the recognition that many Western diets contain low levels of these PUFA have led to numerous recommendations to alter the balance of linoleic acid to n-3 PUFA in our diets. Current evidence suggests that the n-6:n-3 ratio in many Western diets is >16: 1. Some have argued that the ideal ratio of n-6:n-3 FA should be based on that found in human milk or that of the food selected by primitive man and animals in the wild, which would suggest n-6/n-3 ratios of between 3:l and 1O:l (188). It has been argued that humans have evolved eating a diet low in fat and with a lower n-6:n-3 ratio than the modem-day diet; therefore the recommendations for an increase in the proportion of n-3 FA in our diet have the weight of an evolutionary precedent (188,189). From the evidence presented in this chapter, it is clear that the inclusion of greater levels of n-3 FA in the diet, including EPA, will likely have positive benefits for the health of the population. Some of the data presented here show that the beneficial effects of EPA are achieved at very high intakes (g/d), suggesting the need for EPA at a pharmacologic dose rather than the intake that might be reasonably achieved through current dietary practices. References 1. Sayanova, O.V., and J.A. Napier, Eicosapentaenoic Acid: Biosynthetic Routes and the Potential for Synthesis in Transgenic Plants, Phytochernistry 65: 147-158 (2004). 2. Meyer, B.J., N.J. Mann, J.L. Lewis, G.C. Milligan, A.J. Sinclair, and P.R. Howe, Dietary Intakes and Food Sources of Omega-6 and Omega-3 Polyunsaturated Fatty Acids, Lipids 38: 391-398 (2003). 3. UK Department of Health, Nutritional Aspects of Cardiovascular Disease, Report on Health and Social Subjects, No. 46, HMSO London, 1997, p. 132.
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4. Din, J.N., D.E. Newby, and A.D. Flapan, Omega 3 Fatty Acids and Cardiovascular Disease-Fishing for a Natural Treatment, Br. Med. J. 328: 30-35 (2004). 5. Budowski, P., Omega-3 Fatty Acids in Health and Disease, World Rev. Nutr. Diet. 57: 214-274 (1988). 6. Crawford, M.A., and A.J. Sinclair, Nutritional Influences in the Evolution of the Mammalian Brain, in CIBA Foundation Symposium on Lipids, Malnutrition and the Developing Brain, Associated Scientific Publishers, Amsterdam, 1972, pp. 267-287. 7. Sinclair, A.J., Long Chain Polyunsaturated Fatty Acids in the Mammalian Brain, Proc. Nutr. SOC.34: 287-291 (1975). 8. Crawford, M.A., N.M. Casperd, and A.J. Sinclair, The Long-Chain Metabolites of Linoleic and Linolenic Acid in Liver and Brain in Herbivores and Carnivores, Comp. Biochem. Physiol. 54B :395-401 (1976). 9. Svennerholm, L., Distribution and Fatty Acid Composition of Phosphoglycerides in Normal Human Brain, J. Lipid Res. 9: 570-579 (1968). 10. Neuringer, M., G.J. Anderson, and W.E. Connor, The Essentiality of n-3 Fatty Acids for Brain Development and Function of the Retina and Brain, Annu. Rev. Nutr. 8: 517-541 (1988). 11. Sinclair, A.J., N.M. Attar-Bashi, and D. Li, What is the Role of Alpha-Linolenic Acid for Mammals? Lipids 37: 1113-1 123 (2002). 12. Watanabe, S., M. Doshi, and T. Hamazaki, n-3 Polyunsaturated Fatty Acid (PUFA) Deficiency Elevates and n-3 PUFA Enrichment Reduces Brain 2Arachidonoylglycerol Level in Mice, Prostaglandins Leukot. Essent. Fatty Acids 69: 51-59 (2003). 13. Sinclair, A.J., Commentary on the Workshop Statement, Prostaglandins Leukot. Essent. Fatty Acids 63: 135-137 (2000). 14. Kurlack, L.O., and T.J. Stephenson, Plausible Explanations for Effects of Long Chain Polyunsaturated Fatty Acids on Neonates, Arch. Dis. Child Fetal Neonatal Ed. 80: 148-154 (1999). 15. Lauritzen, L., H.S. Hansen, M.H. Jorgensen, and K.F. Michaelsen, The Essentiality of Long Chain n-3 Fatty Acids in Relation to Development and Function of the Brain and Retina, Prog. Lipid Res. 40: 1-94 (2001). 16. Salem, N., Jr., B. Litman, H.-Y. Kim, and K. Gawrisch, Mechanisms of Action of Docosahexaenoic Acid in the Nervous System, Lipids 36: 945-959 (2001). 17. Litman, B.J., S.L. Niu, A. Polozova, and D.C. Mitchell, The Role of Docosahexaenoic Acid Containing Phospholipids in Modulating G Protein-Coupled Signalling Pathways: Visual Transduction, J. Mol. Neurosci. 16: 237-242 (2001). 18. Zimmer, L., S . Dellion-Vaancassel, G. Durand, D. Guilloteau, S . Bodard, J.C. Besnard, and S. Chalon, Modification of Dopamine Neurotransmission in the Nucleus Accumbens of Rats Deficient in n-3 Polyunsaturated Fatty Acids, J. Lipid Res. 41: 3 2 4 0 (2000). 19. Bowen, R.A., and M.T. Clandinin, Dietary Low Linolenic Acid Compared with Docosahexaenoic Acid Alters Synaptic Plasma Membrane Phospholipids Fatty Acid Composition and Sodium-Potassium ATPase Kinetics in Developing Rats, J. Neurochem. 83: 764-774 (2002). 20. Vaidyanathan, V.V., K.V.R. Rao, and P.S. Sastry, Regulation of Diacylglycerol Kinase in Rat Brain Membranes by Docosahexaenoic Acid, Neurosci. Lett. 179: 171174 ( 1994).
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Mononuclear Cell Fatty Acid Compositions but Not Mononuclear Cell Functions, Eur. J. Clin.Investig. 30: 260-274 (2000). 107. Wallace, F.A., E.A. Miles, and P.C. Calder, Comparison of the Effects of Linseed Oil and Different Doses of Fish Oil on Mononuclear Cell Function in Healthy Human Subjects,Br. J. Nutr. 89: 679-689 (2003). 108. Kew, S . , T. Banerjee, A.M. Minihane, Y.E. Finnegan, R. Muggli, R. Albers, C.M. Williams, and P.C. Calder, Lack of Effect of Foods Enriched with Plant- or MarineDerived n-3 Fatty Acids on Human Immune Function, Am. J. Clin. Nutr. 77: 1287-1295 (2003). 109. Bonham, M., J.M. O’Connor, H.D. Alexander, J. Coulter, P.M. Walsh, L.B. McAnena, C.S. Downes, B.M. Hannigan, and J.J. Strain, Zinc Supplementation Has No Effect on Circulating Levels of Peripheral Blood Leucocytes and Lymphocyte Subsets in Healthy Adult Men, Br. J. Nutr. 89: 695-703 (2003). 110. Bechoua, S . , M. Dubois, E. Vericel, P. Chapuy, M. Lagarde, and A.F. Prigent, Influence of Very Low Dietary Intake of Marine Oil on Some Functional Aspects of Immune Cells in Healthy Elderly People, Br. J. Nutr. 89: 523-531 (2003). 111. Harvald, B., Genetic Epidemiology of Greenland, Clin. Genet. 36: 364-367 (1989). 112. Darlington, L.G., and T.W. Stone, Antioxidants and Fatty Acids in the Amelioration of Rheumatoid Arthritis and Related Disorders, Br. J. Nutr. 85: 251-269 (2001). 113. Cleland, L.G., M.J. James, and S.M. Proudman, The Role of Fish Oils in the Treatment of Rheumatoid Arthritis, Drugs 63: 845-853 (2003). 114. James, M.J., R.A. Gibson, and L.G. Cleland, Dietary Polyunsaturated Fatty Acids and Inflammatory Mediator Production,Am. J. Clin. Nutr. 71: 3438-348s (2000). 115. Kremer, J.M., n-3 Fatty Acid Supplements in Rheumatoid Arthritis, Am. J. Clin. Nutr. 71: 349s-51s (2000). 116. Volker, D., P. Fitzgerald, G. Major, and M. Garg, Efficacy of Fish Oil Concentrate in the Treatment of Rheumatoid Arthritis, J. Rheumatol. 27: 2343-2346 (2000). 117. Watanabe, T., and Y. Kuroda, The Effect of a Newly Developed Ointment Containing Eicosapentaenoic Acid and Docosahexaenoic Acid in the Treatment of Atopic Dermatitis, J. Med. Investig. 46: 173-177 (1999). 118. Mayser, P., K. Mayer, M. Mahloudjian, S . Benzing, H.J. Kramer, W.B. Schill, W. Seeger, and F. Grimminger, A Double-Blind, Randomized, Placebo-Controlled Trial of n-3 Versus n-6 Fatty Acid-Based Lipid Infusion in Atopic Dermatitis, J. Parenter. Enteral Nutr. 26: 151-158 (2002). 119. Woods, R.K., F.C.K. Thien, and M.J. Abraham, Dietary Marine Fatty Acids (Fish Oil) for Asthma in Adults and Children (Cochrane Review), Cochrane Database System Issue 4, John Wiley & Sons, Chichester, UK, 2002. 120. Surette, M.E., I.L. Koumenis, M.B. Edens, K.M. Tramposch, B. Clayton, D. Bowton, and F.H. Chilton, Inhibition of Leukotriene Biosynthesis by a Novel Dietary Fatty Acid Formulation in Patients with Atopic Asthma: A Randomized, Placebo-Controlled, Parallel-Group, Prospective Trial, Clin.Ther. 25: 972-979 (2003). 121. Yu, G., and B. Bjorksten, Serum Levels of Phospholipid Fatty Acids in Mothers and Their Babies in Relation to Allergic Disease, Eur. J. Pediatr. 157: 298-303 (1998). 122. Dunstan, J.A., T.A. Mori, A. Barden, L.J. Beilin, A.L. Taylor, P.G. Holt, and S.L. Prescott, Maternal Fish Oil Supplementation in Pregnancy Reduces Interleukin- 13 Levels in Cord Blood of Infants at High Risk of Atopy, Clin. Exp. Allergy 33: 4 4 2 4 8 (2003).
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123. Novak, T.E., T.A. Babcock, D.H. Jho, W.S. Helton, and N.J. Espat, NF-Kappa B Inhibition by Omega-3 Fatty Acids Modulates LPS-Stimulated Macrophage TNFAlpha Transcription, Am. J. Physiol. 284: L84-L89 (2003). 124. Sethi, S., 0. Ziouzenkova, H. Ni, D.D. Wagner, J. Plutzky, and T.N. Mayadas, Oxidized Omega-3 Fatty Acids in Fish Oil Inhibit Leukocyte-Endothelial Interactions Through Activation of PPAR a, Blood 100: 1340-1346 (2002). 125. Van den Berghe, W., L. Vermeulen, P. Delerive, K. De Bosscher, B. Staels, and G. Haegeman, A Paradigm for Gene Regulation: Inflammation, NF-KB and PPAR, Adv. Exp. Med. Biol. 544: 181-196 (2003). 126. Calder, P.C., Dietary Modification of Inflammation with Lipids, Proc. Nutr. SOC. 61: 345-358 (2002). 127. Klerman, G.L., and M.M. Weissman, Increasing Rates of Depression, J. Am. Med. ASSOC.261: 2229-2235 (1989). 128. Hibbeln, J.R., and N. Salem, Jr., Dietary Polyunsaturated Fatty Acids and Depression: When Cholesterol Does Not Satisfy, Am. J. Clin. Nutr. 62: 1-9 (1995). 129. Adams, P.B., S. Lawson, A. Sanigorski, and A.J. Sinclair, Arachidonic Acid to Eicosapentaenoic Acid Ratio in Blood Correlates Positively with Clinical Symptoms of Depression, Lipids 31: S157-Sl61 (1996). 130. Maes, M., A. Christophe, J. Delanghe, C. Altamura, H. Neels, and H.Y. Meltzer, Lowered Omega3 Polyunsaturated Fatty Acids in Serum Phospholipids and Cholesteryl Esters of Depressed Patients, Psychiatry Res. 85: 275-291 (1999). 131. Stoll, A.L., W.E. Severus, M.P. Freeman, S. Rueter, H.A. Zboyan, E. Diamond, K.K. Cress, and L.B. Marangell, Omega 3 Fatty Acids in Bipolar Disorder: a Preliminary Double-Blind, Placebo-Controlled Trial, Arch. Gen. Psychiatry 56: 407-412 (1999). 132. Peet, M., and D.F. Horrobin, A Dose-Ranging Study of the Effects of EthylEicosapentaenoate in Patients with Ongoing Depression Despite Apparently Adequate Treatment with Standard Drugs, Arch. Gen. Psychiatry 59: 913-919 (2002). 133. Nemets, B., Z. Stahl, and R.H. Belmaker, Addition of Omega-3 Fatty Acid to Maintenance Medication Treatment for Recurrent Unipolar Depressive Disorder, Am. J. Psychiatry 159: 4 7 7 4 7 9 (2002). 134. Su, K.P., S.Y. Huang, C.C. Chiu, and W.W. Shen, Omega-3 Fatty Acids in Major Depressive Disorder. A Preliminary Double-Blind, Placebo-Controlled Trial, Eur. Neuropsychopharmacol. 13: 267-271 (2003). 135. Noaghiul, S., and J.R. Hibbeln, Cross-National Comparisons of Seafood Consumption and Rates of Bipolar Disorders, Am. J. Psychiatry 160: 2222-2227 (2003). 136. Puri, B.K., S.J. Counsell, G. Hamilton, A.J. Richardson, and D.F. Horrobin, Eicosapentaenoic Acid in Treatment-Resistant Depression Associated with Symptom Remission, Structural Brain Changes and Reduced Neuronal Phospholipid Turnover, Int. J. Clin.Pract. 55: 560-563 (2001). 137. Marangell, L.B., J.M. Martinez, H.A. Zboyan, H. Chong, and L.J. Puryear, Omega-3 Fatty Acids for the Prevention of Postpartum Depression: Negative Data from a Preliminary, Open-Label Pilot Study, Depression Anxiety 19: 20-23 (2004). 138. Penninx, B.W., A.T. Beekman, A. Honig, D.J. Deeg, R.A. Schoevers, J.T. van Eijk, and W. van Tilburg, Depression and Cardiac Mortality: Results from a CommunityBased Longitudinal Study, Arch. Gen. Psychiatly 58: 221-227 (2001).
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139. Hibbeln, J.R., Seafood Consumption, the DHA Content of Mothers’ Milk and Prevalence Rates of Postpartum Depression: a Cross-National, Ecological Analysis, J. Affect. Disord. 69: 15-29 (2002). 140. Marangell, L.B., J.M. Martinez, H.A. Zboyan, B. Kertz, H.F. Kim, and L.J. Puryear, A Double-Blind, Placebo-Controlled Study of the Omega-3 Fatty Acid Docosahexaenoic Acid in the Treatment of Major Depression, Am. J. Psychiatry 160: 996-998 (2003). 141. Carpenter, W.T., Jr., and R.W. Buchanan, Schizophrenia, N . Engl. J. Med. 330: 681-690 (1994). 142. Fukuzako, H., T. Fukuzako, T. Hashiguchi, S . Kodama, M. Takigawa, and T. Fujimoto, Changes in Levels of Phosphorus Metabolites in Temporal Lobes of DrugNai’ve Schizophrenic Patients, Am. J. Psychiatry 156: 1205-1208 (1999). 143. Pettegrew, J.W., M.S. Keshavan, K. Panchalingam, S. Strychor, D.B. Kaplan, M.G. Tretta, and M. Allen, Alterations in Brain High-Energy Phosphate and Membrane Phospholipid Metabolism in First-Episode, Drug-Naive Schizophrenics. A Pilot Study of the Dorsal Prefrontal Cortex by In Vivo Phosphorus 31 Nuclear Magnetic Resonance Spectroscopy, Arch. Gen. Psychiatry 48: 563-568 (1991). 144. Shioiri, T., T. Someya, J. Murashita, T. Kato, H. Hamakawa, K. Fujii, and T. Inubushi, Multiple Regression Analysis of Relationship Between Frontal Lobe Phosphorus Metabolism and Clinical Symptoms in Patients with Schizophrenia, Psychiatry Res. 76: 113-122 (1997). 145. Puri, B.K., A.J. Richardson, D.F. Horrobin, T. Easton, N. Saeed, A. Oatridge, J.V. Hajnal, and G.M. Bydder, Eicosapentaenoic Acid Treatment in Schizophrenia Associated with Symptom Remission, Normalisation of Blood Fatty Acids, Reduced Neuronal Membrane Phospholipid Turnover and Structural Brain Changes, Int. J. Clin. Pract. 54: 57-63 (2000). 146. Peet, M., J. Laughame, N. Rangarajan, D. Horrobin, and G. Reynolds, Depleted Red Cell Membrane Essential Fatty Acids in Drug-Treated Schizophrenic Patients, J. Psychiatr. Res. 29: 227-232 (1995). 147. Richardson, A.J., T. Easton, and B.K. Puri, Red Cell and Plasma Fatty Acid Changes Accompanying Symptom Remission in a Patient with Schizophrenia Treated with Eicosapentaenoic Acid, Eur. Neuropsychopharmacol. 10: 189-193 (2000). 148. Assies, J., R. Lieverse, P. Vreken, R.J. Wanders, P.M. Dingemans, and D.H. Linszen, Significantly Reduced Docosahexaenoic and Docosapentaenoic Acid Concentrations in Erythrocyte Membranes from Schizophrenic Patients Compared with a Carefully Matched Control Group, Biol. Psychiatry 49: 510-522 (2001). 149. Hibbeln, J.R., K.K. Makino, C.E. Martin, F. Dickerson, J. Boronow, and W.S. Fenton, Smoking, Gender, and Dietary Influences on Erythrocyte Essential Fatty Acid Composition Among Patients with Schizophrenia or Schizoaffective Disorder, Biol. Psychiatry 53: 431441 (2003). 150. Reddy, R.D., and J.K. Yao, Environmental Factors and Membrane Polyunsaturated Fatty Acids in Schizophrenia, Prostaglandins Leukot. Essent. Fatty Acids 69:385-391 (2003). 151. Yao, J.K., S. Leonard, and R.D. Reddy, Membrane Phospholipid Abnormalities in Postmortem Brains from Schizophrenic Patients, Schizophr. Res. 42: 7-17 (2000). 152. Warner, R., J. Laughame, M. Peet, L. Brown, and N. Rogers, Retinal Function as a
Marker for Cell Membrane Omega-3 Fatty Acid Depletion in Schizophrenia: A Pilot Study, Biol. Psychiatry 45: 1138-1 142 (1999).
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153. Peet, M., Nutrition and Schizophrenia: Beyond Omega-3 Fatty Acids, Prostaglandins Leukot. Essent. Fatty Acids 70: 417-422 (2004). 154. Peet, M., J. Brind, C.N. Ramchand, S. Shah, and G.K. Vankar, Two Double-Blind Placebo-Controlled Pilot Studies of Eicosapentaenoic Acid in the Treatment of Schizophrenia, Schizophr. Res. 49: 243-25 1 (2001). 155. Peet, M., and D.F. Horrobin for the E-E Multicentre Study Group, A Dose-Ranging Exploratory Study of the Effects of Ethyl-Eicosapentaenoate in Patients with Persistent Schizophrenic Symptoms, J. Psychiatr. Res. 36: 7-18 (2002). 156. Fenton, W.S., F. Dickerson, J. Boronow, J.R. Hibbeln, and M. Knable, A PlaceboControlled Trial of Omega-3 Fatty Acid (Ethyl Eicosapentaenoic Acid) Supplementation for Residual Symptoms and Cognitive Impairment in Schizophrenia, Am. J. Psychiatry 158: 2071-2074 (2001). 157. Emsley, R., C. Myburgh, P. Oosthuizen, and S.J. van Rensburg, Randomized, PlaceboControlled Study of Ethyl-Eicosapentaenoic Acid as Supplemental Treatment in Schizophrenia, Am. J. Psychiatry 159: 1596-1598 (2002). 158. Edwards, R.W., and M. Peet, Essential Fatty Acid Intake in Relation to Depression, Phospholipid Spectrum Disorder in Psychiatry, M. Peet, I. Glenn, and D.F. Horrobin, eds., Marius Press, Lancashire, UK, 1999, pp. 21 1-221. 159. Skosnik, P.D., and J.K. Yao, From Membrane Phospholipid Defects to Altered Neurotransmission: Is Arachidonic Acid a Nexus in the Pathophysiology of Schizophrenia? Prostaglandins Leukot. Essent. Fatty Acids 69: 367-384 (2003). 160. Song, C., and D.F. Horrobin, Omega 3 Fatty Acid Ethyl-Eicosapentaenoate but Not Soybean Oil Attenuates Memory Impairment Induced by Central I L - l P Administration, J. Lipid Res. 45: 1112-1121 (2004). 161. Bosetti, F., J. Rintala, R. Seemann, T.A. Rosenberger, M.A. Contreras, S.I. Rapoport, and M.C. Chang, Chronic Lithium Downregulates Cyclooxygenase-2 Activity and Prostaglandin E(2) Concentration in Rat Brain, Mol. Psychiatry 7: 845-850 (2002). 162. Rapoport, S.I., and F. Bosetti, Do Lithium and Anticonvulsants Target the Brain Arachidonic Acid Cascade in Bipolar Disorder? Arch. Gen. Psychiatry 59: 592-596 (2002). 163. Hillard, C.J., and W.B. Campbell, Biochemistry and Pharmacology of Arachidonylethanolamide, a Putative Endogenous Cannabinoid, J. Lipid Res. 38: 383-398 (1997). 164. Berger, A., G. Crozier, T . Bisogno, P. Cavaliere, S. Innis, and V. DiMarzo, Anandamide and Diet: Inclusion of Dietary Arachidonate and Docosahexaenoate Leads to Increased Brain Levels of the Corresponding N-Acylethanolamines in Piglets, Proc. Natl. Acad. Sci. USA 98: 6402-6406 (2001). 165. Feldberg, W., Possible Association of Schizophrenia with a Disturbance in Prostaglandin Metabolism: A Physiological Hypothesis, Psychol. Med. 6: 359-369 (1976). 166. Horrobin, D.F., Schizophrenia as a Prostaglandin Deficiency Disease, Lancet 1: 936-937 (1977). 167. Molteni, R., R.J. Barnard, Z. Ying, C.K. Roberts, and F. Gomez-Pinilla, A High-Fat, Refined Sugar Diet Reduces Hippocampal Brain-Derived Neurotrophic Factor, Neuronal Plasticity and Learning, Neuroscience 112: 803-814 (2002). 168. Gorski, J.A., S.R. Zeiler, S . Tamowski, and K.R. Jones, Brain-Derived Neurotrophic Factor Is Required for the Maintenance of Cortical Dendrites, J. Neurosci. 23: 68566865 (2003).
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169. Tisdale, M.J., The “Cancer Cachectic Factor,” Support Care Cancer 11: 73-78 (2003). 170. Tisdale, M.J., Cachexia in Cancer Patients, Nut. Rev. Cancer 2: 862-871 (2002). 171. De Wys, W.D., C. Begg, P.T. Lavin, P.R. Band, J.M. Bennett, J.R. Bertino, M.H. Cohen, H.O. Douglas, Jr., P.F. Engstrom, E.Z. Ezdinli, J. Horton, G.J. Johnson, C.G. Moertel, M.M. Oken, C. Perlia, C. Rosenbaum, M.N. Silverstein, R.T. Skeel, R.W. Sponzo, and D.C. Tormey, Prognostic Effect of Weight Loss Prior to Chemotherapy in Cancer Patients, Am. J. Med. 69: 491497 (1980). 172. De Wys, W.D., Anorexia as a General Effect of Cancer, Cancer 45: 2013-2019 (1972). 173. Tisdale, M.J., Clinical Trials for the Treatment of Secondary Wasting and Cachexia, J. Nutr. 129: 2433-246s (1999). 174. Beck, S.A., K.L. Smith, and M.J. Tisdale, Anticachectic and Antitumor Effect of Eicosapentaenoic Acid and Its Effect on Protein Turnover, Cancer Res. 51: 6089-6093 (1991). 175. Fredrix, E.W., W.H. Saris, P.B. Soeters, E.F. Wouters, A.D. Kester, M.F. von Meyenfeldt, and K.R. Westerterp, Estimation of Body Composition by Bioelectrical Impedance in Cancer Patients, Eur. J. Clin. Nutr. 44: 749-752 (1990). 176. Falconer, J.S., K.C. Fearon, C.E. Plester, J.A. Ross, and D.C. Carter, Cytokines, the Acute-Phase Response, and Resting Energy Expenditure in Cachectic Patients with Pancreatic Cancer, Ann. Surg. 219: 325-331 (1994). 177. Lundholm, K., A.C. Bylund, J. Holm, and T. Schersten, Skeletal Muscle Metabolism in Patients with Malignant Tumor, Eur. J. Cancer 12: 465473 (1976). 178. Hussey, H.J., and M.J. Tisdale, Effect of a Cachectic Factor on Carbohydrate Metabolism and Attenuation by EicosapentaenoicAcid, Br. J. Cancer 80: 1231-1235 (1999). 179. Beck, S.A., and M.J. Tisdale, Production of Lipolytic and Proteolytic Factors by a Murine Tumor-Producing Cachexia in the Host, Cancer Res. 47: 5919-5923 (1987). 180. Wigmore, S.J., J.A. Ross, J.S. Falconer, C.E. Plester, M.J. Tisdale, D.C. Carter, and K.C. Fearon, The Effect of Polyunsaturated Fatty Acids on the Progress of Cachexia in Patients with Pancreatic Cancer, Nutrition 12: S27-S30 (1996). 181. Wigmore, S.J., M.D. Barber, J.A. Ross, M.J. Tisdale, and K.C. Fearon, Effect of Oral Eicosapentaenoic Acid on Weight Loss in Patients with Pancreatic Cancer, Nutr. Cancer 36: 177-184 (2000). 182. Barber, M.D., J.A. Ross, A.C. Voss, M.J. Tisdale, and K.C. Fearon, The Effect of an Oral Nutritional Supplement Enriched with Fish Oil on Weight-Loss in Patients with Pancreatic Cancer, Br. J. Cancer 81: 80-86 (1999). 183. Whitehouse, AS., H.J. Smith, J.L. Drake, and M.J. Tisdale, Mechanism of Attenuation of Skeletal Muscle Protein Catabolism in Cancer Cachexia by Eicosapentaenoic Acid, Cancer Res. 61:3604-3609 (2001). 184. Todorov, P., P. Cariuk, T. McDevitt, B. Coles, K. Fearon, and M. Tisdale, Characteriza-tion of a Cancer Cachectic Factor, Nature 379: 739-742 (1996). 185. Smith, K.L., and M.J. Tisdale, Mechanism of Muscle Protein Degradation in Cancer Cachexia, Br. J. Cancer 68: 314-318 (1993). 186. Strassmann, G., and T. Kambayashi, Inhibition of Experimental Cancer Cachexia by AntiCytokine and Anti-Cytokine-ReceptorTherapy, Cytokines Mol. Ther. 1: 107-1 13 (1995). 187. Li, D., 0. Bode, H. Dmmmond, and A.J. Sinclair, Omega (n-3) Fatty Acids, in Lipids for Functional Foods and Neutraceuticals, edited by F.D. Gunstone, The Oily Press, Bridgewater, UK, 2003, Ch. 8, pp. 225-262.
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188. Sinclair, A.J., and K. O’Dea, Fats in Human Diets Through History: Is the Western Diet Out of Step? in Reducing Fat in Meat Animals, edited by J.D. Woods and A.V. Fisher, Elsevier Applied Science, London, 1990, pp. 1-47. 189. Eaton, S.B., and M. Konner, Paleolithic Nutrition: A Consideration of Its Nature and Current Implications, N. Engl. J. Med. 312: 283-290 (1985).
Chapter 17
Lipase Reactions Applicable to Purification of Oil- and Fat-Related Materials Yuji Shimada Osaka Municipal Technical Research institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan
Introduction Much attention has been focused on the application of lipases to the oil and fat industry. Industrial production of functional lipids with lipases historically started with a cocoa fat substitute at the beginning of the 1980s when 1,3-stearoyl-2-oleoylglycerol, which has a sharp melting point around body temperature, was produced by an exchange of fatty acid (FA) at the 1,3-positionsof 2-oleoyl triacylglycerol (TAG) with stearic acid (1). The new process, with a fixed-bed reactor packed with an immobilized 1,3-position specific lipase, attracted much attention and significantly affected subsequent oil processing with lipases. In the 1990s, an oil containing a high concentration of docosahexaenoic acid (DHA) was produced by selective hydrolysis of tuna oil with a lipase that acted poorly on DHA (2); a human milk fat substitute, 1,3-oleoyl2-palmitoyl glycerol, was also developed by exchange of FA at the 1,3-positionsof tripalmitin with oleic acid (3). In addition, diacylglycerol (DAG) (4) and TAG containing medium- and long-chain FA (5) were produced recently through lipase-catalyzed esterification and interesterification,respectively. Selective reactions with lipases are also effective for the purification of oil- and fat-related compounds. Distillation, organic solvent fractionation, and various kinds of chromatography were adopted industrially for purification of useful materials. Among these methods, distillation and organic solvent fractionation are widely used, but have the drawback that fractionation efficiency is not good. This drawback can be overcome by introducing lipase-catalyzed selective reactions. This chapter describes how a process comprising lipase-catalyzed reactions and distillation (or organic solvent fractionation) is effective for large-scale purifications of unstable FA and of oil- and fatrelated compounds. Lipase: Substrate Specificities and Reactions
Lipases have FA specificity, alcohol specificity,positional specificity (1,3-position specific and nonspecific), TAG specificity, and acylglycerol specificity (6).Although lipases are enzymes that catalyze the hydrolysis of long-chain FA ester bonds, the reaction is reversible, and they also catalyze esterification and transesterification (Fig. 17.1). In general, hydrolysis occurs preferentially in a system containing a large amount of water, and esterification proceeds effectively in a system containing only a small 395
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1. Hydrolysis ROCOR’ + H 2 0
2. Esterification R’OH + R2COOH
ROH + R‘COOH R10COR2+ H 2 0
3. Transesterification 3-1 Acidolysis
ROCOR + R2COOH 3-2 Alcoholysis R ~ O C O R+ R ~ O H
ROCOR2 + R’COOH
R ~ O C O R+ R‘OH
3-3 lnteresterification R’OCOR + R30COR4
R10COR4+ R30COR2
Fig. 17.1. Lipase reactions applicable for purification of useful materials.
amount of water. Transesterification (acidolysis, alcoholysis, and interesterification) is catalyzed efficiently in a mixture without water using an immobilized enzyme. Oil- and fat-related compounds can be converted to the desired molecular forms by a combination of the specificity and reaction. It should be kept in mind, however, that the enzymes act strongly on liquid-state substrates but poorly on those that are solid state. Strategy for Purification of Useful Materials
There are only a few reports on the purification of useful compounds using lipase reactions. If a substance in a raw material is converted to different molecular forms by lipase-catalyzed selective reactions, relatively easy purification can be achieved by distillation or organic solvent fractionation of the reaction mixture. The strategies for construction of the reaction system are illustrated schematically in Figure 17.2. The first strategy is selective decomposition (hydrolysis) of contaminants in a raw material with a lipase (Fig. 17.2A). Change in the physical properties (e.g., molecular weights, boiling points, solubility in organic solvents) of contaminants results in the straightforward purification of the desired compound by distillation or organic solvent fractionation. The second strategy is conversion of a desired compound or contaminants to different molecular forms by a lipase-catalyzed reaction, which is performed after the addition of another substrate (Fig. 17.2B). The conversion also promises an uncomplicated purification of the desired compound by distillation or organic solvent fractionation. This strategy has another advantage. At this point, the compound has been converted to its value-added form after the purification from a raw material. However, according to this strategy, the enzyme reaction plays the role of both conversion of the compound and one process of the purification because only the compound in the raw material can be converted to its value-added form by taking advantage of the substrate specificity of the enzyme.
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A Degradation of Contaminants B Conversion of Desired Compound
Contaminants
Desired compound
Contaminants
Desired compound
Fig. 17.2. Strategy for purification of useful materials by a process comprising enzymatic reaction and distillation (or organic solvent fractionation).
Sfrafegies for Increasing Rea cfion Efficiency
When enzyme reactions are introduced into a process of purification of a useful material, the key is to construct a reaction system in which a high yield is achieved. The strategies for increasing the reaction yield are summarized in Table 17.1. The most popular procedure is removal of reaction product(s), i.e., after separation of substrates and products, substrates are allowed to react again (Table 17.1; A-1). Adoption of this procedure increases the yield for the raw material, but the processes of removing the products and repeating the reaction are required. On the other hand, successive removal of products (referred to as in situ product removal) achieves a high yield even in a single reaction (Table 17.1; A-2). For example, when one of the products is water (7-9) or short-chain alcohol (lo), the product can be removed successively by conducting the reaction under reduced pressure, resulting in a significant increase in yield (Table 17.1; A-2-1). Furthermore, when the melting point of the product is high, a reaction at low temperatures allows it to solidify. Because solid substrates are poorly recognized by enzymes, the product is eliminated from the reaction system, resulting in a high reaction yield (Table 17.1; A-2-2). As an example, esterification of conjugated linoleic acid (CLA) with glycerol at 5°C produced 93% MAG at 95% esterification due to the solidification of monoacylglycerol (MAG) (1 1). A biphasic system (water/organic solvent) is also effective for increasing a reaction yield (Table 17.1; A2-3): The transfer of products into the organic solvent or water phase achieves a high reaction yield (12,13). In addition, if a product is converted to a molecular form that is poorly recognized by the enzyme, a high yield will be achieved because the reverse reaction scarcely occurs (Table 17.1; A-2-4) (14,15). A reaction system, in which the equilibrium shifts in the direction desired, is effective for achieving high yield (Table 17.1, B). The esterification of FA with
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TABLE 17.1 Strategies for Achieving a High Reaction Yield A. Removal of a product 1. Repeated reaction 2. /n situ product removal 2-1. Evaporation of a product 2-2. Removal of a product as a precipitate 2 - 3 . Removal of a product into the organic solvent phase 2-4. Changing a product to a molecular form that is poorly recognized by the enzyme
6.Adoption of reactions shifting in 1. Reaction synthesizing fatty alcohol ester the desired direction 2. Reaction synthesizing steryl ester
C. Stepwise addition of a substrate 1. Reaction in which a substrate inactivates the enzyme 2 . Multistep in situ reaction
fatty alcohols including short-chain alcohol (16-18) or with sterols (19) reached >80% even in the presence of 50% water. Equilibrium of the reaction is determined by the energy levels of substrates and products in thermodynamics, but is theoretically independent of the enzyme property. However, if it is assumed that the lipase recognizes substrate but not product in these reactions, the construction of a reaction system is simplified. We show in this chapter that products in these reactions are recognized poorly by lipases. Yields in some lipase-catalyzed reactions are very low when a substrate inhibits or inactivates the lipases. Excess amounts of methanol, for example, inactivated a lipase in the methanolysis of TAG. In this reaction, the stepwise addition of methanol increased the reaction yield and the lipase stability (19,21) (Table 17.1; C-1). In a two-step in situ reaction, when a substrate in the second step inhibits the first-step reaction, the yield is increased by the addition of the substrate after the first-step reaction reaches the equilibrium state (22) (Table 17.1, C-2). We developed several purification processes comprising lipase-catalyzed reactions with high yield and distillation (or organic solvent fractionation). Among them, purification processes of unstable FA, tocopherols, sterols, FA steryl esters (referred to as steryl esters), and astaxanthin are described in the following sections.
Purification of FA Purification of FA Through Selective Esterification
A mixture of FFA containing a desired FA is esterified with an alcohol by taking advantage of the FA specificity of lipase. When only contaminating FA are esterified, a desired FA is enriched in the FFA fraction. The resulting reaction mixture is composed of alcohol, FFA, and FA esters. If short-path distillation is adopted for fractionation of these components, the reaction system should satisfy the following
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criteria: (i) high degree of esterification of contaminating FA; (ii) high selectivity of a lipase for FA; and (iii) large differences among molecular weights (boiling points) of alcohol, FFA, and FA esters. Lauryl alcohol (LauOH) was selected as a substrate satisfying these demands (16). This reaction system achieved a high degree of esterification for contaminating FA because the lauryl esters of contaminating FA are poorly recognized by lipase (Table 17.1, B-1); thus, the desired FA was efficiently enriched in the FFA fraction. To further increase the purity of the desired FA, FFA were recovered from the reaction mixture and allowed to react again (Table 17.1, A-1). PUFA and CLA isomers were purified according to this strategy. Purification of GLA. GLA was purified from borage oil by a process comprised of repeated selective esterification and distillation (23). The purification is summarized in Table 17.2. An oil containing 45% GLA (GLA45 oil) was used as a starting material, which was prepared by selective hydrolysis of borage oil with Candida rugosa lipase (24). GLA45 oil was first hydrolyzed with Burkholderia cepacia lipase, which acted strongly on GLA. The reaction mixture was subjected to short-path distillation, and FFA were recovered in the distillate fraction. The FFA mixture was then esterified with LauOH using Rhizopus oryzae lipase, which acted weakly on GLA; 95.9% of contaminating FFA were esterified, and GLA was enriched to 89.5% in the FFA fraction. To further increase the purity of GLA, FFA recovered by distillation were esterified again with LauOH, increasing the purity to 98.1%. In this purification process, short-path distillation was very effective for separation of LauOH, FFA, and FA lauryl esters (FALE). In addition, FALE in the FFA fraction were efficiently removed by urea adduct fractionation. This process is effective for the purification of other PUFA. When tuna oil was used as a starting material, DHA was purified to 91% with 60% recovery (25). In addition, n-6 PUFA were purified from a single-cell oil containing 40% arachidonic acid (AA). Because the C. rugosa lipase used in selective esterification acted weakly not only on AA, but also on GLA and dihomo-GLA, the total content of n-6 PUFA increased to 96% with 52% recovery (AA purity, 81%; AA recovery, 53%) (26). Purification of CLA komers. CLA is a group of C,, FA containing a pair of conjugated double bonds in either the cis or trans configuration. A typical commercial product contains almost equal amounts of 9cis, 1ltrans (9c,l It)-CLA and 10t,12cCLA. The mixture of CLA isomers was reported to have various physiologic activities. Also, it was reported recently that 9c,l It-CLA has anticancer activity (27), and that lOt,12c-CLA decreases body fat content (28,29) and suppresses the development of hypertension (30). These studies called a great deal of attention to the fractionation of CLA isomers. The two CLA isomers can be purified by a process of repeated esterification with LauOH and short-path distillation (Fig. 17.3) (31,32). A mixture of CLA isomers was first prepared by alkali conjugation of linoleic acid, referred to as FFA-
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TABLE 17.2 Purification of GLA from GLA45 Oil GLA in FFA fraction Step GLA45 oil Hydrolysisd Distillatione Esterificationf Distillationg Esterificatiod Distillatione Urea adduct'
Weight
Amount of FFAa
Recovery
(kg)
Content Cs/lOO g)
Amount
(kg)
(kg)
(Oh)
10.00 8.69 7.55 16.54 3.87 7.61 2.67 2.09
9.1 5b 7.95 7.51 3.41 3.1 5 2.53 2.28 2.07
45.1 46.3 46.3 89.5 89.4 97.3 98.1 98.6
4.1 3c 3.68 3.48 3.05 2.82 2.46 2.24 2.04
100 89.1 84.2 73.9 68.2 59.6 54.2 49.4
dThe amount of FFA was calculated from its acid value. 'The amount of FA in GLA45 oil. [The content and amount of CLA in GLA45 oil. dReaction conditions: GLA45 oilwater, 2:l (w/w); Burkhorderia cepacia lipase, 250 U/g mixture; 35°C; 24 h. The degree of hydrolysis was 91.5%. T h e reaction mixture was distilled at 180°C and 0.2 rnrnHg; the residue was then distilled at 20OoCand 0.2 mmHg. The two distillates were combined. keaction conditions: FFNLauOH, 1 :2 (mol/mol); water, 20%; Rhizopus oryzae lipase, 50 U/g-mixture; 30°C; 16 h. The degree of esterificationwas 52.0%. gLauOH was removed by distillation at 120'C and 0.2 mmHg, and FFA were then recovered in the distillate at 1 8 5 T and 0.2 mmHg. hReaction conditions: FFNLauOH, 1 :2 (mol/mol); water, 20%; R. oryzae lipase, 70 U/g mixture; 3 0 T ; 16 h. The degree of esterification was 15.2%. The FFA fraction (400 g) obtained by distillation was completely dissolved at 50°C in a solution of 2 L MeOH, 50 mL water, and 400 g urea. The solution was then cooled gradually to 5°C with agitation over -10 h. After removal of the precipitate, the volume of the filtrate was reduced to -700 mL, and 300 mL of 0.2 N HCI was then added. The oil layer (FFA) was washed three times with 300 mL water.
CLA. FFA-CLA contained 45.1% 9c,l It-CLA, 46.8% lOt,12c-CLA, and 5.3% other CLA isomers. FFA-CLA was esterified with LauOH using C. rugosa lipase, which acts strongly on 9c,llt-CLA and weakly on lOt,l2c-CLA. The FFA fraction containing 78.1% lOt,l2c-CLA and the FALE fraction containing 85.1% 9c,l ltCLA were recovered by short-path distillation. The FFA and FALE fractions were used for further purification of 1 0 ~ 1 2and ~ - 9c,l It-CLA, respectively. To purify 10t,12c-CLA, the FFA fraction was esterified again with LauOH. The FFA fraction recovered by distillation consisted of 0.3% LauOH, 91.6% FFA, and 8.1% FALE; the FA composition in the fraction was 3.4% 9c,llt-CLA, 86.3% 10t,12c-CLA, and 9.7% other CLA isomers. FALE and CLA, except for 9c,11tand 1Of, 12c-CLA, were finally removed by urea adduct fractionation. This fractionation completely eliminated FALE and decreased the content of CLA, except for the 9c,llt- and lOt,12c-isomers,from 9.7 to 1.3 wt%. Consequently, the purity of 10t,12c-CLA reached 95.3% (the content of lOt,12c-CLA based on the total content of 9c,llt- and lOt,l2c-isomers, 96.9 wt%). Recovery of 1Ot,12c-CLA by a series of purification procedures was 31% of the initial content (32).
Linoleic acid Alkali conjugation Sc,llt-CLA
45.1%
Esterification with LauOH Short-path distillation
t
LauOH
t
t
FALE
FFA Esterification with LauOH Short-path distillation
LauOH
FFA
HydroIysis Esterification with LauOH Short-path distillation
FALE LauOH
FFA
FALE Hydrolysis Short-path distillation
LauOH
I
FFA
Purified 9c,l1t-CLA 93.1%
Fig. 17.3. Purification process of CLA isomers. P
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Y. Shimada
Another isomer, 9c,l It-CLA, enriched in the FALE fraction, was purified next. The FALE were hydrolyzed chemically, and FFA were recovered. The FFA were esterified again with LauOH. After the reaction, FALE were recovered by distillation; they were hydrolyzed, and a mixture of LauOH and FFA was recovered. The FFA fraction purified by short-path distillation consisted of 0.3% LauOH, 99.4% FFA, and 0.3% FALE; the FA composition in the preparation was 93.1% 9c,lltCLA, 3.5% lOt,12c-CLA, and 0.4% other CLA isomers (the content of 9c,lltCLA based on the total content of 9c,11t- and lOt,12c-isomers, 96.4%). Recovery of 9c,l It-CLA was 34% of the initial content (32). Success in purification of CLA isomers indicated that repeated esterification with LauOH is effective for achieving a high reaction yield, and that short-path distillation is effective for the separation of LauOH, FFA, and FALE. Purification of DHA fthy/ Fster (DHAf Q through Selective A/coho/ysis. LauOH is also effective for achieving a high reaction yield in selective alcoholysis of FA ethyl esters (FAEE). Alcoholysis of FAEE originating from tuna oil with LauOH was conducted using a lipase that acts weakly on DHA-enriched DHAEE in the FAEE fraction. Rhizopus oryzae and Rhizomucor miehei lipases were effective for achieving a high reaction yield (33-35). When FAEE containing 55% DHAEE underwent alcoholysis with 7 mol of LauOH using immobilized R. miehei lipase, 89% of FAEE other than DHAEE were converted to FALE, and the purity of DHAEE increased from 57 to 90% (35). Short-path distillation was of course effective for the separation of LauOH, FAEE, and FALE. This result also shows how a reaction achieving a high reaction yield is effective for the purification of useful materials. Purification of Tocopherols, Sterols, and Sferyl Esters
The final process in vegetable oil refining is deodorization by steam distillation. The resulting distillate (vegetable oil deodorizer distillate; VODD) contains tocopherols, sterols, and steryl esters, and can be utilized as an important material for purification of tocopherols. In a purification process, however, sterols cause an inefficient yield of tocopherols. Sterols can be purified from the residue obtained after purification of tocopherols, but the yield is not high. In addition, steryl esters in VODD are wasted, although they are useful food materials. We thus aimed to purify these materials by a process including lipase-catalyzed reactions. Purification of Tocophero/s and Stero/s. An outline of purification of tocopherols and sterols is shown in Figure 17.4. VODD was fractionated into a low boiling point fraction (not including steryl esters) and a high boiling point fraction (not including tocopherols and sterols), and tocopherols and sterols were purified from the low boiling point fraction. This fraction contained FFA, tocopherols, sterols, partial acylglycerols, and unknown hydrocarbons. Among these, toco-
Vegetable Oil Deodorizer Distillate (FFA, Tocopherols, Sterols, MAG, DAG, TAG, Steryl esters) Short-path distillation
+
+
Low Boiling Point Substances
High Boiling Point Substances
(FFA, Tocopherols, Sterols, MAG, DAG)
(DAG, TAG, Steryl esters) Hydrolysis of acylglycerols
Hydrolysis of acylglycerols Esterification of sterols
Reaction Mixture (FFA, Steryl esters)
(FFA, Tocopherols, Steryl esters)
1
Methyl esterification of FFA
Reaction Mixture (FAME, Tocopherols, Steryl esters)
I
t
FAME
+
Short-path distillation
Tocopherols
1
Short-path distillation
7 Steryl esters
FFA
MeOH
t
-1
Methanolysis of steryl esters
Reaction Mixture
Steryl esters
(FAME, Sterols) n-Hexane fractionation
FAME
Sterols P
Fig. 17.4. Outline of purification of tocopherols, sterols, and steryl esters in vegetable oil deodorizer distillate.
w 0
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pherols, sterols, and partial acylglycerols are not efficiently fractionated by shortpath distillation. Removal of FFA in the tocopherol fraction also is not straightforward. If hydrolysis of partial acylglycerols, conversion of sterols to steryl esters, and methyl esterification of FFA are achieved, the resulting mixture will be composed of tocopherols, steryl esters, and FAME. The boiling point of FAME is lower than that of FFA, and the molecular weight of steryl esters becomes greater than that of the sterols. Hence, the three components are presumed to be fractionated efficiently by short-path distillation. To construct a system in which the three reactions (hydrolysis of acylglycerols, conversion of sterols to steryl esters, and methyl esterification of FFA) proceed in one batch, C. rugosa lipase was selected because the enzyme efficiently catalyzed esterification of sterols with FA and methyl esterification of FA even in the presence of 50% water (Table 17.1; B-1 and -2). The low boiling point substances were first treated with C. rugosa lipase in a mixture containing 20% water and 2 mol methanol/total FA. Consequently, hydrolysis of acylglycerols and methyl esterification of FA proceeded efficiently, but the conversion of sterols to steryl esters was poor (Fig. 17.5A). This result was assumed to be due to the preference for methyl esterification of FA rather than the conversion of sterols to steryl esters. To solve this problem, a two-step in situ reaction was attempted (Table 17.1, C-2; Fig. 17.5B). The first step was hydrolysis of acylglycerols and conversion of sterols to steryl esters. The low boiling point fraction was treated with the lipase in the presence of 20% water. After the reaction reached the equilibrium state, methanol was added to the mixture and the reaction was continued. This twostep in situ reaction proceeded successfully and achieved complete hydrolysis of
A One-step reaction MeOH
Lipase
Sterols
A
FAME
\*+ Sterols
B Two-step in situ reaction Sterols FFA
Steryl esters FFA
MeOH
/ -
Steryl esters FAME
’
Fig. 17.5. Treatment of a mixture of FFA, sterols, and methanol by C. rugosa lipase.
Purification of Oil- and Fat-Related Materials
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acylglycerols, 80% conversion of sterols to steryl esters, and 78% methyl esterification of FA. The two-step in situ reaction with C. rugosa lipase was adopted for purification of tocopherols and sterols from low boiling point substances (Fig. 17.4). A single in situ reaction attained only 80% conversion of sterols to steryl esters. To increase the conversion, after the single reaction, FAME and steryl esters were removed by short-path distillation, and the fraction containing tocopherols was subjected again to the two-step in situ reaction (Table 17.1; A-1). Consequently, conversion of sterols to steryl esters reached 60% (total conversion, 92%). The resulting mixture was finally applied to short-path distillation and was fractionated into FAME, tocopherol, and steryl ester fractions. Through a series of purification steps, tocopherols were purified to 76% with 90% recovery, and sterols were purified as steryl esters to 97% with 86% recovery (22). Purification of Steyl Eesters. A process of purification of steryl esters from high boiling point substances in VODD is shown in Figure 17.4. The high boiling point fraction was composed of DAG, TAG, and steryl esters. Lipases act strongly on acylglycerols, and weakly on steryl esters; thus, the high boiling point fraction was treated with C. rugosa lipase in the presence of 50% water. The treatment achieved complete hydrolysis of acylglycerols only. The products, FFA, were efficiently removed by short-path distillation, and the purity of the steryl esters increased to 97% with 88% recovery (36). This purification, according to the strategy shown in Figure 17.2A appears to be valuable as a new process for the purification of steryl esters from VODD. Conversion of Steryl Esters to Free Sterols. Tocopherols and steryl esters in VODD were highly purified with high recovery through the processes described above. However, sterols were purified as steryl esters. Because free sterols are also useful food additives, we next attempted to convert steryl esters into free sterols. We screened various industrial lipases to find a suitable lipase for hydrolysis of steryl esters at high yield. C. rugosa, Geotrichum candidum, Pseudomonas aeruginosa, P. stutzeri, B. cepacia, and Burkholderia glumae lipases hydrolyzed steryl esters in the presence of 50% water, but the hydrolysis reached equilibrium state at -50%. Products of the hydrolysis of steryl esters are sterols and FFA. The equilibrium in the reaction should shift in the direction of hydrolysis by removal of FFA. Lipase acts strongly on FFA but weakly on FAME. We therefore attempted to convert FFA into FAME along with hydrolysis of steryl esters (Table 17.1, B-I, C-2; Fig. 17.6; in situ product removal). When a mixture of steryl esterdmethanol (1:2, mol/mol) was treated with P. aeruginosa lipase in the presence of 50% water, 98% of the steryl esters were converted to free sterols. Unlike FAME, FFA, and steryl esters, sterols are not soluble in n-hexane. Hence, n-hexane fractionation of the reaction mixture purified the steryl esters to 99% with 92% recovery (Fig. 17.4) (14).
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Stew' + H,O esters
Sterols + FFA FFA + MeOH
FAME + H,O
Fig. 17.6. High conversion of steryl esters to free sterols by in situ product removal
Astaxanthin (3,3-dihydroxy-P,P-carotene-4,4'-diene) is widely distributed in marine creatures and has various physiologic functions, including being a precursor of vitamin A (37), quenching free radicals and active oxygen species (38), having anticancer activity (39),and enhancing the immune response (40). These activities have attracted a great deal of attention, and astaxanthin has been used as a nutraceutical food and an ingredient in cosmetics. An industrially available preparation of astaxanthin from Haematococcus pluvialis cells contained 42% acylglycerols, 25% FFA, and 15% astaxanthin, which was a mixture of free and FA ester forms (free astaxanthidastaxanthin monoesters/astaxanthin diesters = 590: 15, by mol). Astaxanthin was purified from this preparation using a strategy similar to that used for the purification of sterols from high boiling point substances in VODD. The process is shown in Figure 17.7. Contaminating acylglycerols were first hydrolyzed with C. rugosa lipase in the presence of 50% water, and FFA were removed by short-path distillation. This procedure increased the purity of astaxanthin from 15 to 41%. Insolubility of free astaxanthin in n-hexane was noted, and an attempt was made to convert astaxanthin esters in the concentrate to the free form. When a mixture of astaxanthin concentrate, 5 moles of ethanol to total FA, and 50% water was agitated with P. aeruginosa lipase, 90% of the astaxanthin esters were converted to the free form. The free form was efficiently recovered by precipitation with n-hexane. The purity of astaxanthin was thereby raised to 70% with 64% overall recovery of the initial content in the H.pluvialis cell extracts (15). This result is also a good example showing that the equilibrium of a reaction can be shifted in the desired direction by changing a product to the molecular form on which the lipase acts poorly, thus achieving a high reaction yield.
Conclusion Lipases are good tools for oil processing, and have been used industrially for the production of cocoa fat substitutes, human milk substitutes, an oil containing a high concentration of DHA, DAG, and TAG containing medium- and long-chain FA. There are, however, very few reports of the application of lipase to the purification of oil- and fat-related compounds. As described in this chapter, a relatively straightforward purification can be achieved by taking advantage of the substrate selectivity of lipases, and by converting contaminants or a desired compound to different molecular forms. Construction of lipase-catalyzed reactions with a high reaction yield is a key to the success. In addition, the lipase-catalyzed reactions could be powerful tools for purification of the small amounts of useful materials
Cell extracts of Haematococcus pluvialis (FFA ,Acylglycerols, Astaxanthin, Astaxanthin esters)
1
Hydrolysis of acylglycerols
Reaction mixture (FFA , Astaxanthin, Astaxanthin esters)
d Short-path distillation
FFA
Astaxant hin concentrate
(Free Astaxanthin, Astaxanthin esters, Contaminants)
i , 1 EtOH
Ethanolysis of astaxanthin esters
Reaction mixture
(FAEE, Free Astaxanthin, Contaminants)
n-Hexane fractionation
FAEE contaminants
Purified astaxanthin
P
Fig.17.7. Purification process of astaxanthin from Haematococcuscell extracts.
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that are present in the unused biomass. Because a lipase-catalyzed reaction according to this strategy plays the role of both conversion of a desired compound and one process of the purification, the adoption of enzyme reactions for the purification of useful materials is recommended.
References 1. Yokozeki, K., S. Yamanaka, K. Takinami, Y. Hirose, A. Tanaka, K. Sonomoto, and S. Fukui, Application of Immobilized Lipase to Regio-Specific Interesterification of Triglyceride in Organic Solvent, Eur. J. Appl. Microbiol. Biotechnol. 14: 1-5 (1982). 2. Shimada, Y., A. Sugihara, and Y. Tominaga, Production of Functional Lipids Containing Polyunsaturated Fatty Acids with Lipase, in Enzymes in Lipid Modification, edited by U.T. Bornscheuer, Wiley-VCH, Weinheim, 2000, pp. 128-147. 3. Akoh, C.C., and X. Xu, Enzymatic Production of Betapol and Other Specialty Fats, in Lipid Biotechnology, edited by T.M. Kuo and H.W. Gardner, Marcel Dekker, New York, 2002, pp. 461-478. 4. Matsuo, N., and I. Tokimitsu, Metabolic Characteristics of Diacylglycerol, inform 12: 1098-1 102 (2001). 5. Negishi, S., S. Shirasawa, Y. Arai, J. Suzuki, and S. Mukataka, Activation of Powdered Lipase by Cluster Water and the Use of Lipase Powders for Commercial Esterification of Food Oils, Enzyme Microb. Technol. 32: 66-70 (2003). 6. Shimada, Y., A. Sugihara, and Y. Tominaga, Enzymatic Enrichment of Polyunsaturated Fatty Acids, in Lipid Biotechnology, edited by T.M. Kuo and H.W. Gardner, Marcel Dekker, New York, 2002, pp. 493-515. 7. Kawashima, A., Y. Shimada, M. Yamamoto, A. Sugihara, T. Nagao, S. Komemushi, and Y. Tominaga, Enzymatic Synthesis of High-Purity Structured Lipids with Caprylic Acid at 1,3-Positions and Polyunsaturated Fatty Acid at 2-Position, J. Am. Oil Chem. SOC.78: 61 1-616 (2001). 8. Watanabe, Y., Y. Shimada, Y. Yamauchi-Sato, M. Kasai, T. Yamamoto, K. Tsutsumi, Y. Tominaga, and A. Sugihara, Synthesis of MAG of CLA with Penicillium camembertii Lipase, J. Am. Oil Chem. SOC. 79: 891-896 (2002). 9. Yamauchi-Sato, Y., T. Nagao, T. Yamamoto, T. Terai, A. Sugihara, and Y. Shimada, Fractionation of Conjugated Linoleic Acid Isomers by Selective Hydrolysis with Candida rugosa Lipase, J. Oleo Sci. 52: 367-374 (2003). 10. Irimescu, R., K. Furihata, K. Hata, Y. Iwasaki, and T. Yamane, Two-step Enzymatic Synthesis of Docosahexaenoic Acid-Rich Symmetrically Structured Triacylglycerols via 2-Monoacylglycerols, J. Am. Oil Chem. SOC.78: 743-748 (2001). 11. Watanabe, Y., Y. Yamauchi-Sato, T. Nagao, T. Yamamoto, K. Tsutsumi, A. Sugihara, and Y. Shimada, Production of MAG of CLA in a Solvent-Free System at Low Temperature with Candida rugosa Lipase, J. Am. Oil Chem. SOC.80: 909-914 (2003). 12. Matsumae, H., M. Furui, and T. Shibatani, Lipase-Catalyzed Asymmetric Hydrolysis of 3-Phenylglycidic Acid Ester, the Key Intermediate in the Synthesis of Diltiazem Hydrochloride, J. Ferment. Bioeng. 75: 93-98 (1993). 13. Kobayashi, T., S. Adachi, K. Nakanishi, and R. Matsuno, Synthesis of Alkyl Glycosides Through P-Glucosidase-Catalyzed Condensation in an Aqueous-Organic Biphasic System and Estimation of the Equilibrium Constants for Their Formation, J. Mol. Catal. B: Enzyme. 11: 13-21 (2000).
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14. Shimada, Y., T. Nagao, Y. Watanabe, Y . Takagi, and A. Sugihara, Enzymatic Conversion of Steryl Esters to Free Sterols, J. Am. Oil Chem. SOC.80: 243-247 (2003). 15. Nagao, T., T. Fukami, Y. Horita, S. Komemushi, A. Sugihara, and Y. Shimada, Enzymatic Enrichment of Astaxanthin from Haematococcus pluvialis Cell Extracts, J. Am. Oil Chem. SOC. 80: 975-981 (2003). 16. Shimada, Y., A. Sugihara, H. Nakano, T. Kuramoto, T. Nagao, M. Gemba, and Y. Tominaga, Purification of Fatty Acids from Tuna Oil with Rhizopus delemar Lipase, J. Am. Oil Chem. SOC. 74: 97-101 (1997). 17. Shimada, Y., Y. Watanabe, A. Sugihara, T. Baba, T. Ooguri, S. Moriyama, T. Terai, and Y. Tominaga, Ethyl Esterification of Docosahexaenoic Acid in an Organic Solvent-Free System with Immobilized Candida antarctica Lipase, J. Biosci. Bioeng. 92: 19-23 (2001). 18. Watanabe Y., Y. Shimada, T. Baba, N. Ohyagi, S. Moriyama, T. Terai, Y. Tominaga, and A. Sugihara, Methyl Esterification of Waste Fatty Acids with Immobilized Candida antarctica Lipase, J. Oleo Sci. 51: 655-661 (2002). 19. Shimada, Y., Y. Hirota, T. Baba, A. Sugihara, S. Moriyama, Y. Tominaga, and T. Terai, Enzymatic Synthesis of Steryl Esters of Polyunsaturated Fatty Acids, J. Am. Oil Chem. SOC.76: 713-716 (1999). 20. Shimada, Y., Y. Watanabe, T. Samukawa, A. Sugihara, H. Noda, H. Fukuda, and Y. Tominaga, Conversion of Vegetable Oil to Biodiesel Using Immobilized Candida antarctica Lipase, J. Am. Oil Chem. SOC. 76: 789-793 (1999). 21. Shimada, Y., Y. Watanabe, A. Sugihara, and Y. Tominaga, Enzymatic Alcoholysis for Biodiesel Fuel Production and Application of the Reaction to Oil Processing, J. Mol. Catal. B: Enzym. 17: 133-142 (2002). 22. Watanabe, Y., T. Nagao, Y. Hirota, M. Kitano, and Y. Shimada, Purification of Tocopherols and Phytosterols by a Two-step in situ Enzymatic Reaction, J. Am. Oil Chem. SOC. 81: 339-345 (2004). 23. Shimada, Y., N. Sakai, A. Sugihara, H. Fujita, Y. Honda, and Y. Tominaga, Large-Scale Purification of y-Linolenic Acid by Selective Esterification Using Rhizopus delemar Lipase, J. Am. Oil Chem. SOC. 75: 1539-1544 (1998). 24. Shimada, Y., N. Fukushima, H. Fujita, Y. Honda, A. Sugihara, and Y. Tominaga, Selective Hydrolysis of Borage Oil with Candida rugosa Lipase: Two Factors Affecting the Reaction, J. Am. Oil Chem. SOC.75: 1581-1586 (1998). 25. Shimada, Y., K. Maruyama, A. Sugihara, S. Moriyama, and Y. Tominaga, Purification of Docosahexaenoic Acid from Tuna Oil by a Two-step Enzymatic Method: Hydrolysis and Selective Esterification, J. Am. Oil Chem. SOC. 74: 1441-1446 (1997). 26. Shimada, Y., T. Nagao, A. Kawashima, A. Sugihara, S. Komemushi, and Y. Torninaga, Enzymatic Purification of n-6 Polyunsaturated Fatty Acids, Kagaku to Kogyo 73: 125-130 (1999). 27. Ha, Y.L., J.M. Storkson, and M.W. Pariza, Inhibition of Benzo(a)pyrene-Induced Mouse Forestomach Neoplasia by Conjugated Dienoic Derivatives of Linoleic Acid, Cancer Res. 50: 1097-1 101 (1990). 28. Park, Y., K.J. Albright, J.M. Storkson, W. Liu, M.E. Cook, and M.W. Pariza, Change in Body Composition in Mice During Feeding and Withdrawal of Conjugated Linoleic Acid, Lipids 34: 243-248 (1999). 29. De Deckere, E.A., J.M. Van Amelsvoort, G.P. McNeill, and P. Jones, Effect of Conjugated Linoleic Acid (CLA) Isomers on Lipid Levels and Peroxisome Proliferation in the Hamster, Br. J. Nutr. 82: 309-317 (1999).
41 0
Y. Shimada
30. Nagao, K., N. Inoue, Y.-M. Wang, J. Hirata, Y. Shimada, T. Nagao, T. Matsui, and T. Yanagita, The lotrans, 12cis Isomer of Conjugated Linoleic Acid Suppresses the Development of Hypertension in Otsuka Long-Evans Tokushima Fatty Rats, Biochem. Biophys. Res. Commun. 306: 134-138 (2003). 31. Nagao, T., Y. Shimada, Y. Yamauchi-Sato, M. Kasai, K. Tsutsumi, A. Sugihara, and Y. Tominaga, Fractionation and Enrichment of Conjugated Linoleic Acid Isomers by Selective Esterification with Cadi& nigosa Lipase, J. Am. Oil Chem. SOC. 79: 303-308 (2002). 32. Nagao, T., Y. Yamauchi-Sato, A. Sugihara, T. Iwata, K. Nagao, T. Yanagita, S. Adachi, and Y. Shimada, Purification of Conjugated Linoleic Acid Isomers Through a Process Including Lipase-Catalyzed Selective Esterification, Biosci. Biotechnol. Biochem. 67: 1429-1433 (2003). 33. Shimada, Y., A. Sugihara, S. Yodono, T. Nagao, K. Maruyama, H. Nakano, S. Komemushi, and Y. Tominaga, Enrichment of Ethyl Docosahexaenoate by Selective Alcoholysis with Immobilized Rhizopus delamar Lipase, J. Ferment. Bioeng. 84: 138-143 (1997). 34. Shimada, Y., K. Maruyama, A. Sugihara, T. Baba, S. Komemushi, and Y. Tominaga, Purification of Ethyl Docosahexaenoate by Selective Alcoholysis of Fatty Acid Ethyl Esters with Immobilized Rhizomucor miehei Lipase, J. Am. Oil Chem. SOC. 75: 1565-1572 (1998). 35. Maruyama, K., Y. Shimada, T. Baba, T. Ooguri, A. Sugihara, Y. Tominaga, and S. Moriyama, Purification of Ethyl Docosahexaenoate Through Selective Alcoholysis with Immobilized Rhizomucor miehei Lipase, J. Jpn. Oil Chem. SOC. 49: 793-799 (2000). 36. Hirota, Y., T. Nagao, Y. Watanabe, M. Suenaga, S. Nakai, M. Kitano, A. Sugihara, and Y. Shimada, Purification of Steryl Esters from Soybean Oil Deodorizer Distillate, J. Am. Oil Chem. SOC.80: 341-346 (2003). 37. Matsuno, T., Xanthophylls as Precursors of Retinoids, Pure Appl. Chem. 63: 81-88 (1991). 38. Naguib, Y.M.A., Antioxidant Activities of Astaxanthin and Related Carotenoids, J. Agric. Food Chem. 48: 1150-1 154 (2000). 39. Jyonouchi, H., S. Sun, K. Iijima, and M.D. Gross, Antitumor Activity of Astaxanthin and Its Mode of Action, Nutr. Cancer 36: 59-65 (2000). 40. Jyonouchi, H., L. Zhang, and Y. Tomita, Studies of Immunomodulating Actions of Carotenoids. 11. Astaxanthin Enhances In Vivo Antibody Production to T-Dependent Antigens Without Facilitating Polyclonal B-Cell Activation, Nutr. Cancer 19: 269-280 (1993).
Chapter 18
Enzymatic Synthesis of Symmetrical Triacylglycerols Containing Polyunsaturated Fatty Acids Tsuneo Yamane Laboratory of Molecular Biotechnology, Graduate School of Bio- and Agro-Sciences, Nagoya University, Japan
Introduction Symmetrical triacylglycerols (STAG) are defined in the broadest sense as any triacylglycerols (TAG) that have the same type of FA at the sn-l and sn-3 positions and another type at the sn-2 position of the glycerol backbone. They are generally abbreviated as ABA in this chapter, i.e., FA are shown in the order of their positions located at sn-I,sn-2,and sn-3 of the glycerol backbone. Thus AAB is not identical to BAA, but they are enantiomers. This nomenclature is applied throughout this review. Natural edible fats and oils are simply mixtures of a number of TAG that differ in terms of both FA species and their distribution along the glycerol backbone. In contrast to natural edible lipids, sTAG are TAG that are modified either chemically or enzymatically in both the type of FA and/or the position of the FA. A number of studies were carried out for the synthesis of sTAG having (medium chain)-(long chain)-(medium chain)-type FA (MLM) (1). These sTAG are claimed to provide less metabolizable energy per gram than do traditional fats and oils, and to be efficient food sources for patients with pancreatic insufficiency and other forms of malabsorption. Cocoa-butter substitutes, which consist predominantly of stearoyl-oleoylstearoylglycerol (SOS) or more generally SFA-unsaturated FA-SFA type TAG (SUS), and “Betapol” manufactured by Unilever, which has the structure of oleoyl-palmitoyl-oleoylglycerol(OPO), are included in this category of STAG. sTAG containing PUFA such as EPA, DHA, and arachidonic acid (ARA) have received much attention because of their various pharmacologic effects. These include beneficial effects on cardiovascular diseases, immune disorders and inflammation, renal disorders, allergies, diabetes, and cancer ( 2 4 ) . These FA may also be essential for brain and retina development in humans. Among sTAG containing PUFA, sTAG containing one molecule of PUFA and two molecules of medium-chain fatty acids (MCFA) are noteworthy. Several studies were performed for the synthesis of sTAG containing PUFA at specific sites of the glycerol backbone (5-8). The absorption of PUFA into the body depends upon the position of PUFA along the glycerol backbone, i.e., at the sn-1 41 1
T. Yarnane
41 2
(or 3) or sn-2 position (9). sTAG containing PUFA at the sn-2 position and MCFA at the sn-1 and -3 positions can be hydrolyzed into 2-monoacylglycerol (2-MAG) containing PUFA and FA by pancreatic lipase and are efficiently absorbed into intestinal mucosa cells in normal adults. It is to be noted that mammalian pancreatic lipases hydrolyze the ester linkages at the sn-1 and sn-3 positions with a preference for MCFA over long-chain FA (10,ll). Therefore, as a dietary supplement for adults, sTAG containing PUFA at the sn-2 position and MCFA at the sn-1 and sn-3 positions may be suitable. sTAG can be synthesized either chemically or enzymatically. However, an enzymatic synthesis of sTAG is advantageous compared with a chemical process for several reasons. Enzymes are generally specific, giving rise to few or no byproducts, whereas chemical processes require that the groups that are not targets of the reaction be protected and then that the protection removed after the reaction. Enzymes exhibit catalytic action under mild conditions. Enzymatic reactions have an important advantage for the synthesis of sTAG containing PUFA because PUFA are very unstable. They are easily isomerized, oxidized, and polymerized. These properties necessitate using as mild conditions as possible, especially oxygenfree conditions. In this chapter, on the basis of these perspectives, recent advances on the lipase-catalyzed synthesis of sTAG containing PUFA will be summarized, with a special emphasis on both monitoring the reaction and reaction strategies proposed recently to increase the yield (12-21). Several reviews have been published concerning enzymatic synthesis of structured TAG involving sTAG (22-24).
Monitoring the Reaction For the production of a targeted STAG, it is essential to know which types of TAG are formed and how many FA are incorporated at a specific hydroxyl position of glycerol. When the intention is to synthesize a diacid sTAG containing PUFA, i.e., ABA where A = MCFA and B = PUFA, the ABA and its isomers, AAB (and/or BAA) type sTAG must be separated and determined by a suitable analytical method. Moreover, when the intention is to produce pure C,(EPA)C, by lipase-catalyzed transesterification between trieicosapentaenoylglycerol [(EPA)(EPA)(EPA)] and octanoic acid ethyl ester (C,Et) by the following equation (EPA)(EPA)(EPA) + C,Et
+
C,(EPA)C,
+ (EPA)Et
a number of chemical species may appear during the reaction, including the two substrates, the targeted STAG, its positional isomers [(EPA)C,C, and/or C,C,(EPA)], and the inevitable by-product (EPA)Et. TAG containing 2 mol EPA may occur by imperfect replacement of (EPA)(EPA)(EPA), and hydrolytic byproducts may appear from any esters. The first row of Table 18.1 lists almost all of the possible lipid species that may appear in the course of the reaction according to
TABLE 18.1 Peak Identificationfor the Transesterification Reaction Between EPAEt and C,C,C, Peak no.
Chemical species
Possible isomers
by Silver-Ion HPLCa Molecular weight
Number of double bonds
5 6
Trioctanoylglycerol, octanoic acid ethyl ester Octanoic acid, dioctanoylglycerol 1,3-Dioctanoyl-2-eicosapentaenoylglycerol (TAG-A3) 2,3-Dioaanoyl-leicosapentaenoylglycerol (TAG-AI) 1,2-Dioctanoyl-3-cicosapentaenoylglycerol (TAG-A2) Eicosapentaenoicacid ethyl ester (EPAEt) Eicosapentaenoyloctanoyldiacylglycerol (DAC-A)
331 503
5
1 2 3 4
0
629 629
0 5 5
5
7
Eicosapentaenoicacid (EPA)
303
8
Dieicosapentaenoyloctanoyl triacylglycerol (TAG-B)
787
5 10
9
Dieicosapentaenoyl diacylglycerol (DAG-B)
661
10
10
Trieicosapentaenoylglycerol (TAG-C) . - .
945
15
TAG-A = TAG-At + TAG-A2
+ TAGA3.
41 4
T. Yarnane
Equation 1. It is highly preferable to detect these species as well as the targeted STAG. However, separation of more than 11 chemical species in one analysis is not an easy task and requires advanced analytical techniques. Several methods were reported for the determination of the positional distribution of acyl groups in TAG, including enzymatic hydrolysis (25,26), chemical degradation using Grignard reagent (27,28), followed by analysis of MAG and DAG products by chromatographic techniques, 13C NMR (29,30), and silver-ion liquid chromatography (31). However, none of the methods tells us what types of molecular species of TAG are present. Unless each molecular species is separated, only the positional distributions of FA are determined. To know the composition of a sample and its positional distribution of PUFA, the sample must be separated and purified into each molecular species using a chromatographic technique, after which the purified species must be analyzed by enzymatic, chemical, or NMR methods. Accordingly, an easy, simple, and accurate method is required to determine simultaneously both the molecular species composition of TAG and the positional distribution of FA of the components. Our group found that silver-ion liquid chromatography best met these requirements. Silver-ion chromatography is a technique that utilizes the property of silver ions to form reversible polar complexes with double bonds in organic molecules such as unsaturated lipids. This technique enables the separation of unsaturated species according to the number, geometric configuration, and position of the double bond. Some researchers demonstrated the separation of positional isomers of TAG that contain FA having the same number of double bonds (31-35). However, no separation of isomeric TAG containing PUFA was reported. Our group succeeded in separating C,(EPA)C, and (EPA)C,C, [andor C,C,(EPA)] by using 2-propanol as a modifier in a hexane-acetonitrile-based mobile phase for silver-ion HPLC (13,21). In silver-ion HPLC with spectrophotometric detection at 206 nm, a hexaneacetonitrile-based mobile phase is generally used. However, the mobile phase has a solubility limit of acetonitrile in hexane (35). 2-Propanol can serve as a third solvent ensuring good solubility of acetonitrile in hexane. Figure 18.1A and B show the silver-ion HPLC charts of reaction mixtures produced by a 1,3-specific lipase (Rhizomucor rniehei lipase, LipozymeTMRM-IM) and a nonspecific lipase (Pseudomonas pseudoalkali lipase, LiposamTM),respectively, from the interesterification between trioctanoylglycerol(C8C8C8)and (EPA)Et after a 12-h reaction: (EPA)Et + C8C,C,
--+
(EPA)C,C,
+ C,Et
PI
A solvent gradient program was applied (for details see Ref. 12), which allowed the separation of TAG containing EPA. This depends on the number of EPA molecules incorporated into the glycerol backbone and their isomeric distribution. The molecular weight of each peak was determined by HPLC followed by an atmospheric-pressure chemical ionization-MS (APCIMS) assay (as shown in Table 18.1), together with their peak number. The compounds that eluted as peaks 3 and 4 had the same molecular mass; therefore, the two compounds were isomers of
41 5
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
TAG-A. This identification was further confirmed by the observation of fragment ions analyzed by HPLC-APCWS, as shown in Figure 18.1. The identification of peaks 3 and 4 as TAG-A3 and TAG-A1 (andor TAG-A2) (see Table 18.1), respectively, was made on the regioselectivities of the enzymes used. Only peak 4
A
6
I
I
I 8
4
I
8
mln
Fig. 18.1. Silver-ion HPLC chromatogram of the 12-h reaction mixture by two different lipases (12). The reaction was [2]. (A) Reaction mixture with a 1,3-specific lipase (Rhizomucor miehei lipase); and (B) reaction mixture with a nonspecific lipase (Pseudomoms pseudoalkali lipase). The peak numbers are identical to those shown in Table 18.1, Molecular ions and fragmentation of peaks 3 and 4 analyzed by HPLCAPCIIMS are also shown.
T. Yamane
41 6
appeared in the reaction using the 1,3-specific lipase, whereas peaks 3 and 4 were observed when the nonspecific lipase was used. Thus, it was concluded that peak 4 corresponded to TAG-A (TAG-A1 and/or TAG-A2) containing EPA at the sn-1 (or 3)-position, and peak 3 shows the sn-2 positional isomer (TAG-A3). The gradient of hexane/2-propano~acetonitrileas the mobile phase was also successfully applied to monitor the transesterification reaction between the DHA ethyl ester, (DHA)Et, and c&& (Equation3) (as shown in Fig. 18.2), although the acetonitrile content of solvent A and the mobile phase gradient program were slightly different (13). (DHAEt) + C,C,C, + (DHA)C,C,
+ C,Et
[31
Again, only peak 4 appeared with the 1,3-specific lipase and both peaks 3 and 4 were obtained with nonspecific lipase. The identification of each peak is given in Table 18.2. The following points should be noted with regard to the silver-ion HPLC for Equations 1-3: (i) Separation of positional isomers is critically affected by the acetonitrile content of solvent A so that it must be optimized for each pair of TAG. (ii) One isomer with an unsaturated FA at sn-2 position elutes faster than the other with unsaturated FA at the sn-1 or sn-3 position. (iii) (DHA)C,C, elutes later than (EPA)C,C,. (iv) The stereoisomers, TAG-A1 and TAG-A2 (see Table 18.1), and (DHA)C,C, and C,C,(DHA) (Table 18.2) cannot be separated. The positional isomers, TAG-B (Table 18. l ) , and [(DHA)C,(DHA), (DHA)(DHA)C, and C,(DHA)(DHA)] (Table 18.2), also cannot be separated. Silver-ion HPLC proved to be useful in the analysis of the TAG positional composition of more complicated mixtures. The lipase-catalyzed acidolysis of a single-cell oil of high DHA (and DPA) content with octanoic acid (C,) was also investigated (20) according to Equation 4: SCO + C,
+
TAG containing 1 DHA (and DPA) and 2 (2, + Others
[41
The FA composition of SCO was 4.2 mol% myristic acid, 2.5 mol% pentadecanoic acid, 46.3 mol% palmitic acid, 1.3 mol% stearic acid, 10.2 mol% DPA, and 35.5 mol% DHA. The TAG fraction of the reaction mixture was subjected to silver-ion HPLC. In the spectrophotometric detection at 206 nm, the sensitivity of each TAG species depends mainly on its double-bond number. Therefore, the detector shows very weak responses for all of the saturated TAG species. The detector can only estimate the ratio of the desired TAG to their positional isomers that contain the same number of double bonds because the detector’s response is assumed to have the same value for each isomer. The ratio of the positional isomers can be estimated from the corresponding peak areas. Most major peaks (15-17 in number) could be identified. The desired STAG, C,(DHA)C, and C,(DPA)C,, could be separated from their positional isomers, C,C,(DHA) [and (DHA)C,C,] and C,C,(DPA) [and (DPA)C,C,], respectively.
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
A
41 7
5
0)
fn C
0
n
fn
655.4 0)
fn C
op
t
B
d
l
rn
I
min
a
Fig. 18.2. Silver-ion HPLC chromatogram of transesterification shown by reaction [3] (13). (A) Reaction mixture with a 1,3-specific lipase (R. miehei lipase); and (B) reaction mixture with a nonspecific lipase (Ps. pseudoalkali lipase). Peak identification is given in Table 18.2. Mass spectra of the isomers of dioctanoyldocosahexaenoylglycerolare also shown. See Figure 18.1 for abbreviations.
For analyzing the reaction mixture of STAG synthesis by high-temperature gas chromatography (HTGC), precautions should be taken in light of the instability of PUFA at elevated temperatures. On-column injection (36) is the best technique for transferring a sample containing PUFA onto a capillary column to avoid deterioration
P
m
TABLE 18.2 Peak Identificationfor the Transesterification Reaction Between DHAEt and C8C,C, by Silver-Ion HPLC Peak no.
Chemical species Trioctanoylglycerol, octanoic acid ethyl ester Octanoic acid, dioctanoylglycerol 1,3-Dioctanoyl-2-docosahexaenoylglycerol 2,3-Dioctanoyl-l-docosahexaenoylglycerol
Possible isomers
Molecular weight
Number of double bonds
655 655
0 0 6 6
1,2-Dioctanoyl-3-docosahexaenoylglycerol
3
2
3
m
5 6
Docosahexaenoic acid ethyl ester (DHAEt) Docosahexaenoyloctanoyldiacylglycerol
357 503
6 6
7 8
Docosahexaenoic acid (DHA) Didocosahexaenoyloaanoyltriacylglycerol
329 839
6 12
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
41 9
of the sample before entering the column. Careful programming of the column temperature is required to achieve a good peak separation. For quantitative analysis, n-eicosane can be used as an internal standard. Figure 18.3 A shows a HTGC chromatogram of the reaction mixture shown in Equation 1 using a regiospecific lipase (R. miehei lipase, Lipozyme'" RM-IM) (16). By HTGC analysis, changes in the content of TAG-A (the sum of TAG-A1 + TAG-A2 + TAG-A3) during the time course of the reaction could be monitored conveniently. Figure 18.3B shows the result of silver-ion HPLC of the TAG-A fraction, indicating a negligible amount of (TAG-A1 + TAGA2). In HTGC analysis of a TAG fraction obtained from the reaction [4],TAG species are separated depending on their carbon numbers, and the composition of TAG species can then be calculated from the peak areas (20).
Strategy for Enzymatic Synthesis of STAG As is the case with most enzymatic reactions, the performance of enzymatic synthesis of STAG containing PUFA depends on many factors involving the type of 1
?
3
7
Fig. 18.3. HTGC chro-
0
5
J
matogram of the reaction mixture from reaction scheme [ l ] (12). (A) Peak 1, solvent; 2, C,Et; 3, C;, 4, (EPA)Et; 5, EPA; 6, DAG; 7, TAG-A; 8, TAG-B. (B) Silver-ion HPLC of TAG-A fraction (peak 7). Peak 1, C,(EPA)C,; 2 (supposed peak position), C,C,(EPA) and/or (EPA) CC , ., Panel B confirms that isomerically
8 7-
1'0 i5 20 Retention time (min)
25
pure TAGA3, C,(EPA)C,, was synthesized.
42 0
T. Yamane
reaction, the enzyme and its immobilization, temperature, water content, composition of substrates, physical properties of the substrates, reaction time, and mode of operation. Some of these factors are briefly mentioned, and examples of reactions yielding sTAG that contain PUFA are introduced below. Choice of Enzyme and Its Immobilization
Enzymes involved in STAG synthesis are exclusively lipases. Careful exploitation of positional (regional) specificity, FA specificity, and stereospecificity of lipases can provide a maximum yield of the desired STAG.To date, most researchers utilized fungal lipases such as those from R. miehei, R. delemar, R. javanicus, and R. niveus, which are 1,3-specific and hence are effective in synthesizing STAG.For nonregiospecific lipases, a number of microbial lipases are commercially available such as those from Candida sp. (C. antarctica types B, and C . rugosa) and those from Pseudomonas sp. (Ps.fragi, Ps. cepacia, Ps. glumae, and Ps,juorescens).Some lipases also exhibit stereospecificity, e.g., R. miehei and Carica papaya latex lipases were claimed to show sn- 1 and sn-3 preferences, respectively, in interesterification reactions (37,38) although they are not absolute. Immobilization of lipases provides some benefits, including increasing stability and easy recovery and reuse of the enzyme, thereby reducing the production cost. R. miehei, C. antarctica, and Pseudomonas sp. lipases are commercially available in immobilized forms. Chandler et al. (38) prepared immobilized lipase using macroporous polypropylene particles, and Shimada et al. (6,7,39) immobilized Rhizopus delemar lipase onto porous ceramic particles for their studies on enzymatic sTAG production. Our group reported the effective immobilization of fungal and bacterial lipases on fine CaCO, powder (4042). CaCO, powder is used commercially as a food additive, making it a very inexpensive and safe material. The enzymes were effectively immobilized by physical adsorption, which is a straightforward method of immobilization. Due to tight adsorption, leakage of the enzymes was negligible in the neat liquid organic substrate, rendering it completely free from contamination of the protein in the product. The immobilized lipases could be used many times (40). Solvent-Free Systems
Although it was not explicitly recognized since the emergence of “nonaqueous enzymology,” all enzymatic reactions in organic media have been classified into two systems, i.e., solvent systems and solvent-free systems. In the former, the substrate(s) is dissolved in an inert liquid organic solvent. The solvent does not participate in the reaction in any way, but creates an environment in which the dissolved substrate(s) is consumed by the enzymatic action. On the other hand, in the latter system, no organic compounds (except enzyme or immobilized enzyme) other than the substrate(s) exist in a bioreactor. In other words, the bioreactor is occupied with substrate(s) only. In some cases, the reaction system is composed of two or more substrates, one of which exists in great excess (much higher than the stoi-
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
42 1
chiometric molar ratio). In such a case, the excess substrate also works as a bulk solvent for the second substrate. This case is sometimes called reaction-in-neat. There are a number of advantages to solvent-free systems over solvent systems if they work successfully, including very high volumetric productivity, avoidance of enzyme inactivation by the solvent, and preference for safety in the food industry. In addition, the solvent-free system offers a better factory environment; it does not necessitate any explosion-free equipment, and the absence of the solvent is highly desirable for the health of workers engaged in bioprocessing. One of the possible disadvantages in using the solvent-free system, even when it is feasible, is longer reaction times compared with the solvent process, and the enzyme may become inactivated due to the longer duration of the reaction. It is to be noted, however, that a longer reaction time is quite reasonable if one considers the fact that in the solvent-free system, greater absolute amounts of substrate(s) exist in the bioreactor volume than in the solvent system. Volumetric productivity [(kg product formed)/ (L reactor volume-h)] of the solvent-free system may be higher than that of the solvent system if they are compared on the basis of the same volume of the reaction mixture and the same amount of enzyme used. The solvent-free system can be implemented not only in a monophasic system but also in a biphasic system, as shown by our group (15,4042). Public acceptance of the advantages of solventfree biotransformations might also bring a shift from early R&D experiments using organic solvents to industrial implementation of solvent-free bioprocesses. This also holds true for the production of sTAG containing PUFA (7,39). All experiments involving lipase-catalyzed STAG formation described by our group were performed in solvent-free systems (12-21). Increasing the Yield in a Microaqueous System
A reaction scheme of the lipase-catalyzed synthesis of sTAG containing PUFA can be generally formulated as shown in Equations 5 and 6:
S-S’ + S”
STAG + S ’
151
S’ + S” c= sTAG + H,O Equation 5 is a transesterification, which is further subdivided into acidolysis, alcoholysis, and interesterification depending on the acid, alcohol, or ester serving as S ” , respectively (43). Equation 6 represents an esterification between an acid and an alcohol, liberating H,O. There are several strategies that may be successfully applied to reactions [5] and [6] to increase the yield of STAG. Substrate Ratio. In Equations 5 and 6, one of the substrates is a PUFA or a PUFA derivative (e.g., ethyl ester). Pure PUFA are quite expensive; thus, they should be the “limiting substrate” to achieve a total conversion of the substrate, and an excess
42 2
T. Yarnane
molar amount of the other substrate often results in good yields due to a favored equilibrium. Thermodynamic Sbift. Because both reactions in Equations 5 and 6 are reversible, the yield of the targeted sTAG increases as the by-products S’ or H,O are removed from the reaction mixture, by further shifting of the equilibrium (“thermodynamic shift”). The principle is straightforward and can always be applied to any (bio-)chemical reaction. Methods to achieve this include winterization (44), when the solubility of S’ becomes low at reduced temperature, and N, gas bubbling or vacuum (reduced pressure), when S’ is volatile or has a low boiling point (e.g., ethanol in Equation 5 ) or when H,O (Equation 6) is removed by dehydration using activated molecular sieves. Tautomerization of vinyl alcohol is another way of eliminating S‘ from the reaction system (45). When an industrialscale production is considered, a combination of a packed-bed reactor, a substrate reservoir, and a vacuum apparatus may be more realistic than a large stirred tank reactor that is operated under reduced pressure (46). side Reactions fHydro@is andAcy/ Migrafion). Notable side reactions that are concomitant with Equation 1 are hydrolysis and acyl migration. The former depends naturally on the water content. Excess amounts of water always decrease the final yield of the desired product due to hydrolysis of the desired ester. Acyl migration, which is confirmed by the formation of 13-DAG, may (more or less) not be inevitable. This depends on a number of factors such as water content, reaction temperature, enzyme load, reaction time, and substrate ratio (47,48). Xu et al. (49,50) studied the lipase-catalyzed interesterifications of fish oil with octanoic acid, and of medium-chain TAG with sunflower oil, in a solvent-free system in pilot batch and continuous operations. In a pilot-batch production, 0.22-1.37%/h acyl migration occurred in the former operation, whereas in the latter reaction, acyl migration was further reduced fourfold at a similar extent of incorporation. Water Content In applying the effect of the thermodynamic shift, however, one must take special precautions against the presence of trace amounts of H,O in the reaction mixture (43,5 1,52). In a microaqueous solvent-free system, water plays two roles, i.e., water is necessary to maintain the catalytic activity of the lipase and it promotes hydrolysis, an unfavorable side reaction (Equation 5). The water level in the reaction system critically affects the performance of enzymatic reactions in organic media. Water has a profound influence on both the yield and the rate of reactions. Essential water or bound water that is actually in equilibrium with water in the bulk solution must be retained to maintain both the activity and stability of the enzyme molecule. However, an excess amount of water always reduces the final yield of the targeted sTAG due to hydrolysis that results in the formation of by-product(s), When dry N, gas bubbling or vacuum is applied for removing S’ (Equation 5 ) , it may also remove water that is essential for the enzyme’s catalytic
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
42 3
activity. As depicted in Equation 6, the liberated water should be removed, but essential water should be retained. The effect of water on the reaction performance is usually controlled by adjusting the thermodynamic water activity (a,) of the reaction components and the enzyme. The crucial role of trace amounts of water in solvent-free biotransformations makes it a major factor in “microaqueous organic media” systems (43,51,52).
Enzymatic Synthesis of Pure STAG Containing PUFA Pure sTAG can be produced by several methods. Bornscheuer et al. (53,54) proposed a two-step reaction in solvent system as shown in Equations 7 and 8. TAG + Alcohol 2-MAG + FA
-
-
2-MAG + FA Esters
sTAG + H,O
[71
181
The first step (Equation 7) is an alcoholysis (usually ethanolysis) of TAG (triolein, trilinolein, peanut oil, or fish oil) in an organic solvent (e.g., methyl-t-butyl ether or acetone) using a 13-regiospecific lipase. 2-MAG was obtained in up to 80-90% yield at >95% purity by crystallization.The second step (Equation 8) is an esterification of these 2-MAG, in hexane, again with a 1,3-regiospecificlipase, which gave almost pure STAG.For instance, Betapol (OPO) was obtained in >78% yield and 96% purity. Our group reported a chemoenzymatic synthesis of C,(EPA)C, ( 1 3 , applying the organic catalysts, 1,l ’-N-dicyclohexylcarbodiimideand 4-dimethylaminopyridine, for the esterification step in chloroform. The yield after purification by silica gel column chromatography was 42%, and the purity of TAG was 98%, of which 90% was C,(EPA)C,. Thus the chemoenzymatic process was unsatisfactory. Our group then developed a novel three-step enzymatic process that appeared more promising as shown in Equations 9-1 1 (16): (EPA)Et + H,O
-+
Glycerol + 3EPA
EPA + EtOH -+
[91
(EPA)(EPA)(EPA) + H,O
(EPA)(EPA)(EPA) + C,Et
-+
C,(EPA)C,
+ 2(EPA)Et
1101 1111
The starting substrate, (EPA)Et, is currently being sold as a medicine so that a pure substance is available industrially on a large scale. The first step is a hydrolysis of (EPA)Et to give rise to free EPA. The second step (Equation 10) is an esterification in a solvent-free system with nonregiospecific lipase. When immobilized, C. antarctica lipase was used under optimal conditions at appropriately reduced pressure, >90% yield of the targeted product was achieved from stoichiometricratios of the substrates. Although (EPA)(EPA)(EPA)may be produced from a one-step reaction
424
T. Yarnane
Glycerol + 3(EPA)Et -+ (EPA)(EPA)(EPA) + EtOH free EPA is a better acyl donor than (EPA)Et for the enzymatic esterification (55); therefore, hydrolysis of (EPA)Et (the first step [ 9 ] ) is required. Hydrolysis was performed with C. antarctica lipase, which was also used for (EPA)(EPA)(EPA) synthesis. The two steps, 9 and 10, were performed in the same flask without any separation. The reaction mixture from the step [lo] was then subjected to the third step (Equation 11) without any purification after separation of the immobilized enzyme. The third step [ 111 is an interesterification in a solvent-free system with 1,3regiospecific lipase ( R . miehei lipase). When an excess molar ratio of C,Et to (EPA)(EPA)(EPA) (1OO:l) was used, -88% of the overall yield of the targeted product was obtained (Fig. 18.4). The unreacted C,Et and the by-products [free C,, (EPA)Et, and free EPA] can be removed fractionally by short-pass distillation from the acylglycerol fraction, which contains >90% C,(EPA)C,. The regioisomeric purity of the product was 100% by silver-ion HPLC (see Fig. 18.3B). It is to be noted that although only 1 of the 3 mol of EPA was incorporated into the glycerol backbone in a single cycle of the reaction, the 2 mol remaining can be reused in the first step [9] of the next cycle of the reaction so that all of the (EPA)Et is eventually converted to the desired STAG. The advantages of this process are that no organic solvent is used, isolation and purification of the intermediates are not necessary, and the liberated (EPA)Et (and small amounts of free EPA) and remaining C,Et are reusable. When free C, was used (acidolysis), instead of C,Et at the final step, the reaction rate was slower than C,Et, i.e., interesterification had clear advantages over acidolysis (17). However, because C,Et is 10 times more expensive than C,, C, is favored for economic reasons. This problem was overcome by enzymatically synthesizing C,Et from c, and ethanol in a separate reaction, and using it for the final reaction step [ 111. The general scheme for the enzymatic synthesis of pure sTAG includes preparation of monoacid TG, having the same acyl groups at all of the positions of the glycerol backbone, such as (EPA)(EPA)(EPA), and then replacement of FA residues specifically at the 1,3-positions leaving the one at the 2nd position unchanged. However, this strategy cannot be applied for the synthesis of sTAG with a DHA residue at the 2nd position, because the DHA residue is resistant to fungal lipase. To solve this problem, our group applied reactions [7] and [8], but in a solvent-free system using the immobilized C. antarctica lipase. It was believed to act on DHA residues of TG in a position-nonspecific manner. However, when subjected to ethanolysis of (DHA)(DHA)(DHA) in ethanol, it reacted only at the 1,3-position, giving (OH)(DHA)(OH) and (DHA)Et (18). Irimescu et al. (56) further examined ethanolysis of monoacid TAG to optimize the reaction. The 2-MAG yield increased to nearly 100% when the molar ratio of ethanol/monoacid TAG was large (-80%) at ambient temperature. (DHA)(DHA)(DHA) was then converted to
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
+
425
normal pressure
100
8
h
E z C
75
0, c
C
-8
$0
50
-
> -w
2
.-
25
0
Time (h)
.,
Fig. 18.4. Time course of reaction [ I ] with a 1,3-specific lipase, R. miehei lipase (16). Molar ratio of (EPA)(EPA)(EPA)/C,Et was 1:lOO. The reaction was performed with 2% initial water content at 40°C at normal pressure for 10 h, followed by 3 h at 3 mmHg. TAG content [O, C,(EPA)C,; (EPA)(EPA)(EPA);0,C,(EPA)(EPA) and/or (EPA)(EPA)C,; and 0, C,C,C,]. DAG content [A, C,C,OH; A, C,(EPA)OH; and x, (EPA)(EPA)OHl. Note that no MAG were detected. See Figure 18.1 for abbreviation.
426
T. Yamane
(OH)(DHA)(OH), which was then reesterified with C,Et by immobilized R . miehei lipase to afford C,(DHA)C, (Fig. 18.5) (1836). When (EPA) (EPA)(EPA) was the starting TAG, the total time required to obtain C,(EPA)C, was much shorter for the ethanolysis route than reaction [ 111 making the former preferable. Our group recently studied the effect of the water content on the ethanolysis of trioleoylglycerol; we tested four different lipases and found that C. antarctica lipase B was anomalously active even under ultramicroaqueous condition (57) (Fig. 18.6). This characteristic contributes greatly to the effectiveness of its ethanolysis of TAG because there is no need to optimize the water content.
Enzymatic Synthesis of STAGContaining PUFA from Natural Oils To use STAG as nutraceutical fats and oils on a large scale, their production from natural fats and oils may be the primary choice. The following reactions [13] or
100
80
60
40
20
0
Reaction time (h)
Reaction time (h)
Fig. 18.5. Time course of C,(DHA)C, synthesis from (DHA)(DHA)(DHA)by the two-step process via 2-MAG in a nonsolvent system (1 8). Immobilized Candida antarctica lipase B was used in reaction scheme [7] [weight ratio of ethanol/(DHA)(DHA)(DHA)= 31, followed by reaction scheme [8] [molar ratio of C,Et/initial (DHA)(DHA)(DHA)= 201 using
immobilized R. miehei lipase. I, (DHA)(DHA)(DHA);0, (OH)(DHA)(OH);0,DAG; 0, A,C,C,(OH); V, (DHA)(DHA)C,; A,C,(DHA)(OH); and V,CC ,C , .,
C,(DHA)C,;
427
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
1000-
I
I
0
I
I
I
800-
600-
400-
200-
0.1 1 10 Free water content in the reaction mixture (wto/o) Fig. 18.6. Initial formation rate of 2-monooleoylglycerol(2-MO)by the immobilized lipases in ethanolysis of trioleoylglycerol at different water contents (57). OI C. antarctica lipase; W, R. miehei lipase; A,Burkholderia cepacia lipase; V, Therrnomyces lanuginosus lipase.
[ 141 show typical synthetic schemes for MLM-type STAG synthesis from natural fats and oils:
Natural Fat + MCFA
-
MLM + free FA
Natural Fat + (MCFA)Et -,MLM + FAEt
[I31 ~ 4 1
The method comprises an acyl exchange of oils with an excess of MCFA (for acidolysis, reaction [ 131) or its ethyl ester (MCFA)Et (for interesterification, reaction [14]). The strategy is to substitute the FA residues specifically at the sn-1 and -3 positions of the oils with the desired ones by a 1,3-specific lipase (especially of fungal origin such as R . miehei and Rhizopus delemar), keeping the FA residues at the sn-2-position unchanged. Intensive studies of acidolysis reactions were made by Shimada et al. (6,7,39). Rhizopus delemar lipase immobilized on a ceramic carrier was employed for acidolysis of various oils with C,. Hydrolysis of the substrate is a side reaction that should be minimized. They found that the enzyme, which was used first in the
42 8
T. Yarnane
presence of a certain amount of water for activation, after recovery and reuse, did not hydrolyze TAG further in the subsequent reactions. With this “activated enzyme,” modification of oils containing DHA (6), y-linoleic acid (7), and ARA, EPA, and DHA (39) was successfully achieved without the formation of partial acylglycerols. In addition, to enhance the incorporation of C,, the reaction was repeated for three cycles (7). After each cycle, the TAG fraction was recovered and reacted with fresh C, in the subsequent cycle. Consequently, the FA residues at the sn-1 and -3 positions could be replaced completely, whereas those at the sn-2 position remained unchanged. The final products were quite pure with respect to the heterogeneity of the constitutive molecular species (i.e., comprising only a few TAG species). Thus, depending on the choice of the oils as starting material, various kinds of MLM-type STAG can be obtained by 1,3-specific-lipase-catalyzedacidolysis. In spite of the versatility of the lipase-catalyzed acidolysis, DHA-containing oils such as tuna oil are exceptional (6). Because fungal lipases scarcely act on
100
80
60
40
20
0 0.0
0.5
1.0
Reaction time (h) Fig. 18.7. C,(DHA)C,-rich
1.5
2.0 0.0
0.5 1.0 1.5 Reaction time (h)
oil production from bonito oil via 2-MAG (19). First, ethanolysis was carried out with the initial weight of ethanolhonito oil = 3 by C.antarctica lipase B. Then reesterification was performed with the initial weight ratio of C8Et/bonito oil = 3 by R. miehei lipase under vacuum. Temperature was 35°C throughout. W, bonito oil; 0, (OH)(DHA)(OH);0,DAG; 0, C38 [mainly C,(DHA)C,l; A,other TAG; A,C36 [mainly C,(EPA)C,I; and V,c&&. See Figures 18.1 and 18.5 for abbreviations.
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
429
DHA residues of TAG (39), the DHA residues at the sn-1 and 3-positions of the starting material remained. Our group also pointed out a similar problem in acidolysis of single-cell oil (so-called DHA oil) rich in DHA and DPA both of which were resistant to the common fungal lipases (reaction [3]) (20). When immobilized R . miehei lipase was used, the degree of acidolysis was very low (only 23%), leaving a large amount of DHA and DPA residues unexchanged. Our group then reported an alternative strategy for synthesis of DHA-containing STAG (19). The new method includes 1,3-position-specific ethanolysis of DHA-containing TAG by immobilized C. antarctica lipase B to form DHA-containing 2-MAG, followed by reesterification of 2-MG by R . miehei lipase. Thus, the serial reaction was the same as reactions [7] and [8], but without any organic solvent. This strategy enabled the effective preparation of DHA-containing MLMtype STAG from bonito oil (Fig. 18.7) (19),
Summary Because suitable reaction systems using efficient immobilized lipases commercially available were found and optimized, it is now possible to produce a high yield of pure STAG containing PUFA at the sn-2 position and MCFA at the sn-1 and -3 positions without using any organic solvent. Pure C,(EPA)C, and C,(DHA)C, can be produced first by ethanolysis using immobilized C. anturctica lipase B of the corresponding monoacid TAG to yield the corresponding 2-MAG, followed by its reesterification with C,Et by R. miehei lipase. The same strategy can be applied to produce STAG-rich oil from natural edible fats and oils that contain PUFA, i.e., a C,(PUFA)C,-nch oil can be produced from a PUFA-rich oil via the 2-MAG/reesterificationroute. References 1. Christophe, A.B., ed., Structural ModiJied Food Fats: Synthesis, Biochemistly, and Use, AOCS Press, Champaign, IL, 1998. 2. Akoh, C.C., Structured Lipids-Enzymatic Approach, INFORM 6: 1055-1061 (1995). 3. Gill, I., and R. Valivety, Polyunsaturated Fatty Acids, Part 1: Occurrence, Biological Activities and Applications, Trends Biotechnol, 15: 401409 (1997). 4. Gill, I., and R. Valivety, Polyunsaturated Fatty Acids, Part 1: Occurrence, Biological Activities and Applications, Trends Biotechnoll5: 470-477 (1997). 5 . Lee, K.-T., and C.C. Akoh, Characterization of Enzymatically Synthesized Structured Lipids Containing Eicosapentaenoic, Docosahexaenoic, and Caprylic Acids, J . Am. Oil Chem. SOC.75: 495-499 (1998). 6. Shimada, Y., A. Sugihara, K. Maruyama, T. Nagao, S. Nakayama, H. Nakano, and Y. Tominaga, Production of Structured Lipid Containing Docosahexaenoic Acid and Caprylic Acids Using Immobilized Rhizopus delernar Lipase, J . Ferment. Bioeng. 81: 299-303 (1996). 7. Shimada, U., M. Suenaga, A. Sugihara, S. Nakai, and Y. Tominaga, Continuous Production of Structured Lipid Containing y-Linolenic and Caprylic Acids by Immobilized Rhizopus delemar Lipase, J.Am. Oil Chern.SOC.76: 189-193 (1999).
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8. Soumanou, M.M., U.T. Bornscheuer,U. Menge, and R.D. Schmid, Synthesis of Structured Triglycerides from Peanut Oil with Immobilized Lipase, J. Am. Oil Chem. SOC.74: 427-433 (1997). 9. Christensen, M.S., C.-E. H@y,C.C. Becker, and T.G. Redgrave, Intestinal Absorption and Lymphatic Transport of Eicosapentaenoic(EPA), Docosahexaenoic(DHA), and Decanoic Acids: Dependence on Intramolecular Triacylglycerol Structure, Am. J. Clin. Nutr. 61: 56-61 (1995). 10. Battino, N.R., G.A. Vandenburg, and R. Reiser, Resistance of Certain Long-Chain Polyunsaturated Fatty Acids of Marine Oils to Pancreatic Lipase Hydrolysis, Lipids 2: 489493 (1967). 11. Yang, L.-Y., A. Kuksis, and J J . Myher, Lipolysis of Menhaden Oil Triacylglycerols and Corresponding Fatty Acid Alkyl Esters by Pancreatic Lipase In Vitro:A Reexamination,J. LipidRes.31: 137-147 (1990). 12. Han, JJ., Y. Iwasaki, and T. Yamane, Monitoring of Lipase-CatalyzedTransesterification Between Eicosapentaenoic Acid Ethyl Ester and Tricaprylin by Silver Ion HighPerformance Liquid chromatography and High-TemperatureGas Chromatography,J. Am. Oil Chem. SOC.76: 3 1 4 0 (1999). 13. Han, J.J., Y. Iwasaki, and T. Yamane, Use of Isopropanol as a Modifier in a HexaneAcetonitrile Based Mobile Phase for the Silver Ion HPLC Separation of Positional Isomers of Triacylglycerols Containing Long Chain Polyunsaturated Fatty Acids, J. High Res. Chromatogr. 22: 357-361 (1999). 14. Han, JJ., and T. Yamane, Enhancement of Both Reaction Yield and Rate of the Synthesis of StructuredTriacylglycerolContaining EPA Under Vacuum with Water Activity Control, Lipids 34: 989-995 (1999). 15. Rosu, R., M. Yasui, Y. Iwasaki, N. Shimizu, and T. Yamane, Enzymatic Synthesis of Symmetrical 1,3-Diacylglycerols by Direct Esterification of Glycerol in Solvent-Free System,J.Am. Oil Chem. SOC.76: 839-843 (1999). 16. Irimescu, R., M. Yasui, Y. Iwasaki, N. Shimizu, and T. Yamane, Enzymatic Synthesis J. Am. Oil Chem. SOC.77: 501-506 of 1,3-Dicapryloyl-2-eicosapentaenoylglycerol, (2000). 17. Irimescu, R., K. Hata, Y. Iwasaki, and T. Yamane, Comparison of Acyl Donors for LipaseCatalyzed Production of 1,3-Dicapryloyl-2-eicosapentaenoylglycerol, J. Am. Oil Chem. SOC.78: 65-70 (2001). 18. Irimescu, R., K. Furihata, K. Hata, Y. Iwasaki, and T. Yamane, Utilization of Reaction Medium-DependentRegiospecificityof Candida antarctica Lipase (Novozym 435) for the Synthesis of 1,3-Dicapryloyl-2-docosahexaenoyl(or Eicosapentaenoyl) Glycerol, J. Am. Oil Chern. SOC.78: 285-289 (2001). 19. Irimescu, R., K. Furihata, K. Hata, Y. Iwasaki, and T. Yamane, Two-step Enzymatic Synthesis of DocosahexaenoicAcid-Rich SymmetricallyStructured Triacylglycerolsvia 2Monoacylglycerols,J . Am. Oil Chem. SOC.78: 743-748 (2001). 20. Iwasaki, Y., J.J. Han, M. Narita, R. Rosu, and T. Yamane, Enzymatic Synthesis of Structured Lipids from Single Cell Oil of High DocosahexaenoicAcid Content, J. Am. Oil Chem. SOC.76: 563-570 (1999). 21. Iwasaki, Y., Y. Yasui, T. Ishikawa, R. Irimescu, K. Hata, and T. Yamane, Optical Resolution of Asymmetric Triacylglycerols by Chiral-Phase High-Performance Liquid Chromatography,J. Chromatogr. A. 905: 111-1 18 (2001). 22. Iwasalu, Y. and T. Yamane, Enzymatic Synthesis of Structured Lipids, J . Mol. Carol. B: Enzym. 10: 129-140 (2000).
Enzymatic Synthesis of Symmetrical TAG Containing PUFA
43 1
23. Yamane, T. Lipase-Catalyzed Synthesis of Structured Triacylglycerols Containing Polyunsaturated Fatty Acids: Monitoring the Reaction and Increasing the Yield, in Enzymes in Lipid Modifcation, edited by U. Bornscheuer, Wiley-VCH Verlag GmbH, Weiheim, 2000, pp. 148-169. 24. Iwasaki, Y., and T. Yamane, Enzymatic Synthesis of Structured Lipids, in Advances in Biochemical EngineeringfBiotechnology , 90, Recent Progress in Biochemical and Biomedical Engineering in Japan I , edited by Takeshi Kobayashi, Springer-Verlag,Berlin, 2 0 0 4 , ~151-171. ~. 25. Luddy, F.E., R.A. Barford, S.F. Herb, P. Magdiman, and R.R.W. Riemenschneider,Lipase Hydrolysis of Triglycerides by a Semimicro Technique, J. Am. Oil Chern. SOC. 41: 693-696 (1963). 26. Foglia, T.A., E J. Conkerton, and P.E. Sonnet, Regioselective Analysis of Triacylglycerols by Lipase Hydrolysis, J . Am. Oil Chem. SOC.72: 1275-1279 (1995). 27. Becker, C.C., A. Rosenquist, and G. Holmer, Regiospecific Analysis of Triacylglycerols Using Ally1 Magnesium Bromide, Lipids 28: 147-149 (1993). 28. Ando, Y., T. Ota, Y. Matsuhira, and K. Yazawa, Stereospecific Analysis of Triacyl-snGlycerols in Dccosahexaenoic Acid-Rich Fish Oils, J. Am. Oil Chem. SOC.73:483-487 (1996). 29. Gunstone, F.D., High-Resolution NMR Studies on Fish Oils, Chem. Phys. Lipids 59: 83-89 (1991). 30. Bergana, M.M., and T.W. Lee, Structure Determination of Long-Chain Polyunsaturated Triacylglycerolsby High-Resolution 13C Nuclear Magnetic Resonance, J. Am. Oil Chem. SOC.73: 551-556 (1996). 31. Dobson. G., W.W. Christie, and B. Nikolva-Damyanova, Silver Ion Chromatography of Lipids and Fatty Acids, J . Chromatogr. B. 671: 197-222 (1995). 32. Christie, W.W., Separation of Molecular Species of Triacylglycerolsby High-Performance Liquid Chromatography with a Silver Ion Column, J . Chromatogr. 454: 273-284 (1988). 33. Jeffley, B.S J., Silver-Complexationof Liquid Chromatography for Fast High Resolution Separationsof Triacylglycerols,J . Am. Oil Chem. SOC.68: 289-293 (1991). 34. Adlof, R.O., Analysis of Triacylglycerol Positional Isomers by Silver Ion High PerformanceLiquid Chromatography,J . High Resolut. Chromatogr. 18: 105-107 (1995). 35. Adlof, R.O., Separation of cis and trans Unsaturated Fatty Acid Methyl Esters by Silver Ion High-PerformanceLiquid Chromatography,J . Chromatogr.A 659: 95-99 (1994). 36. Traitler, H., Capillary GC: Hot Cold On-Column Injection, J . Am. Oil Chem. SOC.65: 1119-1123 (1988). 37. Villeneuve, P., M. Pina, A. Skarbek, J. Graille, and T.A. Foglia, Specificity of Carica papaya Latex in Lipase-Catalyzed Interesterification Reactions, Biotechnol. Tech. I I : 91-94 (1997). 38. Chandler, I.C., P.T. Quinlan, and G.P. McNeill, Lipase-Catalyzed Synthesis of Chiral Triglycerides,J . A m . Oil Chem. SOC.75: 1513-1518 (1998). 39. Shimada, Y., A. Sugihara, H. Nakano, T. Yokota, T. Nagao, S . Komemushi, and Y. Tominaga, Production of Structured Lipids Containing Essential Fatty Acids by ImmobilizedRhizopus delemer Lipase, J . Am. Oil Chem. SOC.73: 1415-1420 (1996). 40. Rosu, R., Y. Uozaki, Y. Iwasaki, and T. Yamane, Repeated Use of Immobilized Lipase for MonoacylglycerolProduction by Solid Phase Glycerolysis of Olive Oil, J . Am. Oil Chem. SOC.74: 445450 (1997). 41. Rosu, R., Y. Iwasaki, N. Shimizu, N. Doisaki, and T. Yamane, Enzymatic Synthesis of Glycerides from DHA-enrichedPUFA Ethyl Ester by Glycerolysis Under Vacuum, J . Mol. Cat. B: Enzym. 4: 191-198 (1998).
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42. Rosu, R., Y. Iwasaki, N. Shimizu, N. Doisaki, and T. Yamane, Intensification of Lipase Performance in a Transesterification Reaction by Immobilization on CaCO, Powder, J . Biotechnol. 66: 51-60 (1998). 43. Yamane, T., Enzyme Technology for the Lipids Industry: An Engineering Overview, J . Am. Oil Chem. Soc. 64:1657-1662 (1987). 44. Yamane, T., T. Suzuki, and T. Hoshino, Increasing n-3 Polyunsaturated Fatty Acid Content of Fish Oil by Temperature Control of Lipase-Catalyzed Acidolysis, J. Am. Oil Chem. SOC. 70: 1285-1287 (1993). 45. Bomscheuer, U.T., and T. Yamane, Fatty Acid Vinyl Ester as Acylating Agents: A New Method for the Enzymatic Synthesis of Monoacylglycerols, J . Am. Oil Chem. SOC.72: 193-197 (1995). 46. Yoshida, Y., M. Kawase, C. Yamaguchi, and T. Yamane, Enzymatic Synthesis of Estolides by a Bioreactor, J . Am. Oil Chem. SOC.74: 261-268 (1997). 47. Xu, X., A.R.H. Skands, C.-E. H@y,H. Mu, S. Balchen, and J. Adler-Nissen, Production of Specific-Structured Lipids by Enzymatic Interestification: Elucidation of Acyl Migration by Response Surface Design, J . Am. Oil Chem. Soc. 75: 1179-1 186 (1998). 48. Xu, X., H. Mu, A.R.H. Skands, C.-E. Hay, and J. Adler-Nissen, Parameters Affecting Diacylglycerol Formation During Production of Specific-Structured Lipids by LipaseCatalyzed Interesterification,J . Am. Oil Chem. Soc. 76: 175-181 (1999). 49. Xu, X., S. Balchen, C.-E. H@y,and J. Adler-Nissen, Pilot Batch Production of SpecificStructured Lipids by Lipase-Catalyzed Interesterification: Preliminary Study on Incorporation and Acyl Migration, J . Am. Oil Chem. SOC.75: 301-308 (1998). 50. Xu, X., S. Balchen, C.-E. H@y,and J. Adler-Nissen, Production of Specific-Structured Lipids by Enzymatic Interesterification in a Pilot Continuous Enzyme Bed Reactor, J . Am. Oil Chem. Soc. 75: 1573-1580 (1998). 51. Yamane, T., Y. Koizumi, T. Ichiryu, and S. Shimizu, Biocatalysis in Microaqueous Organic Solvent, Ann. N.Y. Acad. Sci. 542: 282-293 (1988). 52. Yamane, T., Importance of Moisture Content Control for Enzymatic Reactions in Organic Solvents: A Novel Concept of “Microaqueous,” Biocatalysis 2: 1-9 (1988). 53. Soumanou, M.M., U.T. Bomscheuer, and R.D. Schmid, Two-step Enzymatic Synthesis of Pure Structured Triacylglycerols,J. Am. Oil Chem. SOC.75: 703-710 (1998). 54. Schmid, U., U.T. Bornscheuer, M.M. Soumanou, G.P. McNeill, and R.D. Schmid, Optimization of the Reaction Conditions in the Lipase-Catalyzed Synthesis of Structured Triglycerides, J . Am. Oil Chem. SOC.75: 1527-1531 (1998). 55. Haraldsson, G.G., B.O. Gudmundsson, and 0.Almarsson, The Synthesis of Homogeneous Triglycerides of Eicosapentaenoic Acid and Docosahexaenoic Acid by Lipase, Tetrahedron 51: 941-952 (1995). 56. Irimescu, R., Y. Iwasaki, and C.T. Hou, Study of TAG Ethanolysis to 2-MAG by Immobilized Candida anturctica Lipase and Synthesis of Symmetrically Structured TAG, J. Am. Oil Chem. SOC.79: 879-883 (2002). 57. Piyatheerawong, W., Y. Iwasaki, X. Xu, and T. Yamane, Dependency of Water Concentration on Ethanolysis of Trioeoylglycerol by Lipases, J . Mol. Catal. B: Enzym. 28: 19-24 (2004).
Chapter 19
Patent Review on Lipid Technology Oi-Ming Laid, Seong-Koon Lod, and Casimir C. Akohb aDepartrnent of Bioprocess Technology, Faculty of Biotechnology and Biornolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia and bDepartment of Food Science and Technology, University of Georgia, Athens, GA 30602
Introduction A patent is, in essence, the grant of a monopoly to an inventor who has used his skill to invent something novel. However, the monopoly is not absolute; patents are granted for only a limited time frame and are accompanied by public disclosure to enable others in the field to improve on the invention. Numerous processes and compositions involving lipid components have been patented extensively since the early 1900s. In this chapter, a review of the patent literature on lipid technology ranging from triacylglycerols (TAG) to ubiquinones in the past 5 yr is presented.
Triacylglycerol TAG is the most abundant lipid component found in natural oils and fats. The patent literature on processes and methods involving TAG is summarized in Table 19.1. Patents related to the synthesis of structured TAG (1-9) have been the main focus in the field. Other methods such as TAG hydrolysis (10-14) and extraction or separation of TAG (15-17) were also patented. Table 19.2 summarizes patent publications on novel compositions comprising TAG. Again, patent publications on various types of structured TAG used in oil and fat compositions predominate (20-34). Apart from the usual application of TAG for consumption, a novel application of high-oleic TAG as an electrical insulation fluid was reported (35).
Diacylglycerol In the past several years, patents filed for the processes and compositions involving diacylglycerol (DAG) have been rising, due mainly to the recent discovery that 1,3positional isomers of DAG possess properties that are able to reduce body fat. Patented processes for producing DAG and methods involving the application of DAG, as well as patent publications on DAG compositions are reviewed in Tables 19.3 and 19.4, respectively. Most of the methods for producing DAG are either by esterification (42-46), glycerolysis (47-50), or alcoholysis (42-44,5 1). Kao Corporation of Japan published numerous patents on a variety of compositions comprising DAG such as cooking and frying oils and fats (58-77), emulsified oil composi-
tions (78-84), powdered fat (85), pet food and feed (86), packaged emulsified beverages (87), oil-cooked potatoes (88), fried food, and other food compositions (89,90). 433
434
0.-M. Lai et al.
Monoacylglycerol
There is less patent literature published on monoacylglycerol (MAG) compared with TAG and DAG. The patent literature involving MAG is summarized in Table 19.5. Reactions involved in the synthesis of MAG are similar to those used to produce DAG and TAG, i.e., esterification, transesterification, and alcoholysis. These reactions are generally conducted in the presence of a reaction catalyst, such as an alkali or an enzyme. However, two patent publications by Kao Corporation of Japan reported interesting methods of producing MAG by transesterification and esterification, respectively, without the use of a reaction catalyst (96,97). Patented applications of MAG include their use in emulsified compositions (98,99), and pharmaceutical and clinical preparations (100-103). Polyunsaturated Fatty Acids
Polyunsaturated fatty acids (PUFA), such as DHA, EPA, and arachidonic acid (ARA), have been of tremendous interest to inventors in the field of lipid technology, judging from the number of patent publications filed in recent years (Table 19.6). Numerous methods of production of ARA by means of fermentation using several microorganisms of genera such as Mortierellu, Phytophtheru, Labyrinthlu, and Pythium were patented (1 12-120). A method for cultivating DHA- and EPAenriched radish and bean sprouts was also published (121). Phytosterols, Phytostanols, and Their Esters
Phytosterols and phytostanols were also among the main lipid components that were actively published in patent literature in the recent years, mainly because of their cholesterol-lowering and other beneficial properties. Table 19.7 summarizes the processes and methods involving phytosterol and phytostanols, and Table 19.8 shows the various compositions that comprise these lipid compounds. Several methods of producing phytosterol derivatives, such as phytosteryl FA esters (151-159) and other esters (160,161) were patented. The problem of insolubility of phytosterols and phytostanols was also solved by the invention of several novel compositions (176-1 80). Carotenes
Carotenes comprise another lipid-soluble component that is found predominantly in natural oils such as palm oil. The current processing method for palm oil does not provide a way to obtain the carotenes as a processing by-product. Table 19.9 shows several methods for obtaining carotenes from their natural sources. Two approaches were observed in these methods, i.e., (i) solvent extraction (209-21 l), and (ii) solvent-free extraction (212).
Patent Review on Lipid Technology
435
Vitamin E
In the last 5 years, few patent publications for vitamin E appeared. Several compositions comprising vitamin E ester derivatives are tabulated in Table 19.10. LG Life Sciences Ltd. filed for a patent for its feed additive comprising a polyethoxylated ester of a-tocopherol, which was claimed to improve meat quality and increase body weight (213). A cyclopropylic ester of a-tocopherol compound was also patented by the SK Corporation, with the claim that the ester possesses biological activity equal to that of natural vitamin E (214). Oryzanol
Oryzanol is one of the minor components found in natural oils and fats, and is valued for its cholesterol-lowering property. Patent publications on processes and compositions involving oryzanol are summarized in Table 19.11. Methods of extracting oryzanol from its natural source, which is primarily rice bran oil and its derivatives, include the following: (i) steps of saponification, solvent extraction, and crystallization (218); (ii) steps of removing phospholipids and FFA, alkali neutralization, and separation (219); and (iii) steps of distillation, hydrolysis, and precipitation (220). McNeilPPC Inc. patented a process for producing a water-dispersible oryzanol composition that utilizes a polyfunctional surfactant as the key ingredient (221). Ferulic Acid and Its Derivatives
Patent publications on ferulic acid and its derivatives are tabulated in Table 19.12. Ferulic acid compounds and compositions for treating or preventing hypertension (223-226) and dementia and enhancing cognition (227,228) were found. Cosmetic applications such as reducing wrinkle formation (229), sunscreen (230,23l), controlling oily skin and sebum secretion (232,233) of ferulic acid compounds were also reported. Ubiquinone
Tables 19.13 and 19.14 summarize the patent literature on processes and methods involving ubiquinone, and ubiquinone-containing compositions, respectively. Several methods for enhancing the dissolution and bioavailability of ubiquinone were patented (238-241); these generally involve the incorporation of a ubiquinone solubilizer into the composition. Processes for producing ubiquinone include (i) fermentation using Suitoellu genus (242) and (ii) extraction from deodorizer distillate (243). The patent literature on compositions of ubiquinone revolves around the following areas: (i) composition for treatment of diseases or disorders (258-276); (ii) composition for solubilizing and/or stabilizing ubiquinone (269,277-283); and (iii) composition for cosmetic applications (284,285).
436
0.-M. l a i eta/.
Conclusion With the rapid growth of the food and pharmaceutical industries both in sales and in the number of new product introductions, lipid components are incorporated into a diverse array of products used in our daily lives. It has thus become necessary for the lipid technologist to know much more about patented processes, methods, and compositions involving lipid components. The information compiled in this chapter is by no means exhaustive and is meant to give the lipid technologist a glimpse of the direction in which the lipid industry is heading in the near future.
TABLE 19.1 A 5-Year Patent Literature Survey on Processes and Methods Involving TAG
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Ref.
Dow Global Technologies Inc. (U.S.)
EP1383854
3/21/2002
WO2002USO8708
Process for separating a seed oil into two TAG fractions comprising a seed oil with an adsorbent having particle size > 40 pm in a bed, thereafter contacting the adsorbent with a desorbent material under minimum flow conditions to obtain a raffinate output stream.
(1 5)
Novozymes NS
W003/040091
11/5/2002
PA200101 635
Process for hydrolyzing TAG comprisingcontactingthe TAG
(10)
(Denmark)
and water consecutively or simultaneously with a positional specific microbial lipase and a positionally nonspecific lipase, wherein lipase is immobilized.
BASF AG (Germany) US6666950 Kao Corp. Uapan)
US2003/0013165
-
6/12/2002
US2001 0996489 JP2001190335
Process for deinking paper comprisinga deinking agent containing beef tallow TAG.
9 3 2 ;o
r-0
5. 2 (18)
Process for hydrolyzing TAG by mixing and feeding an oil-phase (11) substrate and a water-phase substrate to a packed layer with an immobilized lipase packed therein, wherein said TAG is subjected to hydrolysis under feeding conditions such that a shear stress factor (w), which is applied to a surface of said immobilized lipase and is expressed by the following formula: zw = (AP/L) x dpx ~ / ( -1E ) , is from 1 x l o l ' to 1.4 x MPa, wherein APrepresents a maximum pressure loss (MPa) through said packed layer during hydrolysis, L represents thickness (m) of said packed layer, dp represents a weight-basis average particle size (m) of particles of said immobilized lipase, and E represents a void volume of said packed layer.
8
t:
3. Q J
0 0
!+i
(Continued W U
w P
TABLE 19.1 (Continued)
0)
Applicant
Publication no.
Date filed (monld/yr)
Priority
Disclosure
University of Missouri Board of Curator (U.S.)
US6547987
1/25/2000
US20000491185
Process for extracting TAG oil comprising the use of a solvent and a polarity below 0.1, and method for extracting said containing hexane and a fluorocarbon and having viscosity 4 . 6 CP TAG oil at 35-55OC, and cooling resulting micella to 15-25'C.
Unilever (U.S.)
US2002031577 SK7392003
711Ol2001
EP20000204709
Process for crystallizinga solid phase from a liquid TAG oil comprisingsubjecting the liquid oil to ultrasound in the absence of transient cavitation.
Unilever PLC (The Netherlands)
WOO3096817
5/5/2003
E P20020076963
Process for preparing a structured TAG comprising interesterifying a natural fat, containing at least 3 5 % stearic ~ acid and <5%wt PUFA and selected from the group comprised of allanblackiafat, pentadesma fat, kokum fat and sonchy fat, with a fat containing at least 40%wt C,,<,, saturated FA.
Suntory Ltd. (lapan)
WOO3004667
7/7/2002
JP20010201357
Process for producing structured TAG having medium-chain FA at sn-1 and sn-3 positions and highly unsaturated FA at sn-2 position comprising interesterifying a medium-chain FA derivative with a fat comprising at least one n-6 and n-9 unsaturated C,, or higher FA using a 1,3-position selective lipase immobilized on a porous ion-exchange resin having a pore diameter 21OOA.
University of Georgia (U.S.)
US6369252
212311999
US19990255749 US19990223 US19980076167P US19980226
Methods of modulatingtotal cholesterol levels, LDL cholesterol, TAG levels, and/or ratio of T-helper cells to T-cytotoxic cells in an individual comprisingadministrating a structured TAG. Enzymatic methods for producing said structured TAG.
3/6/2001
US20010799996 us20010306
Process for hardening unsaturated lipids comprising mixing and interactingsaid lipids with polyamines such that the peak
Kraft Foods Holdings US6479684 Inc. (U.S.) CA2370812
Ref.
melting point of the resulting combination is higher than that of the lipid alone. Livemax Co. Ltd. (Korea)
US2003032672 KR2002081632
4/18/2002
KR20010021201
Process for producing a conjugated linoleic acid (CLA)-enriched structured lipid comprising transesterification of medium-chain TAG with ester or free form of CLA using lipases.
-
US2002/0197687
6/11/2002
EPOl115081.O
Method for enzymatically splittingTAG for obtaining FA and glycerol in the presence of a lipase, comprisingthe following: (i) preparing a mixture containingTAG, water, and lipases; (ii) subjecting mixture to a glyceride splitting reaction until reaching predeterminedsplitting degree at which, for said mixture, the slowdown of the splitting reaction is below a preset value; (iii)separatingthe FA from the mixture, which comprises an aqueous, glycerolcontaining phase and a partially split organic phase containingsplit FFA, by first separating the aqueous phase from said partially split organic phase, and thereafter, separatingthe FFA from the separated partially split organic phase; and (iv) feeding back the obtained residue of the organic phase into the splitting process.
Monsanto Co. (US.)
EP1196518
2/17/2000
WO2OOOUSO4166
Process for separatinga first TAG containing DHA from a second TAG comprising the following: (i)introducing both TAG into a chromatographic separation zone comprising metal ions capable of complexing with a double bond of FA residue of the first TAG; (ii) isolating a fraction of the feed mixture in the separation zone that has a mass ratio of the first and second TAG greater than in the feed mixture.
Forbes Medi-Tech Inc. (Canada)
W001/91587
6/4/2001
US09/586,431
Method for reducingweight gain and maintaining proper body weight via enhanced metabolism of fats and decreased energy expenditure consists of administering a composition comprising (Continued) W P
W
P
P 0
TABLE 19.1
(Continued) ~
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Ref
TAG having short- and medium-chain FA residues derived from C4&, FA and long-chain FA residues derived from C,,-C,, FA. Abbott Laboratories (U.9
US6160007
9/1/1999
US19990388331 us19990901
Method for enhancing absorption of lipophilic compounds comprising administering lipophilic compounds in conjunction with a structured glyceride component characterized in that at least 40% of the glyceride species has the following: (i)33-70Y0wt acyl moiety has C4-C,,; (ii) 30--67%W acyl moiety has X,,; and (iii)equivalent carbon number >30 and <48.
USA
US5,932,458
3/23/1992
-
Method of producing FA and glycerol by hydrolysis of oleaginous materials, utilizing an immobilized lipase in the form of comminuted lipase-containing seeds, comprising the following: (i) comminuting the seeds to form an immobilized lipase; (ii) mixing an organic solvent, water, and an oleaginous material with the immobilized lipase to form a reaction medium; (iii)agitating the reaction medium under conditions effective to hydrolyze said oleaginous material; and (iv) separating the FA and glycerol from the reaction.
Henkel Corp. (US)
EP1006174
10/281993
US40250
Process of splitting of TAG comprising the following: (i)preparing (13) a mixture containing TAG, water, and 1-1 00 ppm total protein by weight of TAG of a 1,3-position-specific lipase; (ii) hydrolyzing TAG to obtain product having acid value of 25-1 00; (iii)mixing partially split TAG in a pressure splitter with
water; and (iv) subjecting mixture to 20G300"C and 3.1-6.55 MPa to substantially complete splitting of TAG into carboxylic acids and glycerine. Atlantis Marine Inc. (Canada)
CA2260397 WOO044862 EP1151067
112911999
CA19992260397 CA19990129
Method of converting marine TAG oil into bland, stable edible oil comprising treating said oil with a silica at low temperature under vacuum and then with bleaching clay under vacuum and at higher temperature.
(20)
Suntory Ltd. (Japan) and Osaka City Clapan)
jP2000004894
111112000
]PI 9980172942
Method for producing structured lipid having a structure similar to human breast milk type TAG, containing C,,-C,, SFA at sn-2 position and at least one unsaturated FA at sn-1 position, and n-3, n-6 andlor n-9 unsaturated FA at 9 - 3 position, comprising transesterifying a TAG having a melting point G5"C and having C,,-C,, SFA at sn-2 position and mediumchain FA at sn-1 and -3 positions with a TAG in the presence of a 1,3-position specific lipase.
(8)
Suntory Ltd. (Japan) jP2000008074
111112000
JP19980173017
Method for producing structured TAG having a structure similar to human breast milk type TAG, containing C,,-C,, SFA at sn-2 position and at least one unsaturated FA at sn-1 position, and n-3, n-6 andlor n-9 unsaturated FA at sn-3 position, comprising esterifying or transesterifying a TAG having C,&, FA at sn-2 position with n-3, n-6 and/or n-9 unsaturated FA or its esters using a 1,3-position-specific lipase.
(9)
P h) P
TABLE 19.2 A 5-Year Patent Literature Survey on Compositions Involving Triacylglycerols
Applicant
Publication no.
Date filed (moddlyr)
Scotia Lipidteknik AB (U.K.)
US6517883
Kao Corp. (Japan)
JP2003204753
Danisco Cultor America Inc. (US.)
Priority
Disclosure
Ref.
71611998
SE19970002630 SE19970707 W01998SE01326
Food composition giving a prolongedfeeling of satiety cornprising an oil-in-water emulsion containing TAG oils having a solid fat content at ambient or body temperature.
(37)
7/22/2003
JP20020114354
TAG composition with excellent foaming power, syneresisresistant property, oil-off resistance, and shape or form retention, comprising 3&8O%wt of the following: (i) a TAG containing a C ,, or higher SFA and a C, unsaturated FA, wherein the total C atoms of constituent FA is at least 50; (ii)a TAG containing a C ,, or higher SFA and a C,&, unsaturated FA, wherein the FA are all constituent SFA and (iii)a TAG containing C,-C, SFA and a C, unsaturated FA, wherein the total C atoms of constituent FA is below 50.
(38)
P
&
US2003198727
8/7/2001
W02001JPO6778
Oillfat composition for reducing body and visceral fat accumulation, blood sugar level, leptin level, improving insulin resistance and has excellent heat stability, comprising the following: (i) 10.1-94.9%wt TAG; (ii) 0.1-30%wt MAG; and (iii) 5-59.9%wt DAG, having a FA constituent thereof 15-90%wt n-3 FA having <20 carbon atoms.
(39)
EP1112000 NZ510342
911011999
US19980099830P
Reduced-energy, edible plastic fat composition comprising a TAG mixture of two or more TAG having the formula SSL, SLS LLS and LSL, where each fatty acyl residue is independently a ,, FA, and the mixture long-chain C,-C, FA, S is a C+ contains 4&95%wt of SSL and SLS.
(23)
Nisshin Oil Mills Ltd. (Japan)
US2003170368
1/27/2003
US20030351400
Oils and fats composition for reducing lipids in blood comprising a structured TAG having saturated medium-chain C& ,, FA at sn-1 and sn-3 positions and monounsaturatedCl6Xl8 FA at the sn-2 position.
(22)
Unilever (U.S.)
US2002018841 CZ20024096
611312001
EP20000202068
TAG fat comprising a stearin fraction of a high-stearic, high-oleic sunflower oil and a margarine fat comprising said stearin in admixture with a liquid vegetable oil in a weight ratio of 2 0 8 0 to 80:20.
(40)
Renewable Lubricants Inc.
US6534454 WOO3093403
5/4/2002
US20020138958
Biodegradablevegetable oil composition comprising the following: (41) (i)at least one TAG containing C,-C, aliphatic hydrocarbyl groups, and (ii) an antioxidant.
US6645404 CA2276406 W0983 102 1 EP0950249 AU72 7832
8110/2001
US19970778608 US19970106
High-oleic acid TAG composition comprising the following: (i)275% oleic acid; (ii)110% diunsaturated FA; (iii)<3% triunsaturated FA and (iv) <8% saturated FA, and having properties of dielectric strength pF at least 35 kV/100 mil gap, dissipation factor of <0.05% at 25"C, acidity of ~ 0 . 0 3mg KOHIg, an electrical conductivity of <1 pS/m at 25"C, a flash point of at least 25OOC and a pour point of at least -1 5"C, for use as electrical insulation fluids.
10/22/2001
US20010014247 us2001 1022
Structured TAG comprising n-3 PUFA and medium-chain FA. Method for producing said TAG comprising interesterifying coconut oil with FFA obtained from hydrolysis of TAG of an animal source.
(US.) ABB Power T & D Co. (Switzerland)
Council for Scientific US6608223 & industrial Research (India)
(Continued)
P W P
P
P P
TABLE 19.2 (Continued) Applicant
Publication no.
Bunge Foods Gorp. (U.S.)
US2003175404 CA2418599
US2003077340
Unilever NV (The Netherlands)
Date filed (modd/yr)
Priority
Disclosure
Ref.
311812002
US20020100449 US20020318
Structured lipids for use in pan-release cooking compositions produced by interesterification of medium-chain TAG oils with long-chain domestic oils.
(21)
1211012001
W02001 IN00182 W020011018
Structured TAG comprising n-6 PUFA and medium-chain FA. Method for producing said TAG comprising interesterifying coconut oil with FFA obtained from hydrolysis of TAG of a vegetable source.
(25)
EP20000204120 EP20001121 EP20010201916 EP20010521 W02001 EP12273 w020011022
Edible water-in-oil composition comprising a hardstock fat containing at least 45%wt SOS TAG, where S is C,-C, FA and 0 is oleic acid.
(26)
P $ r
CA2425591 WOO241 699 WOO241698 US2002122868 US2002114874
10/22/2001
US2002001661
211612001
US20010785807
Low-energy fat composition comprising a TAG having at least one (27) hydroxy FA having melting point >40"C, and having an energy value < 5 kcal/g.
Fujisawa Pharmaceutical Co. Uapan)
US2002061906
11/28/2001
US20010994702
Medicinal aerosol composition comprising a medium-chain TAG (28) and a tricyclic compound dispersed in a liquefied hydrofluoroalkane propellant.
Kida, H., Fuji Oil C o . Ltd. (Japan)
WOO204581
6/28/2001
JP20000205350
TAG composition having excellent cold resistance comprising a liquid fat at room temperature comprising at least 60%wt PUFA, and 0.1 5-4.5%wt SFA.
'. 2 [1,
7
(42)
Ushioda, T. Suntory Ltd. (Japan)
US6248909 EP0965578 JP2000008074 EP0965578
6/18/1999
JP19980173017 jP19980619
Novel TAG and composition containing said TAG having a TAG structure of human breast milk type, prepared by subjecting a glyceride having a C, 6-C18SFA at 57-2 to transesterification using a lipase with an n-6, n-9, or n-3 unsaturated FA.
(29)
Unilever PLC (U.K.)
SG77567
11/18/1993
EP19920311016
Anti-bloom TAG composition comprising at least 30%0SSM TAG, wherein S is saturated C,6&4 FA and M is saturated C,o<,4 FA and SSM:SMS is >3:1.
(30)
Wrigley W. Jun Co.
MAU714444
10/31/1995 WO1994US12548
Improved chewing gum formulations and bases comprising a structured TAG having medium-chain FA.
(31)
US6103292
4/15/1997
US19970843400 US1997041 5
Compositions containing structured TAG of genetically engineered (32) annuals and food products containing the same.
9 in
(U.S.) Calgene Inc. (U.S.)
.-.,
a
Abbott Laboratories (U.S.)
US5962712
4/23/1997
US19970839086 US19970423
Structured TAG for treatment of disease or stress states, comprising a y-linolenic acid or a dihomo-y-linolenic acid residue, an n-3 FA residue, and a medium-chain FA residue.
St Giles Foods Ltd. (U.K.)
(282337528
5/19/1998
GB19980010628 GB199805 19
Oil composition, adapted to be dispensed by spraying, comprises (34) a vegetable oil and a medium-chainTAG, having C6Xl2.
Procter & Gamble
EP0369516 HK1006129 US4996074 JP2209999 EP0369516
11/6/1989
US19880270314 US19881114
A stable P’TAG hardstock comprising the following: (i) 45-98% PSP TAG; (ii)2-55‘%0 PSS TAG; (iii) <7% PPP TAG; (iv) <7% SSS TAG; (v) ~ 3 DAG; % (vi)
(US.)
ir;
(33)
$. 2
(35)
2. 4
2
5 3 0,
5
P P
Ln
P P
m
TABLE 19.3 A 5-Year Patent Literature Survey on Processes and Methods Involving DAG Date filed (mon/d/yr)
Priority
Disclosure
JP2003274987 WOO3060139
12/27/2002
-
Method for producing DAG comprising reacting the FA or its lower alkyl ester with glycerol, while dehydratingthe reaction mixture, in the presence of an immobilized partial glyceride lipase, and finishes the reaction in which an acid value (AV) of the reaction solution satisfies the formula 50R-60 > AV > 70R-150 (AV > 0).
W003/029392
10/3/2002
US60/327,762
Method for producing 1,3-DAG which comprises glycerolysis between TAG and glycerol in the presence of an alkali metal salt and/or alkali earth metal salt of a mono- and/or dicarboxylic acid as catalyst, and under the following dehydration conditions: (i)glycerol:TAG is 0.2:l to 191; (ii)mixing conducted at 17@280"C; (iii)pressure at (2-500 psi in inert gas; (iv) residual glycerol and MAG separated by steam or molecular distillation and recycled; (v) residual TAG separated by molecular distillation and recycled; and (vi) catalyst inactivated by phosphoric acid and removed by filtration and/or centrifugation or absorption.
Archer Daniels US2003104109 Midland Co. (U.S.)
10/3/2002
US20020263331 Method for producing 1,3-DAG from TAG which uses alkali metal US20021003 salts or alkali earth metal salts of mono- or dicarboxylic acids US20010327762P as catalyst for glycerolysis reaction. US20011003
Kao Corp. (Japan)
9/27/2002
JP2001304409 JP2002253928
Applicant
Publication no.
Kao Corp. (Japan)
Archer Daniels Midland Co. (U.S.)
EP1297752
Ref.
Method for producingfried foods that comprises oil or fat cornpo(53) sition containing 215%wt DAG, with the following conditions: (i)N, content in 25 L oil is 10.2Yowt; (ii) Time required to replace the whole oil is 2-25 h and is defined by the equation: Time required to replace whole oil (h) = (Total amount of oil in fryer)/ (Average amount of new oil supplied per hour).
Method produces DAG-containing fried foods that have good appearance and flavor, do not discolor frying oil and emit offensive odor, and are capable of reducing body fat and obesity.
Kao Corp. Uapan) (cont.)
4/25/2002
JP2001-129847
Method for the following: (i)activating lipid catabolism in small intestine epithelium; (ii)promoting accumulation of FA into small intestine epithelium; (iii) inducing expression of a small intestine lipid metabolic gene; (iv) suppressing synthesis of TAG in small intestine epithelium; (v) promoting energy consumption; (vi) lowering serum remnant-like protein particles (RLP) level; and (vii) lowering serum leptin level, which comprises administering an effective amount of DAG comprising; (a) 15-90%wt of FA is n-3 unsaturated FA and (b) 250%wt of DAG is 1,3-DAG.
Huanan Technology CN1438308 University (China)
-
CN20030113862 CN20030306
Method for producing DAG comprisingthe following: (i)conducting an alcoholysis reaction of a monobasic lower alcohol having a C number <3 with a TAG under immobilized lipase and (ii)separating reaction product to obtain DAG.
Kao Corp. (lapan)
711212000
WO2OOOJPO4661 W020000712 JP19990241554 JP19990827
Process for producing fried instant noodles, comprising an oil or fat composition having 25O'hwt DAG used as frying oil, which, when reconstituted with hot or boiling water, have smooth surfaces to give good mouthfeel, not prone to sogginess, do not smell oily, and have an excellent flavor inherent to flour.
Kao Corp. (Japan)
US20030096866
EP1206912
(Continued
P P
u
P
TABLE 19.3
P
W
(Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Kao Corp. (Japan)
US2002/0025370
7/9/2001
JP200Ck212418
Process for fractionatingoil composition containing 25o%wt partial glycerides into solid and liquid fractions, including the following: (i)dissolving a polyol FA ester, having HLB 17 and melting point range 20-40°C and 3-25°C higher than the oil composition, into the oil composition; (ii)cooling the solution to deposit crystals; and (iii)conducting solid-liquid separation.
Kao Corp. (Japan)
US6,361,980 EP1111064
11/30/2000 11/17/2000
JP11-359794 JP35979499
Process for preparing DAG which comprises carrying out immobilized lipase-catalyzed esterification b e e n FA and/or lower alcohol FA ester and glycerol and/or MAC in enzyme-packed tower under dehydratingconditions, as follows: (i) residence time of reaction fluid in tower is 1120s; (ii) dehydration is by feeding reaction fluid through a spray nozzle in a separate dehydration tank; (iii)volumetric mass transfer coefficient, k,a, of the dehydration process is 20.0005 s-’; (iv) lipase is 1,3-position-selective and having esterification activity 2100 unit/g enzyme; (v) Ud2125, where L is packing thickness (m) in flowing direction in tower and d is average particle diameter (mm) of immobilized lipase, and d20.1 mm; and (vi) pressure loss in the tower is S20 kg/cm2.
Kao Corp. (Japan)
US6,337,414
7/6/1999
JP10-194237
Process for preparing DAG, which comprises carrying out lipase-catalyzed glycerolysis, in the presence of water, between TAG and glycerol, wherein FFA, MAG, and glycerol are removed by partial crystal precipitation during reaction, dehydration, or distillation, and having FFA of 25%wt raw oil or fat contains 13O0wt SFA.
Ref.
Kao corp. (Japan)
EPlO94116
7/6/1999
JP19423798
Process for preparing DAG, which comprises carrying out lipase- (51) catalyzed glycerolysis, in the presence of water, between TAG and glycerol, wherein FFA, MAG, and glycerol are removed by partial crystal precipitation during or after reaction, dehydration, or distillation, and having FFA of t5%wt raw oil or fat contains 130°/owt SFA.
Kao Corp. (Japan)
JP2001252090
-
-
Method for producing DAG carried out by using a hydrophilic membrane holding a position-specificlipase.
(46)
Kao Corp. (Japan)
US6,261,812 WO99/09119
4/30/1998 5/2 1/1998
JP9-221502
Process for producing DAG comprising the following: (i)partial hydrolysis of a fat or oil, conducted by steam decomposition at 1 9&240”C or enzyme hydrolysis and in the presence of 20-1 8O”/owt water, to obtain partial hydrolysate comprising 67-96%wt FFA and having a hue such that the 1 OR+Y value is +I0 and (ii) immobilized 1,3-position lipasecatalyzed esterification between partial hydrolysate and glycerol, conducted at 2&70”C, until DAG purity of 280% is reached.
(47, 57)
Process for producing DAG comprising the following: (i)transesterification between a M A G and TAG and (ii)removal of residual FFA and M A G by steaming at reduced pressures, molecular distillation or chromatography.
(58)
Kanegafuchi Chemical Industries Co. Ltd. (Japan)
JP2000345189
11/1/1999
-
9 iii
2
2 z.
20
3
c:
-$ 2
(5,
5
0,
4
P P
W
U PI
0
TABLE 19.4 A 5-Year Patent Literature Survey on Compositions of DAG Applicant
Publication no.
Date filed (mon/d/yr)
Kao Corp. (Japan)
WOO31067999
211 712003
Priority
Disclosure
JP2002-039775 JP2002-372508
Oil or fat powder comprising the following: (i) 15-79.9%wt glyceride mixture comprising 5434.9%wt TAG, 0.1-5%wt MAG and 15-94.9%wt DAG, and having >5oo/owt of all constituent FA as unsaturated FA; (ii) 2&84.9%wt of one or at least two powder forming bases selected from carbohydrates, proteins, and peptides; and (iii) 0.1-5%wt water.
Kao Corp. (Japan)
w0031024237
911212002
JP2001-277669 JP2002-253927
Oil or fat composition comprising the following: Component (A) having 15-7o%wt DAG in which <15%wt of FA is n-3 unsaturated FA; Component (6) having 3@85"/0wt TAG in which t l 5 % w t of FA is n-3 unsaturated FA and 2 50%wt is a-linolenic acid; 0.004-5D/Owt antioxidant, O.O24).5%wt crystalline inhibitor, and 20.050/owt phytosterol.
Kao Corp. (Japan)
US6635777
6/9/2000
US20030459512 US20030612 JP19990170849
Acid oil-in-water composition comprising 3o%wt or more DAG and a yolk, with a ratio of lysophospholipidsto whole phospholipidsof at least 15%.
Kao Corp. Uapan)
US2003068428 EPl245160
312112002
JP20010087242 JP20010326
Pet food or feed comprising an oil or fat as follows: (i) 21O%wt DAG containing 25Oyowt C,-C, unsaturated FA and 140°/owtunsaturated FA having at least C ,, and at least 4 carbon-carbon double bonds and (ii) 1 2 O % w t FFA, wherein DAG:MAG is 21.
I
Ref.
Kao Corp. (Japan)
US2003lOO54082
4/26/2002
JP2001-129437
Oil or fat composition comprising the following: 5-1 OO%wt MAG and/or DAG with oxidation stability index 27, in which 15-9O%wt FA is n-3 unsaturated FA, and satisfies the following equation: 1 I (cis n-3 unsaturated FA)/(& n-6 unsaturated FA + SFA + transunsaturated FA) 56, an antioxidant, and 2 O.O5%wi phytosterol. Reduces body fat, visceral fat, and obesity.
Kao Corp. (Japan)
EPl249173
4112R002
JP2001115001 JP2002041613
Frying oil or fat composition comprising the following: Component (A) having 15-95%wt DAG, component (B) having one or more C 2 X 8hydroxycahxylic or dicarboxylic acids or salts and derivativesthereof at 7&2000 ppm, antioxidant, and silicone.
Kao Corp. (Japan)
ls2003/0044504
3/21/2002
JP2001-087241 JP2002-008884
Packaged emulsified beverage comprising the following: Component (A) having 0.1-8%wt DAC, wherein 15-9O%wt FA is n-3 unsaturated FA, and 25-95%wt of component (A) is DAG, and component (B) having phospholipids and/or I ipoproteins.
Kao Corp. (Japan)
1S2003/0072858
2/4/2002
US2002/0142089
1/2/2002
JP11-220012 JP11-239970 JP11-220012
Oil or fat composition comprising 0.1-59.8%wt TAG, 4099.7%wt DAG, 0.1-1 O%wt MAG, <5%wt FFA, wherein D A C constitutes 15-89.5%wt n-3 unsaturated C2, or larger fatty acyl and 1%84.5%wt monoenoic acyl groups, 0.575%wt n-6 unsaturatedfatty acyl groups, and 0.1-lO%wt glyceride polymer.
Kao Corp. (Japan)
US2002/0119239
12114R001
JP2000-381596
Acidic oil-in-water type emulsion comprising an oil phase containing 220%wt DAG and 0.5-5%wt crystallization inhibitor.
Kao Corp. (Japan)
EP1203534
111612001
J P2000337330
Oil-cooked or baked potatoes comprising 3-5O%wt of oil or fat which contains QY0wt MAG, 15-49.9%wt DAG constituting ,, or lower FA. 15-1 OO%wt of n-3 unsaturated C
(Continued) VI P d
TABLE 19.4
u1 P h,
(Continued) Date filed (mon/d/yr)
Priority
Disclosure
WOO2/11552 CA2418350
8/7/2001
JP2000-239573
Oil or fat composition comprising 60-1 OO%wt DAC, wherein (63, DAC comprises 15-90%wt n-3 unsaturated C,, and lower FA, 64) 10-6O%wt n-9 unsaturated FA, and satisfies the following equation: 1 2 (cis n-3 unsaturated FA)/(cis n-6 unsaturated FA + SFA+ trancunsaturated FA) 16
WOO2/11551 CA2418348
8/7/2001
JP2000-239574
Oil or fat composition comprising 5-99.9"/0wt MAC having 159O%wt n-3 unsaturated C, and lower FA, 14O%wt n-9 unsaturated FA and 2-5Oo/owt n-6 unsaturated FA 0.149.9°/0wt DAC wherein wt DAG: %wt MAG <1 and 120Y0wt PUFA having at least 4 double bonds; >O.O5%wt phytosterol, 0.02-0.5%wt crystallization inhibitor, and 0.01-5%wt antioxidant. Reduces glutamic oxaloacetic transaminase and glutamic pyruvic transaminase levels. Reduces body fat, visceral fat, and obesity.
App Iicant
Publication no.
Kao Corp. (Japan)
Kao Corp. (Japan)
Ref.
(65, 66)
Kao Corp. (Japan)
WOO1/15542
8/21/2000
JP111243432
Water-in-oil type emulsified fat and/or oil composition comprising: (81) Component (A) having 35-95%wt DAG with melting point 4 0 ° C and the balance of TAG having 13&o%wt palmitic acid 2i'70wtC,, or lower FA, and TAG polymorph in p'form, and and Component (B) having an aqueous phase based on water.
Kao Corp. (Japan)
EP1211305
7/6/2000
JP22001299
Oil of fat composition comprising 0.1-59.8o/owt TAG, 40-99.7Y0wt (75) DAC, 0.1 -1 O%wt MAG, 15YOwtFFA, and 0.1 -1 0Y0wt glyceride polymer, wherein DAC comprises 1549.5o/owt n-3 type unsaturatedC ,, and larger FA and 10-84.5%wt monoenoic fatty acyl groups, and 0.5-75Y0wt n-6 type unsaturatedfatty acyl groups.
P
5
P
%.
%
c
Kao Corp. (Japan)
W001/01787 CA2373832
6/30/2000
JP111184762
Water-in-oil type emulsified fat and/or oil composition comprising: Component (A) having aqueous phase based on water, and component (B) having 15-9O0/owt DAG, 85-1 0Y0wt TAG, 0.1-1 OY0wt flavor component(s), and at least one demulsifier capable of phase reversing >3o%wt of emulsified composition within 1 min after being introduced in water at 36°C.
Kao Corp. (Japan)
WOOOl78162
6/9/2000
JP11/170849
Acid oil-in-water type emulsified composition comprising an oil phase having >3oo/owt DAC, a phytosterol, and a yolk, wherein the ratio of 1ysophospholipids:wholephospholipids 215% of phosphorus content.
Kao C o p (Japan)
EP1186648
6/5/2000
JP16940499 jP29530299
Oil or fat composition comprisingb l 5Y0wt DAC, a FA L-ascorbic
(82, 83)
ester, silicone, and a component selected from among catechin, rosemary extract, sage extract, and tumeric extract, wherein the ash content of catechin is <5%wt.
Kao Corp. gapan)
US6,495,536
10/26/1999
JP11-237556
Oil or fat composition comprising b35%wt DAG, 20.05Y0wt phytosterol, and satisfies the folllowing equation: (cisunsaturated FA)/(SFA + trancunsaturated FA) 26, wherein trans unsaturated and saturated FA b5%wt. Oil or fat composition comprising 28O%wt DAC, bO.O5%wt phytosterol, and satisfies the following equation: (cic unsaturated FA)/(SFA + transunsaturated FA) 26, wherein trancunsaturated and saturated FA 15%wt. Reduces PAIL1 activity. Increases HDL cholesterol levels.
Kao Corp. (Japan)
WO99159424
5/21/1998
-
Fried food containing fat composition comprising 55%Wt SDAG <95%wt, wherein DAC comprises 55Y0wt Sunsaturated FA <93%wt. Fat composition comprising S2%wt MAG, 55%wt (Continued
u P l P
TABLE 19.4 (Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure SDAG <95°/0wt, 5°/0wt STAG <45O/owt, I 100 ppm phospholipids, and satisfies requirements (i),(ii), and (iii):(i) DAG comprises 55'0wt I unsaturated FA <8O0/owt,(ii) DAG comprises 0 . 5 % ISS ~ <20%wt, 2 0 % S~ U <55%wt, and 25o/owt I U U <70%wt, wherein S is C,,-C,, saturated FA group, U is C-, C, unsaturated FA group and SS + YoSU + %UU = 100; (iii) ,, + C22:o)in DAG weight ratio of (C14:o+Cleo) to (CIRo + Co is 1.0-8.0, where S represents C,,-C,, saturated FA, and U represents Cl4-C,, unsaturated FA.
Kao Corp. (Japan)
W002/11550 US2003/198727
8/7/2001
JP2000-239575
Oil or fat composition comprising 10.1-94.9%wt TAG, 0.13o%wt MAG, and 5-59.9%wt DAC as follows: (i) DAG comprises 15-90%wt n-3 unsaturated C , , and lower FA; (ii) DAG:MAG (1; (iii) peroxide value (POV) 5 10; and (iv) color (10R+Y) 5 25.
Kao Corp. (Japan)
US2002/0045000
7/19/2001
JP2000-218813
Oil or fat composition comprising 15-95%wt DAG, 2-1 O%wt phytosterol having C,&, hydrocarbon content of I 1"/.wt, wherein oil is transparent liquid at normal temperature.
Kao Corp. (japan)
EP1135991 US6,448,292
3/21/2001 3/16/2001
JP200W78110
Oil or fat composition comprising l0Y0wtlDAC < 4o%wt having 155%wt unsaturatedfatty acyl groups and 15-1 00Y0wt thereof are n-3 unsaturatedC,, or larger fatty acyl groups, and 40.1 %wt
Ref.
P
i
Kao Corp. (Japan)
woo1/13733 US20030096867
8/23/2000 9117/2002
JPlll237556
Kao Corp. (Japan)
US6,326,050 US6,139,897 EP0990391
8/28/2000 211711999 211711999
JPlC75898 JPlCO75898 JP7589898
Kao Corp. (Japan)
WO99l5942 2
998 512 1/I
-
Water-in-oil type emulsified fat or oil composition comprising an oily phase and an aqueous phase, wherein the oily phase comprises 4o%wt SDAC <95%wt, 5%wt a A G <6O%wt, satisfies requirements (i)and (ii): (i)D A G comprises o.5°/~wt S S <20%wt, 20'26wt S U <55%wt, and 25%W I U U <7oy0wt, wherein S is C14-C,, saturated FA group, U is C14-C,, unsaturated FA group and %SS + %SU + %UU = 100; (ii)weight ratio of (C,40 + C,&,) to (C,,,, + C,,:, + C,,:,) in D A C is 1. W . O ; (iii) D A C having melting point from 20 to <50"C, where S represents C14-C2, saturated FA and U represents C,,-C,, unsaturated FA.
Kao Corp. (Japan)
US6,287,624
31611998
lP9-057793
Food composition comprising a food and 2-8.5Y0wt DAG, wherein D A G comprises capric acid. Food composition comprising a food and 4-85Y0wt DAG, wherein D A G comprises capric acid. Food composition comprising 0.5-85%wt DAG, wherein D A G comprises C,-C,, FA.
413011998
-
Oil or fat composition comprising 215%wt D A G and 1.24.7%wt phytosterol, wherein D A G comprises >55o/owt unsaturated FA and weight ratio of D A G to phytosterol is 1C200. Reduces blood cholesterol levels.
EP0970615
Kao Corp. (Japan)
US6,025,348
Oil or fat composition comprisingat least 35%wt D A G having FA satisfying the following equation: (cisunsaturated FA)/(SFA + transunsaturated FA) 2 6 and phytosterol content 10.05%wt. Reduces plasminogen activator inhibitor type I (PAI-1) activity and increases H D L cholesterol levels.
Oil or fat composition comprising 15-95%wt D A G having
t 27o%wt unsaturated FA, 1.2-20%wt phytosterol and 22000 ppm tocopherol. Reduces hemal cholesterol levels.
UI P
(Continued
UI
TABLE 19.4 (Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Ref.
.2 F
2. Maurel Sante (France)
US6,129,924
6/30/1997
FR9608263
Organometallic complex comprising: (i)cation of a metal in oxidation state of 2-5; (ii)sitosterol and/or sitostanol, and (iii)1,2-DAG having C18:, in sn-1 and linear or branched, saturated or unsaturated C+ ,, FA in sn-2 positions. Method for improving bioavailability of metal cation in a mammal via organometallic complex. Method for treating and preventing genetic or acquired deficiencies or dysfunctions of enzymatic systems necessitatingthe presence of metal cations as catalysts of biochemical reactions.
Loders-Croklaan B.V. (The Netherlands)
~55,891,495
4/25/1995
EP94303170
lcecream coating fat composition comprising 5&9OO/0wt DAG and 5&1 O%wt vegetable TAG, wherein: (i) DAG comprises 1&25%wt SU, 75-9O%wt UU and <5%wt SS; (ii) vegetable TAG comprises (UUU + SUU) 250%wt; (iii) fat comprises SFA of 5-35%wt; and (iv) N line of N,, <35, N, 40, and No >35, where S represents Cl,-C,, SFA and U represents C18:,.
(93)
Loders-Croklaan B-V. (The Netherlands)
US5,879,735
21211 995
EP94301161 EN4301734
Water-in-oil type emulsified fat composition comprising 1@80Y0wt of an oily phase and 90-2O”kOwtof an aqueous phase, wherein the oily phase comprises: (i)3&7o%wt DAG as structuring fat in which 25-70“/0wt is SU, 1&7o%wt is UU, and <3o”/owt is SS and (ii) 7@-3o%wtTAGin which 1-700/0wt is SSU, total SFA is <50“!wt, and satisfies the following equation: C,/(N, x SFA) x >3, where S represents C,-€, saturated FA, U represents C, and larger unsaturated FA, C, represents hardness of fat blend at 5”C, and N, represents solid fat content at 5°C.
(94)
Loders-CroklaanB.V. US5,912,042 (The Netherlands)
21211995
Em4301 162
Fat composition comprising: (i) l0-6Oo/0wt DAG, wherein DAG comprises 27O0/0wt SU; (ii)9&4O%wt TAG, wherein TAG has N, 410;(iii)a high melting DAG with melting point >40”C; and (iv) at least one other DAG or TAG so that the melting point of (iii)and (iv) is 25°C lower than that of (iii), and SFA <4o%wt and N, <15 in total fat blend.
(95)
Fat composition comprising 10@-50%wt (A) and &5o%wt (B), wherein: (A) comprises 1@-5o%wt MAG, 25-55%wt DAG, >1 O%wt TAG and having hydroxyl number of 9@-190 and IV of 40-90, produced from hardened and unhardened vegetable fats, and (B) 5-20%wt MAG, 4&6oy0wt DAG, 2&5O”/owt TAG and having hydroxyl number of 50-1 90 and IV <30, produced from hardened vegetable oils.
(96)
Aarhus Oliefabrik A h (Denmark)
US6,217,874
311311992
DKO467191
9 if: 2
F2. T
8 P 3: 4 2 s
r)
3
0,
B
wl P U
l P n
m
TABLE 19.5 A 5-Year Patent Literature Survey on Processes, Methods, and Compositions Involving MAG
App Iicant
Publication no.
Date filed (mon/d/yr)
Rinoru Oil Mills Co. Ltd. (Japan)
lP2003113396
4/24/2002
Composition comprising a MAG having 250% conjugated FA. Method for producing said MAG comprising subjecting FFA, including conjugated FA and glycerol, to an esterification and/or glycerolysis in the presence of a lipase.
Kao Corp. (Japan)
JP2003049192
8/8/2001
A low-cost method for producing MAG comprising transesterifying a glycerol FA ester with glycerol in the absence of any catalyst by keeping an acid value in the reaction system 21 mg KOH/g.
Kao Corp. (Japan)
J P2003034668
-
-
Novel MAG with an ether carboxylate moiety.
Nestle SA (Switzerland)
AU762712
311211999
EP19980104598
Food compositioncomprising a MAG having 90% monoolein and the remainder being monolinolein and/or a saturated MAG.
Kao Corp. (Japan)
JP2003252829
7118/2001
Eurocine A6 (Sweden)
EP1154792
2/9/2000
Priority
Disclosure
A low-cost method for producing MAG directly from glycerol and FA in the absence of a catalyst comprising mixing the substrates with a stirring blade wherein the peripheral speed of the tip of the blade is 3 m/s and the number of passes is 2 0.1 timeh. WO2000EP01046
Composition for tuberculosis vaccine comprising at least 80% MAG and unsaturated FA having CbX24.
Ref.
Cortecs Ltd. (Korea)
KR2000022353
-
GB19960013858
A hydrophobic preparation for pharmaceutical delivery system comprising an oil phase containingone or more medium-chain MAG, at least one amphiphile, and a hydrophilic species.
(102)
Rikebita Malaysia Sdn. Bhd. (Malaysia), Riken Vitamin Co. Ltd.
JP2001031989
7/7/1999
-
MAG powder composition, which is flowable and mixes well into a powdery premix, comprising 85-65%wt saturated MAG, wherein at least 6O%wt is palmitic acid MAG, and 15-35%wt unsaturated MAG, wherein 55-72%wt is a transisomer and 45-28%wt is a cis-isomer. Method for producing said MAG comprising transesterification of glycerol with an oil prepared by direct partial hydrogenation of at least one oil selected from among palm oil, palm stearin, palm olein, and their fractionation oils.
(108)
Huangma Chemical Group Co. Ltd. (China)
CN1283608
-
CN20000125114
Process for producing MAG from epoxy chloropropane and FA involving a KOH-catalyzedopen-loop dewatering reaction comprising addition of water, conducting hydrolysis, vacuum dehydration, and filtration.
(109)
Nestle SA (Switzerland)
EP1008305
12/10/1998
EP19980123554
An aroma product comprising dissolving in water amino acids peptides or hydrolyzed proteins and reducing sugars or dissolving in water aroma precursors, introducing in said solution a C, and C,, MAG, heating the mixture to a mesomorphic phase structure of a microemulsion, maintaining said heating to develop flavoring compounds, and cooling said mixture.
(99)
Procter & Gamble (US.)
W09920111 CA2306913 EPlO24703
10/16/1998
US19970062849P US19971020
Emulsifier-lipidcomposition comprising: (i) 2-50% MAG; (ii) 0.5- (100) 40% polyglycerol ester; and (iii)[email protected]% fat component.
(Continued)
P
cn
0
TABLE 19.5 (Conti nued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Opta Food Ingredients Inc.
US5935828
51111989
US19890345622
Process for producing MAG containing n-3 FA, comprising lipase-catalyzed transesterification of TAG followed by a low-temperature crystallization.
Global Palm Products Sdn. Bhd. (Malaysia), Hai Loo Trading Re. Ltd. (Singapore)
EP1051386
12/10/1998
WO1998SG00101 US19970990004
Process for producing MAG by glycerolysis of methyl ester derived from animal or vegetable oil or fat with glycerol, wherein the ratio of glycerol to methyl esters is from 0.1 to 3 mol, at 130-1 60°C and vacuum of 200-400 mbar, in the presence of alkaline catalysts.
Abbott Laboratories
US5912372
7/29/1997
US19970902366
A water-miscible lipid derivative effective in inhibiting the infectious activity of pathogenic microorganisms comprising diacetyltartaric acid esters of MAG, wherein at least 90% of FA content is accounted for by a single FA.
Cargill Inc. (U.S.)
US5859270
3/13/1996
US19960614468 US199603 13
Method for preparing purified MAG comprising: (i)adding TAG to the crude MAG composition and (ii)extraction involving the alcohollwater phase.
Alza Corp. (U.S.)
W099/32153 AU1728099
1211711998
US19970068411P
Compositions, devices, and methods for transdermal adrninistration of a drug using a permeation enhancer mixture comprising a MAG and ethyl palmitate.
(U.S.)
(U.9
Ref.
TABLE 19.6 A 5-Year Patent Literature Survey on Processes, Methods, and Compositions Involving PUFA
Applicant
Publication no.
Date filed (mon/d/yr)
Laxdale Ltd. (U.K.)
US6689812
Martek Biosciences Boulder Corp. (US.)
Ref.
Priority
Disclosure
12114/2001
US20010014603 CB19990001809
Pharmaceutical composition for the treatment of a psychiatric or central nervous disorder comprising at least 90% FA, wherein at least 90% is EPA and <5% is DHA
US6541049
211912001
US20010789057
Process for producing arachidonic acid using Mortierella sect. schrnuckeri.
DSM NV (The Netherlands)
US6638561 W09965327 AU757616 TR200003712T
1211711999
EP19980304802
Marine feed composition comprising arachidonic acid or TAG containing arachidonic acid derived from Mortierella genus for promoting growth and pigmentation in marine organisms.
124)
Alpha Food Ingredients Inc. (U.S.)
US6608222
11/21/2001
US20010001413
Composition and method for supplementing feed, nutrition, and diet systems with glycerides of CLA.
125)
Pronova Biocare AS (Noway)
CB2388026 WOO3092673
4/2/2003
CB20020010212 CB20020503
Use of EPA and/or DHA in the preparation of an oral medication for preventing cerebral damage in patients having symptoms of atherosclerosis.
Alcon Inc. (Switzerland), Biodar Ltd. (Israel)
WOO3082313
3/28/2003
US20020368301P
Beadlet formulation useful for inclusion in dietary supplements for improving and maintainingocular nutrition comprising DHA, rosemary, and/or its components. (Continued)
TABLE 19.6 (Continued)
Applicant
Publication no.
Date filed (mon/&yr)
Priority
Disclosure
Kyle, D.J. (U.S.)
WOO3079810
311912003
US20020365182P
Aquatic animal feed containing high amounts of arachidonic acid produced from microalgae.
Martek Biosciences Corp. (U.S.)
EP1342787
11311996
EP19960904435 EPI9960103
Method for producing arachidonic acid-containingoils, which are substantially free of EPA, comprising: (i)cultivating Mortierella alpha to obtain TAG oil containing arachidonic acid and (ii)extracting and recovering said TAG oil.
IPK Inst. Fuer Pflanzengenetik (Germany)
WOO3012092
7B 1 I2002
DE20011037374
Method for production of arachidonic acid in transgenic organism, Phytophthera megasperma.
Protarga Inc. (U.S.)
US6602902
5/1/2001
US20010846838 US20010501
Conjugates of cisDHA and pharmaceutical agents useful in treating noncentral nervous system conditions.
US2002137796
312011998
US20000381484
Method for enhancing growth of preterm infants involving administering a combination of DHA and arachidonic acid to the infant in the form of an infant formula containing said DHA and arachidonic acid.
W099/08509
811411998
US60/055,765
Method for increasing n-3 highly unsaturated FA in poultry meat comprising feeding a feed to poultry, wherein said feed comprises: (i)a source of said n-3 highly unsaturated FA (ii)percentage of n-3 highly unsaturated FA in feed is higher
Omegatech Inc.
(U.S.)
Ref.
in a later phase of said poultry's production period than in an earlier phase; and (iii)an amount of any low-quality n-3 highly unsaturated FA source oil is <2%wt of feed. Janiftec Inc., Asahi Food Processing Co. Ltd. (lapan)
US5976606
3R 1/1997
JP19960199969
Process for producing DHAcontaining tofu or soybean milk drink or a dry powder thereof, comprising mixing soybean milk with a DHA-containing fish oil.
Nippon Suisan Kaisha Ltd. (lapan)
EP1340427
11/12/2001
W02001)PO9879 W02001JP11112 JP20000344863 JP20001113
Dairy products containing EPA andlor DHA having oxidation and emulsification stability comprising emulsifying an acidified milk containing EPA and/or DHA as a fish oil.
Omegatech Inc.
EPll88383 US2002037561
1/16/1996
EPl9960200072 EP19960116 US19950377766 US19950124
Process for the production of arachidonic acid comprising culturing Mortierella schrnuckeri in a medium.
US6372460
713 111998
US2000052902 1
Particulate material suitable for use as a nutritional supplement, as aquaculture feed, comprising up to 35% or more of DHA, and having a particle size of 5-1 0 pm.
-
CN20010127722
Process for producing nutrient DHA-containing powder comprising: (i) production of DHA-rich egg; (ii) separation of yolk from said egg; and (iii)spray-dry said yolk into powder.
(US.)
Martek Biosciences (U.S.)
South China Science CNl335094 & Engineering (China)
National Institute of Advanced Industrial and Technology (Japan)
jP2002291464
3/29/2001
Microorganismconsists of Labyrinthla sp. CHN-1, which produces arachidonic acid in a culture medium.
(Continued) P 01 W
P
m
P
TABLE 19.6 (Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Tama Seikagaku KK (Japan)
JP2002136298
11/1/2000
-
Method for efficiently and selectively producing an acyl glyceride containing DHA at 60% or more by using a 0.1-1.5 M buffer.
Quatex NV
EPll57692
5/22/2000
EP20000110811 EP20000522
Composition for treatment and/or prophylaxis of multiple risk factors for cardiovascular diseases and/or other pathologies sensitive to EPA and DHA comprising at least 8O%wt EPA and DHA, where EPA:DHA is between 0.9 and 1.5, and other n-3 C-C ,, FA are <3%wt.
Nihon Shokken Co. Ltd. (Japan)
JP2001061345
8/27/1999
-
Method for cultivating radish and bean sprouts containing high amounts of DHA and EPA acids by soaking the roots of the radish and bean sprouts in an oil containing DHA and EPA.
Ryu, S.G. (Korea)
US6207441
12/10/1999
KR19990014064
Method of producing DHA using Pseudornonas sp. YS-180.
Abbott Laboratories (US.)
US6,200,624
1/26/1996
-
An enteral formula comprising a fat in an amount between 20 and 45 g/L of formula, wherein fat includes a TAG-containing ingredient derived from egg, wherein TAG have moieties in sufficient quantities to provide 0.05-0.5?'0wt DHA and 0.12%wt arachidonic acid, <0.1 %wt phosphorus, and <5%wt cholesterol.
Nestec S.A (Switzerland)
US6,297,279
1/18/2000
EP97202289
Process for preparinga lipid composition comprising: (i)a mixture of lipids containing palm oil enriched with palmitic acid, a vegetable oil high in linoleic and a-linolenic acids, an oil which is a source of arachidonic acid, and oil which is a source of DHA, in defined proportions, is nonregiospecifically interesterified to obtain an FA composition having a random
Ref.
P (122)
3
distribution of FA residues between sn-1, sn-2, and sn-3 positions of TAG; (ii) composition in (i) is interesterified with a mixture of FFA predominantly comprising medium-chain FA and oleic acid, using a 1,3 regiospecific lipase; and (iii) excess FFA are removed from the composition first by partial deacidification with steam distillation under vacuum, and then by controlled neutralization of the partially deacidified composition. Suntory Ltd. (Japan)
Sobremar S.A. (France)
Nestec S.A. (Switzerland)
Martek Corp. (U.S.)
EP1035211 CA2308065 WOO01 2 744 JP2000069987
8/27/1999
W019990827
EP0784694 IL117251
US6,034,130
EP1001034
JP19980243583 JP19980828 W01999JPO4653 FR19950002153
711311998
1/22/1992
EP97202289
EP19920904428 EPl9920122
Process for producing arachidonic acid-containinglipid comprising culturing a mutant Mortierella having inactivated the activity of n-3 unsaturation and capable of producing
(118)
9
arachidonic acid.
;d
Process for obtaining PUFA comprising hydrolyzing fish oil using a nonposition-selective lipase to obtain FFA, MAG, and DAG, and purifying by complexing said FA with urea, and decomplexing the isolated FA by interesterification between the concentrated PUFA with the crude oil by using a lipase.
(143)
I.
2 8
w
6.
Synthetic lipid composition in which the content and distribution (144) of FA mimic those of human milk fat, comprising 4 % w t FFA and FA moieties of TAG as follows: (i) 35-55Ynwt SFA, 1&36% palmitic acid, 240%wt caprylic and capric acids, 51O%wt lauric acid and 51O%wt myristic acid; (ii) 3@45%wt monounsaturated F A and (iii)9-22Ynwt PUFA which comprises <2%wt long-chain n-6 PUFA comprisingarachidonic acid and <1 %OM longchain n-3 PUFA comprising DHA; the n-6:n-3 FA is 5:l to 15:1, palmitic acid i s predominantly at the sn-2 position of TAG, and arachidonic acid and DHA are distributed between the sn-1, sn-2, and 511-3 positions of TAG. Process for producing arachidonic acid containing oils using Pythium insidiosum.
2 rn
n 2 5.
0,
3
(120)
(Continued ul
P
01 01
TABLE 19.6 (Continued)
Applicant
Publication no.
Martek Biosciences Corp. (U.S.)
EP0800.584 W09621037 F1972829 EP0800584 DE800584T
Color Access Inc. (US.)
WOO103665 CA2342802 EP1109.528 US6171605
Date filed (mon/d/yr)
Priority
Disclosure
Ref.
1/3/1996
US19950367881 US19950103 W01996US00182 W019960103
An oil of fungus Mortierella sp. comprising at least 40% arachidonic acid, and no more than one fifth as much EPA as arachidonic acid.
(119)
7/7/2000
US19990349913
Self-tanning composition comprising an effective amount of DHA and propolis extract.
(140)
P
& P
e. 2 ru
Central China Science & Technology (China)
CN1323904
CN20000114.562
A fermentation method for preparing arachidonic acid cornprising: (i)preparing and carrying out a seed expansion culture comprising bean sprouts juice, formula of seed substratum, and seed cultivation conditions; (ii)preparingand carrying out a fermentation culture comprising corn saccharified liquid, fermentation substratum and fermentation conditions; and (iii) extraction, filtration, thallus’ collection, dehydration, and solvent extraction to obtain arachidonic acid.
(139)
Prime Europe Therapeuticals 5. (Italy)
W09856883 NZ501690
W01998EP03466
Composition of wax esters for use in therapy of cardiocirculatory diseases, thrombosis, platelet hyperaggregation, hyperlipidemia, hypercholesterolemia, inflammation, cancer, and diseases of the immune system, comprising >12% DHA and > 18% EPA.
(141)
Loders Croklaan B.V. EP1013178 (The Netherlands)
Abbott Laboratories (US.)
12/9/1999
NZ331084
Em8310627 EP98310626 EP99302067
Edible composition containing petroselinic acid for the prepara- (142) tion of functional food compositions or food supplements, wherein the said composition is used as an anti-inflammatory component that inhibits the production of metabolites of arachidonic acid and/or reduces the formation of intracellular adhesion molecules or is used as an antiaging component with a positive effect on skin conditions such as wrinkling, sagging, photo-damaged skin, dry skin, flaky skin, and age spots.
US19960592832
Enteral formula or nutritional supplement containing arachidonic acid and DHA and production method thereof.
(149)
Beruricchi Sugou KK JP2000217512 Uapan)
1/29/1999
-
DHA capsule capable of giving taste resembling normal boiled rice comprising adding an edible oil and an antioxidant into a suspension of purified DHA-containing oil and a citrus juice.
(145)
University of Cuelph NZ333892 (Canada) W09749297 EP0906031
-
W01997CA00430 WO19970620 US19960020221P
Method for producing DHA-enriched milk comprising feeding a feed containing DHA and feathermeal inhibitors of microbial degradation of DHA.
(146)
JP19860071270 jP19860331
Method of producing arachidonic acid-containing lipid comprising the use of Mortierella elongate SAM0219 or Mortierella exigua lF08571.
(121)
Method for obtaining arachidonic acid-containing lipid cornprising: (i) drying viscera of livestock at -20 to 70°C until moisture content is 2 1 0 % ~ (ii) ; solvent extraction using ethanol and/or hexane in an amount of 3.5 times dried raw material and at 30-7OoC, distilling off solvent under high vacuum of 5-30 mmHg.
(147)
Suntory Ltd. (Japan)
CAI 340433
Bizen Kasei KK Clapan)
JP11035587
P
TABLE 19.7 A 5-Year Patent Literature Survey on Processes a n d Methods Involving Phytosterols and Phytostanols
Applicant
Publication no.
Date filed (mo/d/y)
Priority
Disclosure
Harting (Chile)
EP1285969
8/8/2002
CL20012044
Process for producing steryl esters comprising: (i)conducting lipase catalyzed alcoholysis between phytosterols and/or phytostanols and FA esters of C,<, aliphatic alcohols; (ii) separating of lipase; and (iii) separating steryl or stanyl esters from reaction mixture by distillation, wherein FA is C ,< ,, lipase is derived from Pseudomonas genus, reaction pressure is <300 mbar, and reaction temperature is 3&90"C.
Novartis Nutrition WO02/100412 AC (Switzerland)
6/7/2002
GBOll4014.4
Process for dispersing phytosterols in oil comprising: (i) mixing together oil, phytosterols, FFA, and phospholipid and (ii)heating the mixture to a temperature below melting point of phytosterols, wherein phospholipid comprises a phosphatidylcholine or lecithin. Dispersion comprises: (i)20.3%wt phytosterols; (ii)20.3%wt FFA; (iii) t 0.1 5%wt phosphatidylcholine or L0.6'Y0wt lecithin.
Danisco Sugar OY (Finland)
US2002/0183530
1/4/2002
Fig91533
Process for purifying phytosterol concentrates comprising contacting phytosterol concentrate having phytosterol content of 2&98% with a pressurized fluid wherein: (i)pressurized fluid is mainly CO,; (ii)temperature is -20 to 180°C; and (iii) pressure is 50-600 bar.
Lipton, Div. of Conopco (U.S.)
US2003/0108591
8/1 Ol2001
-
Method for lowering b l d cholesterol in humans comprising ingesting a composition comprising: (i)at least one vegetable protein, (ii) at least one phytosterol, and(iii) at least one isoflavone.
Procter & Gamble co. (U.S.)
US2002/00982 18 WOOO/69404
11/26/2001 5/4/2000
US601134,397 USO9/439,438
Method for (i) regulating the condition of mammalian keratinous tissue; (ii) thickeningthe skin and preventing, retarding, and/or treating skin atrophy of a mammal; (iii) preventing, retarding,
o\
co
Ref,
and/or treating the appearance of dark, under-eye circles and/or puffy eyes; (iv) preventing retarding. and/or treating sallowness of mammalian skin; (v) preventing and/or retardingtanning of mammalian skin; (vi) desquamating, exfoliating, and/or increasing turnover in mammalian skin, (vii) regulating and/or reducing the size of pores in mammalian skin; (viii) regulatingthe oily and/or shiny appearance of mammalian skin; (ix) preventing. retarding, and/or treating post-inflammatory hyperpigmentation; (x) preventing and/or treating the appearance of cellulite in mammalian skin, comprising of topically applying to the skin a safe and effective amount of a composition comprising: (A) one or more phytosterols selected from the group consisting of psitosterol, campesterol, brassicasterol, A5-avenasterol, lupenol, a-spinasterol, stigmasterol, and their derivatives and (6)a dermatologically acceptable carrier for the phytosterol. fnzymotec Ltd. (Israel)
Forbes Medi-Tech
Inc. (Canada)
W001/75083
W002/34241
4/3/2001
10/25/2001
lL135466
US09/985,749
Process for producing a fat containing sterol FA esters comprising: (i)conducting immobilized lipase-catalyzed selective alcoholysis between free sterol and a fat in a microaqueous environment and (ii)removal of immobilized lipase upon completion of reaction, wherein immobilized lipase has high sterol-specific alcoholytic and/or esterification activity and minimal acidolytic and transesterificationactivities, and may be surfactant-coated.
(1 52)
Method for decreasing weight gain in humans comprises adminiistering one or more sterol and/or stanol derivatives having the following formula:
(160)
T
2 c:
-9. Q
2
3 3
0,
wherein R is a phytosterol or phytostanol moiety, R2 is derived from ascorbic acid, and R3 is H or any metal, alkali earth metal, or alkali metal, and salts thereof. (Continued)
8-.
P Q,
a
P
TABLE 19.7 (Continued)
u
0
Applicant
Publication no.
Date filed (mod&yr)
Priority
Disclosure
Forbes Medi-Tech. Inc. (Canada)
US2002/0156051
7/25/2001
US09/339,903
Method for treating and preventingcardiovascular disease and its underlying conditions including arthrosclerosis and hyperlipidemia in humans comprising administeringone or more sterol and/or stanol derivatives having the following formula:
Ref.
0
0 II
I1
Rz-P-OR
I
OR3
Rz’
C ‘OR
9,
(161)
I?
R2
wherein R is a phytosterol or phytostanol moiety, R2 is derived from ascorbic acid and R, is H or any metal, alkali earth metal, or alkali metal, and salts thereof. Kao Corp. (Japan)
Kao Corp. (Japan)
WOOl/32682
W001/32681
11/2/2000
11/2/2000
JP11/313619 JP11/313620
Process for purification of phytosterol comprising: (i)contacting (168) a crude FA product from vegetable oil/fat containing phytosterol with a mixture of organic solvent and water to crystallize and separate phytosterol from the mixed solvent or (ii) mixing a crude FA ester from vegetable oil/fat containing phytosterol and FA ester of a lower alcohol, allowing it to stand at 140°C to precipitate crystals including FA esters, and separating the crystals to take the lower alcohol solution including the phyto sterol, wherein water in mixed solvent is tl%wt, crude FA ester contains t20%wt C&, FA esters of lower alcohols, organic solvent has dielectric constant 217 at 25”C, and vegetable fat is palm kernel, coconut, or palm oils.
JP11/313618
Process for producing purified phytosterols comprising: (i) saponification of FA esters in crude phytosterol composition with an alkali in a mixed solvent of lower alcohol and water until acid
(169)
-.
2
nJ I
value 510; (ii) cool reaction solution to precipitate crystals of phytosterols; and (iii) separation of phytosterol crystals. Cognis Deutschland W000/47570 GmbH (Germany)
21412000
DE19906551.9
Process for producing phytosterols comprising interesterifying residues from the production of methyl esters with methanol, using an alkaline catalyst, neutralizing the catalyst, and separating the unreacted alcohol.
W.K. Kellogg Institute (U.S.)
US6,228,407
1112211999
US601109,773
Method for improving shelf life of a food product comprising: (i) combining an edible diluent fat with 2-95%wt phytosteryl ester to form a plasticized blend agent (ii)adding said plasticized blend agent to a formulation of a f w d product in an amount sufficient to improve shelf life of food product; and (iii)preparing said food product.
Eugene Science Inc. (Korea)
WOO0161694
912 1I1 999
KRl999112965
Process for producing phytosterol and phytostanol unsaturated FA ester comprising: (i)esterification of phytosterol or phytostanol with unsaturated FA in the presence of a basic catalyst and a carboxyl group activating agent, in nonpolar organic solvent and (ii) precipitation of esterified product in methanol or mixture of methanol and acetone, after filtering the esterified product and evaporating the nonpolar solvent under reduced pressure.
B.C. Chemicals Ltd. (Canada)
WO99142471
211911999
CA2,230,373
Process for preparing phytosterols from tall oil pitch containing phytosteryl esters comprising: (i) converting phytosteryl esters to free phytosterols; (ii) removing light ends from (i) by evaporation to produce a bottom fraction containing free phytosterols; (iii)evaporating bottom fraction to produce light phase distillate containing free phytosterols; (iv) dissolving light phase distillate in an alcohol to produce a solution; (v) cooling the solution to produce a slurry containing crystallized free phytosterols; and (vi) washing and filtering the slurry to isolate crystallized phytosterols. (Continued
P N u
TABLE 19.7 (Continued)
Applicant
Publication no.
Date filed (moddlyr)
Priority
Disclosure
Diarmuid Joseph Long (Ireland)
GB2372990
3/9/2001
-
Process for producing phytosterol FA ester comprising: (i) purifying phytosterol mixture to obtain enriched concentration of sitosterol and reduced concentrations of campesterol and other sterols and (ii)interesterification between the purified phytosterol mixture with FA methyl esters to obtain phytosteryl esters, wherein sodium ethoxide is used as interesterification catalyst.
(153)
Method for modulating phytosterol compositions in plants comprising recombinant double-stranded DNA which comprises: (i) a promoter that functions in plants to cause the production of an RNA sequence, operably linked to (ii) a DNA coding sequence, which encodes an enzyme that binds a first sterol and produces a second sterol, operably linked to (iii) a 3’ nontranslated region that causes the polyadenylation of the 3’ end of the RNA sequence, wherein the promoter is heterologous with respect to the DNA sequence.
(167)
Ref.
P
Asgrow Seed Co., L.L.C. (U.S.)
US2002/0148006
2/8/2001
-
3
P
2. 2
y
Forbes Medi-Tech Inc. (Canada)
WOOO/64921
4/27/2000
us09/300,135
Process for isolating and purifying phytosterols from a wood or plant source comprising: (i) obtaining a concentrated extract of phytosterols and a hydrocarbon; (ii)adding to extract a metal salt which complexes with the phytosterols; (iii) separating phytosterol complex from the hydrocarbon; (iv) washing the complex with a solvent mixture comprising a hydrocarbon andlor a ketone; (v) hydrolyzing the washed complex; and (vi) separating the phytosterols therefrom.
Medical Isotopes Inc. WO99118977 (U.S.)
10/14/1998 US60/062,968
Method of treating cholesterolemia by administering a composition comprising at least one glycoside or glycoside ester of a steryl glycoside selected from the group consisting of psitosteryl-P-D-glycoside, stigmasteryl-P-D-glycoside, and campesteryl-Pa-glycosideand saturated steryl glycosides, wherein said steryl glycoside is dissolved or dispersed in a solubilizing macromolecule.
Raisio Benecol Ltd. (Finland)
412211998
Method for producing a fat blend comprising providing a solid fat comprising a hardstock and combining a liquid oil with the solid fat the improvement comprising reducing the ratio of hardstock to liquid oil in the fat blend while maintaining the texture of the fat blend of at least one phytosterol FA ester and the hardstock to produce an improved fat blend, wherein said hardstock is present in an amount of 21 5%wt.
US6,162,483
US09/010,2 1 1 USO8/740,845
P W u
P
u
P
TABLE 19.8 A 5-Year Patent Literature Survey on Compositions of Phytosterols and Phytostanols
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Cargill Inc. (U.S.)
W003/022064
91612002
US601318,162
Composition comprising 21O h phytosteryl FA ester wherein: (i) FA moiety is 80-95% oleic acid; (ii) phytosterol moiety is derived from soy or tall oil sterol; and (iii)steryl ester has a melting point of 17-35°C. A confectionery product comprising the composition. A method for producing steryl esters comprising transesterification of a blend of FA methyl esters having 2 80% oleic acid moiety with soy phytosterols.
Lonza AG (Switzerland)
W003/009854
4/26/2002
EPOl 117587.4 US60/358.808
Composition comprising: (i) at least one phytosterol or phytostanol, or ester derivative thereof and (ii)carnitine or acylcarnitine or salt thereof, wherein phytosterol or phytostanol ester comprises an n-3 PUFA moiety, preferably DHA or EPA. Composition used for treatment of hyperlipidemia, reduction of serum cholesterol and serum TAG.
Nisshin Oil Mills Ltd. (Japan)
WOO21065845
1118/2002
JP2001-12330
Phytosterol-containing fat composition having improved flavor produced by deodorizing, at 10&270"C, a feed material mixture comprising an edible fat and 1-5O%wt phytosterol.
Nisshin Oil Mills Ltd. (Japan)
WOO21060271
111812002
JP2001-12331
Phytosterol-containing edible fat composition with inhibited phytosterol precipitation during storage and effectively inhibits cholesterol absorption comprising: (i) an edible fat comprising l % w t C,&.; (ii)I - 1 0 % ~phytosterol; (iii) 0.01-1%wt tocopherol; and (iv) 0.005-1 O%wt oleophilic emulsifying agent.
Nisshin Oil Mills Ltd. (Japan)
W002/060270
1/1812002
lP2001-012329
Phytosterol-containing fat composition with inhibited phytosterol precipitation during storage Comprising: (i)a phytosterol-
Ref.
containing edible fat and (ii) at least one emulsifying agent having HLB of 16 in an amount of 0.0001-5 parts by weight per parts by weight of free state phytosterols, wherein emulsifying agent is a sucrose FA ester, glycerol FA ester, sorbitan FA ester, or propylene glycol FA ester. Roche Vitamins Inc. F. HoffmannLa Roche AG (Switzerland)
Harting S.A. (Chile)
us2002/0055493 US200210160990 EP1004594
11/20/2001 1112311999 1111911999
EP98122412.4 EP99119337.6
Phytosterol or phytostanol ester compound comprising: (i) phytosterol moiety consisting of p-sitosterol, stigmasterol, campesterol, and mixtures thereof, and/or (ii) phytostanol moiety consisting of f3-sitostanol, campestanol, and mixtures thereof, and (iii) PUFA moiety consisting of C,,-C,, and at least 3 carbon-carbon double bonds, preferably EPA or DHA. Composition comprising said phytosterol and/or phytostanol ester. Method for lowering serum cholesterol and TAG in mammals. Process for preparing said phytosterol and/or phytostanol ester comprising (i) mixing free phytosterol and/or phytostanol, C,& ester of TAG of said PUFA and (ii) interesterification of mixture in presence of sodium alkoxide of C , C , alcohol, at 8CL140"C and 133-6650 Pa.
(156) (157) (158)
US200310108650
311912001
FRO0103522
Vegetable oil comprising concentrated unsaponifiablematters comprising (i) 0.2-1 Oohwtphytosterol, triterpene alcohols and methyl sterols; (ii) 0.1-2%wt tocopherols and tocotrienols; and (iii) 0.1-7%0wt squalene, and 0.1-l%wt carotenes.
(184)
EP1121928 US2002100163 14
1l3Ol2OO1 1I3012001
CL2000209
Composition for lowering LDL cholesterol level and elevating HDL (186) cholesterol level in mammal comprising an ester(s) of polico(187) sanol(s) or phytosterol(s), wherein FA moiety of phytosterol(s) comprises EPA, DHA, linoleic, linolenic, and arachidonic acids.
TABLE 19.8
P
(Continued)
o\
Applicant
u
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Ref. ~~
Forbes Medi-Tech Inc. (Canada)
W001/66560
3/7/2001
US09/519,278
~
Composition comprising one or more of the following: (i)a derivative represented by one or more of the following formulas: 0
II
R2-P-OR
I
OR3
0 II R2/"OR
0
\\
c-c
R2/
(185)
0
N
'OR
wherein R is a phytosterol or phytostanol moiety, R2 is derived from a-lipoic acid and R, is H or any metal, alkali earth metal, or alkali metal, and salts thereof; (ii) a phytosterol and/or phytostanol in combination with lipoic acid or analogs thereof; and (iii)salts of either (i) or (ii).Method of treating or preventing cardiovascular disease and its underlying conditions, including atherosclerosis and hypercholesterolemia by administeringthe said composition. Forbes Medi-Tech Inc. (Canada)
W001/53320
1/22/2001
US09/489,541
Novel composite crystalline structure comprising a phytosterol and a phytostanol, or derivatives of either for use in treating or preventing cardiovascular disease and its underlying conditions, including atherosclerosis and hypercholesterolemia. Process for preparing said structure comprising: (i) dissolving the phytosterol and phytostanol in a solvent at ambient temperature or above ambient but below boiling point of solvent; (ii) cooling solvent to allow crystal formation; and (iii) filtering and washing the crystals so formed, wherein solvent is any C,-C,? hydrocarbon, C,-€,, alcohol, CI-C,, ketone, C,-C,, carboxylic acids, ethyl acetate, or aromatic; temperature for step (i)is 18-8O0C, and temperature for step (ii)is 1-20°C.
P
5
(177)
US2002/0103139
12/1/2000
-
Compositionfor body weight and cholesterol control comprising: (i) at least one phytosterol in an amount of 5-5o%wt; (ii)a physiologically or pharmaceuticallyacceptable surfactant in an amount of 0.1-50%wt; (iii) a lipid phase in an amount of 1-5O%wt; and (iv) a pharmaceuticallyacceptable excipient for facilitating absorption of lipid phase in an amount of 1~ O ~ o w t .
Monsanto Co. (U.S.)
WOO1/32031
11/3/2000
US601163,383 US60/198,449
Composition comprising: (i) a phytosterol and (ii) a surfactant selected from the group consisting of sodium docusate, ammoniated glycyrrhizin, polyoxyethylenecastor oil, polyethylene glycol, diacetyl lactic acid esters of MAG and DAG, monosodium phosphate derivatives of MAG and DAG, ethoxylated MAG and DAG, quillaja saponin, ethylene oxide propylene oxide block copolymers, vitamin E o-a-tocopheryl glycol 1000 polyethylenesuccinate, hydroxylated lecithin, and mixtures thereof.
Forbes Medi-Tech Inc. (Canada)
WOOl/32029
11/3/2000
US09/434,356
Composition comprising an edible oil or fat and one or more phytosterols and/or phytostanols, wherein the phytosterols and/ or phytostanols are substantially completely dissolved therein. Method for preparingsaid composition comprising: (i)heating the phytosterols and/or phytostanols to a molten form beniveen 120 and 145°C; (ii)heating the edible oil or fat between 80 and 150°C; (iii)mixing the molten material with the heated oil or fat; and (iv) cooling the composition so formed.
(180)
Method for dispersing phytosterol comprising: (i)melting an admixture of phytosterol and an emulsifier at 60-200°C; (ii)admixing the molten mixture to an aqueous beverage or emulsifier-containingaqueous beverage; (iii) stirring the ad-
(178)
US2002/0064548
12/20/2000
KR2000-57652
(179)
-$ Q
3
2 3
0,
s
(Continued) P
u u
P
U
03
TABLE 19.8 (Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Ref.
mixture at high speed to disperse or homogenize phytosterol in beverage, or producing a phytosterol-dispersedbeverage; and (iv) drying the dispersion to produce aqueous phytosterol powder, wherein drying step is by evaporating, freeze-drying, or spray-drying; and the emulsifier is preferably a sucrose FA ester. Forbes Medi-Tech Inc. (Canada)
W001/32679
11/1/2000
USO9/432,836
Novel glycoside compound comprising: (i)a carbohydrate moiety consisting of pentose monosaccharides, disaccharides, trisaccharide, and oligosaccharides and their acyl derivatives and (ii) a noncarbohydrate moiety comprising a phytosterol, a phytostanol, or derivatives thereof. Method for lowering serum cholesterol in humans comprises administeringthe said compound. A product comprising said compound that effectively treats and prevents cardiovascular disease and its underlying conditions, including atherosclerosis and hypercholesterolemia. A process for preparing said compound comprising: (i)protecting the carbohydrate moiety; (ii) converting the protected carbohydrate moiety to a halide or halidelacetate derivative, and (iii)adding phytosterol or phytostanol to the derivative under reaction conditions suitable to form a glycoside.
(1 89)
Uni lever Home & Personal Care USA, Div. of Conopco (U.S.)
US6,231,841
411 212000
CB9908208
Antiperspirant composition comprising: (i)an antiperspirant active, (ii) a liquid carrier; and (iii) a structurant consisting of an effective concentration of a combination of at least one sterol and at least one sterol ester, wherein sterol and sterol ester are in an amount from 0.5 to 20%wt and in mol ratio of 6:l to 1 :4.
(1 92)
Forbes Medi-Tech Inc. (Canada)
W001/00653
6/20/2000
US09/339,903
Chemical structure comprising phytosterol or phytostanol and ascorbic acid having one or more of the following formulas: 0
II R2-P-OR I OR3
0 II R2/'\OR
0
0
\\
c-c
R2/
N
'OR
wherein R is a phytosterol or phytostanol moiety, R2 is derived from ascorbic acid, and R, is H or any metal, alkali earth metal, or alkali metal, and salts thereof. Composition and method for treating or preventing cardiovascular disease and its underlying conditions, including atherosclerosis and hypercholesterolemia by administeringsaid derivative.
-
Forbes Medi-Tech Inc. (Canada)
US6,197,832
US5,985,936
9/14/1999
12/18/1997
-
-
9 ir;
Composition for reducing serum cholesterol in humans comprising: (i)5-75%wt phytosterol; (ii)l d O % w t policosanol; and (iii) &65%wt pharmaceuticallyacceptable formulation aids.
(193)
Method for preventing Alzheimer's disease in an animal by administering a composition comprising: (i)50% p-sitosterol, (ii) 1&25% stigmastanol, and (iii)l&25% campesterol.
(204)
A phytosterol or phytostanol derivative having one of the following formulas:
(191)
m
-.
2 8
t:
'0.
P 4
Forbes Medi-Tech Inc. (Canada)
W000/78789
6/20/2000
US09/337,810
m' 3
0 II
R3-P-OR
wherein R is a phytosterol or phytostanol moiety, R, is 0 or H, and R, is an aromatic unit, preferably benzene, or a heterocyclic unit, preferably nicotinic acid, and all salts thereof. A composition, comestible or beverage comprising said derivative for treating and preventing cardiovascular disease and its underlying conditions, including hypercholesterolemia. (continued)
P
2
P a, 0
TABLE 19.8 (Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Forbes Med-Tech Inc. (Canada)
W000115201
9110/1999
USO9/151,385
Composition for use in preventing and treating cardiovascular disease, its underlyingconditions and other disorders comprising: (i)one or more phytosterols, phytostanols, ester derivatives, or mixtures thereof and (ii) one or more tocotrienols or derivatives thereof, wherein underlyingconditions comprise atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, and thrombosis; other disorders comprising diabetes type II and diseases involving oxidative damage as part of the disease pathology.
Forbes Med-Tech Inc. (Canada)
WOO0104887
712011999
US091118,809
Composition for use in preventing and treating cardiovascular disease and other disorders comprising: (i) one or more phytosterols, phytostanols, ester derivatives, or mixtures thereof; (ii) one or more n-3 PUFA or derivatives thereof; and (iii)one or more n-6 PUFA, wherein n-3 PUFA comprises linolenic, EPA, and DHA.
Forbes Medi-Tech Inc. (Canada)
WO99/6384 1
6/7/1999
US09/092,497
Composition suitable for incorporation into foods, beverages, pharmaceuticals, nutraceuticals, and the like comprising one or more phytosterols, phytostanols, or mixtures thereof, treated to enhance solubility and dispersibility via (i) formation of emulsion or microemulsion or (ii) formation of solid dispersions, formation of suspensions, formation of hydrated lipid systems, or inclusion complexations with cyclodextrins, hydrotopes, and bile salts.
Raisio Benecol OY (Finland)
WO99/56558
5/6/1999
F1981011
Composition comprising a sterol and/or stanol FA ester, wherein FA moiety comprises <7% SFA and >50% PUFA. An edible oil or fat blend comprising said composition. Method for preparing said composition comprising: (i) esterifying FA mixture with free sterol and/or stanol by direct catalytic or enzymatic esterification
Ref.
or (ii)interesterifying alcohol ester of FA with free sterol and/or stanol in the presence of interesterification catalyst. US6,113,972
121311998
-
Phytosterol protein complex for increasing bioavailability of a phytosterol comprising: (i)a phytosterol in an amount between 5 and 8O%wt; (ii)a protein in an amount between 20 and 90°/owt; and (iii) an edible oil in an amount between 5 and 20%wt.
(197)
Raisio Benecol Ltd. (Finland)
US6,441,206
91911998
F1973647 F1974563 F1974648
Phytosterol and/or phytostanol ester derivative comprising acid moiety derived from hydroxy acid, dicarboxylic acid, amino acid, or their salts thereof.
(198)
Lipton, Div. of Conopco Inc. (US.)
US6,031,118 EP0897971
811811998 713 111998
EP97202596
Composition comprising 285% stanol FA ester, wherein 285% is SFA.
(199)
Forbes Medi-Tech Inc. (Canada)
US6,087,353
511511998
-
Composition suitable for incorporation into foods, beverages, pharmaceuticals, and nutraceuticals, which comprises one or more esterified and subsequently hydrogenated phytosterols. Method for lowering serum cholesterol in animals by administering the said composition. Process for preparing said composition comprising: (i) condensing an aliphatic acid with one or more phytosterols to form a phytosteryl ester and (ii) hydrogenatingthe phytosteryl ester to form
(200)
Monsanto Co. (U.S.)
9 ii;
2
P 5. 3
s
t.
2. 4
2
30 3
0,
B
hydrogenated phytosteryl ester. US5,952,393
211211998
-
Composition for reducing serum cholesterol levels comprising: (i) 5-75%wt phytosterol (ii)l d O % w t policosanol; and (iii) &65%wt pharmaceutically acceptable aids, wherein phytostero1:policosanol is preferably 3.2:l.
US2002/0035133
1/16/1998
DE19701264.7
Medication for use for inflamed processes of the diseased organism (202) or its organs, particularly of the skin and subcutaneous tissue, comprising: (i)psitosterol and cholic acid, chenodeoxycholic acid, desoxycholic acid, or water-soluble alkali salts as a suspension
(201)
P
(Continued)
2
P 03 N
TABLE 19.8 (Continued) ~~~
_____
~
Applicant
~~~
~~
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure or in physically dissolved form, or (ii)psitosterol ascorbic acid ester, psitosterol tartaric acid ester or p-sitosterol gluconic acid ester.
US6,407,085
I1611998
DE19701264
Method for treating inflammation by administering a composition in an effective amount comprising p-sitosterol ascorbic acid ester in an amount between 0.5 and 2 . 0 % ~ and a pharmaceutically acceptable carrier.
Forbes Medi-Tech Inc. (Canada)
US5,985,936
2/18/1997
-
Method for preventing Alzheimer’s disease in an animal by administering a composition comprising: (i)250% psitosterol, (ii)1&25% stigmastanol, and (iii) 1&25% campesterol.
Henkel Kommanditgesellschaft auf Aktien (Germany)
US6,383,514
1111911997
DE19649286
A hypocholesterolemic composition comprising: (i)a phytostanol ester and (ii) a tocopherol, wherein (i):(ii) is from 60:40 to 4060.
Cognis Deutschland US6,444,659 GmbH (Germany)
1211911997
DE19646286 DE19700796
A hypocholesterolemic composition comprising: (i)a phytosterol, a phytosteryl ester, or mixtures thereof and (ii) a potentiating agent selected from the group consisting of a chitosan, a (deoxy) ribonucleic acid, and mixtures thereof.
Riken Vitamin Co. Ltd. (japan)
US5,998,396
10/24/1997
-
Edible oil composition comprising a sitosterol, a vitamin E, and an emulsifier in an amount sufficient to render sitosterol soluble in vitamin E and edible oil, with the proviso that said sitosterol is not soluble in said vitamin E or edible oil when said emulsifier is absent.
Unilever N.V. (The Netherlands)
EP0962150 EP0960567 WO96/3 8047
513111996
EP95201444 EP95202042
Fat-based food product, wherein the fat comprises (i) at least one tocopherol andlor polyphenol; (ii) at least one phytosterol in an amount of 2O0.25%W;and (iii)at least one oryzanol in an amount of 20.25%wt.
Ref.
TABLE 19.9 A 5-Year Patent Literature Survey on Processes InvolvingCarotenes
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Ref.
University of Maryland, College Park (US.)
WOO2104415
711112001
US60/217,585
Process for isolatingcarotenes from palm oil comprising: (i) dissolving the palm oil in an organic solvent; (ii) saponifying the organic solution of said palm oil with an alcoholic solution of a mineral base; (iii)washing organic layer with water containing 10-20% alcohol until base is removed; and (iv) separating and concentratingthe resultant organic phase comprising isolated carotenes.
(209)
Process for extracting carotenes from carotenecontaining materials comprising: (i) extracting a carotene-containing material with an extractant comprising at least one member selected from the group consisting of acetonitrile, Kmethylpyrrolidone, N , K dimethylformamide, N,N-dimethyl acetamide, 4-formylmorpholine, 4-acetyl morpholine, 4-methylmorpholine, 4-phenylmorpholine; (ii) formation of two liquid phases, one of which is a carotene-depleted raffinate phase and the other is a carotene-enriched extract phase; and (iii)separation of the two liquid phases.
(210)
Siegfried Peter (Germany)
US6,407,306
1211012000
-
9
B
3
2
$ 8 t 3.
3s 0,
3
(Continued) P
m
w
P 0)
P
TABLE 19.9 (Continued)
Applicant
Publication no.
Date filed (mon/&yr)
Priority
Disclosure
Global Palm Products Sdn. Bhd. (Malaysia)
US5,932,261
12/21/1996
PI-9604534
Process for the production of a natural carotene-rich refined and deodorized oil comprising: (i)distilling the oil in a short-path distiller at 160-200°C and 0.003-0.08 mbar and (ii)removing FFA in the oil by condensation within the distiller.
Henkel Kommandit- US5,902,890 gesellschaft auf Aktien (Germany)
3/11/1996
DE19510098
Ref. (212)
Process for recovering carotene from palm oil comprising: (i)trans(211) esterifying the oil with an alkanol having up to C, to form a twophase mixture comprising a glycerol phase and an ester phase, wherein said ester phase comprises FA esters and carotene; (ii) separating the FA esters from the ester phase by distillation or evaporation to form a residue containing carotene; (iii)saponifying the residue with an alkali metal hydroxide; and (iv) extracting the carotene from the saponified residue with an organic solvent to form an extract phase containing carotene, and removing the solvent from the extract phase by evaporation.
P
3 P g. 2
n, I
TABLE 19.10 A 5-Year Patent Literature Survey on Methods and Compositions Involving Vitamin E
Applicant
Publication no.
Date filed (monldlyr)
LG Life Sciences Ltd. (Korea)
KR2002090293
-
SK Corp. (Korea)
jP2000281674
Nisshin Flour Milling US5929057 Co. (Japan)
312311999
61511998
Priority
Disclosure
KR20010029224 KR20010526
Feed additive composition for animals comprising a polyethoxylated a-tocopherol ester derivative as an effective ingredient for improving meat quality, and increasing body weight and the feed conversion ratio.
(213)
a-Tocopherol cyclopropylic ester compound having biological activity equal to that of vitamin E in vivo and production method for said compound.
(2 14)
US19980092 143 US19980605 jPl9960171028
9-Cis-retinoic acid-a-tocopherol ester compound and pharmaceutical compositions comprising said compound for use as treatment of leukemia.
(2 15)
-
Ref.
2 3
3
P $. 0 L3
t. $
3
3-
Degussa AC
jP11246549
12/17/1998
-
Method for producing a-tocopherol comprises reacting a hydroquinone mono- or diester with an ally1 alcohol derivative in the presence of a zinc halide and a proton-releasing acid at 25-1 00°C.
(216)
Eisai Co. Ltd. (lapan)
JP11092474
9/18/1997
-
Method for producing a-tocopherol acidic succinic acid ester calcium salt by preparing a solution by dissolving atocopherol acidic succinic acid ester in hydrous methanol and a second solution by dissolving a calcium acetate in water or 5&70% hydrous methanol, and subjectingboth solutions to salt exchange by gradually dripping the calcium acetate solution to the first solution at room temperature.
(217)
%
6
P ln cn
TABLE 19.1 1 A 5-Year Patent Literature Survey on Processes, Methods, and Compositions Involving Oryzanol
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Lipton, Div. of Conopco Inc. (U.S.)
US2003/0134028
2/24/2003
EP95201444.7
Fat-based food product comprising natural fat components having (222) cholesterol-loweringeffect, wherein fat comprises (i) 20.25% oryzanol and/or (ii) 20.2% tocotrienol, and/or (iii) t l % phytosterol.
Council of Scientific and Industrial Research (India)
US6,410,762
Lipton, Div. of Conopco Inc. (U.S.)
US2001/0047101
3/30/2001
7/3/2001
-
EP96201870.1
Ref.
Process for extracting oryzanol from rice bran oil soapstock com(218 ) prising: (i) saponifyingthe oil present in soapstock with an alkali followed by neutralization of the excess alkali; (ii) converting into anhydrous porous soap noodles; (iii) extracting unsaponifiable matter in soap noodles with organic solvent; (iv) crystallizing crude unsaponifiable matter to-remove impurities; (v) subjecting residues to column chromatography to obtain oryzanol-rich fraction; and (vi) recrystallizingoryzanol-rich fraction using organic solvent to obtain pure oryzanol. Process for obtaining oryzanol concentrate from oryzanolcontaining crude oil comprising: (i) removal of phospholipids present in crude oil, andlor (ii) removal of FFA, preferably by the use of stripping, and (iii) alkali neutralization of the obtained product, and (iv) separation and removal of the obtained oil phase.
(219)
P
5
P
&.
2
?
McNeil-PPC Inc.
US6,054,144
11/4/1998
US09/025,952
Process for preparingwater-dispersible oryzanol comprising: (i) providing an aqueous stream,; (ii) admixing to an aqueous stream of 2-2.5Ohwt of a polyfunctional surfactant to form a water surfactant mixture; (iii) admixing oryzanol to the water surfactant mixture to form oryzanol suspension; and (iv) drying the oryzanol suspension to recove a water-dispersible oryzanol, wherein the said process is performed in the absence of deaeration and homogenizingsteps.
US5,869,708
1/17/1997
-
Process for extracting otyzanol from crude dark acid oil of rice bran comprising: (i) distilling the FFA from the crude dark acid oil; (ii) hydrolyzing the resultant residue in NaOH solution and at 6&9O0C for 0.5A h; (iii) dissolving the hydrolyzed product in water of amount predetermined for micellar aggregate formation in oryzanol, thereby forming oryzanol-containingmicellar aggregates and adding dropwise aqueous solution of CaCI, to form a precipitate; (iv) drying the precipitate; (v) extracting the oryzanol from dried precipitate using polar organic solvent; and (vi) purifying extracted oryzanol by column chromatography.
(US.)
9
is 3
P U m
P Q) Q)
TABLE 19.1 2 A 5-Year Patent Survey on Processes, Methods, and Compositions Involving Ferulic Acid and Ferulyl Compounds
Applicant
Publication no.
Date filed (mon/d/yr)
Bayer Polymers LLC (U.S.)
US2003/0152682
Kao Corp. (Japan)
Priority
Disclosure
12/20/2002
DEl0164263.6 DE10222883.3
Various ferulic acid amide compounds having heat-generating effect.
(234)
EP1264596
6/4/2002
JP2001169261
Ferulic acid derivative with reduced bitterness that prevents hypertension.
(223)
Korea Institute of Science and Technology (Korea)
W002/083625
12/5/2001
KR2001/20411
Ferulic acid dimers and their pharmaceuticallyacceptable salts and their use for treating dementia.
(227)
Kao Corp. (Japan)
EPll86297
9/5/2001
JP2000268100JP2000268104
Compound for improving or treating hypertension comprising: (i) a compound selected from the group consisting of ferulic acid, caffeic acid, and chlorogenic acid, and esters and pharmaceuticallyacceptable salts thereof; (ii) a component selected from the group consisting of central nervous systemstimulating components, food fibers, extracts of perennial evergreen leaves of the genus Camellia, Jheaceae, or Eucommia ulmoides Oliver, Eucommiae, organic acids having a molecular weight of 60-300, and pharmaceutically acceptable salts thereof, and sugar alcohols.
(224)
Kao Corp. (japan)
EP1186294
8/6/2001
JP2000238039
Composition comprising: (i) ferulic acid or ester thereof, or a pharmaceuticallyacceptable salt thereof and (ii) caffeic acid and/or a chlorogenic acid, or a pharmaceuticallyacceptable salt thereof.
(235)
Ref.
P
k I-
2.
2 [I,
I
Hanbul Cosmetics Co. Ltd. (Korea)
US6,503,941
612 1I2001
KfUOO(M034790
A compound comprising 3,9diferulylcoumestrol for use in skin cosmetic products for restraining wrinkle formation.
National Science Council (Taiwan)
US2001/0053781
2/22/2001
TW89111850
A pharmaceutical composition for enhancing cognition, comprising: (i)5C100 m@g ferulic acid or a pharmaceutically acceptable salt or derivative thereof and (ii)a pharmaceutically acceptable carrier or excipient.
Kao Corp. (Japan)
US6,310,100 EPlO90635
9/22/2000 912212000
JP11-268461 JP12-230463
A composition comprisingferulic acid or a derivative thereof in a form and in an amount suitable for treating a subject suffering from hypertension, comprising at least 15%wt of DAG.
Unilever PLC (U.K.)
W001l07004
711412000
US601145,674
A cosmetic composition comprising 10-100% of an aqueous phase comprising 0.01-1 0% ferulic acid and 1-90% of a nonvolatile polyol, with the weight ratio of polyol to ferulic acid of at least 4:1, wherein the pH of the composition is 3-5 and the half-life of the composition is at least 20 d at 50°C.
USA
US6,346,236
3/28/2000
-
A compound comprising a TAG, wherein, sn-1 position is a ferulyl or coumaryl moiety, one of sn-2 or sn-3 positions is a C-,C, FA moiety and the other sn-2 or 511-3 position is either a C& ,, FA moiety or O H . A sunscreen formulation comprising said compound. A method for making said compound comprising (i) lipase-catalyzed transesterification between TAG and ferulic or coumaric acid and (ii) recovering said compound from reaction mixture.
(Continued
P
a W
P
TABLE 19.12 (Continued)
10 0
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Unilever PLC (U.K.)
EP0945425
3/11/1999
US48733 US48738
Ferulic salicylate compound for controlling oily skin and reducing sebum secretion. Method for producing said compound comprising: (i) preparing a salicylic acid halide by reacting salicylic acid with thionyl halide or oxalyl halide and (ii) reacting the salicylic acid halide with ferulic acid to obtain the said compound.
(232)
Elizabeth Arden Co. (US.)
US6,022,548
3/26/1998
-
Skin conditioning composition comprising 0.0001-20%wt of methoxycinnamyloxysalicylate and a cosmetically acceptable carrier. Method for controlling oily skin, reducing sebum secretion, stimulating collagen and glycosaminoglycan synthesis, treating aged, photoaged, dry, lined or wrinkled skin, and shielding the skin from harmful UVA and UVB light by applying to the skin the said composition.
(233)
Ref.
Chesebrough-Pond's US5,824,326 USA Co., Div. of Conopco inc. (US.)
612711997
-
A cosmetic composition comprising: (i)0.01-55%wt ferulic acid; (237) (ii)0.1-20'70wt dimethyl isosorbide; and (iii) a pharmaceutically acceptable carrier.
Tsuno Food industrial Co. Ltd. (Japan)
7/3 1/1996
JP6-097485
A cosmetic containing a base material, and an antioxidant/UV (231) absorbent comprising at least one ferulic acid ester having a C,-C, alkanol group. Method for reducing UV light reaching a human body comprising applying to the body an effective amount of said base material. Method for inhibiting oxidation of a cosmetic product comprising said base material.
US5,908,615
P
3 P
2. R
TABLE 19.1 3 A 5-Year Patent Literature Survey on Processes and Methods, and Compositions Involving Ubiquinones
Applicant
Publication no.
Date filed (mon/&yr)
Priority
Disclosure
Ref.
Kaneka Corp. (Japan) WOO31032967
1011of2002
JP2001-312179 JP2002-114879
Method for stabilizing reduced ubiquinone comprising bringing into contact said ubiquinonewith ascorbic acid or citric acid, or derivatives thereof.
Kaneka Corp. (Japan) W003/008363
711512002
JP2OOl-215804 JP2002-114878
Method for producing reduced ubiquinone having excellent qualities useful in foods, comprising bringing said ubiquinone into contact with a solvent containinga strong acid.
Kaneka Corp. (Japan) WOO31006412
711512002
JP2001-2 14482 JP2002-114877
Method for producing reduced ubiquinone having excellent qualities useful in foods, comprising reducingoxidized ubiquinone in an aqueous medium with the use of hydrosulfite, wherein reduction is carried out with the co-existence of a salt and/or under deoxidized conditions at pH 17.
Kaneka Corp. (Japan) W003/006411
711512002
JP2001-214477 JP2002-114874
Method for crystallizing reduced ubiquinone that is suitable for industrial production thereof, comprising crystallizing reduced ubiquinone from an organic solvent solution containing the ubiquinonethrough the replacement by water, or from an aqueous solution.
Kaneka COT. (Japan) WOO31006410
711Sf2002
JP2001-214475 JP2001-2 14480 JP2002-114873 JP2002-114875
Method for producing reduced ubiquinone having excellent qualities useful in foods, comprising: (i) an oily oxidized ubiquinone is reacted with a reducing agent in water, and the aqueous phase is separated from the obtained reaction mixture or (ii) an organic phase containing reduced ubiquinone is concentrated by distilling off the coexisting organic solvent at the melting point of the reduced ubiquinone or higher. (Continued)
P Q N
TABLE 19.1 3 (Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Kaneka Corp. (Japan)
W003/006409
Kaneka Corp. (Japan)
W003/006408
Priority
Disclosure
7/15/2002
JP2001-214474 JP2002-114872
Method for producing reduced ubiquinone crystals that is suitable for industrial production comprising crystallizing said reduced ubiquinone in a solution of alcohols and/or ketones to obtain a crystallized reduced ubiquinone having excellent slurry properties or crystalline properties.
7/15/2002
JP2001-214471 JP2002-114854
Method for producing reduced ubiquinone having excellent qualities useful in foods comprising reducing oxidized ubiquinone followed by crystallization from at least one solvent consisting of hydrocarbons, fatty acid esters, ethers, and nitriles.
Centre for Molecular W002/43721 Biology and Medicine (Australia)
11/29/2001
AUPRl773
Method of treatment of one or more side effects of statin therapy comprising administeringto a subject an effective amount of uridine or derivatives thereof either simultaneously, sequentially, or separately to administer an effective amount of at least one ubiquinone.
-
us20021012 5 193
3/4/2002
US60/273,684
Apparatus and method for simultaneous and rapid determination of oxidized and reduced ubiquinone concentrations in human samples using HPLC coupled with an electrochemical detector.
The Regents of the University of California (US.)-
W002/14530
8/14/2001
USO9/639,223
Method for synthesis of ubiquinones and ubiquinone analogs by means of a series of chemical reactions.
Ref.
US2002/0098172
Simonelli (Italy)
6/1/2001
US60/263,953
US2002/0156302
4119/2001
US2002/0018772
6/22/2001
A human treatment method to improve absorption of ubiquinone into an intestinal tract and to maintain ubiquinone basal blood levels comprising administering a soft-gel formulation of 3&100 mg/d of ubiquinone, CelOil SC, and vitamin E.
(239)
-
Method for producing an optically pure trans (0 isomer of ubiquinone comprising extracting solanesol from tobacco dust and using said solanesol as starting material for carrying out a series of chemical reactions to produce the said ubiquinone isomer.
(254)
US60/213,337
Process for manufacturing soft-gel capsules containing an improved formulation of ubiquinone comprising: (i)mixing beeswax in rice bran oil, soybean oil and GelOil SC to produce a first mixture; (ii) cooling the first mixture to below 26°C; (iii)adding vitamin E and natural p-carotene to the first mixture to form a second mixture; (iv) maintaining the second mixture at or below 26°C while adding ubiquinone to form the third mixture; (v) mixing the third mixture for a time sufficient to ensure that the third mixture is homogenous; and (vi) encapsulating the third mixture in a gel capsule.
(238)
(255)
2
3 3
?
$ 0 3
c:
3. 4
W001/37851
10/30/2000
ITRM99A000719
Method for the prevention and treatment of pathologies, or incidental or postsurgical trauma, of the anterior chamber of the eye comprisingthe ophthalmic topical use of a drug containing ubiquinone.
US2001/0034372 GI32360706
2/9/2001 2/8/2001
US60/181,314
Method for treating a human patient suffering from fibromyalgia (256) to produce a therapeutic response in said patient comprising (257) administering to patient a composition comprising ubiquinone and succinic acid each at a dose of 5-500 mu70 kg patient. (Continued)
P Q
TABLE 19.1 3
P
(Continued)
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Kaneka Corp. (Japan)
EPll23979
8/24/2000
JP23756199
A DNA sequence for the enzyme synthesizing ubiquinone side chain synthase from a fungal strain of the genus Saitoella and exploit it for the production of ubiquinone.
Biosytes USA Inc. (US.)
US6,056,971
812311997
US60/022,564
Method for enhancing dissolution and bioavailability of ubiquin- (240) one from an orally delivered soft gelatin capsule in unit dosage form comprising filling a soft gelatin capsule with a uniform liquid nonaqueous solution containing an effective amount of ubiquinone, wherein said nonaqueous solution is prepared by the following steps: (i) mixing 2&90%wt of a nonionic surfaceactive agent as a solubilizer with 2-50%wt of a polyhydric alcohol to form a uniform mixture and (ii) adding an effective amount of ubiquinone in said mixture to form the said non-
Ref. (242)
P
5r2.
2 n,
aqueous solution.
?-
Soft Gel Technologies Inc. (U.S.)
EP0888774
612311998
US886122
Method for improved absorption of ubiquinone into an intestinal tract and maintenance of ubiquinone basal blood levels comprising administering -3&100 mg/d of a soft-gel formulation of ubiquinone and vitamin E.
The Board of Regents, The University of Texas System (US.)
W099/43316
212511999
US60/076,159
Process for the production of ubiquinone comprising: (i) obtaining (243) a deodorizer distillate containing tocopherol and ubiquinone; (ii) separating any sterols, phospholipids, and FA from the deodorized distillate to form a mixture, (iii) subjectingthe mixture to a first separating step by molecular distillation to separate tocopherol and form a second mixture; and (iv) subjecting the second mixture to a second separation step by molecular distillation to provide a concentrated ubiquinone fraction.
(241)
TABLE 19.14 A 5-Year Patent Literature Survey on Compositions Involving Ubiquinones ~
Applicant
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Smithkline Beecham PLC
W003/037284
lOl29l2002
CBOl26085.0
An oral hygiene composition for the prophylaxis and/or treatment of halitosis, periodontal disease, and related disorders, comprising ubiquinone and an orally acceptable carrier or excipient, wherein ubiquinone is in an amount from 0.01 to 1O~/Owt.
(258)
Composition for lessening oxidative stress comprising a ubiquinone as the active ingredient.
(259)
Composition of stable reduced ubiquinone comprising an aqueous solution containing reduced ubiquinone and an antioxidant such as vitamin C and/or a chelating agent such as EDTA.
(277)
(U.K.)
Kaneka Corp. (lapan)
W003/032968
Kaneka Corp. (lapan)
W003/033445
10/15/2002
JP2001-314932
Ref.
i? 3 rn f.
T
ldemitsu Technofine W003/007928 Co. Ltd. (japan), Nisshin Pharma Inc. Uapan) US2003/0059418
10/10/2002
7/17/2002
2/20/2001
JP2001-312181
JP2001-216545
Composition for the prevention of ascites in poultry comprising (260) an effective amount of ubiquinone and 0.5-80 parts by weight of a surfactant.
3 t
-2.
0-
?i e
4
1TMR2000A000106 Composition suitable for the prevention and/or treatment of vasculopathic, cardiac, central, and peripheral cerebral disturbances and for the prevention of learning disorders or disorders related to aging Comprising: (i) a ubiquinone and (ii) a propionyl L-carnitine or a pharmacologically acceptable salt thereof. (Continued) 0
cn
TABLE 19.14 (Continued)
Applicant
Publication no.
Date filed (moddlyr)
Priority
Disclosure
Kaneka Corp. Clapan)
EP1281398
5/9/2001
JPOllO3863
A composition for dermal application comprising oxidized and/ or reduced ubiquinone as the active ingredient in an amount from 0.1 to 9 9 % ~ .
ldemitsu Petrochemical Co. Ltd. (lapan)
EPl304041
7/1/1997
JP17808396
A feed composition for poultry comprising a ubiquinone and sodium hydrogencarbonateor an ammonia-generation inhibitor, wherein the ratio of ubiquinone to sodium hydrogencarbonate is from 1:2 to 1:4000, and the ratio of ubiquinone to the ammonia-generation inhibitor is from 100:l to 1:loo.
Triarco Industries Inc. (U.S).
US6,403,116
11/3/2OOo
-
Composition comprising: (i)0.1-1 0% ubiquinone; (ii) 0.1-10% methyl sulfonyl methane; (iii)0.1-1 5% polysorbate material; (iv) 0.1-1 0% citric acid; and (v) 45-99.6% maltodextrin.
-
US2002/0128184
1/9/2001
-
A composition comprising a glycoprotein matrix bound to a ubiquinone, in an amount from 5 to 15%wt.
-
US6,441,050
a/29/2000
-
An orally compatible, palatable composition in liquid dosage form comprising: (i)an effective amount of reduced and/or oxidized ubiquinone; (ii) 0.5-35%wt of a polysorbate surfactant; (iii)0.2-5O%wt of a TAG; (iv) 0.25-2OY0wt of a phospholipid (v) M5o/owt of a sweetener; and (vi) 1-50%wt of water.
Kaneka Corp. Clapan)
W002/090304
5/9/2002
JP2001-138340
Composition of a stable solution of ubiquinone prepared by coating reduced ubiquinone with liposomes made from refined soybean lecithin, and solubilizing or emulsifying reduced ubiquinone by using a surfactant at low concentration.
Ref.
US2002/0198177
5/9/2002
GBOl13104.4 GBO123446.7
Kaneka Corp. Uapan)
W002/092067
5/8/2002
JP2001-139605
Composition for transmucosal administrationcomprising oxidized and/or reduced ubiquinone as the active ingredient in amounts from 0.0001 to 9 9 % ~ .
Kaneka Corp. (Japan)
W002/100359
5/8/2002
jP2001-139606
Preparation for hair and/or scalp which is effective in growing hair and preventing hair removal and has an excellent skin-care effect on the scalp comprising oxidized or reduced ubiquinone as the active ingredient in an amount from 0.0001 to 9 9 % ~ ~
Alberto-Culver Co. (US.)
W002/078651
4/2/2002
US60/281,039 US09/851,882
A cosmetic composition with improved moisturizing properties, improved skin feel and/or improved visual appeal, comprising ubiquinone and urea, wherein ubiquinone:urea is from 5:l to 1:20.
Vesifact (Switzerland)
AGW002l083098 3/4/2002
EPOllO9131.1
Composition containing ubiquinone in the form of microemulsions.
US2002/0058026
11110/2001
US60/246,995
A medicinal composition for treatment of hypercholesterolemia, mixed dyslipidemia, and coronary artery disease comprising: (i) a 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitor component and (ii) a ubiquinone.
US6,417,233
1011911999
ITB098A0596
Composition for the prevention and/or treatment of mitochondriopathies comprising: (i) a reduced and/or oxidized ubiquinone in an amount effective for therapeutic and/or preventive and/or nutritional activity in humans in need thereof, and (ii) at least one n-3 PUFA consisting of EPA and/or DHA, and, (iii) optionally, a non-n-3 FA selected from a saturated, monounsaturated, n-6 FA, n-9 FA, and mixtures thereof, or (iv) one or more additives
Sigma-Tau Healthscience S.p.A. (Italy)
Composition for pharmaceutical or nutritional use comprising: (i) >70% EPA; (ii) <1O0/o DHA; (iii) <1O0/o linoleic acid; and (iv) ubiquinone in any appropriate assimilable form, wherein EPAubiquinone is between 2000:l and 1:l.
9
P
Lo
m
TABLE 19.14 (Continued) ~~
Applicant
~
Publication no.
Date filed (mon/d/yr)
Priority
Disclosure
Ref.
selected from the group consisting of vitamins, mineral salts, antioxidiring agents, amino acids, polysaccharides, and vegetable fibers. ldemitsu Petrochemical Co. Ltd. (Japan)
US6,2.51,442
511411998
JP9-123985
A feed composition for broilers, for the prevention of ascites, having a metabolizable energy value of at least 3150 kcallkg and containing ubiquinone.
(268)
US6,300,377
2/22/2001
-
An orally compatible composition in liquid dosage comprising: (i) an effective amount of ubiquinone; (ii) a primary surfactant in an amount from 0.1 to 5o%wt; (iii) a glyceryl ester in an amount from 0.1 to 6O%wt, wherein sn-1 , sn-2, and sn-3 positions comprise an H or a C,&, acyl group; (iv) a TAG in an amount from 0.1 to 2!i%wt; (v) a phospholipid in an amount from 0 to 25%wt; and (vi) a secondary bioactive agent in an amount from 0 to 25%wt.
(269)
Composition comprising ubiquinone and an amount of a reducing agent effective to reduce said ubiquinone, and further comprising an amount of surfactant or vegetable oil or mixtures thereof and, optionally, a solvent effective to solubilize said ubiquinone and said reducing agent. Method for preparing said composition comprising: (i) solubilizing ubiquinone in a mixture comprising at least one surfactant, a vegetable oil and mixtures thereof, and optionally a solvent, at elevated temperature to form a solution; (ii) adding the solution an amount of reducing agent effective to reduce ubiquinone; and (iii) encapsulating the solution from (ii) in a hard or soft gelatin capsule.
(283)
WOOll52822
1118/2001
US09/488,332 US09/637,559
P
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Kaneka Corp. Uapan)
US6,184,255 EP0956854
811811997
J P8-234729 JP9-173191
Q-pharma Inc.
US6,200,550
1211111998
-
A toothpaste composition comprising: (i)0.014% ubiquinone; (ii)0.1-1.5% polysorbate 80; (iii) 15-35% SYLOID silica gel; (iv) 1.5% sodium lauryl sulfate; (v) 0.5% sodium dodecylbenzene sulfonate, (vi) 25-60% mixture of sorbitol and glycerol; (vii) 0.5-7Oh mixture of water-soluble hydrophilic colloidal carboxyvinyl polymer, xanthan gum and a polyethylene glycol; (viii) 2% water-soluble flavoring; (ix) 0.24% sodium fluoride; and (x) 1% sodium saccharin.
Kaneka Corp. (Japan)
US6,156,802
512711998
JP154390197
A cholesterol-loweringcomposition comprising ubiquinone as an active ingredient.
Q-pharma Inc.
US6,054,261
5l2011998
-
Compositionfor protecting mammalian organs from damage when isolated from the circulatory system, comprising a perfusion solution, -10 m g h L ubiquinone and -2 pUL polysorbate compatible with an electricalhnechanicalperfusion device.
ldernitsu Materials Co. Ltd. (Japan)
EP0913095
71411997
JP178083196
A feed composition for poultry comprising 0.00054.5%wt of a ubiquinone and a substance selected from the group consisting of antioxidants, antacid agents, and ammonia generation inhibitors.
C.S Bioscience Inc. (U.S).
US5,925,335
611211997
-
An orally absorbable dental formulation comprising a base and at least one active ingredient is ubiquinone in an amount from 10 to 25”/0wt.
(U.S.)
(U.S.)
Medicinal composition comprising ubiquinone as the active ingredient wherein reduced form of ubiquinone is in an amount >2Oohwt and <95%wt of total ubiquinone.
P
W W
500
0.-M. Lai et a/.
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Kutney, J.P., R.K. Milanova, Y. Ding, and H. Chen, Int. Patent W000/78789 (2000). Franklin, K.R., L. Grainer, and A.J. Kowalski, U.S. Patent 6,231,841 (2001). Sorkin, H.L., Jr., U.S. Patent 6,197,832 (2001). Stewart, D.J., Int. Patent W000/15201 (2000). Novak, E., Int. Patent W000/04887 (2000). Stewart, D.J., R.K. Milanova, J. Zawisotwski, and S.H. Wallis, Int. Patent W099/63841 (1999). Corliss, G., J.W. Finley, H.N. Basu, F. Kincs, and L. Howard, U.S. Patent 6,113,972 (2000). Mikkonen, H., E. Heikkila, E. Anttila, and A. Lindeman, U.S. Patent 6,441,206(2002). van Amerongen, M.P., and L.C. Lievense, US. Patent 6,031,118 (2000). Stewart, D.J., R. Milanova, J. Zawistowski, and S.H. Wallis, U.S. Patent 6,087,353 (2000). Sorkin, H.L., Jr., U.S. Patent 5,952,393 (1999). Kief, H., US. Patent 2002/0035133 (2002). Kief, H., US. Patent 6,407,085 (2002). Novak, E., US. Patent 5,985,936 (1999). Weitkemper, N., and B. Fabry, U.S. Patent 6,383,514 (2002). Weitkemper, N., and B. Fabry, U.S. Patent 6,444,659 (2002). Nakano, T., H. Kikuchi, and S . Itoh, U.S. Patent 5,998,396 (1999). Lievense, L.C., EP Patent 0962150 (1999). Khachik, F., Int. Patent W002/04415 (2002). Peter, S . , M. Drescher, and E. Weidner, US. Patent 6,407,306 (2002). Nitsche, M., W. Johannisbauer, and V. Jordan, US. Patent 5,902,890 (1999). Unnithan, U.R., U.S. Patent 5,932,261(1999). Park, B.J., B.Y. Jung, and Y.G. Kim, KR Patent KR2002090293 (2002). Lee, S.J., and H.Y. Chon, JP Patent 2000281674 (2000). Makishima, M., Y. Kanatani, Y. Honma, K. Inomata, and T. Kishiye, U.S. Patent 5,929,057 (1999). Krill, S . , F. Huebner, R. Hahn, H. Weigel, and K. Huthmacher, JP Patent 11246549 (1999). Hirose, N., JP Patent 11092474(1999). Rao, K.V.S.A., B.V.S.K. Rao, and N.B.K. Thengumpillil, U.S. Patent 6,410,762 (2002). van Amerongen, M.P., C. Hofman, and A. Zwanenburg, U.S. Patent 2001/0047101 (2001). Das, P.K., A. Chaudhuri, T. Narayana, B. Kaimal, and U.T. Bhalerao, U.S. Patent 5,869,708 (1999). Burmano, B., R.D. Bruce, M.R. Hoy, and J.D. Higgins, 111, U.S. Patent 6,054,144 (2000). Lievense, L.C., U.S. Patent 2003/0134028 (2003). Suzuki, A., R. Ochiai, and I. Tokimitsu, EP Patent 1264596 (2002). Suzuki, A., R. Ochiai, and I. Tokimitsu, EP Patent 1186297 (2002). Suzuki, A., R. Ochiai, and I. Tokimitsu, US. Patent 6,310,100 (2001). Suzuki, A., R. Ochiai, and I. Tokimitsu, EP Patent 1090635 (2001). Yu, J., K.-J. Shin, D.-J. Kim, and K.3. Lee, Int. Patent W002/083625 (2002). Hsieh, M.-T., and Y.-T. Lin, US. Patent 2001/0053781 (2001).
229. Pyo, H.-B., C.-W. Lee, S.-M. Park, Y.-H. Cho, J.-J. Lee, and J.-H. Kim,U.S. Patent 6, 503,941 (2003).
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0.-M. Lai et a/. Compton, D.L., and J.A. Laszlo, US. Patent 6,346,236 (2002). Taniguchi, H., E. Nomura, T. Tsuno, and S. Minami, US.Patent 5,908,615 (1999). Corey, J.M., V. Deflorio, and A. Vargas, EP Patent 0945425 (1999). Corey, J.M., V. Deflorio, and A. Vargas, US. Patent 6,022,548 (2000). Ley, J.P., G. Krammer, S. Muche, G. Kindel, and I. Reiss, U.S. Patent 2003/0152682 (2003). Suzuki, A., R. Ochiai, and I. Tokimitsu, EP Patent 1186294 (2002). Zhou, Y., A. Lips, F.S. Nanavaty, and J.B. Bartolone, Int. Patent W001/07004 (2001). Crotty, B.A., A.P. Znaiden, and A. Johnson, US. Patent 5,824,326 (1998). Udell, R.G., and S.P. Hari, US. Patent 2002/0018772 (2002). Udell, R.G., and S.P. Hari, U.S. Patent 2002/0098172 (2002). Goldman, R., US. Patent 6,056,971 (2000). Steele, D., EP Patent 0888774 (1999). Matsuda, H., M. Kawamukai, K. Yajima, Y. Ikenaka, J. Hasegawa, and S. Takahashi, EP Patent 1123979 (2001). Willis, R.A., Int. Patent W099143316 (1999). Ueda, T., T. Ono, M. Moro, S. Kitamura, and Y. Ueda, Int. Patent W003/032967 (2003). Ueda, T., S. Kitamura, and Y. Ueda, Int. Patent W003/008363 (2003). Ueda, T., S. Kitamura, and Y. Ueda, Int. Patent W003/006412 (2003). Ueda, T., S. Kitamura, and Y. Ueda, Int. Patent W003/006411 (2003). Ueda, T., S. Kitamura, and Y. Ueda, Int. Patent W003/006410 (2003). Ueda, T., S. Kitamura, and Y. Ueda, Int. Patent W003/006409 (2003). Ueda, T., S. Kitamura, and Y. Ueda, Int. Patent W003/006408 (2003). Linnane, A.W., Int. Patent W002/43721 (2002). Tang, P.H., T. de Grauw, and M.V. Miles, US. Patent 2002/0125193 (2002). Lipshutz, B.H., Int. Patent W002/14530 (2002). West, D.D., US. Patent 2002/0156302 (2002). Brancato, R., S. Capaccioli, M.F. Saettone, and N. Schiavone, Int. Patent W001/37851 (2001). Sneed, P.A., U.S. Patent 2001/0034372 (2001). Sneed, P.A., U.K. Patent 2360706 (2001). Hynes, D., Int. Patent W003/037284 (2003). Fujii, K., T. Kawabe, K. Hosoe, and T. Hidaka, Int. Patent W003/032968 (2003). Aoyama, T., H. Kubota, S. Takagi, and T. Minemura, Int. Patent W003/007928 (2003). Fujii, K., T. Kawabe, K. Hosoe, and T. Hidaka, EP Patent 1281398 (2003). Cavazza, C., U.S. Patent 2003/0059418 (2003). Aoyama, T., and Y. Sugimoto, EP Patent 1304041 (2003). Horrobin, D.F., US. Patent 2002/0198177 (2002). Fujii, K., T. Kawabe, K. Hosoe, and T. Hidaka, Int. Patent W002/092067 (2002). Hammerly, M.P., US. Patent 2002/0058026 (2002). Sears, G., and J. Feher, U.S. Patent 6,417,233 (2002). Aoyama, T., and Y. Sugimoto, U.S. Patent 6,251,442 (2001). Chopra, R.K., U.S. Patent 6,300,377 (2001). Mae, T., Y. Sakamoto, S. Morikawa, and T. Hidaka, US. Patent 6,184,255 (2001). Mae, T., Y. Sakamoto, S. Morikawa, and T. Hidaka, EP Patent 0956854 (1999). Masterson, R.V., and L.D. Manning, US. Patent 6,200,550 (2001).
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Mae, T., and T. Hidaka, US.Patent 6,156,802 (2000). Masterson, R.V., US. Patent 6,054,261 (2000). Aoyama, T., and Y. Sugimoto, EP Patent 0913095 (1999). Shuch, D.J., and G.P. Curatola, U.S. Patent 5,925,335 (1999). Fujii, K., T. Kawabe, K. Hosoe, and T. Hidaka, Int. Patent W003/033445 (2003). Anderson, M.L., and A. Kelker, US.Patent 6,403,116 (2002). Chokshi, D., U.S. Patent 2002/0128184 (2002). Chopra, R.K., U.S. Patent 6,441,050 (2002). Fujii, K., T. Kawabe, K. Hosoe, and T. Hidaka, Int. Patent W002/090304 (2002). Supersaxo, A.W., M.A. Weder, and H.G. Weder, Int. Patent W002/083098 (2002). Chopra, R.K., Int. Patent WOOU52822 (2001). Fujii, K., T. Kawabe, K. Hosoe, and T. Hidaka, Int. Patent W002/100359 (2002). Ghosh, D.K., U.V. Murthy, M.H. Randhawa, N. Gurman, P. Romanowski, M. Hernandez, and D.Eagan, Int. Patent W002/078651 (2002).
Chapter 20
Genetic Enhancement and Modification of Oil-Bearing Crops G.K. Ahmad Parveez and Ravigadevi Sambanthamurthi Advanced Biotechnology and Breeding Center, Biological Research Division, Malaysian Palm Oil Board, 50720 Kuala Lumpur, Malaysia
Introduction Plant genetic engineering became a reality with the successful insertion of a bacterial gene into tobacco mediated by a soil bacterium, Agrobacterium tumefaciens (1). Genetic transformation is a process by which foreign DNA is stably and functionally introduced into living cells. The transformed cells are then regenerated into plants. Since that success, many more plants have been transformed with various genes via different methods, The most common transformation methods include protoplast fusion via polyethylene glycol or electroporation ( 2 3 , microprojectile bombardment (4), and tissue electroporation (5). However, Agrobacterium-mediated and microprojectile bombardments remain the most popular methods. Transgenic plants with useful traits have already been produced. Initially, most of the plants were transformed to be resistant to antibiotics (l),herbicides (6), insects (7), fungi (8), and viruses (9). Later, plants with more useful traits were produced such as those with products of a higher nutritional value (lo), and those producing polymers such as polyhydroxybutyrate (PHB) (1 l), oils with different fatty acid (FA) compositions (12), and edible vaccines (13). Modification of the FA composition includes increasing the saturated fatty acids (SFA) (14), reducing polyunsaturated fatty acids (PUFA) (15 ) and synthesizing very-long-chain fatty acids (VLCFA) (16) and unusual FA (17). Recently, more useful transgenic products such as decaffeinated coffee (18), antioxidants (19), reduced allergens (20), and the human lysozyme (21) were produced. In addition to the increasing number of transformation methods and products, the number of transgenic crops is also increasing exponentially. The International Service for the Acquisition of Agri-Biotech Applications (ISAAA) summarized the status of transgenic plants planted commercially up to 2003 (22). The global area under transgenic crops has increased continuously since the first planting in 1995 to 67.7 million hectares in 2003 at a growth rate of 12-15% annually. The three countries with the largest areas are the United States, Argentina, and Canada with 42.8, 13.9, and 4.4 million hectares, respectively. Globally, -30% of transgenic crops is in developing countries, an amount that is increasing annually. The 508
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main crops grown are soybean (41.4 million hectares), corn/maize (15.5 million hectares), cotton (7.2 million hectares), and rape (canola; 3.6 million hectares). The main traits planted for are herbicide (73%) or insect resistance (18%), or a combination of both (8%). The global market value of transgenic crops in 2003 was estimated to be U.S. $4.50-4.75 billion and is expected to reach $5 billion in 2005 (22). The report highlighted the fact that the number of countries planting transgenic crops is increasing annually; Brazil and the Philippines, for example, approved transgenic crops only in 2003. It is expected that the area and countries planting transgenic crops will increase gradually in the years to come. Crops with new traits, in addition to herbicide tolerance and insect resistance, should be approved and planted in larger areas in the near future. In this chapter, we examine the genetic engineering of the following oil-bearing crops: rapeseed, soybean, cotton, maize, flax, and oil palm. The other oil-bearing crops, such as sunflower, groundnut, jojoba, safflower, coconut and olive, are not discussed due to the lack of progresdwork in FA modification or synthesis of novel products. FA in Plants
The physical and chemical properties of a vegetable oil are determined by its FA composition. FA are classified as saturated [for example, capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0), and stearic (18:O)l or unsaturated [for example, oleic (18:1), linoleic (18:2), linolenic (18:3), and erucic (22:1)] (23). The major edible oils have a similar FA composition in which those with 18 carbons and 1-3 double bonds predominate (24). Most seed oils contain predominantly long-chain fatty acyl groups such as palmitic, oleic, linoleic, and erucic. However, a few species (such as coconut) are unusually rich in medium-chain fatty acyl groups such as caprylic (8:0), capric (10:0), and lauric (12:O). Figure 20.1 shows the general plant FA biosynthesis pathway, which can serve as the basis for planning any modification of the oil composition. The oil can be modified for specific purposes. Concern over the possible contribution of saturated fats to heart disease has increasingly driven consumers to use vegetable oils with the lowest level of saturation. For example, the entry of low-erucic rapeseed (canola) oil into the edible oils market was an immediate success simply because of its low saturation (24), notwithstanding the fact that to produce margarine may require it to be artificially saturated. High-oleic acid is required to improve the stability of frying oil. The linolenic acid content has to be reduced for better shelf-life of the oil. Canola oil, which has a low lauric content, would be a better source for the detergent and surfactant industry if that content could be increased. Erucic acid is essential for the production of lubricants and erucamid in plastic manufacturing (25). In the following sections, the present status of genetic engineering for each of the oil crops described above will be explored, with a focus on the most important progress made to date.
C.K.A. Parveez and R. Sambanthamurthi
51 0
C2 FASI
+
_t
7c3
MetoacYl -ACP Stearoyl- ACP Synthase II Desaturase 16:O-ACP C18:OACP Cl8:lACP
I
Palmitoy1 - ACP Thioesterase
I
Stearoyl- ACP Thioesterase
.1
J.
C16:O Palmitic Acid
C18:O Stearic Acid
I Oieoyl-ACP Thioesterase
J. C18:l Oleic Acid
I
OleoylCoA Desaturase
.1 C18:2 C o A Fig. 20.1. General fatty acid biosynthesis pathway in plants.
Brassica napus (Rape). Rapeseed is 46% oil and 3 8 4 1 % protein by dry weight (26). Among all of the oil crops, rape has been the most engineered because of its easy transformation by Agrobacterium. Kridl and colleagues (12) altered the oil composition by antisensing the stearoyl-ACP desaturase gene to increase stearate, making the oil more suitable for margarine. The stearate content was increased from 1.8% (w/w) (normal) to 39.8% with a concomitant reduction in oleate. The high-stearate plants were morphologically normal and exhibited normal yield. To further increase the stearate content, site-directed mutagenesis of the Garcinia mangostana acyl-ACP thioesterase gene was carried out to change its substrate specificity to stearate. Transforming this mutant gene, which became very highly specific to stearoyl-ACP, into the rape plant drastically increased stearate accumulation (27) to 55-68% more than in the normal plant oil. This was a successful modification of the thioesterase substrate specificity to synthesize more of a novel product. A 12:O-ACP thioesterase from California bay (Umbellullaria californica) was isolated (28) and successfully expressed in Brassica napus (29). The plants produced up to 30 mol% laureate (-26% on a weight basis) in their oil. Interestingly, up to 56 mol% laurate accumulated in the oil of some T, individuals (14). The laurate accumulation resulted in proportionate reductions in the 16- and 18-carbon fatty acyl groups, and a proportionate accumulation of myristic acid (14:O) at a ratio of 1O:l 1aurate:myristate. Further studies showed that most of the laurate in these plants was located at the sn-1 and sn-3 positions of the triacylglycerols (TAG) with 4%at the sn-2 position. It was hypothesized that introduction of a lysophosphatidic acid acyl-transferase (LPAAT) specific for lauric acid would lead to dramatically higher lauric acid in the TAG (30). In a separate experiment, the group transformed a coconut LPAAT (C-LPAAT) with high specificity to laurate, into rape and then crossed the C-LPAAT expressing plant with the above high-laurate rape (31). The progeny showed an increase in laurate at the sn-2 position from
Genetic Modification of Oil-Bearing Crops
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<5 to 30% without a notable overall increase in the laurate content. Production of diploid plants from pollen of the F1 plants showed an accumulation of up to 67 mol% laurate with up to 75% of the laurate at the sn-2 position. In later studies, they demonstrated the effect of C-LPAAT favoring the location of laurate at the sn-2 position. This was surprising because the coconut naturally produces a very high laurate. In another study, Eccleston and Ohlrogge (32) transformed rape with a lauroyl-ACP thioesterase from California bay but could not increase the laurate content above 60 mol% in the TAG. Interestingly, a substantial portion of the FA synthesized in the transgenic seeds was recycled to acetyl-CoA and sucrose through P-oxidation, but the total oil content in the transgenic seeds remained unchanged. They postulated some coordination in the FA pathway compensating for the laurate lost via P-oxidation with the storage of longer-chain FA. They concluded that to produce a novel or specialty oil through genetic engineering, regulating the P-oxidation process may be necessary. In another report, downregulating an endogenous A12-oleoyl-ACP desaturase gene in B . napus substantially increased the oleic acid content in the oil from 63 to 89% (33). The increase was contributed mainly by a reduction in the PUFA (linoleic and linolenic) from 26 to 4%. The plants appeared normal and were able to transfer their traits to their progeny. The Calgene group also demonstrated the accumulation of 8:O (caprylate) and 10:0 (caprate) in rapeseed using an acyl-ACP thioesterase cDNA from a Mexican shrub, Cuphea hookeriana (34).The shrub accumulates up to 75% of both FA in its seed oil. The isolated thioesterase was transformed into rape, enabling the plants to accumulate up to 11,27, and 2 mol% of 8:0, 10:0,and 12:0,respectively, in the seed oil compared with below detection levels in the control plants. The 8:O and 10:0 are desirable because they are easily digested, readily absorbed, and show enhanced delivery of essential unsaturated FA in the sn-2 position. They are also in demand for synthetic motor lubricants (35). In a separate experiment, a palmitoyl-ACP thioesterase was isolated from Cuphea hookeriana and transformed into rape (36). The transgenic plants had increased palmitic acid in their oil (up to 34 vs. 6 mol% in the control), mainly from a reduction in oleic acid. They also had slightly higher stearic (18:0), myristic (14:0),and behenic (20:O)acids. The high-palmitic acid oil is good for improving the texture of margarines and shortenings (37). A cDNA clone for a seed-specific LPAAT from meadowfoam was isolated and transferred into rape (38).The transgenic plants had erucic acid (22:1 ) (absent in untransformed control) at the sn-2 position. Another group at the University of Durham isolated an LPAAT specific to erucoyl CoA from meadowfoam and transformed it into rape (39). The erucic acid at position sn-2 was increased from none up to 28.3% (mol/mol). These experiments demonstrated that LPAAT is capable of putting erucic acid at sn-2 in rapeseed oil. It was also suggested that the introduction of any gene favoring elongation of the FA chain beyond 18 in the presence of LPAAT will lead to dramatically increased levels of 22:1.
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Introduction of a jojoba P-ketoacyl-CoA synthase cDNA (KCS) into canola changed the oil content toward more VLCFA (>18 carbons), i.e., up to 42-52% (16). The transgenic progeny (T,) had higher 20:l (cosenoic acid), 22:l (erucic acid), and 24:l (nirvanic acid) from a reduction in 18:l (oleic acid). This demonstrates the role of KCS in determining the FA chain length in seed oils. Recently, the same group produced transgenic rape accumulating up to 25% nirvanic acid in the oil using the above gene isolated from Lunaria embryos (38). With the ability of LPAAT to introduce erucic acid at the sn-2 position and KCS to increase VLCFA, the group is currently combining both transgenes for developing rapeseed oil higher in VLCFA (38). Preliminary work on combining the transgenes by crossing the plants has had promising results. Progeny transgenic for LPAAT crossed with those transgenic for jojoba KCS resulted in 55% erucic acid accumulation. Interestingly, the progeny of LPAAT transgenic crossed with Lunaria KCS transgenic resulted in an accumulation of 43% erucic and 15% nirvanic acids. Another goal in the genetic engineering of rape is to reduce the polyunsaturates, i.e. linoleic (18:2) and linolenic (18:3) acids, to produce an oxidatively stable oil without the need for hydrogenation. Oleate and linoleate desaturases are involved in the biosynthesis of 18:2 and 18:3, respectively. A group at DuPont has transformed rape with an antisense oleate desaturase cDNA construct (15). The transgenic plants have increased 18:l from 63 to 83.3% and reduced 18:2 and 18:3 from 19.9 to 5% and 10.6 to 5.8%, respectively. Crossing of the transgenic line with a high 18:l mutant increased the 18:l content to 88% and reduced 18:2 and 18:3 to 2.9 and 4%, respectively. The introduction of homologous plant genes involved in FA metabolism is well demonstrated in rape. However, heterologous genes (from bacteria) are now proposed instead for overcoming any likely cosuppression (40). A group in The Netherlands isolated and expressed the bacterial malonyl CoA-acyl carrier protein transacylase (MCAT) gene in rape (41). The MCAT activity was increased 55-fold over that of the endogenous MCAT. However, no significant change in the FA profile of the oil and total seed lipid content was observed, indicating that MCAT is not a rate-limiting step in plant FA biosynthesis. The same group also isolated and transformed a bacterial fabH gene encoding for 3-ketoacyl-ACP synthase I11 (KAS 111) into rape (41). In the transgenic plants, the endogenous KAS I11 expression was increased 3.7-fold. The overexpression resulted in changes in the FA profile, i.e., 18:2 and 18:3 increased at the expense of 18:l. This demonstrates that bacterial FAS protein can interact with the plant FAS system to modify the FA profiles. In another experiment, a research group at Calgene successfully transformed a bacterial phytoene synthase (crtB) gene into rape (42). The carotenoid content in the mature seeds increased drastically (up to 50-fold), resulting in the embryos becoming orange in color. The carotenoids were mainly a- and @-carotenes.In addition to the increase in carotenoids, the plants had less sterol and chlorophyll.
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The FA composition of the plant oil also changed, with oleic acid increasing, likely due to linoleic and linolenic decreasing. In addition to oil modification, better nutritional value of rapeseed is also a goal of genetic engineering. Methionine, cysteine, and lysine are three essential amino acids required by both humans and animals (43). A gene coding for Brazil nut sulfur-rich 2s albumin (which is rich in methionine) was introduced into canola, directly increasing the methionine level in the seed by a third and making the meal a considerably better feed (10). Cruciferin (CruA),a 12s seed storage protein, is a major seed protein that contains low methionine and cysteine (44). A group in Japan transformed rape with an antisense construct containing part of the cruA gene (45). The transgenic plants contained less cruceferin but 10,8, and 32% more lysine, methionine, and cysteine, respectively. The total lipid content and FA composition remained unchanged. Another group at DuPont increased the lysine content in the seed by interrupting the regulation of a single enzyme in the biosynthetic pathway (46). The engineered expression of dihydrodipicolinic acid synthase from Corynebacterium increased the free lysine content in the seed by more than 100-fold, and the total canola seed lysine content almost doubled. Glycine max (Soybean). Soybean is one of the major crops in the United States (47). Transformation methods are available using Agrobacterium and particle bombardment. Genetic engineering to reduce the polyunsaturates, i.e., linoleic (18:2) and linolenic (18:3) acids, for an oxidatively stable oil without the need for hydrogenation was initiated by manipulating the levels of oleate and linoleate desaturases involved in the biosynthesis of 18:2 and 18:3, respectively. A group at DuPont transformed soybean with antisense constructs of linoleate and oleate desaturase cDNAs (15). Transgenic plants with antisense linoleate desaturase had increased 18:2 from 55 to 65% and reduced 18:3 from 8.9 to 1.4%. Introduction of an antisense oleate desaturase increased 18:l from 21.5 to 78.9% and reduced 18:2 to 3 from 55% and 18:3 to 5.8 from 8.9%. The transgene has been stably inherited for at least five generations (48). Crossing the transgenic lines with high-oleic acid mutants increased the oleic acid content up to 84%. Later, a palmitoyl-ACPthioesterase gene was transformed into the transgenic soybean, increasing palmitic acid by 48% (48). One of the goals in genetically engineering soybean is to produce unusual FA for diversifying the oil use. A desired FA is As-eicosenoic acid (20:l A5), which is useful in cosmetics, surfactants, and lubricants (49,50), and usually found in meadowfoam oil. Transferring a Limnanthes dauglassi acyl-CoA desaturase homolog gene into soybean resulted in a maximum 3% A5-hexadecenoic acid in the oil but <1% (wlw) each of A5-octadecenoic and A5-eicosenoic acids (51). Suspecting the low eicosenoic acid to be due to insufficient elongase activity, a L. dauglassi fatty acid elongase (FAEI)was transformed into the plants, giving a total C,, and C,, of up to 18% (w/w). The group later transferred both genes into the same plants and obtained drastic increases in both A5-eicosenoic (10.8%) and A5-docosenoic (22: 1
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A5) (1.3%) acids. The success demonstrates that producing a meadowfoam-like oil in other crops, such as soybean, is possible to enhance the value of their oil. Conjugated double bond FA are useful drying agents and important ingredients in products such as paints and inks. A group at DuPont transformed soybeans with A12-oleic acid desaturase genes from Momordica charantia and Impatiens balsamina, both of which synthesize high-a-eleoteraic (18:3 A9cis,l ltrans,l3trans) and -a-parinaric (18:4 A9cis,l ltrans,l3trans,l5cis) acids (52). They successfully produced as much as 17% (w/w) conjugated FA in the total FA using the first gene alone. Almost 90% of the conjugated FA produced was a-eleoteraic. Oleic acid was also increased in contrast to the reduction in linoleic, linolenic, and palmitic acids. Using the second gene resulted in only 3 and 2% increases in a-eleoteraic and a-parinaric acids, respectively. This report demonstrated another power of genetic engineering in modifying the composition of soybean oil for different industrial and high-value products. Soybean is estimated to cause allergy to 5 4 % children and up to 2% adults, although it is rarely life-threatening (53). Scientists have attempted to reduce the allergens in soybean via genetic engineering. There are as many as 15 proteins in soybean recognizable by immunoglobulin E from sensitive people (54). One of the major allergens is P34/Gly m Bd 30k protein. After identifying its gene, a group at Donald Danforth Plant Science Centre silenced it (20). The completely silent effect was demonstrated in the first transgenic lines and was stably passed on homozygously up to the third generation. The silencing had no adverse effects on the growth of the plants. This success helped reduce the allergenic effect of soybeans. Another group at DuPont improved the nutritional value of soybean meal by increasing its lysine content (46). The expressions of dihydrodipicolinic acid synthase (DHDPS) from Corynebacterium and aspartokinase (AK) from Escherichia coli in the transgenic soybean altered or bypassed the feedback regulation in the biosynthetic pathway, resulting in a several hundred-fold increase in free lysine and a 5-fold increase in total seed lysine. Other progress made includes transgenic plants resistant to the velvet bean caterpillar ( 5 9 , bean pod mottle virus (BPMV) (56),and the herbicide glyphosate (57).
Gossypium hirsutum (Cotton). Cotton is grown mainly for its lint, which provides much of the high-quality fiber for the textile industry. Cottonseed is also a valuable commodity, producing oil, meal, and hull. Genetic engineering of cotton is possible with the successful development of Agrobacterium and particle bombardment transformation methods. Liu and colleagues (58) at CSIRO, Australia, altered the seed oil composition using inverted-repeat gene-silencing on the endogenous A9-stearoyl-ACP desaturase gene. The result was an increase in stearic acid from 2 to 40% from reductions in palmitic, oleic, and linoleic acids. The high-stearic acid plants stably passed down the traits. This success reduced the hydrogenation required for saturating the oil in margarine production. Using the same approach, the group also
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downregulated the endogenous A12-oleoyl-ACPdesaturase gene, thereby drastically increasing the oleic content from 13 to 78% (58). The increase in oleic was interesting because it was at the expense not only of linoleic acid but also of palmitic acid. The group has since crossed the two transgenic types (high-oleic and high-stearic) to produce F, lines with 40% stearic and 37% oleic acid in its oil (59). The group is confident that they can tailor the FA composition in the oil for different uses, such as cocoa butter substitutes. The transgenic plants are now undergoing detailed evaluation for commercialization. Production of transgenic cotton resistant to the herbicide 2,4-D (60), the larvae of Helicuverpa zea (ballworm), Trichoplusia ni (cabbage looper), and Spudoptera exigua (61) and sweet potato whitefly (62) has also been successful.
Zea mays (Corn/Maize). Maize is one of the world’s major crops grown for its starch, which is converted industrially to high-fructose corn syrup. High-oil varieties are of no interest because the oil interferes with the starch production (63). Transformation systems for maize are available via particle bombardment, Agrobacteriurn, electroporation of regenerable tissues, and silicon carbide whisker mediation. Metabolic engineering of corn to modify its vitamin E composition (tocotrienols vs. tocopherols) was conducted at Pioneer Hi-Bred. Tocotrienols and tocopherols are both potent antioxidants, but the former are more efficient in scavenging free radicals (64). They are also beneficial in inhibiting breast cancer cells (65). Expressing a barley homogensitic acid geranylgeranyl transferase (HGGT) gene that codes for the enzyme that catalyzes the first committed step in tocotrienol biosynthesis in maize resulted in up to sixfold increases in both tocotrienols and tocopherols in the oil (19). Interestingly, tocotrienols accounted for 74% of the total vitamin E content. The results demonstrated that a single gene manipulation can stimulate tocotrienol biosynthesis in plants. The success can improve the shelf-life and oxidative stability of the cooking oil and will be useful in the production of lubricating oil. Avidin, a glycoprotein normally found in egg white, was synthesized in maize seed via genetic engineering (66). The transformants showed that avidin can be produced at >2% of the dry seed soluble protein with most of it in the embryo. The trait was stably inherited up to T4, producing avidin at 230 mgkg seed. The protein was identical to the native protein and stable after commercial processing. This study demonstrated that animal protein can be efficiently and stably synthesized in transgenic maize. A group at the University of Minnesota (67) initiated maize genetic engineering to improve the food and feed quality. A dihydropicolinate synthase (DHDPS) gene was transformed into it, and the transgenic cultures had four times higher cellular free lysine. Production of transgenic maize resistant to the European corn borer (68), maize dwarf mosaic virus (MDMV), and maize chlorotic mottle virus (MCMV) (9) was also achieved.
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Linum usitatisimum (Flax). Linseed oil, which is rich in linolenic acid (55%), is produced from flax, and is used in paints, varnishes, inks, and linoleum as a drying oil. Genetic engineering by RNA antisensing to suppress stearoyl ACP desaturase in a high-palmitic/low-linolenic germplasm was suggested to increase stearic and oleic acids to almost the same level as palmitic acid (69). The high-linolenic acid in linseed oil makes flax an ideal target for increasing y-linolenic acid (GLA), one of the essential FA widely used in the nutraceutical and pharmaceutical industries. Transforming a borage A6-desaturase gene construct into flax resulted in up to 2% GLA accumulation in the total FA of the oil (70). The low synthesis of GLA was postulated to be due to the exogenous promoter. When the gene was retransformed using a flax endogenous seed-specific promoter, the production of GLA increased up to 20% of total FA (71). Other progress made is the production of transgenic plants resistant to the herbicides glyphosate (72) and sulfonylurea (73). Elaeis guineensis and E. oleifera (Oil Palms). Palm oil is generally the most price-competitive cooking oil in the world. It is also used in other food products such as shortenings, margarines, and spreads (74). Interestingly, it was shown that palm oil lowers the serum cholesterol level to the same degree as sunflower oil, which is rich in PUFA (75).Furthermore, palm oil has antitumor effects (76), presumably due to its high level of vitamin E, especially tocotrienols (77). The main goal in the genetic engineering of oil palm is to produce a high-oleic oil (78). Such an oil could serve as an industrial feedstock for the oleochemical industry. Other targets of engineering in the oil include higher stearic, palmitoleic, and ricinoleic acids, and the production of biodegradable plastics (79,80). Currently, oil palm genetic engineering is carried out primarily for research. The transgenic palms are planted in a fully contained biosafety greenhouse for evaluation only. A transformation system for oil palm was developed using microprojectile bombardment (81). The production of high-oleic oil palms started off with an analysis of the high level of palmitic acid present in palm oil (44%). From the biochemical studies, the strategy devised was to upregulate KAS I1 and downregulate palmitoyl-ACP thioesterase (82). Downregulating oleoyl-CoA desaturase would then minimize the overflow of oleic to linoleic acid. Transformation of oil palm embryogenic cultures with constructs canying antisense palmitoyl-ACP thioesterase and sense KAS I1 genes was therefore carried out. Currently, the bombarded cultures are at various stages of regeneration with a few hundred plantlets already transferred to soil in the biosafety greenhouse (83). Polymerase chain reaction (PCR) analysis revealed > 90% of the transgenic lines to be positive for the selectable marker and palmitoyl-ACP thioesterase genes. FA analysis, via GC, to assess the FA changes in the young plants has been initiated. This is possible at an early stage because the first batch of plants was transformed with genes driven by a constitutive promoter. Thus, there was no need to wait for the plants to produce fruit (this usually takes 3-4 yr).
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Biochemical studies demonstrated that oil palm contains an active stearoylACP desaturase that can thus be downregulated to increase stearic acid as was done in rape (12). High-stearic palm oil is expected to spawn new applications, such as cocoa butter substitutes and personal-care products including lotions, shaving cream, and rubbing oils (79). Transformation of oil palm embryogenic cultures with constructs carrying an antisense stearoyl-ACP desaturase gene was canied out successfully. Currently, numerous resistant polyembryogenic cultures exist and are undergoing plant regeneration (84). The production of transgenic oil palm synthesizing biodegradable plastics, such as polyhydroxybutyrate (PHB) or copolymer poly-3-hydroxybutyrate-co-3hydroxyvalerate (PHBV), was proposed using the P-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase genes (85). In addition, the threonine dehydratase gene (86) will be used for producing propionyl-CoA, the starting material for PHBV. This work is being carried out under the Malaysia-MassachusettsInstitute of Technology Biotechnology Partnership Programme (79). Production of PHB and PHBV in plants was demonstrated earlier (11,87). Transformation of embryogenic cultures with the above genes has been carried out. Currently, the transformed cultures have been regenerated into a few thousand plantlets maintained in a biosafety nursery (88). Molecular analysis, using PCR, revealed that S O % of the transgenic plantlets were positive for the selectable marker and PHB or PHBV genes. GC analysis for any PHB or PHBV synthesized in the plantlets will be carried out. Future Prospects and Challenges
The previous sections clearly demonstrated the prospects for plant genetic engineering, especially oil-bearing crops. Genetic engineering is now possible for almost all oil crops, including oil palm, a perennial monocot among the last to be transformed. Modifications of the oil FA composition were among the pioneering successes. Later, syntheses of novel FA and products, such as unusual FA, conjugated FA, carotenoids, and biopolymers, were achieved. The successes achieved to date clearly demonstrate that genetic engineering is a powerful tool not only for improving the crop quality and oil composition, but also for synthesizing novel high-value products. It is expected that more and more novel products and FA will be produced in transgenic plants in the near future. Many significant successes have already occurred in the genetic engineering of oil-bearing crops. In addition to the early standard antisense downregulation of genes, new techniques such as inverted-repeat gene-silencing are now powerful tools for this downregulation (58). Site-directed mutagenesis of specific genes resulted in changes in the enzyme substrate specificity and also increased the yields of particular FA or products (27). Modifications by introducing specific acyltransferases can also direct a desired FA to a specific site of the TAG backbone (sn position) and increase its content in the oil (31). Using a gene from an organism that produces a particular product or FA copiously can increase produc-
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tion of the product in a heterologous system (51). Similarly, transforming a plant with nonplant genes, such as the bacterial phytoene synthase gene, has considerably increased the carotenoids (42). Interestingly, the proteins produced by genetic engineering have been stable, even after commercial processing, and identical to the native proteins from heterologous systems (66). In certain cases, manipulating only a single gene sufficed to stimulate the whole pathway in a plant (19). However, despite the successes and prospects, genetic engineering still faces two main challenges, i.e., technical limitation and commercialization. For technical limitation, it was mentioned earlier that when overexpressing an enzyme, it is important to consider whether it is part of a multienzyme complex in equilibrium with other subunits, The overexpression may result in disproportionate changes in the activity of other enzymes (89). In many instances, manipulating one of two genes was insufficient for synthesizing a high level of novel proteidoil in the plants. The transcription factors and/or other nonstructural genes may occasionally also require manipulation. There remain many more unknowns to be uncovered for synthesizing a particular product at a very high level and stably passing on the trait to the following generations. For oil palm, in particular, an additional limitation faced is the long generation cycle with 3-4 yr required before any novel products or modification can be detected in the fruit. The second challenge is the legislative approval for commercial planting and selling of the engineered products. The transgenic plants and their products would have to be safe environmentally and for consumption. For environmental safety, there is a call for the creation of antibiotic marker-free transgenic crops. There are methods for removing selected marker genes, such as using two t-DNA plasmids (90) or even replacing them with nonantibiotic genes such as the phosphornunnose isomerase gene (91). Self-containment of the transgenes via systems such as chloroplast transformation (92) can also be considered. Another concern is the integration of vector DNA into the plant genome in addition to the gene of interest for both direct (93) and Agrobacterium-mediated (94) gene transfer. Using the minimum of transgene cassettes (promoter, open reading frame, and terminator) was shown to be possible, encouraging the integration of low-copy-numbers of transgenes (95). The last and most important issue to consider is whether the food is safe for consumption. Transgenic food must be evaluated for safety in the following areas: the gene and other DNA fragments introduced, the specific product produced by the gene, and the overall product itself (96). The new product is required to be “substantially equivalent” to the traditional product from nontransgenic crops. The questions to be posed to the producer of genetically engineered products are the following: (i) Is the newly produced gene product toxic or potentially toxic? (ii) Has the overall allergenicity of the product changed? (iii) Has the nutrient composition changed? (iv) Is there any unintended effect that can create a new toxicity or allergenicity? and (v) Is any antibiotic marker gene present, and if so, is it safe? (96). Answering all of these questions is not easy because analyses, expertise and,
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most importantly, money, are all required. These are some of the challenges that have to be tackled before any genetically engineered products can be commercialized.
Conclusions In the above sections, successes in the genetic engineering of oil-bearing crops were described to show the potential of the technology. Various products were modified and novel ones synthesized. However, the longer journey remains ahead because higher yields have to be obtained for the products Various approaches have been tried to increase the yields with some promising success, but, nevertheless, still only a promise. Finally, there remain social and legal hurdles to the commercialization of the products. I
Acknowledgments The authors thank the Director-General of MPOB for permission to publish this chapter. We are also grateful to Mr. Andy Chang of MPOB for his invaluable comments on the manuscript.
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58. Liu, Q., S . Singh, and A. Green, High-Oleic and High-Stearic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing, Plant Physiol. 129: 1732-1 743 (2002). 59. Liu, Q., S. Singh, and A. Green, High-Oleic and High-Stearic Cottonseed Oils: Nutritionally Improved Cooking Oils Developed Using Gene Silencing, J . Am. Coll. Nutr. 21: 2058-21 1s (2002). 60. Lyon, B.R., D.J. Llewellyn, J.L. Huppatz, E.S. Dennis, and W.P. Peacock, Expression of a Bacterial Gene in Transgenic Tobacco Plants Confers Resistance to the Herbicide 2,4Dichlorophenoxyacetic Acid, Plant Mol. Biol. 13: 533-540 (1989). 61. Bayley, C., N. Trolinder, C. Ray, M . Morgan, J.E. Quisenberry, and D.W. Ow, Engineering 2,4-D Resistance into Cotton, Theor. Appl. Genet. 83: 645-649 (1992). 62. Thomas, J.C., D.G. Adams, V.D. Keppenne, C.C. Wasmann, J.K. Brown, M.R. Kanost, and H J. Bohnert, Protease Inhibitors of Manduca sexta Expressed in Trangenic Cotton, Plant Cell Rep. 14: 758-762 (1995). 63. Hills, M.J., and D.J. Murphy, Biotechnology of Oilseeds, Biotechnol. Genet. Eng. Rev. 9: 1-46 (1991). 64.Serbinova, E.A., and L. Parker, Antioxidant Properties of a-Tocopherol and aTocotrienol, Methods Enzymol. 234: 354-366 (1994). 65. Nesaretnam, K., R. Stephen, R. Dils, and P. Debre, Tocotrienols Inhibit the Growth of Human Breast Cancer Cells Irrespective of Estrogen Receptor Status, Lipids 33: 461469 (1998). 66. Hood, E.E., D.R. Witcher, S. Maddock, T. Mayer, C. Baszczynski, M. Bailey, P. Flynn, J. Register, L. Marshall, D. Bond, E. Kulisek, A. Kusnadi, R. Evangelista, Z. Nikolov, C. Wooge, R.J. Mehigh, R. Hernan, W.K. Kappel, D. Ritland, C.P. Li, and J.A. Howard, Commercial Production of Avidin from Transgenic Maize: Characterization of Transformant, Production, Processing, Extraction and Purification, Mol. Breed. 3: 291-306 (1997). 67. Bittel, D.C., J.M Shaver, D.A. Somers, and B.G. Gengenbach, Lysine Accumulation in Maize Cell Cultures Transformed with Lysine-Insensitive Form of Maize Dihydrodipicolinate Synthase, Theor. Appl. Genet. 92: 70-77 (1996). 68. Kozial, M.G., G.L. Beland, C. Bowman, N.B. Carozzi, R. Crenshaw, L. Crossland, J. Dawson, N. Desai, M. Hill, S. Kadwell, K. Launis, D. Maddox, K. McPherson, M.R. Meghji, E. Merlin, R. Rhodes, G.E. Warren, M . Wright, and S.V. Evola, Field Performance of Elite Transgenic Maize Plants Expressing an Insecticidal Protein Derived from Bacillus thuringiensis, BiolTechnol. 11: 194-200 (1993). 69. Rawland, G.G., A. McHughen, L.V. Gusta, R.S. Bhatty, S.L. MacKenzie, and D.C. Taylor, The Application of Chemical Mutagenesis and Biotechnology to the Modification of Linseed (Linum usitatissimum L.), Euphytica 85: 317-321 (1995). 70. Qiu, X., H. Hong, S.L. Mackenzie, C.D Taylor, and L.T. Taylor, Expression of Borage A6-Desaturase in Saccharomyces cerevisiae and Oilseed Crops, Can. J . Bot. 80: 42-49 (2002). 71. Qiu, X., H. Hong, N. Datla, D.W. Reed, M. Truksa, Z. Hu, P.S. Covello, and S.L. Mackenzie, Production of Nutraceutical Fatty Acids in Oilseed Crops, in Advanced Research on Plant Lipids, edited by N. Murata, M. Yamada, I. Nishida, H. Okuyama, J. Sekiya, and W. Hajime, Kluwer Academic Press, Dordrecht, 2003, pp. 4 0 3 4 6 .
72. Jordan, M.C., and A. McHughn, Glyphosate Tolerant Flax Plants from Agrobacterium Mediated Gene Transfer, Plant Cell Rep. 7: 281-284 (1988).
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73. McHughen, A., Agrobacterium Mediated Transfer of Chlorsulfuron Resistance to Commercial Flax Cultivars, Plant Cell Rep. 8: 4 4 5 4 9 (1989). 74. Pantzaris, T.P., Trends in Yellow Fats Consumption in EEC, Palm Oil Dev. 18: 3-7 (1993). 75. Qureshi, A.A., N. Qureshi, J.J.K. Wright, S.Shen, G. Kramer, A. Gapor, Y.H. Chong, G. Dewitt, A.S.H. Ong, D. Peterson, and B.A. Bradlow, Lowering of Serum Cholesterol in Hypercholesterolemic Humans by Tocotrienols (Palmvitee), Am. J . Clin. Nutr. 53: 1021s-1026s (1991). 76. Kritchevsky, D., M.M. Weber, and D.M. Klurfeld, Influence of Different Fats (Soybean Oil, Palm Olein or Hydrogenated Soybean Oil) on Chemically-Induced Mammary Tumors in Rats, Nutr. Res. 12: S1754179 (1992). 77. Tan, B., Antitumor Effects of Oil Palm Carotenes and Tocotrienols in HRS/J Hairless Female Mice, Nutr. Res. 12: S163-Sl73 (1992). 78. Cheah, S.C., R. Sambanthamurthi, S.N.A. Abdullah, A. Othman, M.A.A. Manaf, U S . Ramli, and G.K.A. Parveez, Towards Genetic Engineering of Oil Palm (Elaeis guineensis Jacq.), in Plant Lipid Metabolism, edited by J.C. Kader and P. Mazliak, Kluwer Academic Publishers, Dordrecht, 1995, pp. 570-572. 79. Parveez, G.K.A., R. Sambanthamurthi,A. Siti Nor m a r , 0.Rasid, M.M. Masri, and S.C. Cheah, Production of Transgenic Oil Palm-Current Success and Future Considerations, in Proceedings of the 1999 PORIM International Palm Oil Congress, edited by D. Ariffin, K.W. Chan, and S.R.S.A. Sharifah, Kuala Lumpur, Malaysia, 1999, pp. 3-13. 80. Parveez, G.K.A., Novel Products From Transgenic Oil Palm, AgBioTechNet 5: 1-8 (2003). 81. Parveez, G.K.A., Production of Transgenic Oil Palm (Elaeis guineensis Jacq.) Using Biolistic Techniques, in Molecular Biology of Woody Plants, edited by S.M. Jain and S.C. Minocha, Kluwer Academic Publishers, Dordrecht, Vol. 2,2000, pp. 327-350. 82. Sambanthamurthi, R., G.K.A. Parveez, and S.C. Cheah, Genetic Engineering of the Oil Palm, in Advances in Oil Palm Research, edited by B. Yusof, B.S. Jalani, and K.W. Chan, MPOB, Kuala Lumpur, 2000, pp. 284-331. 83. Parveez, G.K.A., R. Omar, A.M.Y. Masani, H.F. Haliza, A.M. Na’imatulapidah, A.D. Kushairi, A.H. Tarmizi, and I. Zamzuri, Transgenic Oil Palm: Where Are We? in Advanced Research on Plant Lipids, edited by N. Murata, M. Yamada, I. Nishida, H. Okuyama, J. Sekiya, and W. Hajime, Kluwer Academic Publishers, Dordrecht, 2003, pp. 415-4 18. 84. Parveez, G.K.A., 0. Abrizah, A.H. Tarmizi, I. Zamzuri, A.D. Kushairi, Y. Salmah, B. Bahariah, and K. Sabariah, Transformation of Oil Palm with Antisense Palmitoyl-ACP Thioesterase Gene for Increasing Oleic Acid Content, in Proceedings of the 2003 MPOB International Palm Oil Congress, 2003, pp. 869-878. 85. Anderson, AJ., and E.A. Dawes, Occurrence, Metabolism, Metabolic Role and Industrial Uses of Bacterial Polyhydroxylalkanoates,Microbiol. Rev. 54: 450-472 (1990). 86. Guillouet, S., A.A. Rodal, G.H. An, P.A. Lessard, and A J . Sinskey, Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production, Appl. Environ. Microbiol. 65: 3100-3107 (1999). 87. Slater, S., T.A. Mitsky, K.L. Houmiel, M. Hao, S.E. Reiser, N.B. Taylor, M. Tran, H.E. Valentin, D.J. Rodriguez, D.A. Stone, S.R. Pedgate, G. Kishore, and K.J. Gruys, Metabolic Engineering of Arabidopsis and Brassica for Poly(3-hydroxybutyrate-co-3hydroxyvalerate) Copolymer Production, Nut. Biotechnol. 17: 1011-1016 (1999).
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88. Parveez, G.K.A., A.M.Y. Masani, Y. Salmah, B. Bahariah, K. Sabariah, G. York, and Y.B. Jo, Transfer of PHB and PHBV Genes into Oil Palm for the Production of Biodegradable Plastics, in Proceedings of the 2003 MPOB International Palm Oil Congress, MPOB, 2003, pp. 859-868. 89. Kridl, J.C., V.C. Knauf, and G.A. Thompson, Progress in Expression of Genes Controlling Fatty Acid Biosynthesis to Alter Oil Composition and Content in Transgenic Rapeseed, in Control of Plant Gene Expression, edited by D.P.S. Verma, CRC Press, Boca Raton, 1993,pp. 481-498. 90. Komari, T., Y. Hiei, Y. Saito, N. Murai, and T. Kumashiro, Vectors Carrying Two Separate T-DNAs for Co-Transformation of Higher Plants Mediated by Agrobacterium tumefaciens and Segregation of Transformants Free from Selection Markers, Plant J . 10: 165-174 (1996). 91. Joersbo, M., I. Donaldson, J. Kreiberg, S.G. Petersen, J. Brunstedt, and F.T. Okkels, Analysis of Mannose Selection Used for Transformation of Sugar Beet, Mol. Breed. 19: 798-803 (1998). 92. Daniell, H., R. Datta, S. Varma, S. Gray, and S.B. Lee, Containment of Herbicide Resistance Through Genetic Engineering of the Chloroplast Genome, Nat. Biotechnol. 16: 345-348 (1998). 93. Kohli, A,, S. Griffths, N. Palacios, R.M. Twyman, P. Vain, D.A. Laurie, and P. Christou, Molecular Characterization of Transforming Plasmid Rearrangements in Transgenic Rice Reveals a Recombination Hotspot in the CaMV 35s Promoter and Confirms the Predominance of Microhomology-Mediated Recombination, Plant J . 17: 59 1-601 (1999). 94. Cluster, P.D., M. O’Dell, M. Metzlaff, and R.B. Flavell, Details of T-DNA Structural Organization from Transgenic Petunia Population Exhibiting Co-Suppression, Plant Mol. Biol. 32: 1197-1203 (1994). 95. Fu, X., L.T. Duc, S. Fontana, B.B. Bong, P. Tinjuangjun, D. Sidhakar, R.M. Twyman, P. Christou, and A. Kohli, Linear Transgene Constructs Lacking Vector Backbone Sequences Generate Low-Copy-Number Transgenic Plants with Simple Integration Patterns, Transgenic Res. 9: 11-19 (2000). 96. Chassy, B.M., Food Safety Evaluation of Crops Produced Through Biotechnology, J . Am. Coll. Nutr. 21: 166s-173s (2002).
Chapter 2 1
Genetically Engineered Oils David Hildebrand and Lewamy Mamadou University of Kentucky, Lexington, KY 40506
Introduction Vegetable oils are the major source of the world’s edible fat and oil production, accounting for -85% of that total; the remainder comes from animal and marine sources. The demand for vegetable oils has increased steadily in recent years not only for food uses but also for industrial purposes. The major oilseed crops supply close to 100 million metric tons of oil in the world. About two thirds of the oil is from soybean, palm, and canola (a genetically altered rapeseed). The major fatty acids (FA) of the world oil supply are palmitic, linoleic, and oleic acids. Advances in understanding lipid biosynthesis have facilitated the genetic engineering of oil seeds as a supplement to breeding oilseeds for improved composition of many of the major oilseeds; new molecular genetic tools have accelerated this progress. The development through plant genetic engineering of functional or “designer” foods represents an opportunity to improve the health and well-being of a vast number of people worldwide. The first reports on genetic engineering of the oil composition in plants appeared in the early 1990s (1,2). An example of molecular genetic improvement is Brassica napus and B. rapa, sources of canoldrapeseed oil. Despite the continuing recalcitrance of soybean transformation and regeneration, considerable progress has been made, including molecular genetic improvement of its oil composition. Edible fat and vegetable oils constitute a major component of human diets (-25% of average energy intake) (3,4). Epidemiologic studies indicate that the prevalence of and mortality from atherosclerotic vascular disease, especially coronary heart disease, are associated with the nature and quality of dietary lipids. Because of the broad uses of plant oils in both food and nonfood applications, metabolic engineering of plant oils has been conducted by academic and industrial researchers (5). As a consequence, plant genetic engineering and its development have opened a window of opportunity to improve the health and well-being of people worldwide through tailoring of the composition and optimization of plantderived lipids with respect to food functionality and dietary needs. Until recently, some modification of the lipid composition of crop plants was achieved through traditional plant breeding techniques by using the natural diversity that exists among plant varieties and closely related species to transfer desirable characteristics from one another as well as chemical or nuclear mutagenesis (3,6,7). The combination of 526
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modern gene technology and plant breeding has provided a powerful tool for qualitatively improving not only the composition of oilseeds to provide the functional properties required for various food oil applications, but also to improve their nutritional value. A major goal in changing the composition of plant storage oil was to change the degree of FA desaturation. Hence, numerous high-oleic, highstearate, high-palmitate, and high-laureate oils were developed to provide highly functional oils. Using a combination of conventional selection, induced mutagenesis, antisense and co-suppression techniques, or post-transcriptional gene silencing (PTGS), some major changes in the proportions of individual FA in a range of oilseeds have been achieved (8-1 0). Several studies demonstrated the positive correlation between the levels of specific FA in plasma and the FA consumed in the diet. However, whether the total fat content of the diet alters the FA composition of plasma phospholipids, cholesteryl ester, triacylglycerol (TAG), and free FA is unknown (1 1-16). Although the relation between dietary fats and chronic disease in humans has long been recognized, certain epidemiologic studies suggested that not only total lipids but also certain types of FA found in the diet can play an adverse as well as a beneficial role in atherosclerosis and in the pathogenesis of different malignancies (17). There have also been indications based on epidemiologic studies of a strong, continuous, and positive relation between total cholesterol levels in blood and the prevalence and incidence of, and mortality from, atherosclerotic vascular disease, especially coronary heart disease (3,18,19). A substantive body of evidence on dietary lipids and blood pressure of humans also suggests that a diet with a high polyunsaturated-to-saturated ratio and low total fat can produce modest reductions in blood pressure in normotensive and hypertensive persons (3). Patterns of fat consumption associated with decreased risk of cardiovascular disease are also associated with a decreased risk of cancer and diabetes (3,20,21). Efforts to predict the effect of diverse fats and oils on degenerative disease risk are complicated by differences in FA profile, FA chain length, degree of saturation, positional isomerization of cis and trans unsaturated bonds, triglyceride structure, presence of lipid oxidation products such as oxysterols, and the presence of antioxidants (22-25). Although metabolic and epidemiologic studies of humans and laboratory animals generally support the concept that a higher intake of polyunsaturated fatty acids (PUFA) is beneficial in terms of lipoprotein metabolism and cardiovascular health (3,26), the consumption of high amounts of PUFA when insufficiently protected by antioxidants led to enhanced susceptibility of membrane lipids to peroxidation. Several studies showed that dietary supplementation with either n-6 or n-3 PUFA is associated with increased susceptibility of tissue lipids to peroxidation both in vivo and in vitro (27). Thus, in view of all the negative effects of lipid oxidation, vegetable oils with reduced levels of PUFA and a higher content of monounsaturated fatty acids (MUFA) have become highly desirable. Nutritionally, the MUFA “oleic acid’ (18;1) appears to have the same low-density lipoprotein (LDL)-lowering effect as linoleic acid and is not as susceptible to in vivo oxidation as linoleic acid (3,28).
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Thus, it was found that a higher ratio of oleic to linoleic acid is inversely correlated with the peroxidation rate of plasma LDL (29-3 1). Because an increase in the ratio of MUFA to PUFA (at least n-6 PUFA) of most vegetable oils appears to be beneficial to health, this goal has been sought by researchers through genetic engineering. Moreover, scientific evidence has accumulated in the past two decades about the beneficial effect of diets with relatively high MUFA contents on a number of cardiovascular risk outcomes, including diabetes and cancers in humans (20,21, 32,33). However, paradoxical results from an experiment in monkeys showed that a diet high in MUFA caused atherosclerosis equivalent to that observed in animals fed a diet high in saturated fatty acids (SFA), apparently due to an increased secretion of cholesteryl oleate-enriched lipoproteins (20,34). Whether this finding is relevant to humans has to be evaluated because it counters the substantive body of evidence showing that MUFA have beneficial effects. There has been a considerable interest recently in increasing the palmitoleic acid (16:l) content in diets for health purposes (35). Thus, it was postulated that palmitoleic acids and lipids incorporating them may be beneficial to health when administrated as foods or pharmaceuticals. MUFA lipids melt at lower temperatures because the double bond is positioned more remotely from the carboxyl group. Thus, one can assume that palmitoleic acid (with a titer of 1 C) shares with oleic acid (16 C titer) the highly beneficial and greater stability of monounsaturated compared with polyunsaturated alkyl structures. FA with a greater fluidity may be expected to be advantageous in several ways. It will increase the activity of the lipases that hydrolyze triglycerides to yield free fatty acids (FFA), which require fluid rather than solid triglycerides as substrates (24). Thus, the greater fluidity of the MUFA and their triglycerides may allow lipoprotein lipase to hydrolyze chylomicrons and very low density lipoproteins (VLDL) more efficiently when they are enriched with 161. In addition this may lower VLDL levels and ultimately lower LDL cholesterol levels. Overall, the enhanced activity of lipases on highly fluid 161 triglycerides may result in a more active release of these FA from adipose tissues, thereby reducing fat deposits (2436). High-Steamte Oils Currently, industries that manufacture shortening, margarine, and confectionery products use large amounts of stearate (18:O) produced mainly from partially hydrogenated plant oils (37). Hydrogenation generates not only extra cost but also significant amounts of truns-FA, which are associated with an elevated risk of heart disease (37-39). Industries manufacturing shortenings and confectionery products could benefit from an oil crop capable of accumulating high levels of stearate. However, stearate does not naturally accumulate to abundant levels in most cultivated oil crops, and the production of a high-stearate phenotype has had only limited success to date through conventional breeding and mutagenesis techniques (37). Although stearic acid is one of the major SFA in most seed oils, its percentages vary among the different oilseed crops from 1.0% in rapeseed oil to 3.6% in sesame
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and corn oils and 4.0% in soybean oil, with a range from 2.2 to 7.2% for the genotypes available in the world germplasm collection (40-42). However, a few plant species, cocoa (Theobroma cacao), shea (Butyrospermumparkii), sal (Shorea robusta),kokum (Garcinia indica), and mangosteen (Garcinia mangostam), were identified as accumulating significant amounts of stearate in their seed oils (43,44). These oils were found to be valuable as cocoa butter or cocoa butter substitutes in many confectionery applications (44). The FA composition of soybeans and safflower has been improved by using selective breeding techniques utilizing natural variants or induced mutagenesis (45-47). Hammond and Fehr (48) were able to increase the amount of stearate produced in soybean oil to levels up to -28.1% of the total FA content using mutagenesis. Rahman et al. (49) reported a novel soybean germplasm with high-stearic levels. This novel soybean was obtained as a consequence of the combination of the loci of highpalmitic and high-stearic acids to determine the effects of altered contents of these two FA on other FA. As a result, two lines (M25 and H P S ) with a fivefold increase in stearic acid (from 34 to 181 and 171 g k g , respectively) were developed. This increase in stearic acid was also associated with a change in the content of oleic and linoleic acids. Furthermore, these authors reported that when oils high in palmitic and stearic acids were combined in the germplasm HPS, this line happened to have the highest known SFA content to date (>380 g k g ) . Thus, such oil might have the potential to increase the utility and improve the quality of soybean oil for specific purposes (49). With the advent of genetic engineering using molecular genetic techniques, several strategies in increasing stearic acid levels in oilseed crops are now possible and the increase in levels of stearic acid is usually at the expense of oleic (18: 1) and linoleic (18:2) acids. Among other strategies, antisense suppression or cosuppression to reduce or knock out the activity of stearoyl-ACP desaturase, which is responsible for converting stearoyl-ACP (saturated) to oleoyl-ACP (unsaturated) (50), is used routinely. Stearoyl-ACP thioesterase is another possible metabolic target. Thus, upregulation of this enzyme by sense-oriented reintroduction of the stearoyl-ACP thioesterase resulted in an increase in the release of free stearate. Knutzon et al. (lo), using a seed-specific antisense mediated PTGS technique, succeeded in downregulating the expression of the Ag desaturase enzyme that converts stearic acid to oleic acid in developing rapeseed. The result was an increase in stearic acid from 2 to -40% at the expense of oleic acid (18: 1) in the transgenic plants, making the oil potentially useful as a cocoa butter substitute (51). Kridl (52) reported transgenic soybeans with stearate levels as high as -53% of the FA content, whereas levels of -4% were observed in nontransformed control plants. In addition, the “said oil composition” of these transgenic plants is unique in that it is low in linoleic and linolenic and high in oleic and stearic acids. This oil should not only have better stability to oxidative degradation but also the change in its triglyceride composition results in a higher melting point than that of common soybean oil. Using chemical mutagenesis, Osorio et al. (53) reported mutant sunflowers with a high concentration of SFA in the oil. Of these reported mutants, mutants CAS-3, CAS-4, and CAS-8 contained stearic acid levels 2-6 times higher than that
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normally observed in the oil of regular sunflowers. Martinez-Force et al. (54)also reported sunflower lines with stearic acid levels between 19 and 35%, obtained through chemical mutagenesis. Using a hairpin RNA-mediated (hpRNA) gene silencing technique, Liu et al. (8) succeeded in transforming cotton (Gossypium hirsutum) with high levels of stearic and oleic acids by downregulating the seed expression of two key FA desaturase genes “ghSAD- 1 encoding stearoyl-acyl carrier protein A9 desaturase and ghFAD2- 1 encoding oleoyl-phosphatidylcholineA12 desaturase .” Cotton is grown primarily for fiber production, but it represents the world’s sixth largest source of vegetable oil. Because of its oil composition (high in palmitic and linoleic, and low in oleic acids, 26,58, and 15%, respectively), cotton oil is nutritionally undesirable because of its LDL-cholesterol raising properties (55). However, cotton is suitable for high-temperature frying applications, although it sometimes has to be hydrogenated to achieve the very high stability needed. Hence, the manipulation of these two key proteins to various degrees could enable the development of cottonseed oils with novel combinations in its FA composition that could be used in margarines, deep frying without hydrogenation, and potentially also in high-value confectionery applications (56). Oils with increased stearic acid content are being developed to enable the production of solid fats without hydrogenation (56).
High-Oleafe Oils As mentioned elsewhere, a major determinant of the quality of edible plant oils is the FA composition. Thus, several studies indicated that oils rich in oleic acid and low in PUFA are commercially and nutritionally desirable. Also, as reported by several studies, the relative susceptibility of FA acyl residues to oxidation, which causes unpleasant odors and tastes commonly associated with rancidity, and the deleterious effects (potential atherogenic effects) of these oxidized products to health were alleviated by the increase of oleic acids. As a result, diets containing oils rich in monounsaturates were reported to be beneficial for health due to their lowering effects on cholesterol levels, without the negative effects of lipid oxidation (3,43758). Several studies also indicated that selective properties of linoleic acid stimulated development of a proinflammatory environment and caused injury to endothelial cells, whereas oleic acid helped to decrease this inflammation (59,60). Moreover, a higher oxidative stability due to reduced oxidation products in the oil without the need for extensive hydrogenation was reported to be achieved as a consequence of the reduced levels of PUFA and the resulting increase in oleic acids. Numerous high-oleic oils were developed to provide not only high-stability cooking oils but also the opportunity to replace the current widespread use of saturated fats that contribute significantly to increased risk of cardiovascular disease. Thus, several authors (8 ,57,61-68) reported the development of breeding material and cultivars with oleate contents of 75 to -90% in most major crops (e.g., rapeseed, soybean, sunflower, peanut, cotton).
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Stoutjesdijk et al. (69), using sense-mediated PTGS (cosuppression) targeted against the A1* desaturase (which converts oleic acid to linoleic acid), reported the development of rapeseed (Brassica napus) and mustard (B.juncea) oils with very high levels of oleic acid (up to 89 and 73%, respectively). Using a similar approach, by suppressing the Fad 2-1 gene, DuPont Co. (9,67) succeeded in producing a soybean with an oxidatively stable oil that had a total polyunsaturated content of 4% and oleic acid content of 85%; in contrast, normal soybeans contain -20% oleic acid (18:l). This increase in oleate levels was accompanied by reduced levels of 18:2 from 55 to e l % and SFA down to 10% (5,9). Furthermore, using antisense construct in a line of canola that carried a mutation that reduced the activity of oleate desaturase, DuPont Co. succeeded in increasing the oleate levels to >88% followed by a decrease in saturate levels to 2% (3,9). The repression of the A'*-oleate desaturase in transgenic rapeseed, as reported by Topfer et al. (51), resulted in an increase in oleic acid of up to 83% in its TAG. A further increase to 88% was observed when this line was crossed with the mutant IMC129, which contains 78% oleic acid. They also reported a similar result with rapeseed lines accumulating up to 87% oleic acid through cosuppression. The silencing of the ghFAD2-1 gene in cottonseed resulted in greatly elevated oleic acid content, up to 77% compared with -15% in the seeds of untransformed plants (8). Peanut (Arachis hypogaea L.) is one of the major oilseed crops, and higholeate mutant varieties with as much as 85% oleate were reported (5839). These were found to rely on the allelic composition of two homologous, microsomal oleolyl-PC desaturase genes (ahFAD2A or ahFAD2B). These authors also reported that the activity of either of these two enzymes was sufficient for a normal oleate phenotype; however, a significant reduction in the levels of ahFAD2B and a mutation in ahFAD2A were responsible for the high-oleate phenotype. These findings were corroborated by the analysis of one chemically induced mutant (M2-225) and one derived from a naturally occurring (M8-2122) mutant. In summary, these improved vegetable oils not only show enhanced nutritional quality and improved stability, but they also do not have to be hydrogenated and provide a suitable substitute for chemically hydrogenated oils, It should be noted that linoleic acid and n-3 FA are essential dietary components so that not all food ingredients should be converted to monounsaturated lipids. The diets of most consumers in developed countries tend to contain excessive linolenic acid and insufficient n-3 FA. Perhaps we should convert most all of our frying and cooking oil sources to high monounsaturated oils (see comments on fried food flavor below) and have a divergent line of vegetable oils for salad dressings and other cold temperature uses that are rich in linoleic acid and n-3 FA. Palmitoleic Acid-Containing Oils
As mentioned elsewhere, vegetable oils rich in MUFA are important not only in human nutrition but also as renewable sources of industrial chemicals (70). Thus,
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by targeting the acyl-acyl carrier protein (ACP)-desaturase, one can potentiate different uses of the oil by manipulating the carbon lengths and the double-bond positions of these MUFA (70). ACP-desaturases (DES) are a class of soluble enzymes associated with the synthesis of MUFA by catalyzing the insertion of a double bond into SFA bound to ACP in the plastids of plant cells (70-72). Work by Shanklin and Somerville (73) revealed that the plant stearoyl-ACP desaturase is unusual in that it is the only soluble A9 desaturase identified to date. This soluble enzyme contains a di-iron (with one interacting with the side chains of El96 and H232 and the other with side chains of El05 and H146) center with ligands from four of the a-helices of the bundle creating a channel. This channel is thought likely to represent a binding pocket for the FA substrate but also to impose a bent conformation on the FA chain at the site of desaturation (70). These authors also reported that with regard to substrate specificity of acyl-ACP desaturases, the geometry of the lower portion of the binding pocket might play a critical role. Thus, the lower pocket is believed to place severe constraints on the length of the acyl chain that can be accommodated beyond the point at which the double bond is to be introduced and to determine the FA chain-length specificity. This idea was supported by the work of Cahoon et al. (74) based on a comparison of predicted amino acid sequences of cat’s claw (which contains nearly 80% palmitoleic acid plus cis-vaccenic acid in its seed oil) A9-l8:O-ACP desaturase with that of castor desaturase. They suggested that, in large part, the difference in substrate specificity between the two desaturases might be explained by a single amino acid (L118W) substitution, in which the conversion of leucine 118 to tryptophan in the mature castor A9- 18:O-ACP desaturase increased the relative specificity of this enzyme to 16:O-ACP by 80-fold. Because the plant A9 desaturase happened to favor 18:O-ACP but not 16:O-ACP as a substrate, only a small amount of 16:l is present in the so-called 18:3 higher plants (75). However, plants such as sea buckthorn and nuts (macadamia nuts) do contain an unusual amount of palmitoleic acid (16:1A9). Thus sea buckthorn was reported to be an important raw material for health and cosmetic products in recent years (76). Sea buckthorn fruit pulp/peel oil contains a high level of palmitoleic acid (16:ln-7, up to 43%) which is not common in the plant kingdom. Furthermore, there is a growing interest in its oil because of the increasing interest in the physiologic role of MUFA. Its seed oil was reported to lower the risk of cardiovascular and cerebrovascular disease as well as regulate immunofunctions and attenuate inflammation (76). Macadamia nut is another source of palmitoleic acid. Its oil is unique in that MUFA are the predominant component (about 80%) and a considerable portion (17-21%) of this is palmitoleic acid (a component not present in substantial amounts in olive oil) (77). Graybum et al. (1) and Wang et al. (75) reported large increases in palmitoleic acid (16:ln-7) after expressing a mammalian or yeast A9 desaturase gene in tobacco or tomato. One particular output trait of current interest is the use of transgenic soybean plants to produce palmitoleic acid FA that have either nutraceutical, pharmaceutical, or even industrial properties. Because soybeans are an important oil source that
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is high in linoleic and SFA (mainly linoleic and palmitic acids, -55 and 15%, respectively), conversion of all or part of these SFA into palmitoleic acid would be beneficial for more than health; converting much of the remaining PUFA into palmitoleic acid could have industrial value. Liu et al. (78) reported converting -50% of the palmitic acid of soybean somatic embryos into palmitoleic acid with good expression of a A9-CoA desaturase.
Why Palmitoleic Acid? A number of studies demonstrated apparently beneficial effects of diets based on high MUFA content primarily derived from olive oil (77,7941). However, few have examined the effects of whole complex foods high in MUFA such as nuts. The macadamia nut is an example of a complex food with good amounts of fiber as well as a number of vitamins and minerals; it also contains -75% oil by weight (77). The health implications of palmitoleic acid were first addressed by Yamori et al. ( 8 2 ) and Abraham et al. (83). The first group reported the preventive role of palmitoleic acid (among five FA tested) in stroke-prone spontaneously hypertensive rats. These authors reported that after feeding these rats diets containing 1% palmitoleic, palmitic, oleic, linoleic, and linolenic acids for 4 wk, only palmitoleic acid significantly improved their survival rate, whereas the control group died of stroke within 2 mon. Furthermore, they found that the mean survival time in the palmitoleic group was 97.7 d longer than the 33.0 d for controls or 37.7 d for the linoleic acid group; the other FA had no effect on survival time. In addition, they reported that (after the experimental period) the levels of palmitoleic acid in the aorta, as well as heart and plasma, were significantly higher than in rats fed the control or linoleic acid diet. Abraham et al. (83) investigated two groups of patients (one retrospectively and the other prospectively) to determine whether an association existed between adipose tissue FA composition and the risk of serious ventricular arrhythmias in acute myocardial infarction; they reported an inverse association between palmitoleic acid and ventricular arrhythmias. The association was stronger in the larger prospective group than in the retrospective group, further suggesting that altered cardiac membrane composition may influence the development of serious arrhythmias. Palmitoleic acid has an unusually low titer, which allows it to be highly fluid in the human body; it is resistant to peroxidation, crosslinking, and free-radical formation. To investigate the effects of a diet containing a high level of palmitoleic acid, Lipotech conducted an animal study at the Case Western Reserve University School of Medicine (36). These authors fed groups of rats diets containing 50% of energy from certain oils for 8 wk. Thus, one group received palmitoleic acid (titer 1 C) supplied as macadamia nut oil, the second group received oleic acid (titer 16 C) supplied as olive oil, and the third group received lauric/myristic acids (titer 44
C and 54 C) supplied as coconut oil. At the end of the experimental period, they reported that the groups did not differ in food intake, weight gain, or cholesterol
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blood levels, but triglyceride levels were increased (not surprising, given the high dietary fat content). However, the group fed the palmitoleic acid-rich diet had significantly lower triglyceride levels than the other two groups, and histopathological analysis revealed some striking differences. Analysis of the FA in the subcutaneous fat of the group fed the palmitoleic acid-rich diet revealed 5 0 4 5 % lower titers than those of the other groups. This group also had normal, healthy livers with densely packed hepatocytes, relatively low tissue fat levels, and very little fat in the peritoneal cavity. In contrast, the groups fed either the oleic- or lauric/myristic acid-rich diets had swollen, pale livers with fewer hepatocytes, high fat levels in tissues, and extensive peritoneal fat deposits (36). A subsequent clinical trial was conducted in 1996 at the University of Hawaii Medical School to evaluate three diets with different FA profiles. In that study (77), 30 men and women (18 to 53 yr old from a free-living population) with normal-tohigh fasting plasma total cholesterol levels were randomly assigned to a three-way crossover of three test diets, so that each subject was exposed to each diet for 30 d. The study compared the effects of a typical American diet (TAD; diet high in saturated fat “37% of energy from fat,” the American Heart Association (AHA) “Step 1” diet “30% of energy from fat” (half the SFA, normal amounts of MUFA and PUFA, and high levels of carbohydrates), and a macadamia nut-based monounsaturated fat diet (MND; 37% of energy from fat). Plasma lipids were evaluated at the end of each test period. Mean total cholesterol level was 201,193, and 191 mg/dL for the TAD, Step 1 diet (AHA) and MND, respectively. The LDL cholesterol level was 130 mg/dL for TAD, 124 and 125 for AHA and MND, respectively, and the HDL cholesterol level was 55 mg/dL, 52 and 53 mg/dL for the TAD, AHA, and MND, respectively. Compared with the typical diet, the Step 1 and macadamia nut diets both had potentially beneficial effects on cholesterol and LDL cholesterol levels. Although hindered by the relatively short test periods and small number of subjects, these results are consistent with previously reported lipid-altering benefits of MWA-rich diets particularly those involving macadamia nut oil (35). Another study by Manohar et al. (84) in hypercholesterolemic men (n = 17; mean age 54 yr) indicated that the plasma MUFA [16:l(n-7), 18:l(n-7), 20:l(n-9)] were elevated, whereas the levels of (n-6) and (n-3) were unaffected by macadamia nut consumption (40-90 g/d). Furthermore, this study indicated that the plasma total cholesterol and LDL concentrations were decreased by 3.0 and 5.3%, respectively, whereas HDL levels were increased by 7.9%, demonstrating that the consumption of macadamia as part of a healthy diet favorably modifies the plasma lipid profile in hypercholesterolemic men despite their high-fat diet. However, work by Nestel et al. ( 8 5 ) , in studying the effects of increased levels of dietary palmitoleic acid compared with palmitic and oleic acids on plasma lipids of hypercholesterolemic men, reported that palmitoleic acid behaved like an SFA and not a MUFA in its effect on LDL cholesterol. Palmitoleic acid was reported to protect rats from stroke (82), apparently by increasing cell membrane fluidity, clearing lipids from the blood, and altering the
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activity of important cell membrane transport systems particularly through inhibition of the Na+, K+-ATPase activity within a narrow range (86). In men and women, elevated bloodtissue levels of palmitoleic acid were positively correlated with protection from ventricular arrhythmias (83) and negatively correlated with markers of atherosclerosis (87). Palmitoleic acid was also reported to inhibit mutagenesis in animals (88) and was negatively correlated with breast cancer incidence in women (21). In the study on the distinct role of SFA and MUFA on p-cell turnover and function, palmitoleic acid improved the p-cell secretory function that was reduced by palmitic acid and also promoted p-cell proliferation at normoglycemic glucose concentrations, counteracting the negative effects of palmitic acid. These authors found that palmitoleic acid or oleic acid did not affect DNA fragmentation and induced P-cell proliferation and furthermore, each of these FA prevented the deleterious effects of both palmitic acid and high-glucose concentration (89,90). Welters et al. (91) reported that MUFA have the unique capacity to antagonize apoptosis induced by the activation of several different pathways in pancreatic p-cells. Most interestingly, these authors reported that palmitoleate (16:ln-7) or oleate (18: 1) markedly inhibited apoptosis induced by exposure of clonal BRIN-BD11 p-cells to serum withdrawal or a combination of interleukin-1P + interferon-y. They further suggested that (16: 1) or (18:1) might have a regulatory function at a distal step common to several apoptotic pathways in these cells. Using Fourier transform infraredattenuated total reflection analysis to study the enhancement of skin distribution of propylene glycol (PG) in the skin of rats, Taguchi et al. (92) found that high-purity cis-palmitoleic acid and oleic acid enhanced PG flux into the dermis and increased the dermal steady level of PG, suggesting their efficacy as skin penetration enhancers. Wille and Kydonieus (93) reported that palmitoleic acid (16: 1A6 cPA) in the human sebum lipid fraction was not only the predominant monoene but also the most active antimicrobial FA. Thus, they further suggested that palmitoleic acid could be useful in topical formulations for treatment of secondary grampositive bacterial infections, as a gram-positive bacteria antimicrobial in wound dressings, as well as a natural gram-positive antimicrobial preservative in skin and hair care products. Anecdotal observations from Lipotech’s animal study suggest that diets rich in palmitoleic acid might also have beneficial effects on skin moisture and suppleness (Robert Butz, personal communication).
High-Laurate Oils Lauric acid and its derivatives are highly valued for their detergent properties; they are used extensively in soaps, shampoos, and detergents. Traditionally, soaps were made from animal fats by converting them to the sodium and potassium salts of stearate or palmitate. Salts of these longer-chain FA are not very soluble in water especially at colder temperatures. Cooler temperatures reduce their detergent properties, and salts of short-chain FA are not hydrophobic enough for efficient cleansing of hydrophobic materials. Salts of medium-chain FA such as dodecanoic acid
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or lauric acid are ideally balanced in hydrophobicity and water solubility for use in cleaning materials. SDS, a very effective and commonly used detergent, is a derivative of lauric acid (Fig. 21.1). Most lauric acid (dodecanoic acid) is derived from plants of the palm family (Palmae) especially coconut and oil palm; the major temperate oilseeds contain little or no laurate. The U S . imports -$4,000,000 worth of lauric oils (coconut and palm) mainly from the Philippines, Malaysia, and Indonesia. Because lauric acid is derived from only two crop species and mainly from one region in the world, climatic conditions and other factors can greatly affect the supply and therefore the price. Hence, there has long been an interest in temperate crop sources of lauric acid that can be grown in places such as the United States, Canada, and Europe (94). A number of plants were identified in nature that accumulate laurate and/or other medium-chain FA in their seed oils including Attalea colendu (95) and Cuphea species (96). Work is in progress on the domestication of Cuphea as a temperate source of medium-chain FA (97-104). Fruits of Laurus nobilis of the Mediterranean region are -45% lauric acid (105). The seeds of the temperate tree, the California Laurel (Umbellularia californica Nutt., Lauraceae), are reported to have as much as 64% oil containing up to 70% lauric acid, with capric acid constituting much of the remaining oil FA (106). Qualea grundiflura (Vochysiaceae) from a Brazilian savannah or “cerrado” can contain >70% lauric acid (107). The enzymatic transesterification of oils with laurate sources can result in oils containing various laurate levels (108), but it still relies on the limited sources of lauric acid. The genetic engineering of temperate oilseeds can provide a new source of laurate; a group at Calgene set out to accomplish this in the 1980s, which culminated in a remarkable scientific success (109). Until the 1980s, little was known about the biochemistry and molecular genetics of oil biosynthesis in plants. Crop genetic engineering techniques also did not become available until the 1980s and rapeseedcanola, Brassica napus, was the first oilseed to be transformed and regenerated. It continues to be the most easily genetically engineered oilseed although it is possible to readily genetically engineer other widely grown oilseeds including soybeans (1 10). Generation and analysis of various genetic mutants at different steps in lipid biosynthesis, particularly by Browse and Somerville (113), enabled the unraveling of much of the biochemistry of plant lipid biosynthesis ( l l l ) , and the cloning of most plant lipid biosynthetic genes was achieved during the 1990s. This is exemplified by the cloning of the first desaturase gene by Shankhin and Somerville (112). It became 0
Na’ O-S-
II I
0
Na+& Fig. 21.1. Sodium dodecyl sulfate, a derivative of lauric acid.
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known that the synthesis of SFA preceded by the chain elongation of two carbons at each step was catalyzed by the FA synthase complex (FAS) with the actual elongation step being catalyzed by 3-ketoacyl-ACP synthases (KAS) with KAS I11 and KAS I plus the other FAS enzymes culminating in palmitoyl-ACP and KAS II/FAS producing stearoyl-ACP ( 113). It was not known how medium-chain FA such as lauric acid were produced until Pollard et al. (114) presented strong evidence that an unusual FA-ACP thioesterase from developing seeds of California bay trees stopped the elongation of the FA$ by releasing medium-chain FA from ACP. This was confirmed by Voelker et al. ( 2 ) , who achieved the accumulation of 20 mol% of laurate by expression of this California bay thioesterase gene, Uc F a t B l , in Arabidopsis seeds. Subsequent work by the Calgene group was able to produce B . nupus seeds with oil having as much as 60% lauric acid with high expression of the Uc FatBl gene; the main limitation to achieving higher levels was the near exclusion of laurate from the sn-2 position by specificity of the lysophosphatidic acid acyltransferase (LPAT) (1 15). Coconut endosperm TAG is typically -50% laurate distributed in all three positions, indicating that coconut endosperm LPAT can accept laurate-CoA as a substrate. The Calgene group therefore purified the coconut endosperm LPAT and cloned its corresponding cDNA (116). Expression of this LPAT together with the California bay thioesterase in canola resulted in an oil with significant amounts of laurate in the sn-2 position but the overall laurate content of the oil was not increased (109). One of the possible limitations to the accumulation of laurate in transgenic oilseeds beyond the 60% achieved in canola by Calgene may be the induction of high P-oxidation in developing seeds of these plants. Eccleston and Ohlrogge ( I 17) reported that -50% of the laurate produced in developing seeds of these plants is catabolized by P-oxidation rather than being incorporated into TAG. Lauryol-CoA oxidase activity was increased severalfold in seeds expressing the California bay thioesterase but palmitoyl-CoA oxidase activity was unchanged. Interestingly, the overall oil content was not significantly reduced apparently due to higher overall TAG biosynthesis induced in these transgenic plants to make up for the laurate lost to P-oxidation (1 17). How to achieve a very high accumulation (i.e., close to 100%)of unusual FA in oilseeds remains a major challenge for plant biochemistry and metabolic engineering. Nevertheless the highlaurate canola is a remarkable achievement; the oil has as high or higher laurate levels than normal sources of detergents. Calgene’s high-laurate canola is marketed under the trade name LauricalTM. Calgene was bought by Monsanto, making LauricalTMnow a trade name of Monsanto/Calgene. Plant breeders have been able to develop LauricalTMcultivars with yields similar to other canola cultivars (Maelor Davies, personal communication). Fomuso and Akoh (118) reported on the production of a trans-FA free margarine from this high laurate canola by enzymatic transesterification with stearic acid. To date, Laurical” is not yet widely grown, but -60% of canola grown in North America is genetically engineered for herbicide tolerance (1 19).
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Trans-Free Oils
Many fats and oils are partially hydrogenated to increase stability and/or raise the melting point, but standard hydrogenation procedures generate significant amounts of trans-FA at up to 45%. There is now sufficient evidence that trans-FA can raise LDL (or "bad") cholesterol levels like dietary saturated fats and cholesterol and increase rhe risk of cardiovascular disease that the FDA will begin requiring transFA levels to be included on food labels by January 1, 2006 (http://www.cfsan. fda.gov/-dms/transfat.html). Because of this, researchers in different areas of oil science .and technology are developing stable vegetable oils with reduced or no trans-FA. Three different approaches can be taken to achieve this goal. One approach is to alter the hydrogenation process itself to minimize the formation of trans-FA (120). Another approach is to transesterify vegetable oils with other FA so that hydrogenation is not required as demonstrated by Fomuso and Akoh (1 18) for stearate. A third approach, which will be briefly reviewed here, is to change the genetic makeup of the oilseeds so that the initial FA composition is similar to that achieved after hydrogenation of normal oilseeds. Many vegetable oils are partially hydrogenated to increase the stability of cooking oils and hydrogenated further for use as margarines and shortenings. To reduce or eliminate the need for hydrogenation of vegetable oils used for margarines and shortenings, the goal of plant geneticists has been mainly to develop high-stearate oils. As described above for these oils, many groups were successful in achieving this goal with a variety of vegetable oils using different approaches. From genetic engineering or mutagenesis, sunflowers were developed with 35% rapeseed with as much as 40% and soybeans with 53% stearic acid (121-123). High-stearate phenotypes can be achieved by reducing stearoyl-ACP desaturase activity, thus decreasing the conversion of stearate into unsaturated FA or highly expressing thioesterases with specificity for stearoyl-ACP, which is removed as a substrate for subsequent desaturation reactions. It is important that these large increases in stearate be seed-specific and more so in TAG than in the membrane lipids because high stearate in membranes can reduce membrane fluidity and result in relatively poor germination rates (124-126). For cooking or frying oils, the goal is to minimize the excessively high oxidation rate that results in rancidity with its associated undesirable olfactory characteristics as well as the production of unhealthy oxidation products. The simple way to achieve this is to reduce or even eliminate the PUFA because PUFA have high autoxidation rates with the rate greatly increasing with larger numbers of double bonds. Vegetable oils with large reductions in linolenic acid can be used in some applications with minimal hydrogenation. Oils that are composed predominately of MUFA with little or no PUFA are sufficiently oxidatively stable to be used without hydrogenation, eliminating the associated trans-FA. Breeders and molecular geneticists have also had great success in developing vegetable oils high in the MUFA, oleic acid. This is discussed above in the section on high-oleate oils and
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just a few highlights will be noted here. An oleic acid content as high as 90% was achieved in many major vegetable oils including soybeans, rapeseed, and sunflower (5,9). This high-oleic acid soybean oil with no hydrogenation is, of course, a liquid at room temperature and has oxidative stability equivalent to that of solid hydrogenated soybean oil (127). The main means for achieving these high-oleate phenotypes is the seed-specific suppression of AI2 desaturase. In the high-oleic acid soybeans, for example, the seed-specific A1* desaturase, Fad2-1, was suppressed, whereas the constitutive A12 desaturase Fud2-2, which supplies linoleic acid for most plant cells but has little contribution to seed oil composition, was unchanged (128). The palmitate content of these high-oleate lines can be reduced by silencing the palmitoyl-ACP thioesterase, thereby reducing the SFA content of the soybean oil to <4% while further increasing the oleate content (127). One potential drawback to very high-oleate oils is that much of the desirable fried-food flavor is derived from linoleic acid during frying (129). Acknowledgments Thanks to Elizabeth Moore, Matt McConnell, Eric Hudson, Keshun Yu, and Hirotada Fukushige for editorial help.
References 1. Grayburn, W.S., G.B. Collins, and D.F. Hildebrand, Fatty Acid Alteration by a A9 Desaturase in Transgenic Tobacco Tissue, BioTechnology 10: 675-678 (1992). 2. Voelker, T.A., A.C. Worell, L. Anderson, J. Bleiaum, C. Fan, D.J. Hawkins, S.E. Radke, and H.M. Davies, Fatty Acid Biosynthesis Redirected to Medium Chains in Transgenic Oilseed Plants, Science 257: 72-74 (1992). 3. Broun, P., S . Gettner, and C. Somerville, Genetic Engineering of Plant Lipids, Annu. Rev. Nutr. 19: 197-216 (1999). 4. Grundy, S.M., Comparison of Monounsaturated Fatty Acids and Carbohydrates for Lowering Plasma Cholesterol in Man, N . EngZ. J. Med. 314: 745-748 (1986). 5 . Thelen, J.J., and J.B. Ohlrogge, Metabolic Engineering of Fatty Acid Biosynthesis in Plants,MetaboZ. Eng. 4: 12-21 (2002). 6. Ohlrogge, J., J. Browse, and C.R. Somerville, The Genetics of Plant Lipids, Biochirn. Biophy~.ACZU1082: 1-26 (1991). 7 . Kessler, D.A., M.R. Taylor, and I.J.H. Maryansk, The Safety of Food Developed by Biotechnology, Science 256: 1747-1749 (1992). 8. Liu, Q., S.P. Singh, and A.G. Green, High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing, Plant Physiol. 129: 1732-1743 (2002). 9. Kinney, A., Development of Genetically Engineered Soybean Oils for Food Applications, J . Food Lipids 3: 273-292 (1996). 10. Knutzon, D.S., G.A. Thompson, S.E. Radke, W.B. Johnson, V.C. Knauf, and J.C. Kridl, Modification of Brassica Seed Oil by Antisense Expression of a Stearoyl-Acyl Carrier Protein Desaturase Gene, Proc. Natl. Acad. Sci. 89: 2624-2628 (1992).
11. Raatz, S.K., D. Bibus, W. Thomas, and P. Kris-Etherton,Total Fat Intake Modifies Plasma Fatty Acid Composition in Humans, J. Nutr. 131: 231-234. (2001).
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12. Manku, M.S., D.F. Horrobin, Y.-S. Huang, and N. Morse, Fatty Acids in Plasma and Red Cell Membranes in Normal Humans, Lipids 18: 906-908 (1983). 13. Ma, J., A.R. Folson, E. Shahar, and J.H. Eckfeldt, for the Atherosclerosis Risk in Communities (ARIC) Study Investigators, Plasma Fatty Acid Composition as an Indicator of Habitual Dietary Intake in Middle-Aged Adults, Am. J . Clin. Nutr. 62:5 564-571 (1995). 14. Lopez, S.M., S.L. Trimbo, E.A. Mascioli, and G.L. Blackburn, Human Plasma Fatty Acid Variations and How They Are Related to Dietary Intake, Am. J . Clin. Nutr. 53: 628-637 (1991). 15. Judd, J.T., M.W. Marshall, and J. Dupont, Relationship of Dietary Fat to Plasma Fatty Acids, Blood Pressure and Urinary Eicosanoids in Adult Men, J . Am. Coll. Nutr. 8: 386-389 (1989). 16. Dougherty, R.M., C. Galli, A. Ferro-Luzzi, and J.M. Lacono, Lipid and Phospholipids Fatty Acid Composition of Plasma, Red Blood Cells, and Platelets and How They Are Affected by Dietary Lipids: A Study of Normal Subjects from Italy, Finland, and the USA,Am. J . Clin. Nutr. 45: 443-455 (1987). 17. Cohen, L.A., and E.I. Wynder, Do Dietary Monounsaturated Fatty Acids Play a Protective Role in Carcinogenesis and Cardiovascular Disease? Med. Hypotheses 31: 81-89 (1990). 18. Ferguson, J., N. Mackay, and G. McNicol, Effect of Feeding Fat on Fibrinolysis, Stypven Time, and Platelet Aggregation in Africans, Asian, and Europeans, J . Clin. Puthol. 23: 580-585 (1970). 19. Jacobs, D., G. Blackburn, M. Higgins, D. Reed, H. Iso, G. McMillan, J. Neaton, J. Nelson, J. Potter, B. Rifkind, J. Rossouw, R. Shekeller, and S. Usuf, Report on the Conference on Low Blood Cholesterol: Mortality Associations, Circulation 86: 1046-1060 (1992). 20. Kris-Etherton, P.M., T.A. Pearson, Y. Wan, R.L. Hargrove, K. Moriarty, V. Fishell, and T.D. Etherton, High-Monounsaturated Fatty Acid Diets Lower Both Plasma Cholesterol and Triacylglycerol Concentrations, Am. J . Clin. Nutr. 70: 1009-1015 (1999). 21. Simonsen, N.R., J.F.-C. Navajas, J.M. Martin-Moreno, J J . Strain, J.K. Huttunen, B.C. Martin, M. Thamm, A.F. Kardinaal, P. van’t Veer, F.J. Kok, and L. Kohlmeier, Tissue Stores of Individual Monounsaturated Fatty Acids and Breast Cancer: The EURAMIC Study. European Community Multicenter Study on Antioxidants, Myocardial Infarction, and Breast Cancer,Am. J . Clin. Nutr. 68: 134-141 (1998). 22. Hu, F.B., J.E. Manson, and W.C. Willet, Types of Dietary Fat and Risk of Coronary Heart Disease: A Critical Review, J . Am. Coll. Nutr. 20: 5-19 (2001). 23. Mozaffarian, D., T. Pischon, S.E. Hankinson, N. Rifai, K. Joshipura, W.C. Willet, and E.B. R i m , Dietary Intake of truns Fatty Acids and Systemic Inflammation in Women, Am. J . Clin. Nutr. 79: 606-612 (2004). 24. Emken, E.A., Biochemistry of Unsaturated Fatty Acid Isomers, J . Am. Oil Chem. SOC. 60: 995-1004 (1983). 25. Yu-Poth, S., T.D. Etherton, C.C. Reddy, T.A. Pearson, R. Reed, G . Zhao, S. Jonnalagadda, Y. Wan, and P.M. Kris-Etherton, Lowering Dietary Saturated Fat and Total Fat Reduces the Oxidative Susceptibility of LDL in Healthy Men and Women, J . Nutr. 130: 2228-2237 (2000). 26. Harris, W., n-3 Fatty Acids and Serum Lipoproteins: Human Studies, Am. J . Nutr. 65 (suppl.): S1645-S1654 (1997).
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81. Spiller, G.A., D.J. Jenkins, L.N. Cragen, J.E. Gates, 0. Bossello, K. Berra, C. Rudd, M. Stevenson, and R. Superko, Effects of a Diet High in Monounsaturated Fat from Almonds on Plasma Cholesterol and Lipoproteins, Am. J . Clin. Nutr. 11: 126-130 ( 1992). 82. Yamori, Y., Y. Nara, T. Tsubouchi, Y. Sogawa, K. Ikeda, and R. Horie, Dietary Prevention of Stroke and Its Mechanism in Stroke-Prone Spontaneously Hypertensive Rats-Preventive Effect of Dietary Fibre and Palmitoleic Acid, J . Hypertens. Suppl. 4: S449-S452 (1986). 83. Abraham, R., R. Riemersma, D. Wood, R. Elton, and M. Oliver, Adipose Fatty Acid Composition and the Risk of Serious Arrhythmias in Acute Myocardial Infarction, Am. J . Cardiol. 63: 269-272 (1989). 84. Manohar, L.G., R.J. Blake, and R.B.H. Wills, Macadamia Nut Consumption Lowers Plasma Total LDL Cholesterol Levels in Hypercholesterolemic Men, J . Nutr. 133: 1060-1 063 (2003). 85. Nestel, P., P. Clifton, and M. Noakes, Effects of Increasing Dietary Palmitoleic Compared with Palmitic and Oleic Acids on Plasma Lipids of Hypercholesterolemic Men, J. Lipid Res. 35: 656-662 (1994). 86. Swarts, H., F.M. Schuumans Stekhoven, and J.J. de Pont, Binding of Unsaturated Fatty Acids to N+, K(+)-ATPase Leading to Inhibition and Inactivation, Biochim. Biophys. Acta 1024: 32-40 (1990). 87. Theret, N., J. Bard, M. Nuttens, J. Lecerf, C. Delbart, M. Romon, J. Salomez, and J. Fruchart, The Relationship Between the Phospholipids Fatty Acid Composition of Red Blood Cells, Plasma Lipids, and Apolipoproteins, Metabolism 42: 526-528 (1993). 88. Hayatsu, H., S. Arimoto, and T . Negishi, Dietary Inhibitors of Mutagenesis and Carcinogenesis, Mutat. Res. 202: 429-446 (1988). 89. Maedler, K., G.A. Spinas, D. Dyntar, W. Moritz, N. Kaiser, and M.Y. Donath, Distinct Effects of Saturated and Monounsaturated Fatty Acids on @Cell Turnover and Function, Diabetes 50: 69-76 (2001). 90. Maedler, K., J. Oberholzer, P. Bucher, G.A. Spinas, and M.Y. Donath, Monounsaturated Fatty Acids Prevent the Deleterious Effects of Palmitate and High Glucose on Human Pancreatic P-Cell Turnover and Function, Diabetes 52: 726-733 (2003). 91. Welters, H.J., M. Tadayyon, J.H.B. Scarpello, S.A. Smith, and N.G. Morgan, Monounsaturated Fatty Acids Protect Against B-Cell Apoptosis Induced by Saturated Fatty Acids, Serum Withdrawal or Cytokine Exposure, FEBS Lett. 560: 103-108 (2004). 92. Taguchi, K., S. Fukushima, Y. Yamaoka, Y. Takeuchi, and M. Suzuki, Enhancement of Propylene Glycol Distribution in the Skin by High Purity cis-Unsaturated Fatty Acids with Different Alkyl Chain Lengths Having Different Double Bond Position, Biol. Pharm. Bull. 22: 407-41 1 (1999). 93. Wille, J.-J., and A. Kydonieus, Palmitoleic Acid Isomer (C16:1A6) in Human Skin Sebum Is Effective Against Gram-Positive Bacteria, Skin Pharmacol. Appl. Skin Physiol. 16: 176-187 (2003). 94. Sayler, T., Laurate Canola Looks to Be the Next Oil Boom, Prairie Grains 6: 1-5 (1997). 95. Blicher Mathiesen, U., and H. Balslev, Attalea colendu (Arecaceae), a Potential Lauric Oil Resource, Econ. Bot. 44: 360-368 (1990). 96. Knapp, S.J., L.A. Tagliani, and W.W. Roath, Fatty Acid and Oil Diversity of Cuphea viscosissima: A Source of Medium-Chain Fatty Acids, J. Am. Oil Chem. SOC.68: 515-517 (1991).
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97. Pandey, V., R. Banerji, B.S. Dixit, M. Singh, S. Shukla, and S.P. Singh, Cuphea a Rich Source of Medium Chain Triglycerides: Fatty Acid Composition and Oil Diversity in Cupheaprocumbens, Eur. J . Lipid Sci. Technol. 102: 463-466 (2000). 98. Knapp, S.J., J.M. Crane, L.A. Tagliani, and M.B. Slabaugh, Cuphea viscosissimus Mutants with Decreased Capnc Acid, Crop Sci. 37: 352-357 (1997). 99. Thompson, A.E., ‘‘Stdire” Cuphea Hybrid, HortScience 30: 166167 (1995). 100. Olejniczak, J., E. Adamska, Z. Przybecki, and A. Wojciechowski, Domestication of Cuphea Through Mutagenesis, M A , Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture,Vienna, 1994. 101. Gonzalez, A.G., E. Valencia, T.S. Exposito, J.B. Barrera, and M.P. Gupta, Chemical Components of Cuphea Species. Carthagenol: A New Triterpene from C . carthagenensis, Planta Med. 60: 592-593 (1994). 102. Ali, M.S., and S.J. Knapp, Heterosis of Cuphea lanceolata Single-Cross Hybrids, Crop Sci. 36: 278-284 (1996). 103. Roath, W.W., M.P. Widrlechner, and J.H. Kirkbride, Collecting Cuphea in Brazil, Mexico and the United States, Plant Genet. Res. Newslett. 93: 29-33 (1993). 104. Crane, J.M., L.A. Tagliani, and S.J. Knapp, Registration of Five Self-Fertile, Partially Nondormant Cuphea Germplasm Lines: VL-90 to VL-95, Crop Sci. 35: 1516-1517 (1995). 105. Kolancilar, H., Preparation of Laurel Oil Alkanolamide from Laurel Oil, J . Am. Oil Chem. SOC.81: 597-598 (2004). 106. Reynolds, T., J.V. Dring, and C. Hughes, Lauric Acid-Containing Triglycerides in Seeds of Umbellularia californica Nutt. (Lauraceae), J . Am. Oil Chem. SOC.68: 976-977 (1991). 107. Mayworm, M.A.S., and A. Salatino, Fatty Acid Composition of “Cerrado” Seed Oils, J . Sci. Food Agric. 72: 226-230 (1996). 108. Forssell, P., P. Parovuori, P. Linko, and K. Poutanen, Enzymatic Transesterification of Rapeseed Oil and Lauric Acid in a Continuous Reactor, J . Am. Oil Chem. SOC. 70: 1105-1109 (1993). 109. Knutzon, D.S., and V.C. Knauf, Manipulating Seed Oils for Polyunsaturated Fatty Acid Content, in Plant Lipid Biosynthesis: Fundamentals and Agricultural Applications, edited by J.L. Harwood, Cambridge University Press, Cambridge,UK, 1998,pp. 287-304. 110. Hildebrand, D.F., S. Rao, and T. Hatanaka. Redirecting Lipid Metabolism in Plants, in Lipid Biotechnology, edited by T.M. Kuo and H.W. Gardner, Marcel Dekker, New York, 2001. 111. Ohlrogge, J.B., and J.G. Jaworski, Regulation of Fatty Acid Synthesis,Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 109-136 (1997). 112. Shanklin, J., and C. Somerville, Stearoyl-Acyl-Carrier-ProteinDesaturase from Higher Plants Is Structurally Unrelated to the Animal and Fungal Homologs, Proc. Nut. Acad. Sci. USA 88: 2510-2514 (1991). 113. Somerville,C., J. Browse, J.G. Jaworski, and J.B. Ohlrogge,Lipids, in Biochemistry and Molecular Biology of Plants, edited by B.B. Buchanan, W. Gruissem, and R.L. Jones, American Society of Plant Physiologists, Rockville,MD, 2000, pp. 456-527. 114. Pollard, M.R., L. Anderson, C. Fan, D.J. Hawkins, and H.M. Davies, A Specific AcylACP Thioesterase Implicated in Medium-Chain Fatty Acid Production in Immature
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115. Voelker, T.A., T.R. Hayes, A.M. Cranmer, J.C. Turner, and H.M. Davies, Genetic Engineering of a Quantitative Trait: Metabolic and Genetic Parameters Influencing the Accumulation of Laurate in Rapeseed, Plant J . 9: 229-241 (1996). 116. Knutzon, D.S., K.L. Lardizaval, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, and J.G. Metz, Cloning of a Coconut Endosperm cDNA Encoding a 1-Acyl-sn-glycerol-3-phosphate Acyltransferase That Accepts Medium-Chain-Length Substrates, Plant Physiol. 109: 999-1006 (1995). 117. Eccleston, V.S., and J.B. Ohlrogge, Expression of Lauroyl-Acyl Carrier Protein Thioesterase in Brassica napus Seeds Induces Pathways for Both Fatty Acid Oxidation and Biosynthesis and Implies a Set Point for Triacylglycerol Accumulation, Plant Cell 10: 613-621 (1998). 118. Fomuso, L.B., and C.C. Akoh, Enzymatic Modification of High-Laurate Canola to Produce Margarine Fat, J . Agric. Food Chem. 49: 4 4 8 2 4 8 7 (2001). 119. Anonymous. History and Prevalence of GE Canola. Available at: http://www.geopie.cornell.edu/crops/canola.html#history(accessed Aug. 2,2004). 120. Beers, A., and G. Mangnus, Hydrogenation of Edible Oils for Reduced trans-Fatty Acid Content, Inform 15: 404-405 (2004). 121. Knutzon, D.S., G.A. Thompson, S.E. Radke, W.B. Johnson, V.C. Knauf, and K.J.C, Modification of Brassica Seed Oil by Antisense Expression of a Stearoyl-Acyl Carrier Protein Desaturase Gene, Proc. Nut. Acad. Sci. 89: 2624-2628 (1992). 122. Kridl, J., Method for Increasing Stearate Content in Soybean Oil, US.Patent 6,380,462 (2002). 123. Martinez-Force, E., V. Fernandez-Moya, and R. Garces, Sunflower Seeds and Oil Having a High Stearic Acid Content, U S . Patents 6,410,831 and 6,486,336 (2002). 124. Thompson, G.A., and C. Li, Altered Fatty Acid Composition of Membrane Lipids in Seeds and Seedling Tissues of High-Saturate Canolas, in Physiology, Biochemistry and Molecular Biology of Plant Lipids, edited by J.P. Williams, M.U. Khan, and N.W. Lem, Kluwer, Dordrecht, The Netherlands, 1997, pp. 313-315. 125. Voelker, T., and A. Kinney, Variations in the Biosynthesis of Seed-Storage Lipids, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 335-361 (2001). 126. Wiberg, E., P. Edwards, J. Byrne, S . Stymne, and K. Dehesh, The Distribution of Caprylate, Caprate, and Laurate in Lipids from Developing and Mature Seeds of Transgenic Brassica rapus L., Planta 212: 3 3 4 0 . 127. Kinney, A.J., Production of Specialised Oils for Industry, in Plant Lipid Biosynthesis: Fundamentals and Agricultural Applications, edited by J.L. Harwood, Cambridge University Press, Cambridge, UK, 1998, pp. 273-285. 128. Heppard, E.P., A.J. Kinney, K.L. Stecca, and G.H. Miao, Developmental and Growth Temperature Regulation of Two Different Microsomal 0 6 Desaturase Genes in Soybeans, Plant Physiol. 110: 311-319 (1996). 129. Warner, K., W.E. Neff, W.C. Byrdwell, and H.W. Gardner, Effect of Oleic and Linoleic Acids on the Production of Deep-Fried Odor in Heated Triolein and Trilinolein, J . Agric. Food Chem. 49: 899-905 (2001).
Chapter 22
Emulsion Technologies to Produce Oxidative Stable Emulsions Containing n-3 Fatty Acids Min Hu, Eric A. Decker, and D. JulianMcClernents Department of Food Science, University of Massachusetts, Amherst, M A 01003
Introduction Evidence that dietary bioactive lipids promote good health continues to grow as shown by the following examples. It has been estimated that one in five men and women has some form of cardiovascular disease, a fact that has made it the number one cause of death in the United States. Coronary heart disease (CHD) has been implicated in 52% of deaths in the United States at an economic cost estimated at $368.4 billion (1). PUFA, and in particular n-3 FA, were shown to affect CHD in a beneficial manner through their actions on lipid metabolism, heart function, vasodilation, platelet aggregation, and blood clotting (2). Type 2 diabetes is the most common form of diabetes, affecting almost 17 million Americans and costing an estimated $100 billion annually (3). Type 2 diabetes results from the inability of the body to make enough or properly use insulin, resulting in hyperglycemia and hypertriglyceridemia as well as the development of vascular complications. The frequency of type 2 diabetes increases with age, and individuals with type 2 diabetes are 2-4 times more likely to have CHD. The ability of dietary PUFA to lower serum triglyceride concentrations without adversely affecting glucose control suggests that individuals with type 2 diabetes could benefit from increased consumption of bioactive lipids (4). The American Cancer Society estimates that >1.2 million new cancer cases occurred in 2002 at an estimated direct cost of $56.4 billion (5). Research continues to show that dietary lipids such as lycopene and conjugated linoleic acid (CLA) can play an important protective role against cancer. Although the incorporation of unsaturated and bioactive lipids into processed food could be beneficial to the health of consumers, the utilization of these lipids as food additives is limited by their susceptibility to oxidative degradation. If lipids oxidize during the processing and storage of food products, this will not only alter the nutritional compositiodbioactivity of the product but will also influence sensory quality because oxidation of unsaturated FA leads to rancid flavors and aromas, and oxidation of carotenoids results in bleaching and thus color changes. Therefore, to produce foods with physiologically bioactive lipid components, methods must be developed to control oxidative reactions. 547
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The oxidation of bulk lipids has been studied extensively, and there is now a fairly good understanding of the factors that affect oxidation in these systems (6,7). Research in this area has elucidated many of the mechanisms by which lipid oxidation proceeds under various conditions and the types of reaction products produced (8). The importance of the physical state and organization of lipids in foods on their susceptibility to oxidation was recognized many years ago (9). Even so, it is only in the last decade that systematic studies of lipid oxidation in food emulsions were undertaken (10-23). This is surprising considering the large number of foods that consist either partly or wholly of emulsions or that were in an emulsified form sometime during their production [e.g., dairy products, mayonnaise, margarine, soups, sauces, baby foods, and beverages; (24)]. There are significant differences between lipid oxidation in bulk fats and in emulsified fats due to the presence of a droplet membrane, the partitioning of ingredients between lipid and aqueous phases, the presence of water-soluble ingredients not present in bulk fats, and the fact that the lipid is in contact with water rather than air.
Lipid Oxidation in Emulsions Emulsions are thermodynamically unstable because of the positive free energy required to increase the surface area between the oil and water phases (24). For this reason, emulsions tend to separate into a layer of oil (lower density) on top of a layer of water (higher density) with time. To form emulsions that are kinetically stable for a reasonable period (a few weeks, months, or even years), chemical substances known as emulsifiers must be added before homogenization. Emulsifiers are surface-active molecules that absorb to the surface of freshly formed droplets during homogenization, forming a protective membrane that prevents the droplets from coming close enough together to aggregate. The most common emulsifiers used in the food industry are surface-active proteins (e.g., from casein, whey, soy, and egg), phospholipids (e.g., egg or soy lecithin), and small molecule surfactants (e.g., Spans, Tweens, FA). An emulsion can be considered to consist of three regions, i.e., the interior of a droplet, the continuous phase, and the interfacial membrane. The interfacial membrane consists of a narrow region surrounding each emulsion droplet, consisting of a mixture of oil, water, and emulsifier molecules. Typically, the interfacial membrane has a thickness of a few nanometers, and often makes up a significant proportion of the total number of molecules present in the droplet (25). The various molecules in an emulsion partition themselves among these three different regions according to their polarity and surface activity. Nonpolar molecules are located predominantly in the oil phase, polar molecules in the aqueous phase, and surface-active molecules at the interface. The precise molecular environment of a molecule may have a significant effect on its chemical reactivity. Therefore, the nature of the emulsion droplet interfacial membrane would be expected to be extremely important in lipid oxidation reactions because it could dictate how lipids
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(e .g., unsaturated FA and lipid hydroperoxides) would interact with aqueous phase prooxidants (e.g., transition metals and reactive oxygen species). Lipid oxidation is a free radical chain reaction between unsaturated fats and oxygen that can proceed in an autocatalytic manner. However, in many foods, lipid oxidation is not truly autocatalytic because it is accelerated by prooxidants such as UV light, photosensitizers, transition metal ions, and certain enzymes (8,26). Research in our laboratory showed that the oxidation of Tween 20-stabilized algal oil emulsions is inhibited by metal-binding agents including ethylene diamine tetraacetic acid (EDTA) and the plasma iron-binding protein, transferrin (1 8). Because these algal oil emulsions do not contain added prooxidants, these results indicate that the lipid oxidation in the emulsion is not truly due to autooxidation but instead is promoted by endogenous transition metals. The most common prooxidant transition metals in foods are iron and copper. EDTA can inhibit both copper- and iron-promoted lipid oxidation. The extremely high iron-binding constant for transferrin [>lo7 higher than other metals; (27)] suggests that transferrin would be effective mainly at inhibiting the prooxidative activity of iron. In addition, chelators such as EDTA and transferrin have limited lipid solubility and reside mainly in the aqueous phase. Therefore, the ability of transferrin to strongly inhibit the oxidation of Tween 20-stabilized algal oil emulsions suggests that iron is the main lipid oxidation catalyst and that prooxidative iron either originates in the aqueous phase or is transported into the aqueous phase from the droplet interface or droplet interior. Iron accelerates lipid oxidation primarily by promoting the breakdown of peroxides into free radicals. Most food-grade lipids contain preexisting lipid hydroperoxides, and hydrogen peroxide can be produced in foods from the spontaneous dismutation of the superoxide anion (26). The ferrous state of iron (Fe2+) will decompose hydroperoxides >lo5 times more quickly than ferric iron (Fe3+) (27). In addition, the reactivity of ferric iron is also limited by its low water solubility which is 1017 and 1013 times lower than that of ferrous iron at pH 7 and 3, respectively (28). Although the ferrous state of iron is more reactive and more soluble, ferric ions can be more common in foods (29). Even with its low reactivity and solubility, ferric iron could be an important lipid oxidation catalyst during the long-term storage of emulsified lipids, especially if it is able to interact with the interfacial membrane of emulsion droplets. As with iron, lipid hydroperoxides exist in essentially all foods containing unsaturated FA (30). High-quality food lipids generally contain lipid hydroperoxide concentrations in the range of 10-100 pmol/g lipid. Although these concentrations may seem low in a food system, they are an estimated 400-1000 times greater than the lipid hydroperoxide concentrations found in vivo [e.g., plasma lipids; (3 1,32)], suggesting that significant oxidation occurs during the refining and storage of food oils. Because both iron and hydroperoxides exist in foods, it is likely that factors that affect their interactions will have a major influence on the oxidative stability of emulsions.
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€ffect of the interfacial Properties of Emulsion Droplets on the Activity of Iron
Research in our laboratory showed that the interaction of iron with the emulsion droplet interface is an important factor determining oxidation rates. By using corn oil-in-water emulsions stabilized with anionic sodium dodecyl sulfate (SDS), cationic [dodecyltrimethylammonium bromide (DTAB)] and nonionic (polyoxyethylene 10 lauryl ether; Brij) surfactants, it was found that iron-promoted lipid oxidation rates were highest with anionic and lowest with cationic emulsion droplets (14). The zeta potential was then used to show that both ferrous and ferric ions readily interacted with SDS- but not with DTAB- or Brij-stabilized hexadecane emulsion droplets ( 15). Factors that decreased iron-emulsion droplet interactions such as increasing pH, chelators (EDTA and phytate), and NaCl resulted in decreased lipid oxidation rates (14,15). In a comparison of iron-promoted lipid oxidation rates in SDS-stabilized emulsions containing low (0.12 pmol/g oil) and high (17 pmol/g oil) lipid hydroperoxide concentrations, oxidation increased with increasing hydroperoxide concentrations, suggesting that peroxides were limiting oxidation rates (15). These data suggest that an important factor in the oxidation of emulsified lipids is the ability of iron to interact with lipids and/or lipid hydroperoxides at the interfacial membrane of emulsion droplets.
Effect of Emulsion Interfacial Characteristics on Lipid Oxidation and Lipid Hydroperoxide Stability As mentioned earlier, iron is a major lipid oxidation promoter in oil-in-water emulsions. The ability of iron to accelerate lipid oxidation is due primarily to its interactions with hydroperoxides, resulting in the formation of free radicals. These free radicals will in turn oxidize unsaturated FA, leading to the formation of additional lipid hydroperoxides. Iron-promoted hydroperoxide decomposition is common in many foods because both iron and hydroperoxides are ubiquitous to lipid-containing food systems. Reactions between metals and lipid hydroperoxides are involved in the rapid exponential increase in oxidation observed during the propagation step of lipid oxidation and lead to b-scission reactions that decompose FA into the lowmolecular-weight compounds responsible for rancidity [for review see (30)]. Incorporation of oxygen into an unsaturated FA during the formation of a lipid hydroperoxide results in an increase in the polarity of the lipid molecule. Research during this project showed that incorporation of oxygen into FA in the form of hydroperoxides increases surface activity, and FFA hydroperoxides are more surface-active than triacylglycerol hydroperoxides (33). Because the lipid hydroperoxides are surface-active, they would migrate to the surface of the emulsion droplet where they could interact with prooxidants such as aqueous phase iron and/or iron associated with the emulsion droplet interfacial membrane. This was observed in model systems consisting of Tween 20 (nonionic)-, SDS (anionic)-, or
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DTAB (cationic)-stabilized hexadecane emulsions containing cumene hydroperoxide. Hexadecane was used in this model as a nonoxidizable lipid so that additional hydroperoxides would not be formed from the free radicals originating from the breakdown of the cumene peroxide as would occur in an emulsion containing unsaturated FA. In this model, ferrous ions were able to break down hydroperoxides in an equal molar fashion in the SDS-stabilized emulsion (e.g., 500 pM Fe2+ decomposed -500 pM hydroperoxide). However, when DTAB or Tween 20 was used as the emulsifier, hydroperoxide decomposition was more than threefold lower, with 500 pM Fe2+decomposing 4 5 0 pM hydroperoxide. The higher reactivity of iron in SDS-stabilized emulsions could be due to increased iroddroplet interactions because of electrostatic attraction between the positively charged iron and the anionic droplet surface. The importance of emulsion droplet-iron interactions is especially relevant for the oxidized states of the iron. Ferric ions decomposed cumene hydroperoxide only in the anionic SDS-stabilized emulsion droplets, suggesting that the ferric ions must be in direct contact with the emulsion droplet to be an active prooxidant. This is likely due to the lo5 times slower reaction rates of ferric compared with ferrous ions (7), making physical concentration of Fe3+ at the droplet surface an important factor in the ability of ferric ions to decompose hydroperoxides . Another potential difference between the surfactants used in this study is the size of their hydrophilic head groups, with SDS and DTAB producing an interfacial thickness of -0.5 nm compared with 1.4 nm for Tween 20 (34). Therefore it is possible that differences in lipid hydroperoxide decomposition could also be due to the large hydrophilic head groups of Tween-20, which provide a physical barrier that could prevent lipids from reacting with aqueous phase prooxidants. For instance, if an emulsion droplet could be prepared with a large interfacial membrane layer, it may be difficult for aqueous phase iron to interact with lipid hydroperoxides. The ability of iron to promote cumene hydroperoxide decomposition as well as the oxidation of salmon oil was lower in emulsion droplets stabilized by Brij 700 than Brij 76. These two surfactants have the same hydrophobic tail group length [CH,(CH,),, -1, but different length polar head groups; Brij 700 contains 100 compared with Brij 76’s 10 oxyethylene groups. Decreasing lipid oxidation rates in the Brij 700-stabilized salmon oil-in-water emulsions suggests that the thicker interfacial layer provided by Brij 700 was able to act as a physical barrier to decrease lipid-prooxidant interactions (17). Research in our laboratory also showed that increasing surfactant hydrophobic tail group size can decrease lipid oxidation as can be seen in salmon oil-in-water emulsions stabilized by polyoxyethylene 10 lauryl ether (Brij-lauryl, C12) or polyoxyethylene 10 stearyl ether (Bnj-stearyl, C18). Oxidation of salmon oil was greater in emulsions stabilized by Brij-lauryl than Brij-stearyl as determined by both lipid hydroperoxides and headspace propanal (35). However, the ability of hydrophobic tail group size to alter oxidation rates is much less than alterations in oxidation rates observed in emulsions stabilized by surfactants with varying hydrophilic head group
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size or charge. This may not be due to the type of banier provided by the hydrophobic tail group, but instead the magnitude of the barriers formed, because it is possible to produce oil-in-water emulsions with much greater variations in interfacial membrane properties by altering hydrophilic head properties. A somewhat obvious factor that will affect lipid oxidation rates is the concentration of lipid hydroperoxides because decomposition of hydroperoxides is what leads to off-flavor development in oxidized oils. Although lipid hydroperoxide concentrations are routinely monitored in food oils, little attention has been paid to emulsifiers. Data from our laboratory indicate that Tweens and lecithin can contain from 12 to 35 pmol hydroperoxide peroxide/g surfactant (19). Iron is capable of decomposing Tween 20 hydroperoxides, with this reaction causing the destruction of the antioxidant a-tocopherol (19). Increasing Tween 20 hydroperoxide from 3.5 to 15.6 pmol/g surfactant can decrease the lag phase of the oxidation of a salmon oil-in-water emulsions 50% as determined by both lipid hydroperoxide and headspace propanal formation (36). Brij hydroperoxides were also shown to promote iron-accelerated oxidation in corn oil-in-water emulsions (37). Because the surfactant hydroperoxide concentrations used in these studies are within the levels commonly seen in commercial surfactants, minimizing the concentrations of hydroperoxides in emulsifiers could have a beneficial effect on the shelf-life of food emulsions. Effect of Surfactant Micelle Solubilizationon Lipid Oxidation in Oil-in- Water Emulsions
Surfactants are normally used to stabilize oil-in-water emulsions against flocculation and coalescence by forming a protective membrane around the droplets. After homogenization, often significant quantities of nonadsorbed surfactant molecules are present in the aqueous phase of emulsions. Above a certain concentration, known as the critical micelle concentration, the nonadsorbed surfactant forms micelles . Micelles are aggregates of surfactant molecules in which the nonpolar tails form the interior and the polar head-groups form the exterior (25). Surfactant micelles are capable of incorporating nonpolar molecules within their hydrophobic core and polar molecules within the palisade layer formed by the surfactant head groups. Micelles may therefore be able to incorporate lipids, antioxidants or pro-oxidants, which may alter the stability of a system to lipid oxidation. Surfactants can potentially influence the physical location of antioxidants in oil-in-water emulsions by causing solubilization of lipid-soluble antioxidants into the aqueous phase. Excess Brij micelles in an oil-in-water emulsion increased the partitioning of phenolics into the continuous phase with polar antioxidants (propyl gallate) partitioning more than nonpolar antioxidants butylated hydroxytoleune (BHT). Solubilization of propyl gallate was rapid, coming to equilibrium in 4 min. Increasing surfactant micelle concentrations from 0.3 to 2.8% increased the solubilization of propyl gallate 2.3-fold (38). In oil-in-water emulsions, the physical location of lipid hydroperoxides could affect their ability to interact with both
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unsaturated FA and prooxidants such as iron. Interfacial tension measurements show that linoleic acid, methyl linoleate, and trilinolein hydroperoxides are more surfaceactive than their nonperoxidized counterparts. In oil-in-water emulsions containing surfactant micelles (Brij 76) in the continuous phase, linoleic acid, methyl linoleate, and trilinolein hydroperoxides were solubilized out of the lipid droplets into the aqueous phase. Brij 76 solubilization of the different hydroperoxides was in the order of linoleic acid > trilinolein 2 methyl linoleate (33). Surfactant micelles could also affect the physical location of prooxidant metals. To test this possibility, lipids containing femc ions were used to produce oil-in-water emulsions, and continuous phase iron concentrations in emulsions were measured as a function of varying continuous phase polyoxyethylene 10-law1 ether (Brij) concentrations.Continuous phase iron concentrations increased with increasing surfactant micelle concentrations (0.1-2.0%) and storage time (1-7 d). Micellular solubilization of iron into the continuous phase iron was higher at pH 3.0 than at pH 7.0 (37). Although the above experiments show that surfactant micelles can affect the physical location of both prooxidants and antioxidants, their net effect on lipid oxidation rates can vary depending on the characteristics of the emulsion systems. In general, the addition of surfactant micelles to emulsions prepared with a typical food oil results in inhibition of lipid oxidation (33,37,38). Solubilization of phenolic antioxidants into the aqueous phase by Brij micelles did not alter the oxidative stability of salmon oil-in-water emulsions (38). However, if an oil is high in lipid hydroperoxides or iron, Brij micelles will inhibit lipid oxidation (33,37), suggesting that the ability of surfactant micelles to inhibit lipid oxidation is due primarily to their ability to alter the physical location of prooxidants. Lipid Oxidation in Protein-StabilizedOil-in- Water Emulsions
Research with synthetic surfactants showed that emulsions droplets with thick, cationic interfacial membranes will have improved oxidative stability due to decreased interactions between aqueous phase prooxidants and lipid phase oxidation substrates. Unfortunately, the synthetic surfactants used in these studies are not approved for food applications. Proteins represent a potential emulsifier that could be used to produce cationic emulsion droplets with thick interfacial membranes. Research in this project showed that when salmon or corn oil emulsions are stabilized with proteins, oxidation rates are dramatically slower when the pH is below the PI of the protein and thus the emulsion droplet is cationic (39,40). Although the existence of a cationic charge is critical to decrease lipid oxidation rates, the charge density does not seem to be directly related to oxidative stability. For instance, the cationic charge density of whey protein-stabilized salmon oil emulsions at pH 3.0 was in the order of b-lactoglobulin > a-lactalbumin > whey protein isolate > sweet whey, whereas inhibition of lipid oxidation was in the order of P-lactoglobulin 2 sweet whey > whey protein isolate L a-lactalbumin. Similarly, the fact that the cationic charge (as determined by the zeta potential) of corn oil
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emulsion droplets stabilized by whey protein isolate (55.9 mV) was almost twice as high as the casein (29.9 mV)- and soy protein isolate (29.4 mV)-stabilized emulsions droplets; the oxidative stability of the whey protein isolate-stabilized emulsions was intermediate among the three proteins, suggesting that the magnitude of the positive charge of the emulsion droplet charge did not have a major effect on lipid oxidation rates. The lack of correlation between emulsion droplet charge density and oxidative stability suggests that additional factors are affecting lipid oxidation rates in proteinstabilized emulsions. As described above, increasing the thickness of the interfacial membrane of emulsion droplets can decrease oxidation rates, a factor that may help explain why casein, which can form a thick interfacial layer around dispersed oil droplets of up to 10 nm compared with 1-2 nm for whey proteins, was more effective at decreasing lipid oxidation rates when it was used to stabilize corn oilin-water emulsions (40). An additional factor that could be involved in differences in the oxidative stability of the different emulsions is the difference in amino acid composition between the proteins. The free sulfhydryl group of cysteine can inhibit lipid oxidation. When whey protein isolate was treated with N-ethylmaleimide to block free sulfhydryls before the formation of emulsions, no alteration in oxidation rates was observed, suggesting that free sulfhydryls at the emulsion interface do not inhibit lipid oxidation rates (39). It is possible that other antioxidative amino acids, such as tyrosine, phenylalanine, tryptophan, proline, methionine, lysine, and histidine, could be responsible for differences in the oxidative stability of emulsions stabilized by various proteins. In addition to the effect of proteins at the interface of oil-in-water emulsions droplets, aqueous phase proteins can also influence lipid oxidation rates. The addition of whey proteins to the continuous phase of Tween 20-stabilized salmon oilin-water emulsions results in inhibition of lipid oxidation (21). The free sulfhydryls of the continuous phase whey proteins are involved in this antioxidant activity because blocking sulfhydryls with N-ethylmaleimide decreased antioxidant activity, and increasing sulfhydryl exposure by thermal processing increased antioxidant activity. In addition, proteins can chelate iron and potentially remove it from the surface or interior of oil-in-water emulsions. This potential of proteins to change the physical location of iron suggests that chelation could also be involved in the antioxidant activity of continuous phase proteins (41).
Summary Numerous physical properties affect the chemistry of lipid oxidation in oil-in-water emulsions. The primary mechanism of lipid oxidation in emulsions is the metalcatalyzed decomposition of lipid hydroperoxides into free radicals. Finding technologies to minimize transition metaulipid hydroperoxide interactions may be an additional strategy for increasing the oxidative stability of food lipids. This could be accomplished by alterations in emulsion droplet charge and thickness, removal
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of metals from the emulsion droplets by chelators, and/or removing lipid hydroperoxides from emulsion droplets with surfactant micelles. These strategies may not only decrease lipid oxidation rates but may also increase the effectiveness of traditional antioxidant additives, thus providing a multihurdle strategy to increase substantially the oxidative stability of lipids in food dispersions. Through such technologies, it may be possible for food processors to incorporate oxidatively unstable bioactive lipids (e.g., n-3 FA) into functional food products. References 1. American Heart Association, Heart and Stroke Statistical Update, American Health Association, Dallas, 2004. 2. Simopoulos,A.P., Essential Fatty Acids in Health and Chronic Disease, Am J . Clin. Nutr. 70 (3 Suppl.): 560s-569s (1999). 3. American Diabetes Association, www.diabetes.org (accessed 2004). 4. Luo, J., S. W. Rizkalla, H. Vidal, J. M. Oppert, C. Colas, A. Boussairi, M. Guerre-Millo, A S . Chapuis, A. Chevalier, G. Durand, and G. Slama, Moderate Intake of n-3 Fatty Acids for 2 Months Has No Detrimental Effect on Glucose Metabolism and Could Ameliorate the Lipid Profile in Type 2 Diabetic Men. Results of a Controlled Study, Diabetes Care 21: 717-724 (1998). 5. Brown, M.L., J. Lipscomb, and C. Snyder, The Burden of Illness of Cancer: Economic Cost and Quality of Life, Annu. Rev. Public Health 22: 91-1 13 (2001). 6. Fritsch, C.W., Lipid Oxidation-The Other Dimensions, INFORM 5: 423-436 (1994). 7. Halliwell, B., M.A. Murcia, S. Chirico, and 0.1. Aruoma, Free Radicals and Antioxidants in Food and In Vivo: What They Do and How They Work, Crit. Rev. Food. Sci. Nutr. 35: 7-20 (1995). 8. Nawar, W.W., Lipids, in Food Chemistry, edited by O.R. Fenema, Marcel Dekker, New York, 1996,pp. 225-319. 9. Labuza, T., Kinetics of Lipid Oxidation in Foods, Crit. Rev. Food Sci. Technol. 10: 355405 (1971). 10. Frankel, E.N., S.W. Huang, J. Kanner, and J.B. German, Interfacial Phenomena in the Evaluation of Antioxidants: Bulk Oils vs. Emulsions, J . Agric. Food Chem. 42: 1054-1059 (1994). 11. Roozen, J.P., E.N. Frankel, and J.E. Kinsella,Enzymic and Autoxidation of Lipids in Low Fat Foods: Model of Linoleic Acid in Emulsified Hexadecane, Food Chem. 50: 33-38 (1994). 12. Roozen, J.P., E.N. Frankel, and J.E. Kinsella, Enzymic and Autoxidation of Lipids in Low Fat Foods: Model of Linoleic Acid in Emulsified Triolein and Vegetable Oils, Food Chem. 50: 39-43 (1994). 13. Huang, S.-W., E.N. Frankel, R . Aeschbach, and J.B. German, Partition of Selected Antioxidants in Corn Oil-Water Model Systems, J . Agric. Food Chem. 45: 1991-1994 (1997). 14. Mei, L., D.J. McClements, J. Wu, and E.A. Decker, Iron-Catalyzed Lipid Oxidation in Emulsions as Affected by Surfactants,pH and NaCI, Food Chem. 61:307-312 (1998). 15. Mei, L., E.A. Decker, and D.J. McClements, Evidence of Iron Association with Emulsion Droplets and Its Impact on Lipid Oxidation, J . Agric. Food Chem. 46: 50725074 (1998).
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16. Donnelly, J.L., E.A. Decker, and D.J. McClements, Iron-Catalyzed Oxidation of Emulsified Menhaden Oil as Affected by Surfactants, J . Food Sci. 63: 997-1000 (1998). 17. Silvestre, M.P.C., W. Chaiyasit, R.G. Brannan, D.J. McClements, and E.A. Decker, Ability of Surfactant Head Group Size to Alter Lipid and Antioxidant Oxidation in Oilin-Water Emulsions, J . Agric. Food Chem. 48: 2057-2061 (2000). 18. Mancuso, J.R., D.J. McClements, and E.A. Decker, Ability of Iron to Promote Surfactant Peroxide Decomposition and Oxidize a-Tocopherol, J . Agric. Food Chem. 47: 41464149 (1999). 19. Mancuso, J.R., D.J. McClements, and E.A. Decker, The Effects of Surfactant Type, pH, and Chelators on the Oxidation of Salmon Oil-in-Water Emulsions, J . Agric. Food Chem. 47: 41124116 (1999). 20. Mancuso, J. R., D.J. McClements, and E.A. Decker, Iron Accelerated Cumene Hydroperoxide Decomposition in Hexadecane and Trilaurin Emulsions, J . Agric. Food Chem. 48: 213-219 (2000). 21. Tong, L.M., S. Sasaki, D.J. McClements, and E.A. Decker, Antioxidant Activity of Whey in a Salmon Oil Emulsion, J . Food Sci. 65: 1325-1329 (2000). 22. Jacobsen, C., J. Adler-Nissen, and A.A. Meyer, Effect of Ascorbic Acid on Iron Release from the Emulsifier Interface and on the Oxidative Flavor Deterioration in Fish Oil Enriched Mayonnaise, J . Agric. Food Chem. 47: 4917-4926 (1999). 23. Jacobsen, C., K. Harvigsen, M.K. Thomsen, L.F. Hansen, P. Lund, L.H. Skibsted, G. Homer, J. Adler-Nissen, and A S . Meyer, Lipid Oxidation in Fish Oil Enriched Mayonnaise: Calcium Disodium Ethylenediaminetetraacetate, but Not Gallic Acid, Strongly Inhibited Oxidation Deterioration,J . Agric. Food Chem. 49: 1009-1019 (2001). 24. Dickinson, E., An Introduction to Food Colloids, Oxford University Press, Oxford, 1992. 25. Dickinson, E., and D.J. McClements, Advances in Food Colloids, Blackie Academic & Professional, Glasgow , 1995. 26. Kanner, J., J.B. German, and J.E. Kinsella, Initiation of Lipid Peroxidation in Biological Systems, Crit. Rev. Food Sci. Nutr. 25: 319-364 (1987). 27. Dunford, H.B., Free Radicals in Iron-Containing Systems, Free Radic. Biol. Med. 3:405-421 (1987). 28. Zumdahl, S.S., Chemistry, 2nd ed., D.C. Heath and Co., Lexington, MA, 1989. 29. Clydesdale, F.M., Mineral Interactions in Foods, in Nutrient Interactions, edited by C.E. Bodwell and J.W. Erdman, Marcel Dekker, New York, 1988. 30. Decker, E.A., and D.J. McClements, Transition Metal and Hydroperoxide Interactions: An Important Determinant in the Oxidative Stability of Lipid Dispersions, inform 12: 251-255 (2001). 31. Girotti, A.W., Lipid Hydroperoxide Generation, Turnover and Effector Action in Biological Systems, J . Lipid Res. 39: 1529-1542 (1998). 32. Patel, R.P., and V.M. Darley-Usmar, Molecular Mechanisms of the Copper Dependent Oxidation of Low-Density Lipoprotein, Free Radic. Res. 30: 1-9 (1999). 33. Nuchi, C.D., P. Hernandez, D.J. McClements, and E.A. Decker, Ability of Lipid Hydroperoxides to Partition into Surfactant Micelles and Alter Lipid Oxidation Rates in Emulsions, J . Agric. Food Chem. 50: 5445-5449 (2002). 34. McClements, D.J., Food Emulsions: Principles, Practice and Techniques, CRC Press, Boca Raton, Florida, 1999.
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35. Chaiyasit, W., M.P.C. Silvestre, DJ. McClements, and E.A. Decker, Ability of Surfactant Tail Group Size to Alter Lipid Oxidation in Oil-in-Water Emulsions, J. Agric. Food Chem. 48: 3077-3080 (2000). 36. Nuchi, C.D., D J . McClements, and E.A. Decker, Impact of Tween 20 Hydroperoxides and Iron on the Oxidation of Methyl Linoleate and Salmon Oil Dispersions, J . Agric. Food Chem. 49: 4912-4916 (2001). 37. Cho, Y.-J., D J . McClements, and E.A. Decker, Ability of Surfactant Micelles to Alter the Physical Location and Reactivity of Iron in Oil-in-Water Emulsions, J. Agric. Food Chem. 50: 5704-5710 (2002). 38. Richards, M.P., W. Chaiyasit, D J . McClements, and E.A. Decker, Ability of Surfactant Micelles to Alter the Partitioning of Phenolic Antioxidants in Oil-in-Water Emulsions, J . Agric. Food Chem. 50: 1254-1259 (2002). 39. Hu, M., D J . McClements, and E.A. Decker, Impact of Whey Protein Emulsifiers on the Oxidative Stability of Salmon Oil-in-Water Emulsions, J . Agric. Food Chem. 51: 1435-1439 (2002). 40. Hu, M., D.J. McClements, and E.A. Decker, Lipid Oxidation in Corn Oil-in-Water Emulsions Stabilized by Casein, Whey Protein Isolate and Soy Protein Isolate, J . Agric. Food Chem. 51: 16961700 (2002). 41. Tong, L.M., S . Sasaki, D.J. McClements, and E.A. Decker, Mechanisms of Antioxidant Activity of a High Molecular Weight Fraction of Whey, J . Agric. Food Chem. 48: 1473-1478 (2000).
Chapter 23
Chemistry for Oxidative Stability of Edible Oils Eunok Choed, JiyeunLeed,and David B. Minb aDepartmentof Food and Nutrition, The lnha University, Incheon, Korea, and bDepartment of Food Science and Technology, The Ohio State University, Columbus, OH 43210
Introduction The oxidative stability of oils is the resistance to oxidation (1). Resistance to oxidation can be expressed as the period of time necessary to attain the critical point of oxidation, whether it is a sensorial change or a sudden acceleration of the oxidative process (2). Oxidative stability is an important indicator of oil quality and shelf-life (3) because oxidation produces low-molecular-weight off-flavor compounds in the oils. The off-flavor compounds make the oil less acceptable or unacceptable to consumers or for use as a food ingredient. Oxidation of the oil also destroys essential fatty acids (FA) and produces toxic compounds and oxidized polymers. The oxidation of oil is very important in terms of the palatability, nutritional quality, and toxicity of edible oils. Different chemical mechanisms are responsible for the oxidation of edible oils during processing and storage, depending upon the types of oxygen. Two types of oxygen react with edible oils. One is called atmospheric triplet oxygen and the other is singlet oxygen. The important oxidation mechanisms in edible oil are autoxidation and photosensitized oxidation. Autoxidation is a free radical chain reaction, in which atmospheric triplet oxygen, 302, reacts with a lipid radical. The chemical properties of atmospheric triplet oxygen easily can be explained by the molecular orbital of the oxygen as shown in Figure 23.1. The 302in the ground state with two unpaired electrons has a permanent magnetic moment with three closely grouped energy states in a magnetic field and is termed triplet oxygen. Triplet oxygen is a radical compound with two unpaired orbitals in the molecules. It reacts with radical food compounds under normal reaction conditions according to spin conservation. Photosensitized oxidation of edible oils occurs in the presence of light, sensitizers, and atmospheric oxygen, in which singlet oxygen is produced. The electron configuration in the 2pn antibonding orbital of singlet oxygen, lo,, is shown in Figure 23.2. Singlet oxygen with one empty orbital at 2pn antibonding orbitals is an electrophilic molecule and has one energy level in a magnetic field. Nonradical electrophilic readily reacts with compounds containing 558
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Energy
0
Fig. 23.1. Molecular orbital of triplet oxygen, 30,.
high densities of electrons, such as the double bonds of unsaturated FA. The lo, has energy of 93.6 kJ above the ground state ( 4 3 . The lo, in solution deactivates by transferring its energy to the solvent, and its lifetime depends on the solvent. The lifetime of lo, is -2 ps in water ( 6 ) and 17 and 700 ps in hexane and carbon tetrachloride, respectively (7). Oxidation of edible oils is influenced by the reaction energy, FA composition, types of oxygen, and minor compounds such as metals, pigments, phospholipids, free fatty acids (FFA), mono- and diacylglycerols (MAG/DAG), thermally oxidized compounds, and antioxidants. Many efforts have been made to improve the oxidative stabilities of oils by studying the effects of these factors systematically. This chapter reviews the reaction mechanisms and kinetics, factors, and oxidation products of autoxidation and photosensitized oxidation, and antioxidants naturally present in edible oils.
Fig. 23.2. Electron Configuration of 2px antibonding orbital of singlet oxygen, lo2.
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Mechanisms of Autoxidation in Edible Oil Autoxidation of oils, a free radical chain reaction, includes the initiation, propagation, and termination steps: Initiation
RH
Propagation
R* + 30, ROO* + RH
Termination
ROO* + R* Re + R*
-
R* + H* ROO* ROOH + R* ROOR RR
The reaction of nonradical singlet state FA with radical state atmospheric 30,is thermodynamically unfavorable due to electronic spin conservation (8). The hydrogen atom in the FA in edible oil is removed, and lipid alkyl radicals are produced in the initiation step. Heat, metal catalysts, and UV and visible light can accelerate free radical formation of FA. The energy required to remove hydrogen from lipid is dependent on the hydrogen position in the molecules. A hydrogen atom adjacent to the double bond. especially hydrogen attached to the carbon between two double bonds, is removed easily. Hydrogen at C11 of linoleic acid is removed at 50 kcdmol. The energy required to remove hydrogen in C8 and C14 of linoleic acid is 75 kcdmol and the homolytic dissociation energy between hydrogen and C17 or 18 is -100 kcaUmol(9). The double bond adjacent to the carbon radical in lipid molecules shifts to the more stable next carbon and from the cis to the trans form. Autoxidation of linoleic and linolenic acids produces only conjugated products. The hydroperoxide positional isomers formed in the autoxidation of oleic, linoleic, and linolenic acids are shown in Table 23.1. The lipid alkyl radical reacts with diradical atmospheric 30,and forms the lipid peroxy radical. The reaction between the lipid alkyl radical and 30,occurs very TABLE 23.1 Hydroperoxides of FA by AutoxidatiorP FA Oleic acid
Linoleic acid Linolenic acid
aSource: Reference 10.
Hydroperoxide
Relative amount (%)
8-00H 9-00H 10-OOH 11-0OH 9-00H 13-00H 9-00H 12-00H 13-00H 16-00H
2 6-2 8 22-25 22-24 2 6-2 8 48-53 48-53 2 8-3 5 8-1 3 10-1 3 2 8-3 5
Oxidation of Oils
561
quickly at normal oxygen pressure; consequently, the concentration of the lipid alkyl radical is much lower than that of the lipid peroxy radical (1 1).The lipid peroxy radical abstracts hydrogen from other lipid molecules and reacts with the hydrogen to form hydroperoxide and other lipid alkyl radicals. These radicals catalyze the oxidation reaction; the free radical chain reaction is called autoxidation. The rates for the formation of lipid peroxy radical and hydroperoxide depend only on oxygen availability and temperature (12). When radicals react with each other, nonradical species are produced and the reaction stops. Figure 23.3 shows the formation of hydroperoxide in the autoxidation of linoleic acid. The primary oxidation products, lipid hydroperoxides, are relatively stable at room temperature and in the absence of metals. However, in the presence of metals or at high temperature they are readily decomposed to alkoxy radicals and then form aldehydes, ketones, acids, esters, alcohols, and short-chain hydrocarbons. The most likely pathway of hydroperoxide decomposition is a homolytic cleavage between oxygen and the oxygen bond, in which alkoxyl and hydroxyl radicals are produced. The activation energy to cleave the oxygen-oxygen bond is 46 kcal/mol
-He
. .
i
i CH3(CH2)4
(CH 2)7COOH
CH dCH W2)4
C
H &COOH
+so2,H. O , OH
CH3(CH2)4
13-Hydroperoxide
9-Hydroperoxide
Fig. 23.3. Hydroperoxide formation in the autoxidation of linoleic acid.
562
E. Choe e t a / .
R~-CH
=CH
- FH-R1
OOH
Fig. 23.4. Mechanisms of hydroperoxide decomposition to form secondary oxidation compounds.
lower than that to cleave the oxygen-hydrogen bond (13). The alkoxyl radical undergoes homolytic @-scissionof the C-C bond and produces 0x0 compounds and an alkyl or alkenyl radical (Fig. 23.4). The ultimate secondary oxidation products of lipid are mainly low-molecular-weight aldehydes, ketones, alcohols, and shortchain hydrocarbons as shown in Table 23.2. The time for secondary product formation from the primary oxidation product, hydroperoxide, differs for different oils, Secondary oxidation products are formed immediately after hydroperoxide formation in olive and rapeseed oils. However, in
Oxidation of Oils
563
TABLE 23.2
Decomposition Compounds of FAME by Autoxidationa Class
Oleic acid
Linoleic acid
Linolenic acid
Aldehydes
Octanal Nonanal 2-Decenal Decanal
Pentanal Hexanal 2-Octenal 2-Nonenal 2,4-Decadienal
Propanal Butanal 2-Butenal 2-Pentenal 2- Hexena I 3,6-Nonadienal Decatrienal
Carboxylic acid
Methyl heptanoate Methyl heptanoate Methyl heptanoate Methyl octanoate Methyl octanoate Methyl octanoate Methyl nonanoate Methyl 8-oxooctanoate Methyl 8-oxooctanoate Methyl 9-oxononanoate Methyl 9-oxononanoate Methyl 9-oxononanoate Methyl 1O-oxodecanoate Methyl 1O-oxodecanoate Methyl 1O-oxodecanoate Methyl 1O-oxo-8-decenoate Methyl 1 1-oxo-9-undecenoate
Alcohol
1-Heptanol
1-Pentanol 1-0ctene-3-01
Hydrocarbons
Heptane Octane
Pentane
Ethane Pentane
aSource:Reference 10.
sunflower and safflower oils, secondary oxidation products are formed when there is an appreciable concentration of hydroperoxides (1). Most decomposition products of hydroperoxides are responsible for the offflavor in the oxidized edible oil. Aliphatic carbonyl compounds have more influence on the oxidized oil flavor due to their low threshold values. Threshold values for hydrocarbons, alkanals, 2-alkenals, and trans, trans-2,4-alkadienals are 90-2150,0.0&1,0.04-2.5, and 0.04-0.3 ppm, respectively (10).Hexanal (23.5%), 2-decenal (34.3%), 2-heptenal (29.5%), and trans-2-octenal (18.1%) were the major volatile compounds detected by a solid-phase microextraction method in soybean and corn oils (PV of 5 ) , respectively (14). Pentane, hexanal, propenal, and 2,4-decadienal were present in high amounts in canola oil stored uncovered at 60°C (15). Frankel (10) reported that trans, cis-2,4-decadienal was the most important compound in determining the oxidized flavor of oil, followed by trans, trans2,4-decadienal, trans, cis-2,4-heptadienal, 1-octen-3-01, butanal, and hexanal. Hexanal, pentane, and 2,4-decadienal were suggested and used as indicators to determine the extent of the oil oxidation (16-19). Trans-2-hexena1, and trans, cis, trans-2,4,7-decatrienal and 1-octen-3-one were reported to give grass-like and fishlike flavors in oxidized soybean oil, respectively (8). No single flavor compound is responsible for the oxidized flavor of vegetable oils.
E. Choe et a/.
5 64
'Sen'
Excited -as t
-
k = 1 20 x 10'h
k = 2 x 10'ls
k = 10 - 1 O'/S Singlet oxygen
Ground
formation Fig. 23.5. Excitation and deactivation of sensitizer.
Mechanisms for Singlet Oxygen Formation and Photosensitized Oxidation in Edible Oil Light accelerates the oxidation of oil, especially in the presence of sensitizers. Chlorophylls are common sensitizers in edible vegetable oils. Sensitizers absorb light energy very rapidly, in picoseconds, to enter an excited singlet state and then
3~en'
ROOH
m0,
+ .Sen+
Fig. 23.6. Reaction of triplet sensitizer with substrates.
ROOH
Oxidation of Oils
565
return to the ground state via the emission of light, internal conversion, or intersystem crossing (Fig. 23.5). Fluorescence and heat are produced by the emission of light and internal conversion, respectively. Intersystem crossing results in the excited triplet state of the sensitizers. An excited triplet sensitizer may accept hydrogen from the substrate or donate an electron to it and produce radicals (type I) as shown in Figure 23.6. An excited triplet sensitizer reacts with 30,and produces a superoxide anion by electron transfer. The superoxide anion produces lo, by spontaneous dismutation. O,*-
H,O,
+ O,*- + 2H+ + O,*-
___3)
H,O,
+ lo,
HO* + OH- + '0,
The excitation energy of a triplet sensitizer can be transferred onto an adjacent 30,to form lo, by triplet-triplet annihilation, and the sensitizer returns to its ground singlet state (type a).Kochevar and Redmond (20) reported that a sensitizer molecule may generate 103-105 molecules of 10, before becoming inactive. The rate of the type I or II process depends on the kinds of sensitizers (21) and substrates, and the concentrations of substrate and oxygen (22). Compounds that are readily oxidized (phenols or amines) or readily reduced (quinones) favor type I. On the other hand, olefms, dienes, and aromatic compounds, which are not so readily oxidized or reduced, more often favor type II. Photosensitized oxidation of edible oil follows the lo, oxidation pathway. lo, was suggested to be involved in the initiation of lipid oxidation (23). lo, either reacts chemically with other molecules, or transfers its excitation energy. When '0, reacts with unsaturated oils, primarily ally1 hydroperoxides are formed by ene reaction (24), as shown in Figure 23.7. Electrophilic lo, can react directly with high electron density double bonds without the formation of alkyl radical and form hydroperoxides at the double bonds. The migration of the double-bond positions and trans FA occurs when hydroperoxide is formed. This results in the production of both conjugated and nonconjugated hydroperoxides as shown in Table 23.3, which differs from autoxidation. Figure 23.8 shows the oxidation of linoleic acid by lo,.
I 2
~ ( C H? Z ) - ~ C O cO HH
Hzb
s ( c H ~ ~ 2 ~ c o o H
$--n'
Fig. 23.7. Formation of ally1 hydroperoxide of oleic acid by ene reaction.
E. Choe et a/.
566
TABLE 23.3 Hydroperoxides of FA by Singlet Oxygen Oxidation Relative amounta (YO)
Oleic acid Linoleic acid
Linolenic acid
9-00H 10-OOH 9 - 0 0 H (Qb 10-OOH (NC) 1 2 - 0 0 H (NC) 1 3 - 0 0 H (C) 9 - 0 0 H (C) 1 0-OOH (NC) 1 2 - 0 0 H (C) 1 3 - 0 0 H (C) 1 5 - 0 0 H (NC) 1 6 - 0 0 H (C)
48 52 32 17 17 34 23 13 12 14 13 25
aSource: Reference 10. bC, conjugated; NC, nonconjugated.
Hydroperoxides formed by lo, oxidation are decomposed by the same mechanisms for the hydroperoxides formed by 30,in autoxidation. Frankel (10) reported that the amount of 2-decenal and octane was higher in '02-oxidized oleate than that in autoxidized oleate. The contents of octanal and 10-oxodecanoate in autoxidized oleate were higher than those of lO,-oxidized oleate. 2-Heptenal and 2-butenal were
I
Nonconjupaled
Fig. 23.8. Hydroperoxide formation of linoleic acid by '0, oxidation.
Oxidation of Oils
567
present in 'O2-oxidized linoleic and linolenic acids, whereas they were negligible in autoxidized linoleic and linolenic acids. Heptenal was formed in soybean oil only in the presence of chlorophyll and light (25). A beany flavor, which is a unique and undesirable flavor in soybean oil with a low peroxide value (PV), has been a problem for the last 70 yr, one that has been studied extensively, both nationally and internationally (26,27). 2-Pentylfuran and
Fig. 23.9. Formation of 2-pentyl-
furan from linoleic acid by '0, oxidation.
568
E. Choe et a / .
pentenylfuran were reported to be responsible for the beany flavor in soybean oil (26-30). Min el al. (25) reported the detailed chemical mechanisms for the formation of 2-pentylfuran and 2-(2-pentenyl) furan by lo, oxidation of linoleic and linolenic acids present in soybean oil as shown in Figures 23.9 and 23.10, respectively. Min et al. (25) strongly indicated that the reversion flavor of soybean oil can be decreased or eliminated by removing chlorophyll from the oil during pro-
0 II
CH~-CH,-CH=CH-CH~-CH=CH-CH~-~H-CH=CH-(CHZ)CI-CCIH
t
+
'02
f
Fig. 23.10. Formation of 2-pentenylfuran from linolenic acid by '0, oxidation.
Oxidation of Oils
569
cessing. The soybean oil industry currently removes chlorophyll effectively from soybean oil using a bleaching material during the refining process, and the beany flavor is no longer a serious flavor problem in soybean oil.
Factors Affecting the Oxidation of Edible Oil The oxidation of oil is influenced by the FA composition of the oil, oil processing, energy in heat or light, the concentration and type of oxygen, FFA, MAG and DAG, transition metals, peroxides, thermally oxidized compounds, pigments, and antioxidants. It is not easy to differentiate the individual effects of these factors because interactions exist among them. FA Composition of Oils
Oils that are more unsaturated oxidize more quickly than less unsaturated oils (31). Soybean, safflower, or sunflower oil (iodine values >130) stored in the dark had a significantly ( P < 0.05) shorter induction period than coconut or palm kernel oil whose iodine value is <20 (32). High-oleic and high-stearic oils from gene silencing of the oilseeds or hydrogenated soybean oil had higher autoxidative stability (33,34). The autoxidation rate greatly depends on the rate of alkyl radical formation in the lipid, and the formation rate of FA alkyl radical depends mainly on the types of FA. The relative autoxidation rate of oleic, linoleic, and linolenic acid was reported as 1:40-50: 100 on the basis of oxygen uptake (8). The difference in singlet oxygen oxidation rate among FA is lower than that for autoxidation. The reaction rates between '0, and stearic, oleic, linoleic, and linolenic acids are 1.2 x lo4, 5.3 x lo4, 7.3 x lo4, and 10.0 x lo4 M-'.s-', respectively (35). The relative '0, reaction rates for oleic, linoleic, and linolenic acids are 1.0:1.4:1.9,respectively. Soybean oil reacts with lo, at a rate of 1.4 x lo5M-'. s-l in methylene chloride at 20°C (36). The type of PUFA, nonconjugated or conjugated dienes or trienes, has little effect on the reaction between the lipid and lo, (37). Oil Processing
The oil-processing method affects the oxidative stability of the oil. Crude soybean oil was the most stable to oxidation followed by deodorized, degummed, refined, and bleached oil during 6 d of storage at 55°C in the dark (38). The induction time in hexane-extracted rapeseed oil oxidation was 10.5 f 1.9 h compared with an induction time of 8.1 f 0.7 h for pressed rapeseed oil at 90°C (39). Roasting of safflower and sesame seeds before oil extraction improved the oxidative stability of the oils (40,41). The oxidative stability increased as the roasting temperature of the seeds increased. Some Maillard reaction products were reported to be antioxidants. The Maillard products may have contributed to the oxidative stability of the roasted safflower and sesame oils.
570
E. Choe e t a / .
Storage Temperature and Light The autoxidation of oils and the decomposition of hydroperoxides increase as the storage temperature increases (42,43). The formation of autoxidation products during the induction period is slow at low temperature (44). The concentration of the hydroperoxides increases until the advanced stages of oxidation. The content of polymerized compounds increases significantly at the end of the induction period of autoxidation (45). The hydroperoxide degradation rate of crude herring oil stored at 50°C in the dark was higher than the formation rate of hydroperoxide. The reverse phenomena were observed in the same crude hemng oil at 0 or 20°C in the dark (11). The reaction temperature has little effect on lo, oxidation due to the low activation energy of 0-6 kcal/mol(37,46). Light is much more important in lo, oxidation. Light of shorter wavelength had more detrimental effects than longer wavelengths (47). Reportedly, the effect of light on the oil oxidation becomes less as the storage temperature increases (44). Because lo, oxidation occurs in the presence of light, the packaging of the oils is very important. Transparent plastic bottles increase oil oxidation. The incorporation of Tinuvin 234 [2-(2-hydroxy-3,5-di(1,Idimethylbenzy1)phenyl)benzotriazole], which is a UV light absorber (48), into the transparent plastic bottles improved oxidative and sensory stability of soybean oil under light at 25°C (49). Oxygen The oxidation of oil can take place when oil, oxygen, and catalysts are in contact. Both concentration and type of oxygen affect the oxidation of oils. The oxygen concentration in the oil is dependent on the oxygen partial pressure in the headspace of the oil (50).A higher amount of oxygen is dissolved in the oil when the oxygen partial pressure in the headspace is high. Oxidation of the oil increased with the amount of dissolved oxygen (5 1). The heat of solubilization of oxygen in the oil was reported to be -3.7 kcal/mol (52). The solubility of oxygen is higher in oil than in water, and in crude oils than in refined oils (53). One gram of soybean oil dissolves 55 pg oxygen at room temperature (50).The amount of oxygen dissolved in oil is sufficient to oxidize the oil to a PV of -10 meq/Kg in the dark (54). Min and Wen (51) reported that the rate constants for oxygen disappearance in soybean oil containing 2.5,4.5,6.5, and 8.5 ppm dissolved oxygen during storage at 55°C in the dark were 0.049,0.058,0.126, and 0.162 p p d h , respectively. The effect of oxygen concentration on the oxidation of oil increased at high temperature and in the presence of light and metals. Higher oxygen dependence of oil oxidation at high storage temperature is due to low oxygen solubility in the oil at high temperature (50).The oxygen has to be transported into the oil by diffusion when the oil is not stirred, such as during storage at low temperature. Convection is another important pathway for oxygen penetration into the oil from the surface when the oil is stirred, for example, during processing at high temperature.
Oxidation of Oils
571
The oil oxidation rate is independent of oxygen concentration at sufficiently high oxygen concentrations. The oxidation rate is dependent on oxygen concentration when oxygen content is low and is independent of lipid concentration (50). The autoxidation rate of oil at >4-5% oxygen in the headspace was independent of oxygen concentration and directly dependent on lipid concentration (55). However, the reverse was true at low oxygen pressure ( ~ 4 %oxygen) in the headspace (56). The oxidation of rapeseed oil at 50°C in the dark, measured as oxygen consumption or PV, was strongly influenced by oxygen concentration <0.5%, whereas the oxidation rate decreased with oxygen concentration at >1% oxygen (57). Oxygen and edible oil can react more efficiently when a small oil sample size or an oil sample with a high ratio of surface to volume was used (32). When the surface:volume ratio increases, the relative rate of oxidation is less oxygen-dependent with a low oxygen content. The container surface can act as a reduction catalyst, and its effect was proportional to the area of container in contact with oils (58). Volatile formation in rapeseed oil in the dark was reported to be influenced by the interaction between temperature and oxygen concentration. Production of 2-pentenal and 1-pentene-3-one was positively correlated with oxygen concentration at 50"C,but negatively at 35°C (57). The reaction rate of lo, with lipids is much higher than that of 302. Linoleates react with lo, at a 1450 times faster rate than with 302 (23). Minor Compounds
Edible oil consists mainly of TAG, but it also contains minor components such as FFA, MAG and DAG, metals, phospholipids, peroxides, chlorophylls, carotenoids, phenolic compounds, and tocopherols. Some of them accelerate the oil oxidation, and others act as antioxidants. FFA and MAG and DAG. Edible oil contains FFA, and processing decreases the FFA contents. Crude soybean oil contains -0.7% FFA; however, refined oil contains 0.02% (38). Crude sesame oil contains 2.26% FFA (59), and bleached oil contains 0.56% (60).FFA are more susceptible to the autoxidation than esterified FA (61). FFA act as prooxidants in edible oil (62,63). They have hydrophilic and hydrophobic groups in the same molecule and prefer to be concentrated on the surface of edible oils. The hydrophilic carboxyl groups of the FA will not easily dissolve in the hydrophobic edible oil and are present on the surface of edible oil. Mistry and Min (63) reported that the FFA decrease the surface tension of edible oil and increase the diffusion rate of oxygen from the headspace into the oil to accelerate oxidation of the oil. MAG and DAG, usually present at 0.07-0.11 and 1.05-1.20% in soybean oil, respectively, acted as prooxidants to increase the oxidation at 55°C in the dark (64,65). MAG and DAG decrease the surface tension of edible oil and increase the diffusion rate of oxygen from the headspace into the oil to accelerate oxidation of the oil. Mistry and Min (65) reported that MAG and DAG should be removed from the oil during the oil-refining process to improve the oxidative stability of edible oils.
E. Choe eta/.
572
Metals. Crude oil contains transition metals such as iron or copper, but the refining process reduces their concentrations. Edible oils manufactured without refining, e.g., extra virgin olive oil and sesame oil, contain relatively high amounts of transition metals (Table 23.4). Metals increase the rate of oil oxidation due to the reduction of activation energy of the initiation step in the autoxidation down to 63-lo4 kJ/mol (69). Metals also react directly with lipids to produce lipid alkyl radicals; reactive oxygen species such as lo,, the hydroxyl radical from 30,,and hydrogen peroxide are also produced (50). Lipid alkyl radical and reactive oxygen species accelerate the oxidation of oil, Copper accelerates hydrogen peroxide decomposition 50 times faster than ferrous ion (Fez+), and Fez+ acts 100 times faster than ferric ion (Fe3+).
Fe3++ RH Fez+ + 30,
O,*-
+ O,*- + 2H+
-
Fe2++ Re + Ha
__5
Fe2++ O,*-
lo, + H20,
Metal catalyst H,O,
+ O,*-
Fe2++ H,O,
HO* + OH-
+ '0, Fe3++ OH- + HO.
u
Metals also accelerate autoxidation of oil by decomposing hydroperoxides (70). Fez+ is much more active with a rate of 1.5 x lo3 M-' .~ ' ( 7 1 than ) Fe3+in decomposition of lipid hydroperoxides during the propagation step to catalyze autoxidation (72). ROOH + Fez+ (Cu') ROOH + Fe3+(Cu2+)
-
ROO+ Fe3+(Cu2+)+ OHROO.
+ Fe3+(Cu2+)+ H+
TABLE 23.4 Copper and Iron Contents in Edible Oils Metal content
Oil
Copper (ppb)
Iron (ppm)
Cold-pressed sesame oila Crude soybean oilb Virgin olive oila Cold-pressed sunflower oila Refined olive oilc Refined soybean oilb
16 (3.0-38) 13.2 9.8 (1 .O-79) 5.2 (2.2-8.5) 15 2.5
1.1 6 (0.1 8-1.52) 2.80 0.73 (n.d.-9.79) 0.26 (0.22-0.31) 0.08 0.20
aSource: Reference 66. bource: Reference 67. cSource: Reference 68.
Oxidation of Oils
57 3
Fe3+also cause decomposition of phenolic compounds such as caffeic acid in olive oil and decrease the oxidative stability of the oil (73). Phospholipids. Crude soybean oil contains phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA). Deodorized soybean oil contains only PC and PE at 0.86 and 0.12 ppm, respectively (74). Phospholipids act as antioxidants and prooxidants depending on their concentration and the presence of metals. Phospholipids reduce the oxidation of oil by sequestering metals; the concentration for the maximal antioxidant activity was between 3 and 60 ppm. Soybean oil oxidation decreased with 5-10 ppm phospholipids, and higher amounts of phospholipids acted as prooxidants. Yoon and Min (75) reported that phospholipids acted as antioxidants in the presence of iron, by chelating iron. The antioxidant effect in soybean oil in the dark at 60°C was the highest with PA and PE followed by PC, phosphatidylglycerol, and PI. Yoon and Min (75) reported that phospholipids worked as prooxidants in purified soybean oil, which does not contain metals. Phospholipids have hydrophilic and hydrophobic groups in the same molecule. The hydrophilic groups of the phospholipids are on the surface of the oil, and the hydrophobic groups are in the edible oil. Phospholipids decrease the surface tension of edible oil and increase the diffusion rate of oxygen from the headspace into the oil to accelerate oxidation of the oil (75). Chlorophylls. Chlorophylls are the most common pigments present in edible vegetable oils. Virgin olive oil and rapeseed oil contain chlorophyll at 10 f 4.9 and 5-35 ppm, respectively (76). Virgin olive oil also contains 4.1-15.1 ppm pheophytin (77). The chlorophyll concentrations in crude and bleached soybean oil are 0.30 and 0.08 ppm, respectively (38). Chlorophylls are generally removed during oil processing, especially during the bleaching process (Table 23.5), and refined oil produces less lo,. Chlorophylls and their degradation products, pheophytins and pheophorbides, act as sensitizers to produce lo, in the presence of light and atmospheric oxygen and accelerate the oxidation of oil (79-8 1). Pheophytins have a higher sensitizing activity than chlorophylls, but lower than that of pheophorbides (37,82). Soybean oil containTABLE 23.5
Chlorophyll Contents in Canola Oil During Processinga ~
~
Oil
~
~
~
~
~
~
~
~
~~~~~~~~
Chlorophyll a (ppm)
Pheophytin a (ppm)
Pheophytin b (ppm)
Pyropheophytin a (ppm)
Pyropheophytin b (ppm)
i.aa
3.31
7.16 6.27
1.34 1.07 1.12 0.32
16.57 9.40 9.13 0.21
3.13
0.27 0.22
Extracted Degummed Refined Bleached aSource: Reference 78
-
0.56
1.a4 1.79 0.25
5 74
E. Choe eta/.
ing chlorophyll produced volatiles during storage under light, but not in the dark (83). The headspace volatile formation in soybean oil under light increased as the concentration of chlorophyll increased. The chlorophyll in soybean oil was completely removed by silicic acid column chromatography, and the oil was called purified soybean oil. Chlorophyll-free purified soybean oil did not produce headspace volatiles under light, but the original soybean oil with chlorophylls did so under the same experimental conditions. Rahmani and Csallany (37) also showed that the oxidation of virgin olive oil containing pheophytin was accelerated by the illumination of fluorescent light. Although chlorophylls are strong prooxidants under light, they act as antioxidants in the dark, possibly by donating hydrogen to free radicals (84,85). Thermally Oxidized Compounds. The processing of oils can produce oxidized compounds such as cyclic carbon-to-carbon linked dimers ,noncyclic hydroxyl dimers, carbon-to-carbon linked trimers, dimers and trimers joined through carbon-to-carbon or carbon-to-oxygen linkage, and trimers joined through carbon-to-oxygen linkage. Refined, bleached, deodorized (RBD) soybean oil contains 1.1% thermally oxidized compounds (86). Thermally oxidized TAG accelerated the oxidation of soybean oil during storage at 55°C for 6 d (86). The acceleration of the oil oxidation increases with the concentration of thermally oxidized compounds. Lipid hydroperoxides also acted as prooxidants (87). Antioxidants. Edible oils naturally contain antioxidants such as tocopherols, tocotrienols, carotenoids, phenolic compounds, and sterols. Antioxidants are sometimes intentionally added to the oil to improve the oxidative stability. Antioxidants are the compounds that extend the induction period of oxidation or slow down the oxidation rate. Antioxidants inactivate free radicals, control transition metals, quench lo2,and inactivate sensitizers. Antioxidants can donate hydrogen atoms to free radicals and convert them to more stable nonradical products (88). The major hydrogen-donating antioxidants are monohydroxy or polyhydroxy phenolic compounds with various ring substitutions. Any compound whose reduction potential is lower than that of a free radical can donate a hydrogen to that radical unless the reaction is kinetically unfavorable. Standard l-electron reduction potentials of alkoxyl, peroxyl, and alkyl radical of PUFA are 1,600, 1,000, and 600 mV, respectively (89). The standard reduction potential of antioxidants is -500 mV or below. This clearly shows that antioxidants react with lipid peroxy radicals before the peroxyl radicals react with other lipid molecules to produce another free radical. An antioxidant radical produced from the reaction with lipid peroxy radical has lower energy than the peroxyl radical itself due to resonance structure (Fig. 23.1 1). Metal chelators such as phosphoric acid, citric acid, ascorbic acid, and EDTA can convert iron or copper ions into insoluble complexes or sterically hinder formation of the complexes between metals and lipid hydroperoxides (90). For this reason, citric acid is sometimes added to the oil to reduce oxidation during storage.
Oxidation of Oils
575
Fig. 23.1 1. Resonance stabilization of an antioxidant radical.
Citric acid improved the sensory quality of soybean oil containing 1 ppm iron at 55OC (91). Min and Wen (91) reported that the antioxidant effects increased as citric acid content increased, and >150 ppm citric acid was necessary to overcome the catalytic effect of 1 ppm iron. When the oil contains 10.1 ppm iron such as an RBD oil, the common practice of adding -150 ppm citric acid to the oil for improvement of oxidative stability is not necessary (91). Some antioxidants quench lo, or an excited sensitizer. lo, is quenched physically and chemically. In physical quenching, singlet oxygen is converted into 30, by either energy transfer or charge transfer, and there is no oxidation of the antioxidant. In chemical quenching, antioxidants react with lo, to produce oxidized antioxidants (9). Tocopherols. Tocopherols are the most important antioxidants in edible oils. Soybean, canola, sunflower and corn oils contain relatively high amounts of tocopherols (Table 23.6). Although palm oil does not contain large amounts of tocopherols (118-146 ppm), it has higher concentration of tocotrienols, i.e., a-, y-, and 8-tocotrienols at 21 1, 353-372, and 56-67 ppm, respectively (95). Safflower oil also contains y- and 8-tocotrienols at 3.8-7.0 and 7.5-8.4 ppm, respectively (41). TABLE 23.6
Tocopherol Contents of Edible Oil Tocopherol content (ppm) Oil
a
P
Y
6
Total
275.0
1162 695.4 648.9 603 597 566 397-540 168- 226
~~
Soybeana Canolaa Sunflowera Corna Roasted sesameb RapeseedC SaffIowerd Olivee a50urce: Reference 92. bSource: Reference 93. C50urce:Reference 12. dSource: Reference 41. e50urce: References 76,94.
116.0 272.1 61 3.0 134.0 4 252 386-520 168- 226
34.0 0.1 17.0 18.0 -
8.6-12.4
-
737.0 423.2 18.9 412.0 584 314 2.4-7.7
-
-
39.0 9
-
-
F. Choe et a/.
576
The tocopherol contents in edible oils are affected by the cultivar and processing, and storage of the oil (96). The tocopherol contents of sesame oil range between 404 and 540 ppm, depending on the cultivars (97). The tocopherol contents in rapeseed oil are 793.6 f 107.9 and 748.9 f 93.2 ppm for hexane-extracted oil and pressed oil, respectively (39). The refining process, especially deodorization, reduces tocopherol contents (38 98,99). Crude, bleached, and deodorized soybean oil contains tocopherols at 1,670, 1,467, and 1,138 ppm, respectively (38). In virgin olive oil, the a-tocopherol concentration decreased with storage time of the oil (96). There was no tocopherol left in olive oil stored in the dark at room temperature for 12 mon (100). Tocopherols compete with unsaturated oils for lipid peroxy radicals. Lipid peroxy radicals react with tocopherols much faster at 104-109 M-' * s-l than with lipid (10-60 M-'. s-'). One tocopherol molecule can protect -103-108 PUFA molecules at a low PV (101). Tocopherols can transfer a hydrogen atom at the 6hydroxyl group on its chroman ring to a lipid peroxy radical and scavenge the peroxyl radicals. Tocopherol, with a reduction potential of 500 mV, donates hydrogen to lipid peroxyl radicals that have a reduction potential of 1,000 mV and produces lipid hydroperoxide and tocopheroxyl radicals. Tocopheroxyl radicals are more stable than peroxyl radicals due to resonance structures. This ultimately slows down the oil oxidation rate in the propagation stage of autoxidation. Reaction rates of peroxy radicals of stearic and oleic acids with a-tocopherol were reported to be 2.8 x lo6 and 2.5 x lo6 M-'.s-', respectively (102). Tocopheroxyl radicals can interact with other compounds or each other, depending on the oxidation rates. Tocopheroxyl radicals react with each other at low lipid oxidation rates and produce tocopheryl quinone and tocopherol, At higher oxidation rates, tocopheroxyl radicals may react with lipid peroxyl radicals and produce tocopherol-lipid peroxy complexes, which can hydrolyze to tocopheryl quinone and lipid hydroperoxide (103). T + ROO* T* + TTO+ ROO.
- [T-OOR]
T* + ROOH
T + Tocopheryl quinone Tocopheryl quinone + ROOH
The effectiveness of tocopherols as antioxidants depends on the isomers and concentration of tocopherols. The free radical-scavenging activity of tocopherols is the highest in &tocopherol followed by y-, p-, and a-tocopherol (98). It was also reported that the antioxidant activity of tocopherol isomers varied with tocopherol concentration. a-Tocopherol was more effective in reducing the oil oxidation than y-tocopherol up to 200 ppm and less effective above this concentration (104). The optimal concentration of tocopherols as antioxidants is dependent on their oxidative stability, i.e., the lower the oxidative stability of the tocopherol, the lower the optimal concentration of tocopherol for maximal antioxidant activity.
Oxidation of Oils
577
The optimal concentration of a-tocopherol, the least stable among tocopherol isomers, to increase the oxidative stability of soybean oil at 55°C in the dark was 100 ppm and that of y- and 6-tocopherols was 250 and 500 ppm, respectively (105). Tocopherols, particularly a-tocopherol, act as prooxidants when present in high concentrations in vegetable oils via propagation of free radicals, depending on the hydroperoxide concentration (105-107). When the concentration of lipid peroxy radical is very low, the tocopheroxyl radical abstracts hydrogen from the lipid and produces tocopherol and lipid alkyl radical; however, the reaction rate is very low. Formation of lipid alkyl radicals by tocopherols accelerates lipid oxidation; this is called tocopherol-mediated peroxidation (108,109). The addition of 100 ppm a-tocopherol increased the oxidation of purified olive oil at the early stage of autoxidation; however, a-tocopherol added to moderately oxidized (PV = 15) purified olive oil or lard significantly decreased oxidation of the oil (1 10). The threshold value for a-tocopherol as a prooxidant in virgin olive oil oxidation was 60-70 ppm; when less a-tocopherol was initially present in the oil, the threshold value for prooxidant activity was reached more quickly (96). Prooxidants activity of a-tocopherol decreased as the temperature increased even at a high concentration (111). a-Tocopherol demonstrated the highest prooxidant activity followed by y- and 6tocopherol in soybean oil (105). Oxidized tocopherols increased the oxidation of soybean oil, and prooxidant activity was the highest in oxidized a-tocopherol followed by oxidized y- and 6-tocopherol (1 12). Prevention of tocopherol oxidation and removal of oxidized tocopherols during oil processing are strongly recommended to improve the oxidative stability of the oil. Tocopherol ( r ) can quench lo, at a rate of 2.7 x lo7 M-' s-l . The 0, quenching effect is in the decreasing order of a - , p-, y-, and 6- tocopherols (1 13,114). Tocopherol forms a charge transfer complex with lo, by electron donation to lo,. The singlet state of the tocopherol-'02 complex undergoes intersystem crossing into the triplet state and produces less reactive 302 and tocopherol. When there is no chemical reaction between tocopherol and lo,, it is called physical quenching.
T+Q,
-
- -
[T-+-~O,I~
[T-+-~o,I,
T+~o,
Tocopherols react irreversibly with lo2in chemical quenching and produce tocopherol hydroperoxydienone, tocopheryl quinone, and quinone epoxide (Fig. 23.12). Carotenoids. Carotenoids are a group of tetraterpenoids consisting of isoprenoid units. The double bonds in carotenoids are conjugated forms, usually all trans forms. p-Carotene is one of the most studied carotenoids. Edible oils, especially unrefined oils, contain p-carotene. Crude palm oil and red palm olein contain 500-700 ppm carotenoids (1 15). Virgin olive oil contains 1.O-2.7 ppm p-carotene as well as 0.9-2.3 ppm lutein (77).
E. Choe et a/.
578
'0,
H3C
ClBH39
H @ ;b *cl
CH3
OOH
CH3
a-Tocopherol
Hydroperoxydienone
H
O
H3C
/
W
cI6H33
a-Tocopherol endoperoxide
CH3
a-Tocopherol quinone
a-Tocopherol quinone epoxide
Fig. 23.12. Singlet oxygen oxidation of a-tocopherol.
p-Carotene can slow down the oil oxidation by light filtering, lo, quenching, sensitizer inactivation, and free radical scavenging. Fakourelis et al. (79) reported that oxidation of olive oil containing only p-carotene under light at 25OC was decreased by the filtering out of some of light energy mainly between 400 and 500 nm. In the presence of chlorophylls, p-carotene decreased the oxidation of soybean oil stored under light by '0, quenching (83). '0, quenching by carotenoids is mainly by energy transfer from singlet oxygen to carotenoids, without generating oxidized products. Excited carotenoids return to the ground state by giving off heat.
'0,+ 'Carotenoid 3Carotenoid*
-
___3L
30,+ 3Carotenoid* Carotenoid + heat
Oxidation of Oils
5 79
One mole of p-carotene can quench 250-1,000 molecules of lo, at a rate of 1.3 x 1O’O M-’ s-I (22). lo, quenching of carotenoids is dependent on the number of conjugated double bonds (1 16). Carotenoids having at least 9 conjugated double bonds act as an effective lo, quencher, and quenching activity increases as the number of double bonds increases (9). Carotenoids inactivate excited sensitizers physically by absorbing energy from sensitizers. The excited carotenoids return to the ground state by transfering the energy to the surrounding solvent, the oil (1 17). Lee et al. (118) reported that it was very difficult for p-carotene with the high 1-electron reduction potential of 1060 mV to donate hydrogen to the alkyl ( E O ’ = 600 mV) or peroxy radical (EO’ = 770-1440 mV) of PUFA. An NMR study showed that p-carotene did not donate a hydrogen atom to quench free radicals (118). p-Carotene can donate hydrogen to a hydroxy radical, which has a high reduction potential (2310 mV) and produce a carotene radical (Car.). A carotene radical is a fairly stable species due to delocalization of unpaired electrons in its conjugated polyene system. A carotene radical may react with other radicals such as lipid peroxy radicals at low oxygen concentration and form nonradical products (116,119).
-
Car + HO*
___3c
Car.
Car* + ROO*
___3c
Car-OOR
+ H,O
A lipid peroxy radical may be added to p-carotene and produce a carotene peroxy radical, especially at >150 mmHg of oxygen (1 19). Carotene peroxy radicals react with 30,and then with lipid molecule and produce lipid alkyl radicals, which propagate the chain reaction of lipid oxidation (120). Car + ROO*
ROO-C~+ ~ .3 0 , ROO-Car-00.
+ R’H
-
ROO-Car. ROO-Car-OO* ROO-Car-OOH + R’.
The antioxidant activity of P-carotene was not seen in soybean oil stored in the dark (1 18). During photosensitized oxidation of soybean or rapeseed oil in opened vessels, p-carotene increased the oil oxidation, but the copresence of tocopherols decreased oxidation of the oil (121). The antioxidant activities of carotenoids by hydrogen donation remain controversial, p-Carotene may also donate electrons to free radicals and become p-carotene cation radicals (122,123). The transfer of hydrogen or electrons from carotenoids to free radicals depends on the reduction potentials of the free radicals and the chemical structures of carotenoids, especially the presence of oxygen-containing functional groups (124). The p-carotene cation radical has a relatively high standard 1-electron reduction potential (1060 mV) and does not readily react with oxy-
E. Choe et a / .
580
gen to form peroxide (125). The p-carotene cation radical may react with alkyl, alkoxyl, or peroxyl radicals formed in soybean oil during oxidation. Lee et al. (1 18) reported that p-carotene was prooxidant in soybean oil due to the electron transfer mechanism in their 2,2-diphenyl- 1-picry1 hydrazyl and NMR studies. Other Phenolic Compounds. Phenolic compounds other than tocopherols in edible oils also exert antioxidant activity. Sesame oil, which contains a high amount of unsaturated FA (iodine value = 109), has good oxidative stability (32). The autoxidation rate of sesame oils at 60°C was much lower than those of corn oil, safflower oil, and a mixture of soybean and rapeseed oils (126). Roasted sesame oil had greater oxidative stability than unroasted sesame oil (127,128). The remarkable oxidative stability of sesame oil is due to the presence of lignan compounds as well as tocopherols (129,130). Lignan compounds in sesame oil include sesamin, sesamol, sesamolin, sesaminol, and sesamolinol (Fig. 23.13). Sesamin is the predominant lignan compound in unroasted sesame oil (474 ppm), followed by sesamolin (159 ppm). Sesamol concentration in unroasted sesame oil was 4 - 7 ppm (13 1,132); however, roasted sesame oil contains a higher concentration (>20 ppm) of sesamol (60). Sesamol is produced by hydrolysis of sesamolin during oil
ow
Q3
sesamol
sesamin
sesaminol Fig. 23.13. Lignan compounds present in sesame oil.
Oxidation of Oils
sai
processing (133,134). Sesamol is converted to a sesamol dimer and then to sesamol dimer quinone (135,136). Sesamol and sesaminol demonstrated higher antioxidant activity than sesamin in sunflower oil autoxidation by scavenging radicals (132). Sesame oil also contains phytosterols such as campesterol, stigmasterol, psitosterol, and 4,5-avenasterol, with p-sitosterol as the predominant compound (132). Sitosterol behaves partly as a prooxidant by increasing the solubilization of oxygen (137) and partly as a weak antioxidant in sunflower oil and lard by competing with lipid molecules for oxidation at the oil surface (58,138). Olive oil, which is very stable to autoxidation (l), contains phenolic compounds and tocopherols. Phenolic compounds in olive oil include tyrosol (4hydroxyphenylethanol) , hydroxytyrosol (3,4-dihydroxyphenylethanol),hydroxybenzoic acids, oleuropein, and derivatives of tyrosol and hydroxytyrosol (139,140) (Table 23.7). The amount of tyrosol, hydroxytyrosol, and phenolic acids in olive oil was 34.9, 37.8, and 36.3 ppm, respectively (73). The phenolic compounds in olive oil acted as antioxidants mainly at the initial stage of autoxidation (96) by scavenging free radicals and chelating metals (141). Hydroxytyrosol was the most effective antioxidant in olive oil oxidation (139,140,142). Antioxidant Interactions. Edible oil often contains multicomponent antioxidants and demonstrates interactions among them. Photosensitized oxidation of soybean oil containing 8 ppm chlorophyll was lower when a-tocopherol and p-carotene were used together than when each was used alone (144). The phenomenon that a combination of two or more antioxidants works better than the equivalent quantity of any one antioxidant is known as synergism. A combination of metal chelator and free radical-scavenging antioxidants decreases oxidation of the oil mainly by TABLE 23.7 Phenolic Compounds in Virgin Olive Oila Phenolic alcohols
Phenolic acid and its derivatives
Secoiridoids
3,4-Di hydroxyphenyl ethanol (3,4-DHPEA, hydroxytyrosol), phydroxyphenylethanol (pHPEA, tyrosol), 3,4-di hydroxyphenylethanol-glucoside Vanillic acid, syringic acid, pcoumaric acid, o-coumaric acid, gallic acid, caffeic acid, protocatechuic acid, phydroxybenzoic acid, ferulic acid, cinnamic acid, 4-(acetoxyethil)-l,2-dihydroxybenzene, benzoic acid Dialdehydic form of elenolic acid linked to 3,4-DHPEA, dialdehydic form of elenolic acid linked to pHPEA, oleuropein aglycon, ligstroside aglycon, oleuropein, pHPEA derivative
Lignans
1-Acetoxypinoresinol, pinoresinol, 1-hydroxypinoresinol
Flavones
Apigenin, luteolin
5ource: Reference 143.
582
E. Choe e t a / .
the action of the chelator at the initiation step and that of the free radical scavenger at the propagation step. Because tocopherols are the most common antioxidants in edible oil, they were used in research on interactions among antioxidants. a-Tocopherol had synergistic effects with p-carotene to decrease the autoxidation (145) and photosensitized oxidation of soybean oil (144). The synergistic effects of tocopherol and @-carotenewere suggested to be due to protection of @-carotenefrom degradation under light by tocopherols. a-Tocopherol had synergism with ascorbic acid and phospholipids. Ascorbic acid gives hydrogen to the tocopheroxyl radical produced from the reaction between the lipid peroxy radical and tocopherol, and regenerates tocopherol. The nitrogen moiety of phosphatidylethanolamine and phosphatidylcholine, or the reducing sugar of phosphatidylinositol, donates hydrogen or electron to tocopherols and delays the oxidation of tocopherols to tocopheryl quinone (75). Sesamol and sesaminol added to sunflower oil had synergistic antioxidant activities with y-tocopherol(l32). The hydroperoxide concentration sometimes affects the synergistic effects of antioxidants. Olive oil autoxidation was lower when a-tocopherol and phenolic compounds were used together than when they were used separately (44). However, there was no significant synergism between a-tocopherol and phenolic compounds in olive oil at a low hydroperoxide concentration (1 10).
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101. Kamal-Eldin, A., and L.-A. Appelqvist, The Chemistry and Antioxidant Properties of Tocopherols and Tocotrienols, Lipids 31: 671-701 (1996). 102. Simic, M.G., in Autoxidation in Food and Biological Systems, edited by M.G. Simic, and M. Karel, Plenum, New York, 1980, pp. 17-26. 103. Liebler, D.C., P.F. Baker, and K.L. Kaysen, Oxidation of Vitamin E: Evidence for Competing Autoxidation and Peroxyl Radical Trapping Reaction of the Tocopheroxyl Radical, J . Am. Chem. SOC.112: 6995-7000 (1990). 104. Yanishlieva, N.V., A. Kamal-Eldin, E.M. Marinova, and A.G. Toneva, Kinetics of Antioxidant Action of a-and y-Tocopherols in Sunflower and Soybean Triacylglycerols, Eur. J. Lipid Sci. Technol. 104: 262-210 (2002). 105. Jung, M.Y., and D.B. Min, Effects of a - , y-, and &Tocopherols on the Oxidative Stability of Soybean Oil, J . Food Sci. 55: 1464-1465 (1990). 106. Cillard, J., P. Cillard, and M . Cormier, Effect of Experimental Factors on the Prooxidant Behavior of a-Tocopherol, J . Am. Oil Chem. SOC.57: 255-261 (1980). 107. Terao, J., and S. Matsushita, The Peroxidizing Effect of a-Tocopherol on Autoxidation of Methyl Linoleate in Bulk Phase, Lipids 21: 255-260 (1986). 108. Bowry, V.W., and R. Stocker, Tocopherol-Mediated Peroxidation. The Prooxidant Effect of Vitamin E on the Radical-Initiated Oxidation on Human Low-Density Lipoprotein, J . Am. Chem. SOC.115: 6029-6044 (1993). 109. Yamamoto, Y., Role of Active Oxygen Species and Antioxidants in Photoaging, J . Dermatol. Sci. 27 (Suppl. I ) : 1 4 (2001). 110. Blekas, G., M. Tsimidou, and D. Boskou, Contribution of a-Tocopherol to Olive Oil Stability, Food Chem. 52: 289-294 (1995). 111. Marinova, E.M., and N.V. Yanishlieva, Effect of Temperature on the Antioxidative Action of Inhibitors in Lipid Autoxidation, J . Sci. Food Agric. 60: 313-318 (1992). 112. Jung, M.Y., and D.B. Min, Effects of Oxidized a - , y,and &Tocopherols on the Oxidative Stability of Purified Soybean Oil, Food Chem. 45: 183-187 (1992). 113. Jung, M.Y., E. Choe, and D.B. Min, Effects of a - , fL, y-, and &Tocopherols on the Chlorophyll Photosensitized Oxidation of Soybean Oil, J . Food Sci. 56: 807-815 (1991). 114. Mukai, K., K. Daifuku, K. Okabe, T . Tanigaki, and K. Inoue, Structure-Activity Relationship in the Quenching Reaction of Singlet Oxygen by Tocopherol (Vitamin E) Derivatives and Related Phenols. Finding of Linear Correlation Between the Rates of Quenching of Singlet Oxygen and Scavenging of Peroxyl and Phenoxyl Radicals in Solution, J . Org. Chem. 56: 41884192 (1991). 115. Bonnie, T.Y.P., and Y.M. Choo, Valuable Minor Constituents of Commercial Red Palm Olein: Carotenoids, Vitamin E, Ubiquinone and Sterols, J . Oil Palm Res. 12: 1424 (2000). 116. Beutner, S., B. Bloedom, S. Frixel, I.H. Blanco, T. Hoffmann, H.-D. Martin, B. Mayer, P. Noack, C. Ruck, M. Schmidt, I. Schulke, S. Sell, H. Emst, S. Haremza, G. Seybold, H. Sies, W. Stahl, and R. Walsh, Quantitative Assessment of Antioxidant Properties of Natural Colorants and Phytochemicals: Carotenoids, Flavonoids, Phenols and Indigoids. The Role of p-Carotene in Antioxidant Functions, J . Sci. Food Agric. 81: 559-568 (2001). 117. Stahl, W., and H. Sies, Physical Quenching of Singlet Oxygen and cis-trans Isomerization of Carotenoids,Ann. N.Y. Acad. Sci. 691: 10-19 (1992). 118. Lee, J.H., B. Ozcelik, and D.B.Min, Electron Donation Mechanisms of p-Carotene as a Free Radical Scavenger, J . Food Sci. 68: 861-865 (2003).
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Chapter 24
Structured and Specialty Lipids Casimir C. Akoh Department of Food Science and Technology, The University of Georgia, Athens, GA 30602
Introduction The study of structured lipids (SL) has come of age. The industry is slowly but certainly appreciating the possible role and use of SL in foods. SL can be generally defined as lipids that are chemically and/or enzymatically modified from their natural biosynthetic form (1). When applied to foods, they include flavor, fragrances, acylglycerols, glycerophospholipids, and other esters. Lipids can also be modified through plant and animal breeding and genetic engineering. Any alteration in the structure of a naturally occurring lipid is considered a modification. Modification of lipids with lipase provides a useful way in which to improve their physical and/or chemical characteristics such as melting properties, solid fat contents (SFC), saponification number, viscosity, iodine value, and oxidative stability (1-7). Some modification may involve the incorporation of new and “beneficial” fatty acids (FA) or simply the movement of FA among the sn-l,2, and 3 positions in the case of TAG or among the sn-1 and -2 positions of glycerophospholipids. By using sn-13-specific lipases and -specific phospholipases A,, A,, C, and D, desirable SL products can be obtained. On the other hand, physical processing may change the physical property of lipids but not the structure. The term “specialty lipids” refers to fats with special functional or nutritional properties for both edible and pharmaceutical uses (8). With growing consumer and industry interest in healthier foods, trans-free fats, SL, and lipase catalysis have become the subject of a great deal of research in academia and industry (food, nutritional, and even pharmaceutical industries). Little work has been done on proving that these modified lipids can actually function well in the food or medium for which they are intended. It appears that the rapid growth of the market for functional and nutraceutical foods and consumer demand for healthier fats will prompt industry to invest money in the use of lipases as “green” catalysts for the production of SL and specialty lipids. The time has come for the consumer and the food industry to reap the benefits of decades of study on lipases and phospholipases as biocatalysts for lipid modifications and transformations. Excellent reviews on the enzymatic processes and strategies for the synthesis of SL and lipid modifications are available elsewhere (1-8). Some of the applications of the SL and specialty lipids are discussed. 591
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Functional Applications of SL A functional lipid is similar to or may be a conventional fat or oil that is consumed as part of a normal diet, but is demonstrated to have physiologic benefits, i.e., beyond serving a nutritional function, it may reduce the risk of chronic disease. The functionality of the lipids in food products varies from one product to another. For instance, high and sharp melting fats are preferred in confectioneries, whereas liquid and odorless oils, such as soybean oil, are favored in salad dressing; olive, canola, and high-oleic oils are favored in frying applications. The variation in functionality demands specific types of lipid for particular applications. Plastic Fats for Foods. SL are texturally important in the manufacture of plastic fats such as margarine, modified butters, and shortenings. The physical property of any fat or oil is determined by the chain length and unsaturation of the FA and their distribution among the three hydroxyl groups of glycerol. Fats with a higher percentage of saturated fatty acids (SFA) tend to be solid at room temperature, and those with a higher percentage of unsaturated FA tend to be liquid. Interesterification alters the original order of distribution of FA in the glycerol moiety producing fats with different melting and crystallization characteristics than the parent fat (9). This allows the production of tailor-made fats to suit particular foods. In the manufacture of margarine, the objective is to produce a fat mixture with a steep SFC curve to obtain a stiff product in the refrigerator that spreads easily upon removal and melts quickly in the mouth (10). When short- (SCFA) or medium-chain fatty acids (MCFA) and long-chain fatty acids (LCFA) are interesterified, they can produce TAG with good spreadability and temperature stability (10). Due to the growing concern about the health implications of trans fatty acids (TFA) in margarine, research interest has been generated in the production of zero TFA margarine or shortenings. Interesterification of the butter stearin fraction and liquid oils such as sunflower and soybean oils yield products with desirable spread characteristics and almost zero TFA (1 1). Palm stearin-sunflower oil (4050 and 50:50) blends subjected to transesterification using Pseudomonas lipase were shown to be suitable for table margarine formulation (12). Seriburi and Akoh (13) were able to produce a soft-type margarine blend by interesterifying lard and high-oleic sunflower oil (60:40) using SP 435 lipase from Cundidu antarctica. Palm stearin and palm kernel olein were interesterified using sn-l,3-specific Rhizomucor miehei at a 40:60 ratio to produce an experimental table margarine (14). Rousseau and Marangoni (15) interesterified butterfat and butterfat-canola blends enzymatically to produce a cold spreadable butter. Interesterification was found to be a highly useful method for improving butter spreadability, although it also resulted in loss of some butter flavor. Fomuso and Akoh (16) modified laurate canola enzymatically with stearic acid to produce a transfree margarine. Unilever was granted a patent (17) on a similar SL made with interesterified lauric rapeseed oil (65%) and fully hydrogenated soybean oil (35%). The interesterified oil can be used as a hardstock for blending with desirable liquid oils.
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Trans-Free Fat Alternatives. In the fats and oils industry, TFA are formed during partial hydrogenation of PUFA. Hydrogenation is used to improve the consistency and oxidative stability of refined, bleached, and deodorized dietary fats. These fats are used mainly in margarines, shortenings, spreads, and confectioneries to improve their textural properties, modify their melting and crystal behavior, and enhance stability. Modification to improve the functional and nutritional properties of fat is of interest to the food industry. Modification in structure can be achieved through hydrogenation, chemical and enzymatic interesterification, genetic engineering, and plant breeding techniques (1,5,6). Nonstructural modification approaches to producing fats with desirable or improved nutritional and physical properties include blending, such as in the production of Appetizer shortening and Good-Fry oil (18). Currently, about one third of the world’s edible fats and oils is hydrogenated, whereas -10% is either fractionated or interesterified (19). Hydrogenation is common in highly unsaturated oils, such as soybean, rapeseed, cottonseed, and fish oils, whereas fractionation is better applied to palm oil and other more saturated oils (18). Hydrogenation will raise the melting point and reduce the iodine value of TAG as the oil is converted from a liquid at room temperature to semisolid plastic fats. The SFC of the modified fat as determined by pulsed NMR increases. TFA have higher melting points than cis FA, and therefore contribute considerably to the melting and plastic properties of fats, including baking performance. The physiologic role of TFA has been controversial since the epidemiologic study of Willett et al. (20) suggested a positive correlation between the intake of TFA and the risk of coronary heart disease. Because of health and nutritional concerns raised by previous studies, there has been renewed interest in dietary TFA in Europe and North America (21-25). The U S . Food and Drug Administration (FDA) issued a final rule on the labeling of the TFA contents of conventional foods and dietary supplements (26) to be effective in January 2006. It is estimated by the FDA that adding trans fat information on the label would save between $900 million and $1.8 billion due to reduced medical costs, decreased pain and suffering, and greater productivity (27). The food industry is responding by repositioning themselves through research, reformulations, blending, and seeking of alternative fats, such as SL and genetically engineered fats. PepsiCo’s Frito-Lay division has already removed trans fat from their snacks and chips. Kraft said it would make its cookies and snacks with less fat and sugar, and McDonald’s promised to reduce fats in fried foods (27). SL are tailor-made fats and oils or TAG mixtures modified to approximate a particular FA or TAG composition to attain some desired property, such as reduced energy value, or a nutritional, physical, or functional property (1-3,5,18). Examples include Appetizer shortening (a blend of animal fat, 85-95%, and vegetable fat, 5-15%), Good-Fry oil (a high-oleic corn or sunflower oil), Bohenin (glycerol 1,3-dibehenate 2-oleate), Caprenin (contains octanoic, decanoic, and behenic acids), Salatrim (contains SCFA and LCFA), ARASCO (arachidonic acid
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single-cell oil, TAG produced by Mortierella alpina and containing -40% arachidonic acid), and DHASCO (DHA single-cell oil, TAG produced by Crypthecodinium cohnii and containing -40% DHA) oils. SL combines the unique characteristics of component FA, such as melting behavior, digestion, absorption, and metabolism, to enhance their use in foods, nutrition, and therapeutics. Indeed, the nutritional value of any TAG or SL and their physicochemical properties are determined by the FA composition and the positional distribution of acyl groups in the glycerol molecules. Exploration of lipase-catalyzed reactions, which can play a considerable role in the modification of fats and oils to produce trans-free alternatives to hydrogenated fats for various food applications, is warranted. List (28) reviewed the reformulation and processing strategies to increase the health value of food fats without TFA. Cocoa Butter and Cocoa Butter Alternatives. Cocoa butter is the fat of choice in the confectionery industry. Its polymorphism greatly affects the physical properties of chocolate products, such as gloss, snap, contraction, heat resistance, quick and sharp melting in the mouth, and bloom resistance (29,30). The limited availability of cocoa butter, which affects its cost, has prompted much research on alternatives that can be used as cocoa butter replacements or extenders in chocolate and confectionery coatings. There are no naturally occurring fats with physical properties similar to those of cocoa butter; all alternatives are made by blending and/or modifying fats. When using enzymatic processes for producing cocoa butter alternatives, several factors must be considered. The melting behavior must be very similar to that of cocoa butter to achieve the same cooling effect in the mouth. An alternative fat that is to be used in conjunction with cocoa butter should not interfere with the correct crystallization of the cocoa butter during tempering. The p crystals are the desirable polymorph in the confectionery industry. The most common cocoa butter equivalents to date include palm oil, palm mid-fractions, illipe (Shorea stenoptera) fat, shea (Butyrospermum parkii) butter, sal (Shorea robusta) fat, and kokum (Garcinia indica) butter. Also some commercially blended alternatives are available. When these natural fat sources are modified by incorporating either palmitic or stearic acid using sn-l- and sn3-selective lipases, it is possible to produce a cocoa butter-like fat whose FA composition closely resembles that of cocoa butter. An extensive review of cocoa butter alternatives is available (31). Foglia et al. (32) suggested beef tallow as a possible base fat for producing SL that could be used as cocoa butter alternatives. Osborn and Akoh (33) reported that SL made enzymatically by randomizing beef tallow or by incorporating stearic acid into beef tallow may be useful as a cocoa butter extender, because “chocolates” produced with the SL had some physical properties similar to those of chocolates produced with only cocoa butter. At present, efforts are underway in several laboratories to synthesize enzymatically cocoa butter alternatives with functionality identical to that of natural cocoa butter.
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Healthier and More Stable Frying Fats. With the recent developments in genetic engineering, oilseeds can be genetically modified to produce fats and oils with modified FA composition. These genetically modified fats and oils have various applications in the food industry, such as in deep frying. High-oleic sunflower oil demonstrates excellent behavior with respect to thermoxidation and frying stability (34). To determine frying stability and performance, the characteristic odors and flavors of genetically modified soybean and canola oils were reported by Warner and Mounts (35). They found that flavor characteristics were significantly higher in potatoes fried in modified oils than those fried in standard oil. High-oleic corn oil has significantly better flavor and lower “room odor intensity” than normal or hydrogenated corn oil (36). Other genetically modified frying oils with improved frying characteristics include low-linolenic rapeseed and soybean oils (37,38). Enova oil, described below, can also be used as healthy frying oil. Coating Lipids. SL can be used in coating applications (39). Coating is applied on food products for a variety of reasons. The edible coating is prepared basically from polysaccharides, proteins, and lipids (40). The first two components are effective in preventing or minimizing the transport of gases under conditions of relatively low humidity. Unlike these hydrophilic components, lipids are very effective as moisture barriers. Lipids can control mass transfer by preventing the movement of moisture, permanent gases, and aromas between the food and the external environment. Lipids are preferred as coating ingredients mainly to inhibit the loss of moisture and to improve the surface appearance of the finished products. Many types of lipids are used as coating materials, including waxes, fats, oils, and chocolate. Other than waxes, acetylated glycerol monoesters of LCFA are used. For many applications, waxes were used predominantly as coating materials to protect against moisture loss (41) especially for edible coatings. Other than moisture prevention, lipids are coated as a carrier for other ingredients such as nuts, or for protecting encapsulated materials from moisture absorption. Food products such as fruits and vegetables, confectioneries, nuts, refrigerated and frozen meat, baked products, and moisture-sensitive ingredients are coated with lipids (42). Waxes form flexible films at room temperature; they retain their flexibility at low temperature and are fairly effective barriers to moisture. TAG from various sources and compositions are also used for this purpose. Hydrogenation of many unsaturated oils such as coconut, cottonseed, peanut, soybean, or other vegetable oils makes them solid or wax-like substances with physical characteristics suitable for many applications. This increase in melting point also improves their viscoelastic and mechanical properties and imparts greater chemical stability and increased moisture barrier properties (43,44). Hydrogenation improves the melting point but at the same time, it increases the TFA content of fats. Alternatively, lipase-catalyzed modification can be employed for the same purpose but without TFA formation. Many high value-added products have been obtained by enzymatic modification of lipids. The palm oil mid-fraction
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was interesterified to obtain a cocoa butter equivalent by Rhizopus arrhizus lipase, which closely resembles cocoa butter in FA composition (45). Mixtures of palm kernel oil, cocoa butter, and anhydrous milk fat were studied for compound coating applications (46). We produced enzymatically large quantities of SL that were suitable for coating applications, specifically as a moisture barrier. Lipozyme IM60 lipase was used to conduct the acidolysis reaction of tristearin/lauric acidoleic acid at a 1:4:1 molar ratio. The melting peak of the SL product was 31.4"C. The effectiveness of the SL was compared with cocoa butter-coated crackers and uncoated crackers as a control. The synthesized SL was better in preventing moisture absorption than cocoa butter (39). The potential for using SL in such applications appears to be great and warrants further study, Enova Oil. Enova oilTMis a cooking oil marketed by Archer Daniels Midland Company Kao LLC (Decatur, IL) as a joint venture between ADM and Kao Corporation (Japan). The oil was developed in Japan by Kao and first introduced as Enova Healthy Econa Cooking Oil in Japan in 1999. Enova oil was produced through a patented enzymatic process using a special blend of soybean and canola oils that results in an increased concentration (80% by weight) of DAG. Enova oil is cholesterol-free and an excellent source of vitamin E. The DAG in Enova contains 2 0 4 5 % by weight oleic acid, 15-65% linoleic acid, and <15% linolenic acid. The oil can be used in cooking, frying, salad dressing, and baking operations. Because most of the FA occur at the sn-1,3 positions of the glycerol, Enova is believed to be metabolized differently than conventional vegetable oils. Consequently, the oil is burned directly by the body to produce energy and is not stored as fat in the adipose tissue. Because the MAG produced as a result of lipase hydrolysis are poorly reassembled into chylomicrons, dietary FA move to the liver for P-oxidation. The oil is said to help with the fight against body fat and obesity and may help people lose weight when included in the diet. Enova oil can help lower serum triacylglycerols (47). The health benefits of DAG are discussed in Chapter 28 of this book. Enova oil was granted GRAS (Generally Recognized As Safe) by the U.S. FDA in 2000 for use in margarine-like spreads, and as a salad and cooking oil.
Lorenzo's Oil. Lorenzo's oil (LO) was invented by Michaela and August0 Odone, the parents of Lorenzo who had the rare genetic disorder called X-linked adrenoleukodystrophy (X-ALD). The neurological disorder causes the breakdown of the myelin that insulates the nerve fibers. Up to 17,000 males worldwide are affected with the disease and many die within 2 yr of manifestation (48). Lorenzo's oil is based on 22:1n-9 (erucic acid) and 18:ln-9 (oleic acid). The U.S. FDA is currently reviewing the possibility of using this oil for the treatment of X-ALD. The potency of LO as a specialty oil in the treatment of X-ALD requires further study. Using lipases to combine the emcic and oleic acids with a medium-chain or n-3 PUFA in the same glycerol backbone (also known as SL), as a delivery system, is
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worthy of investigation. Certainly the development of new oils to help with the management of this particular disease merits attention. Nufraceufical Applications
A nutraceutical lipid is a lipid isolated, concentrated, or synthesizedmodified from natural fats and oils that provides medical or health benefits, including the potential for the prevention of and/or treatment of disease. BetapolTMis a successful example of a nutraceutical lipid synthesized with enzymes. The enzymatic synthesis of Betapol was reviewed (8). Infant Formulas and Betapol. It is expected that the fat component of infant formulas should contain FA, such as MCFA, linoleic acid, linolenic acid, and PUFA, in the same position and amount as those found in human milk. Human milk is comprised of 20-30% palmitic acid, with 33% at the sn-2 position (4). The fat in most infant formulas is of vegetable origin and tends to have unsaturated FA in the sn-2 position. Betapol is the brand name for a human milk fat (HMF) substitute developed by Loders Croklaan (8). It is produced from vegetable oils using sn-l,3specific lipases to mimic HMF. Betapol resembles human breast milk but with a high content of palmitic acid at the sn-2 position where the acids are better absorbed (8). Innis et al. (49) found that infants fed human milk had 26% palmitic acid in their plasma TAG compared with 7.4% in infants fed vegetable oil-based infant formula with the same total concentration of palmitic acid, but not at the sn-2 position. When rats were fed a coconut oil and palm olein SL, absorption was increased due to the increased proportion of long-chain saturates at the sn-2 position (50).Therefore, SL with high proportions of palmitic acid at the sn-2 position would provide a fat with improved absorption capability in infants (4). Care must be taken with regard to the concentrations of SFA at the sn-2 position, because palmitic acid is the only saturate that has been studied extensively and other longchain saturates may have hypercholesterolemic effects (5 1). Enteral and Parenteral Application. Initially, SL were designed for use in parenteral and enteral nutrition to replace fat emulsions containing predominantly LCFA (52). Although medium-chain triacylglycerols (MCT) have many advantages including being a quick energy source, a minimum amount of LCT is still necessary to provide essential fatty acids (EFA). Physical mixtures of MCT and long-chain triacylglycerols (LCT) have proven useful in the past for enteral (oral tube feeding) and parenteral (intravenous feeding) nutrition. More recently, structured TAG comprised of LCFA and MCFA have emerged as the preferred alternative to physical mixtures for treatment of patients, although both products provide identical fat contents. SL comprised of both LCFA and MCFA are designed to provide simultaneous delivery of the FA and a slower, more controlled release of the LCFA into the bloodstream (52). The advantages of enterally fed SL may well
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relate to differences in absorption and processing. SL containing MCFA may provide a vehicle for rapid hydrolysis and absorption due to their smaller molecular size and greater water solubility compared with long-chain TAG (53). SL offer several advantages over native oils and physical mixtures, including improved immune function, decreased cancer risk, thrombosis prevention, cholesterol lowering, improved nitrogen balance, and no risk of reticuloendothelial system impairment (54). Increases in protein synthetic rates in both skeletal muscle and liver were also demonstrated in patients administered SL (55). The more recent studies on SL for medical applications are an attempt to define the factors affecting the absorption and metabolic fates of a structured TAG and the effects they have in vivo. The results of several such studies were summarized by Osborn and Akoh (5). Lee et al. (56) showed that although they have a similar total FA composition, enzymatically modified lipids and a physical mixture of lipids have different metabolic pathways based on the structure and due to the difference in FA composition at the sn-2 position of lipid molecules. In this study, soybean oil was modified by incorporating caprylic acid (8:O) at the sn-1 and sn-3 positions; the LCFA remained intact at the sn-2 position. This SL was compared with a physical mixture of soybean oil and tricaprylin. Caprylic acid was found in the livers and inguinal adipocyte TAG of rats fed SL, whereas it was not present in those fed a physical mixture. This suggests that the positional distribution of 8:O is important in the metabolism of TAG and may lead to different physiologic influences. Conflicting results regarding the influence of positional distribution on absorption and metabolism were reported, but may be due to differences in the oils and experimental conditions used (57-59). Straarup and Hoy (60) extended this study by comparing the lymphatic transport of a specific SL comprised of rapeseed oil and decanoic acid (10:0), a randomized fat, and a physical mixture in normal and malabsorbing rats. All three lipids compared in that study had the same FA profile. Rapeseed oil was used as the control. In that study, the decanoic acid was located mainly in the sn-1 and sn-3 positions in the specific SL, but at all positions in the randomized lipid, which was synthesized chemically. In normal rats, faster absorption rates of 18:ln-9 and 18:2n-6 FA were obtained from the specific SL and the physical mixture compared with the randomized and control oils. The authors concluded that this was a result of faster hydrolysis of the specific TAG compared with the randomized and native oils. The MCFA and the sn-2 MAG released after hydrolysis of MCT in the physical mixture may improve the emulsification of LCT and therefore account for the increased absorption rate of the physical mixture. The recoveries of 18:3n-3 were similar for the physical mixture and randomized oils, but significantly lower than for the specific oil. This indicates that the TAG structure of the oil and the presence of 10:0, not the level of individual FA, were the major determinants of the amount of FA absorbed and transported. The specific SL had low amounts of 10:0 at the sn-2 position, and dietary 10:0 from the sn-1 and sn-3 positions was thought to be absorbed directly through the portal vein. Therefore, less 10:0 was expected
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in the lymph as a result of SL feeding compared with the randomized oil and physical mixture diets. However, similar amounts of 10:0were observed in the mesenteric lymph from the three oils, indicating better hydrolysis and higher absorption rates of the specific SL, as well as acyl migration in the TAG during hydrolysis. The intragastric administration of fat to the rats excluded the activation of lingual lipase in this study, and rats have only trace amounts of gastric lipase. Therefore, all hydrolysis of test oils measured in this study was the result of pancreatic lipase. The preduodenal lipases preferentially hydrolyze SCFA and MCFA from the sn-3 position; thus, improved recovery from the specific SL would be expected in patients when these lipases are also contributing to hydrolysis. Clinical treatment of short-bowel patients is one promising area for this type of SL because these patients frequently have compromised EFA status (61,62). Mu and Hoy (63)compared the intestinal absorption of different SL in vivo. They studied the lymphatic transport of FA from specific SL containing different MCFA, varying from caprylic acid (8:O) to lauric acid (12:O)in the sn-1,3 positions and LCFA in the sn-2 position, to investigate the effect of chain length of MCFA on the absorption of LCFA and the distribution of MCFA between the portal vein and lymphatics in rats with normal fat absorption. The results of this study showed that the chain length of MCFA located in the primary positions does not affect the lymphatic transport of LCFA in the sn-2 position. This result suggests that similar type SL may be used to provide different LCFA according to clinical demand. Similar intestinal absorption of different LCFA can be expected in other SL-containing MCFA at the sn-1 and sn-3 positions. It is therefore possible to manufacture SL with different MCFA to direct the FA from the sn-1 and sn-3 positions toward the liver through the portal vein or toward the muscle or adipose tissues through formation of chylomicrons, without affecting the transport of the LCFA in the sn-2 position. The TAG in total parented nutrition (TPN) is normally administered as an emulsion. These emulsions are suspected of suppressing immune function because pneumonia and wound infection often occur in patients treated with TPN. However, the influence of various lipid emulsions on leukocyte function is still unclear. Kruimel et al. (64)attempted to explain this phenomenon through in vitro studies of lipid emulsions containing FA with different chain lengths on the production of radicals by polymorphonuclear leukocytes. Emulsions made from LCT, a physical mixture of LCT and MCT, and an SL were compared in this study. The results indicated that physical mixtures caused higher peak levels and faster production of oxygen radicals, compared with LCT and SL emulsions. Chambrier et al. (65)conducted a similar study comparing the effect of physical mixtures and SL on postoperative patients. They did not observe hepatic function disturbances in patients given the SL, which are often observed with TPN. The plasma TAG levels remained normal in patients given SL, whereas they increased significantly with the physical mixture. Bellantone et al. (66) gave lipid emulsions to patients after colorectal surgery. The differences between the SL and physical mixture groups were less marked in that study.
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SL synthesized from fish oil and MCT were administered to patients undergoing surgery for upper gastrointestinal malignancies. The diet was compared with a control diet that differed only in its fat source. The SL diet was tolerated significantly better, led to improved hepatic and renal function, and reduced the number of infections per patient (67).In our laboratory, we fed female mice diets supplemented with an SL containing n-3 PUFA and caprylic acid or soybean oil for 21 d (68). The effect of the diet on serum lipids and glucose concentrations was determined at the end of the feeding period. In spite of the higher content of caprylic acid in the SL, 8:0 was not detected in the livers of the SL-fed mice. High amounts of n-3 PUFA were found in the livers of the SL-fed mice (68).These findings suggest that the 8:0was metabolized quickly for energy and that different FA in the diet may eventually lead to a change in the FA composition of the liver. SL containing MCFA and n-3 PUFA could be a therapeutic or medical lipid source and may be useful in enteral and parenteral nutrition. This SL could decrease serum cholesterols and TAG (68).It may also reduce the rate of body weight gain because the MCFA were metabolized more rapidly in the body compared with soybean oil. Swails et al. (69)demonstrated significant reductions in prostaglandin production in postsurgical patients enterally administered an SL formula containing EPA and DHA. This downregulation in prostaglandin production did not predispose the patients to any postoperative events such as a higher incidence of infection or an inability to heal wounds. In fact, improved liver function was reported in the SL patients. The physiologic function of the SL appears to be due in part to the n-3 FA acting through altered prostaglandin release from mononuclear cells. Diets containing an SL composed of MCFA and linoleic acid led to improved absorption of EFA in patients with cystic fibrosis. These patients experience malabsorption as a result of impaired pancreatic function. However, the SL provided an efficient way to supplement linoleic acid and to provide energy from the MCT, which were the preferred substrate for oxidative metabolism (70).No signs of central nervous system toxicity were noted in patients given the SL, and there was no tendency to ketosis (71).Recently, a 4-wk study was conducted on the efficacy and safety of an SL prepared from coconut oil and soybean oil (72).Patients receiving the SL were compared with other patients given LCT. This double-blind, randomized, crossover study indicated that the SL was safe and efficient when provided to patients receiving home parental nutrition on a long-term basis and that it may be associated with a possible reduction in liver dysfunction (72). Reduced-Calorie Fats. SL may be designed to serve as low-energy fats. When a poorly absorbed long-chain SFA is interesterified with an SCFA or MCFA, a lowor reduced-energy SL is usually produced. SCFA are useful in synthesis of lowenergy SL because they provide fewer calories per unit weight than LCFA (73). This technique of interesterifying long-chain SFA and MCFA or SCFA was utilized in industry by Procter & Gamble to interesterify coconut, palm kernel and rapeseed oils chemically to produce Caprenin@,an SL containing 8:0, 10:0, and
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22:O. Because of the presence of behenic acid, Caprenin@is only partially absorbed by the body; therefore it supplies 5 kcal/g vs. the 9 kcal/g supplied by conventional fats. The principle of combining SCFA and LCFA was also used by Nabisco Foods Group in the chemical synthesis of Salatrim@,now marketed as Benefat@by Cultor Food Science. Benefat is produced by base-catalyzed interesterification of highly hydrogenated vegetable oils with TAG of acetic and/or propionic and/or butyric acids (73). The resulting TAG can include an infinite number of low-energy fat products with various applications in foods, such as cocoa butter substitutes, baked products, salad dressings, and filled dairy products. The available energy of Salatrim@ molecules was determined to be -5 kcal/g. Butyric and caproic acids were interesterified with triolein to form an SL containing 4:0,6:0, and 18:l; because the average energy value of 4:O and 6:O is 6.8 kcal/g, the overall energy value of the SL is reduced (74,75). Bohenin is an SL designed to prevent or slow down bloom and stickiness in chocolates by promoting the development of stable crystal polymorphs. The energy density of Bohenin is 5 kcal/g (76). It is composed of behenic acid (22:O) and oleic acid (18:l). Stepan Company (Maywood, NJ) ingredient division recently released two types of MCT marketed under the Neobee brand name for use in nutritional bar processing. Neobee M-5 migrates to the top of an uncoated bar to prevent sticking to packaging material. Neobee 1095 prevents the formation of bloom in chocolate coatings. Others have studied the enzymatic synthesis of low-energy SL (77,78). Kanjilal et al. (79) synthesized SL from vegetable oils that contained EFA and natural antioxidants. Behenic acid was incorporated into the sn-1 and sn-3 positions of sunflower oil using lipase as the catalyst. The SL products contained 5.4 kcal/g and had an improved plastic nature, which increases the potential food applications for such a product, especially because it is a trans-free solid fat. The SL was fed to rats and was compared with the control group fed sunflower oil. No differences were observed in the amount of food consumed, indicating that the palatability and taste of the SL were very similar to the natural sunflower oil. In addition, no differences were observed between the groups regarding the levels of major FA of the plasma total lipids.
Conclusions Consumers are becoming increasingly aware of the potential health benefits of consuming healthy oils such as canola and olive oils and as well as consuming n-3 PUFA from fish and fish oils. The issue of TFA in our foods and their health implications is prompting the food industry, the FDA, and consumers to change strategies in the manufacture, regulation, and consumption of fats, respectively. Modified fats such as SL, genetically engineered fats, and reformulated natural fats and oils have a role to play in the future of consumer well-being and the functionality of fats and oils in food and nutrition. It is not necessarily the amount of fat, but the type of FA and their positions in the glycerol backbone, that may determine
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the functional or nutraceutical applicationshses of the fats of the future. The time is ripe for the industry to adopt these new and existing technologies to provide consumers with safe and nutritious foods.
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38. Mounts, T.L., K. Warner, G.R. List, W.E. Neff, and R.F. Wilson, Low-Linolenic Acid Soybean Oils Alternatives to Frying Oils, J . Am. Oil Chem. SOC.71: 495499 (1994). 39. Sellappan, S . , and C.C. Akoh, Enzymatic Acidolysis of Tristearin with Lauric and Oleic Acids to Produce Coating Lipids, J . Am. Oil Chem. SOC.77: 1127-1 133 (2000). 40. Gennadios, A., M.A. Hanna, and L.B. Kurth, Application of Edible Coatings on Meats, Poultry and Seafoods: A Review, Lebensrn.-Wiss. Technol. 30: 337-350 (1997). 41. Guilbert, S., Technology and Application of Edible Protective Films, in Food Packaging and Preservation: Theory and Practice, edited by M. Mathlouthi, Elsevier Applied Science, London, 1986,pp. 371-394. 42. Shellhammer, T.H., and J.M. Krochta, Edible Coatings and Film Barriers, in Lipid Technologies and Applications, edited by F.D. Gunstone and F.B. Padley, Marcel Dekker, New York, 1997,pp. 453479. 43. Nesaretnam, K., N. Robertson, Y. Basiron, and C.S. Macphie, Application of Hydrogenated Palm Kernel Oil and Palm Stearin in Whipping Cream, J. Sci. Food Agric. 61: 401-407 (1993). 44. Warner, K., E.N. Frankel, J.M. Snyder, and W.L. Porter, Storage Stability of Soybean OilBased Salad Dressings-Effects of Antioxidants and Hydrogenation, J . Food Sci. 51: 703-708 (1986). 45. Mojovic, L., S.S. Marinkovic, G. Kukic, and G.V. Novakovic, Rhizopus arrhizus LipaseCatalyzed Interesterification of the Midfraction of Palm Oil to a Cocoa Butter Equivalent Fat, Enzyme Microb. Technol. 1.5: 4 3 8 4 3 (1993). 46. Williams, S.D., K.L. Ransom-Painter, and R.W. Hartel, Mixtures of Palm Kernel Oil with Cocoa Butter and Milk Fat in Compound Coatings, J . Am. Oil Chem. SOC.74: 357-366 (1997). 47. Katsuragi, Y., Effects of Dietary Diacylglycerol on Obesity and Hyperlipidemia, The Jpn. SOC.Human Dry Dock 14: 12-16 (1999). 48. Watkins, C., Lorenzo’s Oil Vindicated, inform 14:38-39 (2003). 49. Innis, S.M., C.M. Nelson, M.F. Rioux, and D.J. King, Development of Visual Acuity in Relation to Plasma and Crythrocyte n-6 and n-3 Fatty Acids in Healthy Term Gestation Infants,Am. J . Clin. Nutr. 60: 347-352 (1994). 50. Lien, E.L., R.J. Yuhas, F.G. Boyle, and T.M. Tomarelli, Corandomization of Fats Improves Absorption in Rats, J . Nutr. 123: 1859-1867 (1993). 51. Pai, T., and Y.Y. Yeh, Stearic Acid Modifies Very Low-Density Lipoprotein Lipid Composition and Particle Size Differently from Shorter Chain Saturated Fatty Acids in Cultured Rat Hepatocytes, Lipids 32: 143-149 (1997). 52. Babayan, V.K., Medium Chain Triglycerides and Structured Lipids, Lipids 22: 417420 (1987). 53. Jensen, G.L., and R.G. Jensen, Specialty Lipids for Infant Nutrition. 11. Concerns, New Developments, and Future Applications, J . Pediatr. Gastroenterol. Nutr. 1.5: 382-394 (1992). 54. Kennedy, J.P., Structured Lipids: Fats of the Future, Food Technol. 45: 76-83 (1991). 55. DeMichele, S.J., M.D. Karistad, V.K. Babayan, N.W. Istfan, G.L. Blackburn, and B.R. Bistrian, Enhanced Skeletal Muscle and Liver Protein Synthesis with Structured Lipid in Enterally Fed Burned Rats, Metabolism 37: 787-795 (1988). 56. Lee, K.T., C.C. Akoh, W.P. Flatt, and J.H. Lee, Nutritional Effects of Enzymatically Modified Soybean Oil with Caprylic Acid versus Physical Mixture Analogue in Obese Zucker Rats, J . Agric. Food Chem. 48: 5696-5701 (2000).
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57. Jensen, G.L.,N. McGarvey, R. Taraszewski, S.K. Wixon,D.L. Seidner, T. Pai, Y.Y. Yeh, T.W. Lee, and S.J. DeMichele, Lymphatic Absorption of Enterally Fed Structured Triacylglycerol Compared with Physical Mix in a Canine Model, Am. J . Clin. Nutr. 60: 5 18-524 (1994). 58. Christensen, M.S., A. Mullertz, and C.E. Hoy, Absorption of Triglycerides with Defined or Random Structure by Rats with Biliary and Pancreatic Diversion, Lipids 30: 521-526 (1995). 59. Tso, P., M. Karistad, B.R. Bistrian, and S J. DeMichele, Intestinal Digestion, Absorption, and Transport of Structured Triglycerides and Cholesterol, Am. J. Physiol. 268: G568G577 (1999). 60.Straarup, EM., and C.E. Hoy, Structured Lipids Improve Fat Absorption in Normal and Malabsorbing Rats, J . Nutr. 130: 2802-2808 (2000). 61. Jeppesen, P.B., M.S. Christensen, C.E. Hoy, and P.B. Mortensen, Essential Fatty Acid Deficiency in Patients with Severe Fat Malabsorption, Am. J . Clin. Nutr. 65: 837-843 (1997). 62. Jeppesen, P.B., C.E. Hoy, and P.B. Mortensen, Essential Fatty Acid Deficiency in Patients Receiving Home Parented Nutrition,Am. J . Clin. Nutr. 68: 126-133 (1998). 63. Mu, H., and C.E. Hoy, Effects of Different Medium-Chain Fatty Acids on Intestinal Absorption of Structured Triacylglycerols,Lipids 35: 83-89 (2000). 64. Kruimel, J.W., A.H. Naber, J.H. Curfs, M.A. Wenker, and J.B. Jansen, With MediumChain Triglycerides, Higher and Faster Oxygen Radical Production by Stimulated PolymorphonuclearLeukocytes Occur, J . Parenter. Enteral Nutr. 24: 107-1 12 (2000). 65. Chambrier, C., M. Guiraud, J.P. Gibault, H. Labrosse, and P. Bouletreau, Medium- and Long-Chain Triacylglycerols in Postoperative Patients: Structured Lipids versus Physical Mixture, Nutrition 15: 274-277 (1999). 66. Bellantone, R., M. Bossola, C. Carriero, M. Maleaba, P. Nucera, C. Ratto, P. Crucitti, F. Pacelli, G.B. Doglietto, and F. Crucitti, Structured Versus Long-Chain Triglycerides: A Safety, Tolerance, and Efficacy Randomized Study in Colorectal Surgical Patients, J . Parenter. Enteral Nutr. 23: 123-127 (1999). 67. Kenler, AS., W.S. Swails, D.S. Driscoll, SJ.DeMichele, B. Daley, T.J. Babienead, M.B. Peterson, and B.R. Bistrian, Early Enteral Feeding in Postsurgical Cancer Patients: Fish Oil Structured Lipid-Based Polymeric Formula Versus a Standard Polymeric Formula, Ann. Surg. 223: 316-333 (1996). 68. Lee, K.T., C.C. Akoh, and D.L. Dawe, Effects of Structured Lipid Containing Omega-3 and Medium Chain Fatty Acids on Serum Lipids and Immunological Variables in Mice, J . Food Biochem. 23: 197-208 (1999). 69. Swails, W.S., A S . Kenler, D.F. Driscoll, S.J. DeMichele, J. Babineau, T. Utsunamiya, S. Chavali, R.A. Forse, and B.R. Bistrian, Effect of Fish Oil Structured Lipid-Based Diet on Prostaglandin Release from Mononuclear Cells in Cancer Patients After Surgery, J . Parenter. Enteral Nutr. 21: 266-274 (1997). 70. Bell, S J.,D. Bradley, R.A. Forse, and B.R. Bistrian, The New Dietary Fats in Health and Disease, J . Am. Diet. Assoc. 97: 280-286 (1997). 71. Sandsrom, R., A. Hyltander, U. Korner, and K. Lundhom, Structured Triglycerides to Postoperative Patients: A Safety and Tolerance Study, J . Parenter. Enteral Nutr. 17: 153-157 (1993). 72. Rubin, M., A. Moser, N. Vaserberg, F. Greig, Y. Levy, H. Spivak, Y. Ziv, and S. Lelcuk, Structured Triacylglycerol Emulsion Containing both Medium- and Long-Chain Fatty
606
73. 74.
75.
76. 77. 78. 79.
C.C. A koh
Acids, in Long-Term Home Parenteral Nutrition: A Double-Blind Randomized CrossOver Study,Nutrition 16: 95-100 (2000). Smith, R.E., J.W. Finley, and G.A. Leveille, Overview of SALATRIM, a Family of Low Calorie Fats, J . Agric. Food Chem. 42: 432434 (1994). Fomuso, L.B., and C.C. Akoh, Enzymatic Modification of Triolein: Incorporation of Caproic and Butyric Acids to Produce Reduced-Calorie Structured Lipids, J . Am. Oil Chem. SOC.74: 269-272 (1997). Fomuso, L.B., and C.C. Akoh, Structured Lipids: Lipase-Catalyzed Interestenfication of Tricaproin and Trilinolein,J . Am. Oil Chem. SOC.75: 405409 (1998). Auerbach, M.H., L.P. Klemann, and J.A. Heydinger, Reduced-Energy Lipids, in Structured and Modified Lipids, edited by F.D. Gunstone, Marcel Dekker, New York, 2 0 0 1 , ~485-510. ~. Akoh, C.C., and L.N. Yee, Enzymatic Synthesis of Position-Specific Low-Calorie Structured Lipids, J . Am. Oil Chem. SOC.74: 1409-1413 (1997). Kaimal,T.N.B., S. Kanjilal, and R.B.N. Prasad, US.Patent 6,617,141 (2003). Kanjilal, S., R.B.N. Prasad, T.N.B. Kaimal, Ghafoomnissa, and S.H. Rao, Synthesis and Estimation of Caloric Value of Structured Lipid-Potential Reduced Calorie Fat, Lipids 34:1045-1055 (1999).
Chapter 2 5
Lipids in Infant Formulas and Human Milk Fat Substitutes Nikolaus Weber and Kumar D. Mukherjee Federal Research Center for Nutrition and Foods, Institute for Lipid Research, D-48147 Munster, Germany
Introduction The benefits of breast milk for both term and preterm infants are well-known, and breast-feeding remains the optimum and ultimate method of feeding infants until at least 4-6 mon of age; however, medical, metabolic, and economic reasons may lead to a lower incidence and shorter duration of breast-feeding (1,2). Particularly in affluent countries such as the United States, the incidence of nursing is as low as 55% and the duration of breast-feeding is only 4 wk, whereas in poorer countries infants are breast-fed for up to 2 yr (2). Hence, there is the need for infant formulas in many parts of the world. Moreover, special formulas are required for preterm infants and those with impaired digestion and absorption. Lipids are the richest source of energy in human milk (2,3). For newborns, lipids in human milk are the most suitable source of energy, providing per gram more than twice as much energy as carbohydrates and/or proteins. The energy requirement of newborns is -420 I d k g body weight, whereas that of the adults is generally <170 kJkg. Due to the high energy requirement of newbornskg body weight, compared with adults, lipids are very important components of infant food. Moreover, lipids in human milk contain a wide variety of fatty acids (FA), including long-chain PUFA (LC-PUFA), which have a structural role in brain development and function of the retina of the infant and in the production of bioactive substances such as prostaglandins and leukotrienes (4-6). This chapter is devoted to the composition of the lipids in human milk, the physiologic role of lipids in infant nutrition, knowledge of the lipid requirement in infant food formulations, and the lipid composition of commercial infant food formulations.
The Physiologic Role of Lipids in Infant Nutrition Lipids of human milk comprise -98% triacylglycerols (TAG), -0.5-1 % phospholipids and glycolipids, and -0.5% cholesterol (7). The FA composition of breast milk fat shows (Table 25.1) that TAG containing long- and medium-chain FA (MCFA) are the major constituents along with phospholipids and cholesterol. The medium-chain TAG (MCT) containing C4-C,, FA are readily digested because of 607
TABLE 25.1 FA Composition of Human Milk Fat FA compositionb (yo)
Total fata Reference
($g) 36 34.2
40.3
41 48
10:0 5.4= 1.0 1.4 1.2 1.0 1.5 44.3c 0.9 1.1
12:O 4.4 5.7 5.2 4.4 7.3 4.7 4.9 4.3 6.9 5.5
14:O 5.7 6.3 6.1 6.9 6.3 11.4 5.5 6.2 4.7 8.0 7.5
16:O 29.gC 22.0 18.3 21.6 22.0 24.1 18.6 19.8 19.3 22.7 25.9
18:O 8.1 6.2 7.6 8.1 5.4 7.2 7.2 8.0 5.8 7.1
16:ld 3.3 2.3 2.2 3.3 3.5
2.6 2.3 2.6
18:lc9 35.5 31.3 32.7 32.2d 31.3 32.6 31.5 33.6 35.0 33.2 26.7 33.2
18:2n-6
18:3n-3
20:4n-6
20:5n-3
22:6n-3
15.6 10.9 10.5 14.7 10.9 10.5 11.3 14.6 13.3 15.6 14.1 10.2 11.8
1.o 1.o 1.2
0.6 0.5 0.4
0.3e 0.1 <0.1
0.3 0.1
0.7 1.o 0.6 0.9 1.2 1.o 1.l 0.7 0.7 0.6
0.5 0.5 0.1 0.1 0.5 0.4 0.5 1 .o 0.4 0.2
<0.1 0.1
0.3 0.3
0.1 0.1 0.1 0.1 0.1 0.05 <0.1
0.2 0.2 0.2 0.2
z 2 % 2 (u
4
3
P
3
3 rn a. m
0.2 0.1
Tomisting of -98% TAG (7). '+A are designated by number of carbon atoms:number of double bonds; n-x indicatesthe position of the first double-bond counted from the methyl end. Values are rounded. l o t a l saturated (10 0 + 12:O and 16:O + 18:0, respectively)fatty acid. dl 8:1 isomers. Total 205n-3 + 22:6n-3 fatty acids.
Lipids in lnfant Formulas
609
their preferential cleavage by preduodenal lipases, such as lingual and gastric lipases. The MCFA formed are readily absorbed without formation of bile salt-dependent micelles and are transported to the liver via the portal blood. A part of the MCT is also directly absorbed without prior hydrolysis and transported to the liver. Thus, MCT are a rapidly and readily absorbable source of energy for infants. The long-chain TAG, however, are hydrolyzed in infants predominantly by pancreatic lipase in the intestine, as in adults, and the resulting FA and 2-monoacylglycerols are absorbed via the intestinal lumen and are transported to various tissues via the lymph and blood. Fatty A cids
TAG and other lipids of human milk contain long-chain n-6 and n-3 PUFA, which are derived partly from the diet of the mother directly and partly from the maternal metabolism of linoleic (LA) and a-linolenic (ALA) acids, as shown in Figure 25.1. The long-chain n-6 and n-3 PUFA of human milk, i.e., di-homo-y-linolenic acid (20:3n-6), arachidonic acid (ARA, 20:4n-6), docosatetraenoic acid (22:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (22:5n-3), and docosahexaenoic acid (DHA, 22:6n-3), are important for infant growth and development because they are precursors of the bioactive prostaglandins, thromboxanes, and leukotrienes (19-22). These compounds have a regulatory function in various biochemical processes including immune response. They are formed in several tissues by the action of lipoxygenase and cyclooxygenase. Like adult humans, infants are unable to synthesize LA and ALA; therefore these essential fatty acids (EFA) have to be provided with the diet to ensure growth and development. Term infants are able to convert LA to ARA and ALA to EPA andDHA(4). ARA is the most important n-6 LC-PUFA, because it is the direct precursor of the eicosanoids of the 2-series (Fig. 25.1), which are powerful physiologic regulators, and because it is an abundant constituent of the central nervous system (23). ALA is converted to n-3 LC-PUFA including EPA, which is a precursor of eicosanoids of the 3and 5-series, and DHA (Fig. 25.1), which is a major structural and functional LCPUFA of the central nervous system (23). An adequate intake of both n-6 and n-3 EFA in a particular ratio is important for the formation of bioactive compounds from AA and EPA. Because LA and ALA compete for the same enzymes in the pathways of formation of n-6 and n-3 LC-PUFA, respectively, they inhibit each other’s desaturation and chain elongation (24). The excessive availability of LA compared with ALA in the infant diet, both from human milk and formula, is expected to promote the conversion of LA to n-6 LC-PUFA at the expense of the conversion of ALA to n-3 LCPUFA. Therefore, an adequate DHA status of the infant might require the ingestion of preformed DHA (25). Others, however, consider that adequate and recommended availability of LA and ALA in most infant formulas does not necessitate the supplementation of LC-PUFA that are not essential in the strict classical sense (2627).
9
12
COOH
18:2
II
Linoleic acid
9
12
18:3 A6-
15
6
COOH
11
12
15
y-Linolenic acid
+
8
COOH 17
11
8
-
20:4
5
-
Arachidonic acid (20:4 06) 244
(2-series)
J
Leukotrienes (4-series)
11
8
20:5
piq
>COOHj
Prostanoids
14
all-cis8.11,14,17-Eicosatetraenoicaicd
/ Prostanoids J (1series)
-
6
20:4
Dihomoy-linolenic acid
14
9
Stearidonic acid
205
-
COOH
12 9 a-linolenic acid
1a:4
18:3 COOH -
14
II
1
24:s-
f-
1
1~ 1 1 1
22:4
22:s-
17
11
8
COOH
5
Eicosapentaenoic acid (205 03. EPA)
\ Prostanoids
245
Leukotrienes (5-series)
A6-Desaturase
22:s
14
(3-series)
22:6 -246
COOH 19
16
13
10
7
4
Docosahexaenoic acid (22:6 03. DHA)
Fig. 25.1. Biosynthesis of long-chain PUFA of the w3- and w6-series (03 and w6 LC-PUFA)as well as the formation of eicosanoids.
Lipids in Infant Formulas
61 1
Both ARA and DHA are structural components of cell membranes. The concentration of DHA is particularly high in the retina and cerebral cortex. Therefore, DHA is considered to be essential to the function of the brain and the retina. Newborns have a limited capacity to produce LC-PUFA, including DHA, in particular when they consume a diet low in LC-PUFA precursors. The hypothesis that a balanced addition of LC-PUFA to preterm infant formula during the neonatal period of life confers long-term developmental advantage was tested in two doubleblind, randomized, controlled trials with preterm (28) and term infants (29). No significant beneficial effects of LC-PUFA supplementation on general and neurodevelopment, visual acuity, or other variables were found in these studies during the first 18 (preterm) or 14 mon (term infants), yet a nonsignificant positive effect beyond this period of time was not excluded for preterm infants (28). In contrast, positive influences on development were found for both low-birth-weight (30) and term infants (31) fed LC-PUFA formulas. The critical period during which the dietary supply of LC-PUFA can influence the maturation of cortical function extends beyond 6 wk of age as was found in a randomized controlled trial with 65 healthy term infants (32). Moreover, a prospective clinical study showed that supplementation of healthy term infants with LC-PUFA during the first 2 mon of life has positive effects on the quality of general movement (33). The effects of dietary DHA supply during early infancy on later cognitive development indicated significant correlations between plasma and erythrocyte DHA levels and improved performance on mental development index (34). LC-PUFA and Their Dietary Precursors in formulas
freterm Infants. Most neonatal LC-PUFA start to decline after birth even after the ingestion of human milk (35,36). This has considerable implications, especially for preterm infants whose LC-PUFA status is lower than that of term infants. Feeding preterm infants formula diets supplemented with LC-PUFA results in plasma LCPUFA levels similar to those of breast-fed infants, without evidence of adverse effects (37). Intake of additional ARA and DHA by preterm infants has developmental benefits (38-41). Prolonged differences in the LC-PUFA status in breast- and formula-fed term infants are often observed ( 4 2 4 ) , yet many term-infant formulas do not contain LC-PUFA. This is possibly due to inconsistent results reported to date on the beneficial effects of LC-PUFA supplementation in preterm and term infants (38-4O,45). Thus, in preterm infants, beneficial effects on visual evoked potential acuity were reported after the addition of n-3 LC-PUFA to formulas with or without added ARA (4649). Other studies assessing visual acuity in preterm infants also reported beneficial effects upon supplementing the formula with n-3 LC-PUFA (50-52), whereas in another study, there was no benefit from feeding n-3 LCPUFA (53). Studies on visual recognition memory and visual attention of preterm infants after DHA supplementation showed that both familiar and novel stimuli
61 2
N.Weber and K.D. Mukherjee
evoked more and shorter duration looks compared with control groups (5435). No effect of dietary LC-PUFA was observed on brain stem auditory evoked potential (52,53) or somatosensory evoked potential (53). The addition of ARA and DHA to preterm infant formula leads to immune consequences, such as lymphocyte populations, cytokine production, and antigen maturity that are similar to those of infants fed human milk (56). On the other hand, it was shown that breast-fed infants and infants fed a formula diet not supplemented with LC-PUFA do not differ in immunocompetent cell cytokine production despite significantly lower concentrations of LC-PUFA in erythrocyte cell membranes (57). Fish oil supplementation to preterm formula demonstrated either negative effects (58-60) or no effect (37,53,61-63) on growth, whereas other studies reported no adverse effects on the growth of preterm infants upon supplementation of the formula with both n-3 and n-6 LC-PUFA (52,64-67). A meta-analysis of data from three randomized trials and one nonrandomized study concluded that supplementation of formula with LCPUFA has beneficial effects on the visual acuity of preterm infants at 2- and 4-mon corrected age (40). Modulation of the LC-PUFA status of infants by reducing the LA:ALA ratio in formula, as suggested by Gibson et al. (68), increased the DHA concentration of plasma phospholipids of preterm infants with a concomitant reduction in ARA levels (69). Supplementation of preterm infant formula containing EPA and DHA with additional ALA maintained plasma and erythrocyte DHA levels similar to those found in breast-fed infants without altering n-6 PUFA status (70). Fish-oil formulas increased n-3 LC-PUFA status in low-birth-weight infants with a concomitant decrease in n-6 LC-PUFA, whereas GLA did not augment ARA status compared with breast-fed infants (71). Term Infants. Some studies reported benefits for visual acuity from supplementing term-infant formula with DHA, with or without added ARA (72,73), whereas in others, there was no difference in this regard between LC-PUFA-supplemented and -unsupplemented formulas (74-76). A meta-analysis of the above data showed no effect of LC-PUFA supplementation of formula on the development visual acuity in term infants; however, an improvement in visual acuity by LC-PUFA was observed for infants only at the age of 2 mon (77). No effect of LC-PUFA supplementation on development (45,74,75,78) and no negative effects on growth (72,74,76,79) of term infants were reported. No difference in memory function was observed in term infants after feeding formula with ARA and DHA (80). Increasing the n-3 LC-PUFA contents of plasma and erythrocyte phospholipids by decreasing the LA/ALA ratio of formulas was also reported for term infants; this was accompanied by a significant reduction of ARA in erythrocyte phospholipids, although no difference in visual acuity and growth between the various formula-fed groups and breast-fed group was found (81). No difference in visual function (82,83)was observed in term infants fed breast milk or formulas with varying proportions of LA and ALA. Decreasing the ratio of LA:ALA in the
Lipids in Infant Formulas
613
formula of term infants reduces to some extent the postnatal decline in the DHA content of plasma phospholipids; however, this is associated with a postnatal decrease in plasma ARA (84). No difference in the visual function of the various groups was observed (85). A novel infant formula milk with an added single-cell oil (SCO) containing LC-PUFA did not have any detrimental effects on growth and general health of term infants (86). Triacylglycerols
Human milk contains -3-5% total lipids. The main components are TAG, which comprise -98% or more of human milk fat, whereas phospholipids are present in low proportions only (0.61%) in human milk fat. Unlike vegetable oils, human milk TAG and TAG of some animal species contain palmitic acid esterified predominantly in the sn-2 position, whereas oleic, linoleic, and linolenic acids are preferentially located at the sn- 1,3 position (Table 25.2). Human milk and the milk of some animal species contain bile salt-stimulated lipase, which, in conjunction with gastric lipase and pancreatic lipase, is generally considered to be responsible for complete hydrolysis of milk-fat TAG and high absorption of the resulting FA in the infant (87,88). Table 25.3 summarizes the molecular species composition of human-milk TAG (15,91,92). It is obvious from the table that human-milk TAG consist of many molecular species, with >50% of them containing palmitoyl moieties (15,16). The results reported by Dotson et al. (15) indicate very high proportions of triacylglycerol molecular species with medium-chain acyl moieties (>45%). Somewhat different results were obtained by Winter et a1. (16), who separated the TAG of human milk fat by silver-ion HPLC (Ag+-HPLC), followed by reversedphase HPLC as well as GC and calculated as many as 170 different types of TAG present in human milk fat. The two major types of TAG, i.e., palmitic-oleic-oleic (POO; 11.8 mol%) and palmitic-oleic-linoleic (POL; 10.0 mol%), are almost twice as abundant as expected from random FA distribution. The same is true for molecular triacylglycerol species containing mediumchain acyl moieties (>I8 mol%). The authors speculated that the apolarity of TAG containing 16:O or 18:O acyl moieties might be modulated by incorporation of unsaturated or medium-chain acyl moieties (16). Moreover, appreciable amounts of the latter, MCFA, are expected to be esterified at the sn-3 position, leading to excellent substrates for the lingual and gastric lipases that produce FA, MAG, and DAG, which assist hydrolytic activity of pancreas lipase in the duodenum (16). Most preterm-infant formulas contain MCT, but their effects on the metabolism of PUFA are controversial. Studying the metabolism of [I3C]LA in preterm infants fed a formula with MCT, Rodriguez et al. (70) found that oral MCT effectively reduced the oxidation of PUFA and LC-PUFA without compromising the biosynthesis of endogenous n-6 LC-PUFA. The feeding behavior in neonates
TABLE 25.2 Regiospecific Distribution of FA in Human Milk TAG as Compared with Those of Plant Oils Human-milk
TAG^ FAd 16:O 18:O 18:ln-9 18:2n-6 18:3n-3
Total
21 .o 7.1 40.2 13.4 1.5
Soybean oiP
(Yo) sm2
Total
(Yo) sn-2
54.2 2.9 17.1 8.1 0.9
9.9 3.6 25.0 53.6 9.3
2.6 0.5 26.6 64.6 10.8
Sunflower oilc Total
(Yo) sm2
7.4 4.8 24.3 62.4
aFA are designated as given in Table 25.1; m = stereospecific numbering of glycerol backbone. bSourcer Reference 89. cSource:Reference 90.
1.5 0.9 25.1 72.1
Low-erucic rapeseed oilc Total
6.5 1.6 51 .O 29.0 8.6
(YO)
Olive oilc
sn-2
Total
(YO)sm2
1.8 0.8 43.4 21.8 6.2
10.2 2.7 64.7 19.1 0.8
2.2 0.9 70.6 23.9 0.9
z
f 4
> 2
p 5
s
5
rn
2.
8
Lipids in Infant Formulas
61 5
TABLE 25.3 Molecular Species of Major Human Milk TAGdfb Molecular species Reference
16:0-18:1-18:1 (POO) 16:0-18:1-18:2 (POL) 14:0-14:0-18:1 (MMO) 14:0-14:0-16:0 (MMP) 16:0-16:0-18:1 (PPO) 18:1-18:1-18:2 (OOL) 14:0-16:0-18:1 (MPO) 16:0-18:2-18:2 (PLL) 12:O-16:O-18:l (LaPO) 12:O-16:O-16:O (LaPP) 12:O-18:O-18:l (LaSO) 16:0-18:0-18:1 (PSO) 14:0-18:1-18:1 (MOO) 16:0-16:0-18:2 (PPL) 14:0-18:1-18:2 (MOL) 18:1-18:1-18:1 (000) 18:1-18:2-18:2 (OLL) 16:0-16:1-18:1 (PPoO) 16:0-18:0-18:2 (PSL) 14:0-16:0-18:2 (MPL) 12:0-18:1-1 8:l (LaOO) 12:O-16:O-78:2 (LaPL) 12:O-18:l-18:2 (LaOL) 12:O-14:O-18.1 (LaMO) 18:0-18:1-18:2 (SOL) 12:O-18:O-18:0 (LaSS) 18:0-18:0-18:1 (SSO)
Molecular acyl composition (1 6) (1 5)
11.8 10.0
4.4 3.3 3.3 3.2 3.1
3.1 2.8 2.4 2.3 2.1 1.9 1.9 1.7 1.5 1.4 1.3 1.2 1.1 1 .o
3.2 4.7 4.5 12.0
8.8 4.8 14.7 6.2 1.3
2.2 3.3
2.0 6.1
7.4 3.4
dMolecular species of TAG are designated by their FA composition. The FA sequences listed do not necessarily correspond with the actual TAG structures. bFA are designated as given in Table 25.1 as well as using the following abbreviations: L, linoleic acid; La, lauric acid; M, myristic acid; 0, oleic acid; P, palmitic acid; Po, palmitoleic acid; S, stearic acid.
whose formula diet contained MCT indicated that the MCT:LCT ratio mohfies physiologic functions involved in energy-balanceregulation and sleep time (93).
Phospholipids and Glycolipids The phospholipid content in human milk fat frequently ranges from 0.4 to 1% (Table 25.4). In milk, including human milk, phospholipids are associated mainly with the membrane of the fat globules. The main phospholipid components consist of phosphatidylcholines (PC) and phosphatidylethanolamine (PE) as well as sphin-
N. Weber and K.D. Mukherjee
61 6
TABLE 25.4 Composition of Complex Polar Lipids in Human Milka
Complex polar lipids
Concentration of complex polar lipids in total milk lipidsb (@loog,
Phospholipids Total phospholipids PC PE
0.4-1 13.1-32.2 7.4-2 7.7 3.7-9.3 5.3-13.1 29.0-54.8
PS PI SphC Clycolipids Cangliosides Cerebrosides
Composition (YO, range) of the various phospholipid classes
0.4-0.5 0.5-0.6
dSource: Reference 7; values rounded. bOn a basis Of 4% total lipids in human milk. CAbbreviation:Sph, sphingomyelins.
gomyelins, whereas phosphatidylserines (PS) and phosphatidylinositol (PI) are present as minor constituents (7,30,94,95). In addition to phospholipids, some other complex polar lipids, particularly glycolipids, such as neutral cerebrosides (glycosylceramides) and acidic gangliosides, are present in human milk each with a concentration of -20 mgL. Some of these glycolipids of human milk, e.g., the ganglioside GM1 and the globosides, Gb3 and Gb4, may have an important role in the protection of the infant against bacteria-mediated diseases; however, clinically relevant effects of these glycolipids have not yet been demonstrated (7,30,94,96).
Source of Lipids for Infant Formula Fa t t y Acids
Formulas for healthy term infants usually contain 3.3-3.8% TAG (97). In view of the essentiality of LA in humans (98), numerous national and international regulatory agencies recommend specific levels of LA in infant formula (300-500 mg/100 kcal; 99). Although the essentiality of ALA has also been established (19), the recommendations concerning its level in infant formula are rather limited. LA:ALA ratios of 4-10 (100) and 5-15 (101) in infant formula were suggested. The TAG contained in vegetable oils and fats are common sources of saturated, monounsaturated, and PUFA in infant formulas. Table 25.5 shows the FA composition of oils and fats used for infant formulas. Data on butter fat is included because butter is used in some infant formulas in Europe. The widely varying FA
Lipids in Infant Formulas
61 7
TABLE 25.5 FA Composition of Oils a n d Fats Used for Infant Formulationsa FA composition (%) Oil/fat Soybean Low-erucic rapeseed Corn Sunflower High-oleic sunflower High-oleic safflower Palm olein Coconut Butter
12:O
44.6 2.3
14:O
16:O
18:O
18:l
18:2n-6
18:3n-3
3.8 1.8 2.3 3.5 11.3 1.5 4.4 32.6 9.8
22.8 56.1 17.0 45.3 75.2 77.0 42.5 2.3 20.4
51 .O 20.3 51.5 39.8 8.1 15.0 11.2
6.8 9.3
6.0 16.8 8.2
10.2 4.0 22.7 5.4 6.5 4.5 39.0 18.2 21.3
1.8
1.2
aSource: Reference 2.
composition of the oils and fats (Table 25.5) gives the manufacturer the ability to alter the FA composition of the lipids in the formula as desired from the physiologic point of view. Most of these oils and fats, properly blended, provide adequate amounts of LA and ALA in suitable ratios. Corn oil is commonly used for infant formula due to its oxidative stability (102); however, the level of ALA is low and the LA:ALA ratio high in corn oil. Both soybean oil and low-erucic canola-type rapeseed oil are more suitable for infant nutrition because they contain a more balanced proportion of LA and ALA; although the latter oil is acceptable in Europe, the US. Food and Drug Administration has precluded its use in infant formulas in the United States (103). Sources of LC-PUFA (DHA and ARA) in infant formula include egg yolk lipids, marine oils, and single cell oils (SCO), such as microalgal and fungal oils (104,105). Although both DHA and ARA occur in egg-yolk phospholipids (Table 25.6), as opposed to marine oils and SCO in which they occur in the TAG, egg- yolk lipids in formulas were found to be effective in raising the erythrocyte and plasma level of DHA and ARA in term infants (59,74,106). Compared with egg yolk lipids, some marine oils contain relatively large proportions of EPA in addition to DHA (Table 25.6). EPA, which is an antagonist of ARA, increased erythrocyte levels of EPA, reduced ARA, and resulted in poorer growth of preterm infants fed formulas containing high-EPA marine oil compared with those fed standard formula (5058). LowEPA marine oils, such as tuna oil, which are rich in DHA (-7% EPA; -21% DHA) are now commercially available (Table 25.6) as a source of DHA for supplementation of infant formula. Safety evaluation of DHA-rich tuna oil for use in infant formulas showed no adverse effects in newborn piglets (107). Unlike marine oils, SCO do not contain EPA. Several species of microalgae and fungi produce large amounts of n-3 and n-6 PUFA in their biomass (1 15,116). Selected fungi were used for the production of oils containing y-linolenic acid (n-6 18:3, GLA) at levels similar to those in some plant oils, such as seed oils of borage
TABLE 25.6 FA Composition of Egg-Yolk Lipids as Compared with Fish Oils and a Fish-Oil Concentrate (Marinol D40TM) FA composition of lipids (%)
Egg yo1k
Fish oils
Regular
Regular
Regular
"n-3
eggs
eggs
eggs
eggs"
Menhaden
Bluefin tuna
Californian sardine
Japanese sardine
Salmon
Mackerel
Marinol
(109)
(110)
(110,111)
(112)
(113)
(1 12)
(112)
(114)
(112)
D40a
32.0 12.5 2.8
24.8 7.7 4.2
32.4'
33.4'
19.6-24.0 2.4-3.4 11.2-1 7.9
29.3'
16.2 3.5 9.2
15.5 3.7 9.5
15 6 6
15.9 1.7 9.0
6.7 2.4 4.3
34.5 14.5 1.4
42.0 17.0 0.9
49.8' 13.3 0.5
10.7-23.4 0.%1.7 0.43.7 1.l-2.7 0.6-2.3 10.2-1 4.1 0.2-1 .o 1.1-2.5 3.3-1 0.6
21.5 1.3
11.4 1.3 0.9 3.2 1.6 16.9 3.6 2.5 12.9
17.3 2.5 1.3 8.1 2.5 9.6 7.8 2.8 8.5
23 3 1 10
12.9 1.3 1.o 11.9 0.4 7.6 13.9 0.6 7.7
15.6 1.2
FA (Ref.)
(108)
16:O 18:O 16:l 18:l 18:2n-6 18:3n-6 201 20:4n-6 205n-3 22:l 22:5n-3 22:6n-3
3
5
0-
3.8
0.7
0.2 0.6
47.4b 10.1 3.6
NDC
2.0
0.8 0.3
0.9
3.6
aProductof Lcders Croklaan, Wormerveer, The Netherlands. 9otal saturated or monounsaturated FA. =ND, not determined.
3.8
6.4 1.o 6.6 5.5 2.9 20.7
Trace
9 9 3 8
?
su Q
3
P
z
0.8
C
2.0
m q
7.2 2.7 4.1 38.4
fi
3
m m
Lipids in Infant Formulas
61 9
(Borago oflicinalis; 117), evening primrose (Oenothera biennis; 118), black currant, and other Ribes spp. (1 19) that also contain 10-25% GLA. Typically, an SCO from the biomass of a commercial Mucor circinneloides strain contains -1 6% GLA (120). A commercially available fungal oil from the biomass of Mortierella isabellina contains -10% GLA (Table 25.7). Some benefits, such as an increase in the plasma DHA level without affecting the ARA level of term infants, were reported (121); these involved the incorporation of GLA from black-currant seed oil into an infant formula containing tuna oil at a level of 0.15% DHA and 0.5% GLA. The FA composition of several other commercially available single-cell TAG oils that can be used in infant formula is given in Table 25.7. SCO containing ARA, such as ARASCO@(40% ARA) and SUN-TGA25@(25% ARA), are produced from the biomass of the psychotropic fungus Mortierella alpina. Both oils contain minor proportions ( 2 4 % ) each of GLA and dihomo-y-linolenic acid (n-6 20:3), but no EPA. Large-scale production of ARASCO@(123) is carried out in fermentors using sugars and vegetable protein hydrolyzate as carbon and nitrogen sources, respectively. The biomass is separated from the culture broth by centrifugation, dried, and subjected to oil extraction with hexane. The oil is subsequently refined, bleached, and deodorized essentially in the same manner as in industrial production of plant oils. The refined, bleached, and deodorized oil is mixed with high-oleic sunflower oil to a standardized level of 40% ARA and packaged after the addition of antioxidants, such as a-tocopherol and ascorbyl palmitate. Safety evaluation of an ARA-rich oil from M . aZpina (SUN-TGA40S@)for use in infant formulas showed no adverse effects in newborn piglets (107). In a manner similar to that described above, a SCO containing DHA, DHASCOB (40% DHA), is produced from the biomass of a heterotrophic, nonphotosynthetic marine alga, Crypthecodinium cohnii (122). DHASCO@,an orange-colored oil mixed with high-oleic sunflower oil to a standardized level of 40% DHA, contains essentially no other PUFA than DHA (Table 25.7). The safety of both ARASCO@ and DHASCO@was evaluated (125-127). The Ministry of Health in The Netherlands granted a similar safety approval to include ARASCO@and DHASCO@in infant formulas. Many term and preterm infant formulas containing ARASCO@and DHASCOB are now marketed in Europe, the Middle East, South-East Asia, and Australia. Recently, a microalga (Schizochytriurn sp.) was grown on a commercial scale to yield a biomass containing 48% fat with a DHA content of 25% (128). An oil containing -40% DHA was extracted recently from the above microalga, refined, and sold in bulk to the supplement industry (129).
LC-PUFA Concentrates. The commercial SCO available to date contain up to -40% ARA or DHA and -10% GLA (Table 25.7). Recently, enzymatic methods were applied to enrich the constituent PUFA of plant oils, marine oils, and SCO via kinetic resolution, catalyzed by lipases (130). GLA is of considerable commercial interest due to its beneficial biomedical properties (121,131). The ability of lipases to discriminate against GLA was uti-
N o \ 0
TABLE 25.7 Composition of Major Constituent FA of CommerciallyAvailable Single Cell Oils Containing PUFA
Organism
Carbon/n itrogen source
Trade name /manufacturer, supplier
Mortierella alpinad
Dextrosefyeast extract
ARASCO/Martek
or vegetable protein hydrolyzate
FA (& ol0 ' 14:O
16:O
18:O
18:l
18:2n-6
8
10
21
7
9, 18:3n-6
204n-6 40
22:6n-3
.2
5
Bioscience, Columbia, MD
0-
2 nJ
M. alpina
D e x t r w e a s t extract
SUN-TGA25/Suntory, Osaka, japan
Cypthecodinium cohniib
Dextrosdyeast extract or vegetable protein hydrolyzate
DHASCO/Martek Bioscience
Mortierella isabellina'
Sugars
Fungal oil/ Sigma, St. Louis, MO
"Source:Reference 122. bSource:Reference 123. cSource:Reference 124.
14
6
14
26
25
4
3
P 15
16
<1
21
1
21
4
37
22
10
Lipids in Infant Formulas
62 1
lized for the enrichment of this FA from FA mixtures, derived from a commercial
M . isabellina oil, by selective esterification of the FA, other than GLA, with nbutanol (124). Thus, using lipases from Rhizomucor miehei and Candida rugosa, the GLA content is increased from 10% in the starting FA mixture to >60% in the FA that remain unesterified. Lipase-catalyzed selective esterification of FA derived from evening primrose oil or borage oil with n-butanol leads to conversion of most of the FA to butyl esters, with the exception of GLA, which is enriched as a concentrate (up to 75%) in the unesterified FA (132,133). Selective hydrolysis of the TAG of evening primrose oil or borage oil, catalyzed by lipases from R. miehei and C. rugosa, also leads to enrichment of ylinolenoyl moieties in the unhydrolyzed acylglycerols (-48%), whereas the other acyl moieties are cleaved to yield FA containing lower levels of GLA (134). The ability of the lipase from R. miehei to discriminate against DHA was utilized for the enrichment of this acid to -50% from a mixture of FA derived from marine oils via lipase-catalyzed selective esterification with n-butanol (132) or methanol (135). Selective hydrolysis of marine oil TAG, catalyzed by lipases from Aspergillus niger and C. rugosa, was employed to concentrate n-3 PUFA in the unhydrolyzed acylglycerols (136). In a commercial process (137), a marine oil was partially hydrolyzed by C. rugosa lipase to yield an acylglycerol fraction enriched in EPA and DHA; the acylglycerol fraction was subsequently isolated by evaporation and converted to TAG via hydrolysis and reesterification, both catalyzed by R . miehei lipase. Selective esterification of FA from tuna oil with lauryl alcohol, catalyzed by R . delemar lipase yielded an unesterified FA fraction containing 73% DHA compared with 23% DHA in the starting material (138). Using the same lipase, selective esterification of tuna oil FA with lauryl alcohol, extraction of the unreacted FA, and their repeated esterification with lauryl alcohol resulted in a FA fraction containing as much as 91% DHA (139). By using lipase from Pseudomoms sp., marine oil TAG were interesterified with marine oil FA fractions that were enriched with n-3 PUFA, thereby raising the level of total n-3 LC-PUFA in the TAG to 65% (140). Selective alcoholysis of ethyl esters of tuna oil FA with ethanol by using R. delemur and R. miehei lipases as biocatalysts led to the enrichment of DHA in the ethyl ester fraction from 23 to -50 mol% (141) and from 60 to 93 mol% (142). Selective interesterification of tuna oil TAG with ethanol by using R. miehei lipase yielded an acylglycerol fraction containing 49% DHA, whereas selective esterification of tuna oil FA with ethanol yielded a FA fraction containing 74% DHA (143). A TAG mixture containing -40% DHA (Marinol D40@,Loders Croklaan, Wormerveer, The Netherlands) for use in infant-food formulation (Table 25.6) was produced commercially from fish oil by lipase-catalyzed reactions. It is conceivable that such PUFA-enriched products from SCO will also be available in the future. FA generated by lipase-catalyzed hydrolysis of a commercial SCO from M . alpina were subjected to selective esterification with lauryl alcohol, catalyzed by lipase from C.
622
N. Weber and K.D. Mukherjee
rugosa. In this way, the ARA content was increased from 25% in the starting FA mixture to >50% in the FA that remain unesterified (144).
Triacylglycerols Various approaches can be adopted to formulate fats and fat blends suitable for infant formulas having absorption and other nutritional properties similar to those of human milk fat. Infant formulas containing blends of plant oils to give a 16:O level similar to that in human milk fat, however, with 16:O esterified predominantly at the sn-1,3 positions of TAG, may result in lower fat absorption and reduced calcium absorption in infants (145-152). This is due in part to the release of the high-melting, less-soluble 16:O FA from the sn-1,3 positions of TAG in the intestinal lumen (153) and the formation of insoluble calcium salts of 16:O that are excreted in the stool and thus unavailable for absorption (154). As a result, part of the dietary energy and minerals, such as calcium, are lost. Similar levels of 16:O in plasma TAG and phospholipids of breast-fed infants and those fed formula containing low levels of 16:O suggest that adequate proportions of 16:O are endogenously synthesized and incorporated into membrane lipids in formula-fed infants ( 155,156). Chemical randomization of suitable oils and fats, such as palm olein, leads to even distribution of all the fatty acyl moieties across all three positions of TAG, resulting in an elevation in the level of palmitoyl moieties in the sn-2 position to -35% (154). However, incorporation of randomized palm olein into infant formula does not significantly improve fat absorption; this was attributed to increased proportions of tripalmitin with lower digestibility, which is also formed by the randomization process (154).
Structured TAG. The general strategy for the preparation of structured TAG utilizes the sn- 1,3 regiospecificity of TAG lipases in lipase-catalyzed esterification and transesterification reactions (157). Similar to human milk TAG, structured TAG suitable for infant formulas contain palmitic acid esterified predominantly at the sn-2 position and unsaturated FA at the sn-1,3 positions. Such TAG are produced commercially by transesterification of tripalmitin, derived from palm oil, with oleic acid or PUFA, obtained from plant oils, using sn-1,3-specific lipases as a biocatalyst (158; Fig. 25.2). Immobilized lipases used successfully include those from microbial sources, such as R . miehei (Lipozyme RM IM@; 158) and Thermomyces Zanuginosus (Lipozyme TL IM@; 159) and plants such as papaya (Carica papaya) latex (160). Deacidification and fractionation of the products of interesterification yielded structured TAG consisting predominantly of molecular species that are also abundant in human milk TAG (15,91,92). It was demonstrated very recently in a double-blind, prospective clinical trial that palm olein-containing formula may lead to significantly reduced bone mineralization and bone mineral density in healthy term infants (161). Table 25.8 shows the regiospecific distribu-
Lipids in lnfant Formulas
623
c18:l O-Ci6o
+
Cigp acids c103
Tripalmitin
Tripalmitin FA
+
____.)
cl69 acid
sn-1,3 specific llpase
Structured TAG
Palmltic acid
Fig. 25.2. Preparation of structured TAG for use as human milk fat replacers by lipasecatalyzed transesterification of tripalmitin with unsaturated FA catalyzed by sn-l,3specific lipase.
tion of acyl moieties in commercial structured triacylglycerol products (BetapolTM; Loders Croklaan) prepared by lipase-catalyzed interesterification compared with that of human milk-fat TAG. Such products can be blended with suitable plant oils to yield a product resembling the FA composition and their regiospecific distribution in human-milk TAG. Structured TAG containing 16:O at the sn-2 position and ARA at the sn-l,3 positions, which should be suitable as additives to infant formulas, were prepared by regiospecific interesterification (acidolysis) of tripalmitin with ARA, catalyzed by Rhizopus delemar lipase (164). Structured TAG containing 16:O at the sn-2 position and medium-chain acyl moieties (4:O-1O:O) in the sn-l,3 positions were prepared by sn-1,3 regiospecific incorporation of the medium-chain acyl moieties via interesterification of tripalmitin with ethyl esters of MCFA (165) or with caprylic acid and its alkyl esters (166). Such structured TAG might provide a source of instant energy in special infant formulas due to rapid cleavage of the MCFA from the sn-l,3 positions and their direct catabolism in the liver. Initial studies with term infants, either breast-fed or fed conventional formulas containing -22% 16:0, indicated similar levels (-26%) of 16:O in total plasma TAG, but distinctly higher levels of 16:O at the sn-2 position of these TAG in breast-fed infants (-23%) compared with the formula-fed infants (-7%), suggesting a higher absorption of 16:O as sn-2 MAG from human milk compared with conventional formula (167). The level of 16:O in the sn-2 position of chylomicron TAG of term infants that were breast-fed (human milk containing -56% 16:O in the sn-2 position of TAG) or given formulas containing structured TAG (Betapol or Betapol-2 containing 29% 16:O in the sn-2 position, each; cf. Table 25.8) or palm olein oil (containing 5% 16:O in the sn-2 position) was found to be related to the level of 16:O in the sn-2 position of dietary TAG, i.e., -28, 16.3, and 8%, respectively (89,163). Plasma TAG of preterm infants fed structured TAG (Betapol containing 58% 16:O in the sn-2 position) contained higher proportions (-29% compared with -25%) of 16:O than those fed a conventional preterm formula (containing -10% 16:O in the sn-2 position; 168). Neither the plasma phospholipids of preterm
N m
P
TABLE 25.8 Positional Distribution of Acyl Moieties in Human-Milk TAG and Betapol" TAG Stereospecific position of acyl moieties at the glycerol backbone Betap01-2~r~ ( d l 00 g)
Betapolbrc (g/lOOg)
Human-milk TAGa (mol O h ) Acyl moieties
SG-1
sm2
sm3
1o:o 12:o 14:O 16:O 16:l 18:O 18:l 18:2n-6 18:3n-3
0.2 2.3 3.5 12.4 1.6 15.2 46.4 14.4 0.9
0.2 7.8 12.5 51.2 2.4 1.5 11.5 8.5 0.8
1.8 13.9 10.7 11.7 3.2 5.2 31.8 16.7 1.4
20:l 204n-6 22:6n-3
0.5 0.05 ND
0.3 0.4 0.3
0.3 0.4 0.1
Total TAG
sn-2
Total TAG
5m2
?
F m 23.9-25.4
47.1-58.0
3.7 24.8
1.6 29.1
2.5-3.7 34.8-36.7 12.5-1 2.6 1.1-2.7
2.4-2.5 13.2-1 8.9 5.1-1 1.4 0.5-1.5
5.2 39.5 23.4 2.6
2.2 34.6 28.4 2.6
NDe ND
0.1 ND
U
2
2 Q
dSource.-Reference 7. bBetapl and Betapol-2 are products of Loders Croklaan, Wormerveer, The Netherlands. cSourcerReference 162. dSource: Reference 163. %D, not determined.
3 D
2%
a.
8
Lipids in lnfant Formulas
62 5
infants (169) nor the chylomicron phospholipids of term infants (89) contained lower levels of ARA or DHA after consumption of formulas containing structured TAG compared with conventional oils. The absorption of total fat, 16:0, 18:0, and calcium was significantly higher in term infants fed Betapol (containing -24% 16:0, -47% in the sn-2 position of TAG) than in those fed a conventional formula (containing -20% 16:0,-8% in the sn-2 position of TAG; 170). Significantly higher 16:O and 18:O absorption and lower calcium excretion were also observed in preterm infants fed formula with structured TAG (containing 58% 16:O in the sn-2 position) than in those fed a standard formula (169). An investigation of the toxicity of Betapol in terms of general, reproductive, and postnatal development did not indicate the presence of an unexpected toxicant (17 1).
Phospholipids Human milk formulas contain 2.3-4.3% total lipids as an oil-in-water emulsion, stabilized by added MAG, DAG, or phospholipids and bovine milk whey proteins. These lipids and proteins form a membrane that surrounds the TAG globules. For example, one brand of formula contains 24 mg/100 mL phospholipids as well as choline (2). The lipid globules formed in this formula are -0.8 pm in diameter, which is much smaller than those observed in human milk (-4 pm). PC and sphingomyelins are the predominant phospholipids in human milk (Table 25.4). The dietary intake of both choline-containing phospholipids determines the availability of choline to organs and tissues that require large amounts of choline for membrane biosynthesis during the neonatal period (172). Recently, choline was shown to be an essential nutrient in human-milk and infant formulas (173). Various human-milk formulas used in nutritional studies of infants were supplemented with egg yolk phospholipids that provide LC-PUFA, e.g., ARA and DHA (28,31,94,174). The nutritional effects of including egg yolk from n-3 PUFAenriched eggs in an infant formula were studied in a randomized controlled trial demonstrating significantly higher DHA concentration in erythrocyte lipids after feeding this diet compared with nonsupplemented formulas (1 10). Intestinal absorption of n-3 LC-PUFA was found to be higher from phospholipids than from TAG in preterm infants fed breast milk or formula (174).
Lipid Composition of Commercial Infant-Food Formulations Table 25.9 shows the FA composition of lipids of several commercial infant-food formulas. Most of the formulas contain minor proportions (<2%) of MCFA and moderate proportions (-5-13%, each) of lauric and myristic acids (Table 25.9) similar to human-milk lipids (Table 25.1; 94). The levels of palmitic and stearic acids in most of the formulas (Table 25.9) are quite similar to those of the humanmilk lipids (Table 25.1). Oleic and linoleic acids are by far the major unsaturated
TABLE 25.9 FA Composition of Commercial Milk Fat Formulas for Infant Nutrition FA composition (%)
Total fat Formula
(YO)
PrematiP Prematil + Milupand AptarniIb Aptamil with Milupanb
35 35
Aptarnil with MilupanC Enfarnil with irond Nutrilon Premiume CaIIia-1‘ Preemie SMAg Betapolh Betapol-2’ Alrniron AW Nestec formulak a Milupa,
40 37.5
34.5 33.8
10:0
12:O
14:O
16:O
18:O
1.2 1.1
6.3 4.9 4.8 4.95.6 5.3
5.6 5.6 5.3
25.8 26.3 25.0 26.126.8 26.6
8.2 8.5
5.6-
1.4 20.1 1.9
(C6412)
4.6
12.7
5.9 5.4 34.4 4.5 46.5* 5.9
2.9 2.4
13.0 1.6
3.7 5.7 8.6
11.5
7.4
(‘l4<24)
22.7
3.3
10.8 23.925.4 24.8 21.4 23.3
6.2 2.53.7 5.2 3.8 10.2
161
1.0 29.4* 0.2 34.0* 0.8
0.3 3.6
18:lc9
18:2n-6
18:3n-3
20:4n-6
32.6 32.9 36.0 30.232.2 32.5
10.6 12.0 11.4 11.512.8 11.9 14.6 11.6 17.8 12.8 12.512.6 23.4 16.5 12.4
0.8 0.6 0.7
ND 0.3 1 <0.1 0.30.4 0.4
-
39.0 33.0 34.s 36.7 39.5 30.6 32.7
0.60.65 1.o 1.5 1.3 1.65 1.4 1.12.7 2.6 0.3 1.1
20511-3
22:6n-3
ND
0.04
ND 0.17
0.03
0.1 50.25 0.24
ND ND
ND
ND ND
ND ND
ND
ND
Friedrichsdorf, Germany; preterm infant formula. Source: Reference 28. bMilupa, Friedrichsdorf, Germany (containingLC-PUFA from egg phospholipids, milk fat, and vegetable oils). Source: Reference 31. ‘Milupa, Friedrichsdorf, Germany (containingLC-PUFA from egg phospholipids, milk fat, and vegetable oils). Source: Reference 174. dMead Johnson Nutritional Group; Evansville, IN. Source: Reference 32. Wutricia, Zoetermeer, The Netherlands; 6:0-8:0, 3.3%. Source: Reference 33. ‘Bl&ldina-sa, Croupe Danone, Villefranchesur-Saijne, France. Source: Reference 175. WyethAyerSt Laboratories, Radnor, PA; preterm formula; 8:0,9.3%.Source: Reference 65. Loders Croklaan, Wormerveer, The Netherlands; synthesized TAG formulas with high proportions of 16:O in sm2 position (Betapl’M). Source: Reference 162 Loders Croklaan, Wormerveer, The Netherlands; synthesized TAG formulas with high proportions of 160 in sm2 position (Betapol-2). Source: Reference 163. j Nutricia, Zoetermeer, The Netherlands. Source: Reference 176. Nestec, Vevey, Switzerland; 4@&0,4.0%. Source: Reference 45. ‘Total saturatedor monounsaturatedFA. ND, not detected.
Lipids in Infant Formulas
62 7
FA of both infant formulas (Table 25.9) and human-milk lipids (Table 25.1). Most of the commercial formulas contain levels of a-linolenic acid (Table 25.9) similar to those in human-milk lipids (Table 25.1). In contrast, most of the formulas contain distinctly lower levels of ARA, and n-3 EPA and DHA (Table 25.9) compared with human-milk lipids (Table 25.1). Infants, particularly preterm infants have a limited ability to synthesize LCPUFA. However, these FA are important for normal development of infants, e.g., visual and brain development. Although present in human milk, LC-PUFA have been almost completely excluded from infant formulas for quite a long time. Recently, several formulas were propagated for both preterm and healthy term infants containing GLA and/or LC-PUFA (Table 25.10; 121,177-179).
Perspectives It appears that randomized trials with standard methodologies and follow-up of preterm infants beyond 12 mon are necessary to assess more precisely the extent of benefit offered by LC-PUFA supplementation (26,27). It is generally accepted that in both preterm and term infants, the LC-PUFA status at birth affects postnatal LCPUFA changes (175,180-182); however, this has not been taken into account in most of the studies designed to assess the benefits of various ratios of LA:ALA and the presence of LC-PUFA in infant formula. Thus, further research is warranted (25). The studies carried out to date suggest (25) that to prevent postnatal LCPUFA deficiency, formula-fed infants should be given formulas with a lower LA:ALA ratio than currently in use. Moreover, supplementation of formula with DHA appears to be necessary to attain a DHA status comparable to that of breastfed infants. The addition of ARA to the formula should also be considered because increasing levels of ALA and DHA tend to reduce the ARA status of infants. A few international advisory bodies recommend the supplementation of formulas with LC-PUFA (101,183,184), and the European Union Directive as well as the current draft standard of the Australian and New Zealand National Food Authority allow the addition of LC-PUFA as “optional” nutrients (27). A group of investigators recommends that formula for term infants should contain at least 0.2% of total FA as DHA and 0.35% as ARA to keep the biochemical LC-PUFA status comparable to that of breast-fed infants (177,179). However the FDA considers that more research is required before a decision on recommended supplementation of infant formula with LC-PUFA can be taken (185). Very recently, the plausible mechanisms for effects of LC-PUFA on the growth of infants were discussed in a critical review ( 186). Concentrates of LC-PUFA, prepared enzymatically from plant, marine, and microbial oils via lipase-catalyzed kinetic resolution (130) would be useful additives for the fortification of infant formulas with physiologically active FA. Enzymatically synthesized structured TAG containing 16:O at the sn-2 position and unsaturated fatty acyl moieties, including ARA (164) and DHA, would be generally beneficial for use
N 0
TABLE 25.10 Proportionsof y-Linolenic Acid (GLA) and/or Long-Chain PUFA (LC-PUFA) in Commercial Infant Formulasa
m
Proportion of CLA and LC-PUFA (YO) infant formulas
Lipids added as supplements for GLA or LC-PUFA
Preterm and low birthweight infants Humana Ob Humana 0 HAb Aletemil preterm formulaC Nutriprem LBWd Prematile Frisopre' Beba preterm formula, PreNidal + LC-PUFAg OsterPrem with LC-PUFA" PreCallia au DHA' BleviPrend Nenatalk PremiIon' SMA Low BirthweighP
sco sco sco
Healthy term infants Humana ESb Blernil 1 plud Beba Start HAg Karicare' Adapta 90 lniciom PreAptamile
Marine oils Egg lipids E g g lipids SCO, tuna oil Marine oil Evening primrose oil,
Borage oil,
egg lipids
18:3n-6
20:4n-6
20:5n-3
0.2
0.2
Black currant seed oil, egg lipids, marine oil Borage oil, egg lipids, marine oil Evening primrose oil, egg lipids Borage oil, marine oil Black currant seed oil, egg lipids, marine oil
0.3 0.4 0.2 0.35 0.3
Borage oil, marine oil Marine oil Egg lipids
0.9
0.1 0.4 0.4 0.2 0.1 0.1
0.1 0.3 0.6 0.45 0.6
0.3 0.3 0.4
0.2
egg lipids
0.2
22:6n-3
0.4
0.3 0.35 0.2 0.3 0.3 0.5 0.4 0.15 0.4 0.3 0.4 0.2 0.15 0.15 0.2 0.2 0.2
aSource: Reference 179. See b r n for suppliers: bHumana, Herford, Germany; cAlete, Munich, Germany; *owe & Gate, Trowebridge, U.K.; eMilupa, Friedrichsdorf;Germany; 'Friesche Flag, Leeuwarden, The Netherlands; gNestl6, Vevey, Switzerland; "arley, Nottingham, U.K.; 'Gallia, Paris, France; jLaboratoriosOrdesa, Barcelona, Spain: kNutricia. Zoetermeer. The Netherlands; 'Nutricia Ltd., Auckland, New Zealand; Wander, Bern, Switzerland.
Lipids in lnfant Formulas
629
in formulas for term and preterm infants. Structured TAG containing 16:O at the sn-2 position and medium-chain acyl moieties (4:O to 1O:O) in the sn-1,3 positions could provide a source of rapid energy in special infant formulas due to rapid cleavage of t h e MCFA f r o m t h e sn- 1,3 positions a n d their direct catabolism i n t h e liver ( 165,166).
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183. British Nutrition Foundation, Unsaturated Fatty Acids: Nutritional and Physiological Significance, Chapman & Hall, London, UK, 1992. 184. FAO/WHO Expert Committee: Fats and Oils in Human Nutrition, Food and Nutrition Paper No. 57,FAO, Rome, Italy, 1994. 185. Raiten, DJ., J.M. Talbot, and J.H. Waters, Assessment of Nutrient Requirements for Infant Formulas, J . Nutr. 128: 20593-22938 (1998). 186. Lapillonne, A., S.D.Clarke, and W.C. Heird, Plausible Mechanisms for Effects of Long-Chain Polyunsaturated Fatty Acids on Growth, J . Pediatr. 143: S9-Sl6 (2003).
Chapter 26
Cocoa Butter, Cocoa Butter Equivalents, and Cocoa Butter Replacers Kazuhisa Yamada, Masahisa Ibuki, and Thomas McBrayer Fuji Vegetable Oil, Savannah, CA 31408
Introduction Chocolate and confectionery compounds are loved by people throughout the world. However, these products contain >30% of fats such as cocoa butter (CB), CB equivalents (CBE), CB replacers (CBR), CB substitutes (CBS), and milk fat. With more attention to a healthier lifestyle, people tend to avoid oils and fats due to their association with the development of obesity and cardiovascular disease. Evidence exists concerning the relation of cardiovascular disease and an increase in LDL cholesterol concentration when people consume too much fat. On the other hand, a number of interesting papers and findings exist regarding the effect of the fats used in chocolate (compounds) and positive health benefits. In this chapter, we review the following: (i) classification of fats for chocolate (compound); (ii) the effect of CB and CBE on health; and (iii) the healthy fats for chocolate (compound) and their applications.
The Classification of Fats for Chocolate (Compound) In this section, the chemical and physical properties, the characteristics, and production processes of the four types of fats for chocolate (compound), i.e., CB, CBE, CBR, and CBS, are examined. Cocoa Butter
CB is obtained through the crushing and grinding of cocoa beans, which are grown in countries close to the equator. The functions of CB include the following: (i) it is an important ingredient in chocolate, accounting for almost 30%; (ii) the cocoa aroma spreads smoothly in the mouth because of rapid melting at near body temperature; (iii) it preserves the oil-soluble aroma; (iv) it is a source of energy; and (v) it is a carrier of oil-soluble vitamins. Composition of Cocoa Butter. CB consists mainly of sn-1,3 distearoyl sn-2 oleoyl acyl glycerol (SOS), sn-1 palmitoyl sn-2 oleoyl sn-3 stearoyl acyl glycerol (POS), 642
Chocolate Fats
643
TABLE 26.1 Major Symmetric TAG of Cocoa Butters (CB) and Typical Cocoa Butter Equivalent (CBE)a Symmetric TAG Brazilian CB Ivory Coast CB Malaysian CB Typical cocoa CBE
25 26 27 33
36 39 38 12
18 19 18 35
5OS, sn-1,3 distearoyl sn-2 oleoyl acyl glycerol; POS, sn-1 palmitoyl sn-2 oleoyl sn-3 stearoyl acyl glycerol; POP, sn-1,3 dipalmitoyl sn-2 oleoyl acyl glycerol.
and sn-1,3 dipalmitoyl sn-2 oleoyl acyl glycerol (POP), which are represented by symmetric triacylglycerols (TAG). These symmetric TAG cause CB to melt rapidly at body temperature. Table 26.1 shows the typical composition of major TAG. Brazilian CB contains less SOS and POS than Ivory Coast and Malaysian CB. This means that Brazilian CB is softer than the others. Usually CB has excellent oxidative stability because it contains >90% fatty acids (FA) that have high oxidative stability. Typical FA data are shown in Table 26.2. The sum of palmitic, stearic, and oleic acids, which have high stability, is >90%. Physical Properties and Characteristics of CB. Figure 26.1 shows the solid fat content (SFC) of CB. Malaysian CB is the hardest, and Brazilian CB is the softest. The SFC profile indicates the heat resistance of the chocolate. Chocolate made with Brazilian CB has poor heat resistance; however, it has good mouth melting behavior. Table 26.3 shows the characteristics of each CB. Cocoa Butter Equivalents
The major TAG and FA of a typical CBE are shown in Tables 26.1 and 26.2, respectively. The only difference between a typical CBE and CB is the ratio of symmetrical TAG. CBEs contain higher levels of SOS and POP than CB because CBEs are a combination of palm mid-fraction and SOS fats. The commercially TABLE 26.2 Major FA Composition of CB and a Typical CBEa Stearic
Palmitic
Oleic
Major FA
(YO)
(YO)
(%)
Brazilian CB Ivory Coast CB Malaysian CB Typical cocoa CBE
30 32 34 31
26 26 26
38 36 36
31
34
5 e e Table 26.1 for abbreviations.
K. Yarnada eta/.
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100
I
I
80 h
8
v
70
c-'
6
c)
c
60
8
50
5
40
0
5 30
Fig. 26.1. Solid fat content of cocoa butters (CB) and a typical cocoa butter equivalent (CBE): (+), Brazilian CB; (A), Malaysian CB; (W), Ivory Coast CB; and (O),typical CBE.
v)
20 10
0 15
20
25
30
35
40
Temperature ("C)
available sources of SOS fats are fractionated enzymatically interesterified sunflower and/or safflower oil, fractionated shea nut oil, and fractionated sal fat.
CBE by Enzymatic Interesterification. CBE, especially fractionated enzymatically interesterified CBE, have many advantages: Reliable supply and stable price. Sunflower and/or safflower oil, the raw materials of enzymatic interesterification, are readily available. Therefore, the supply is reliable and the price is stable. On the other hand, shea nut oil and sal fat are wild plants, and the supply and price are not stable. Stable quality. The quality of CBE produced by the enzymatic interesterification method is quite stable because of the quality of the raw-material oils and the adjustable enzymatic interesterification reaction. Therefore, quality aspects such as acid value or cooling curve, which are critical for the subsequent processes, are very consistent. Excessive processing cost is thus avoided. Safety. In Japan, certain enzymes are permitted to be catalysts of the interesterification reaction by Agricultural Standard (JAS). CBE have been produced by TABLE 26.3 The Characteristics of CBa ~
Brazilian CB Ivory Coast CB Malaysian CB aA, excellent;
~~~~~~~~~
Mouth feel
Snap
Tempering property
Heat resistance
A B
B A
B
C
A
B
A
A
A A
B, good; C, insufficient. See Table 26.1 for abbreviations.
Chocolate Fats
645
enzymatic interesterification for >15 years since the Fuji Oil Company made the process available in 1988. In the United States, the use of high-oleic sunflower and safflower oils in CBE was granted “generally recognized as safe” (GRAS) status in 1996, and lipase from Rhizopus niveus was granted GRAS status by the U.S. Food and Drug Administration (FDA) in 1998. In Canada, the lipase R. niveus was approved by Health Canada in 1998. This lipase has sn-1,3 specific activity, which is the same selectivity as human pancreatic lipase. Therefore, CBE by enzymatic interesterification can be safely hydrolyzed in the human body. In addition, the raw materials, sunflower and safflower oil, are domestic oils that have been consumed for a long period of time and are quite familiar to consumers compared with shea nut, sal fat, or illipe fat.
Process of CBE by Enzymatic Interesterification. Generally, lipase works as the catalyst for hydrolysis and splits TAG into FA and MAG similar to what occurs when lipase and oil exist under high water-content conditions such as in the human small intestine. However, when lipase and oil exist under quite low moisture conditions, lipase works as the catalyst for interesterification, which is a reversible reaction (Fig. 26.2). Enzymatic interesterification is catalyzed by an immobilized lipase under quite low levels of moisture. Enzymatic interesterification and the CBE production process are summarized in Figure 26.3. Characteristics of CBE. Although CBE resemble CB, they are not only less expensive but also make up for the deficiencies of CB. The merits of CBE are as follows: (i) lower costs; (ii) improvement in the heat resistant, of chocolate; and (iii) improvement in the bloom resistance of chocolate. The occurrence of bloom can be significantly
Fig. 26.2. Hydrolysis and interesterification reaction by lipase.
K. Yamada et a/.
646
lnteresterification
E!+s-
Immobilized sr1-1~3Specific Lipase
High-Oleic Sunflower Oil andor Safflower 011
E% E:
0
+s,o+
I
t-O FractlonaUon
Bleaching, and Deodorlzatlon
I I
Fig. 26.3. The process scheme of CBE by enzymatic interesterification. See Figure 26.1 for abbreviation.
delayed with the addition of CBE at 5% of the chocolate recipe. For example, in large countries that have substantial differences in temperature between the northern and southern regions, CBE are quite effective in preventing bloom. Cocoa BufferReplacers
CBR are defined as nonlauric vegetable fats with physical, but not chemical, characteristics similar to those of CB. They are nontempering alternatives to CB because they readily form stable crystals without tempering. However, chocolate made from CBR has inferior mouth-feel characteristics compared with chocolate made from CBE.
Production Process and Characteristics of CBR. The raw materials include palm oil, rice bran oil, soybean oil, and cottonseed oil. After hydrogenation, they are fractionated to achieve a suitable melting profile for chocolate compounds. Their merits include elimination of the tempering process, reduced costs compared with CB, heat resistance for compound chocolate, and high oxidative stability. However, fat compatibility with CB is only 25% at a maximum of the total fat content, not in the compound recipe, whereas CBE are 100%compatible. Therefore, the compound chocolate made from CBR will be deficient in cocoa flavor.
Chocolate Fats
647
The Chemical and Physical Properties of CBR. Most unsaturated FA of CBR convert to the trans-form from the cis-form during the hydrogenation process. Typical TAG and SFC of CBR are shown in Table 26.4 and Figure 26.4. The Regulation of Trans Fat. Conventional CBR are partially hydrogenated and fractionated soybean oil, cottonseed oil, or palm oil. Typically, they contain significant levels of trans FA due to partial hydrogenation. The FDA announced the final regulation regarding the labeling of trans fat content on July 11, 2003. According to the regulation, mandatory declaration of the trans content must be listed on the nutrition facts panel of food products. The regulation has a compliance date of January 1, 2006, with the following key points: (i) Separate line item below the declaration for saturated fat with trans fat to be listed in grams per serving; (ii) no % daily value (DV) required; (iii) trans fat content to be expressed to the nearest 0.5-g increment c5 g and the nearest gram increment >5 g; (iv) if the amount of trans fat is c0.5 g, the amount will be expressed as “0”;(v) the definition of trans fats is all unsaturated FA that contain one or more isolated (i-e., nonconjugated) double bonds in a trans configuration. Trans vaccenic acid and other trans acids of ruminant origin are included in the definition (1). Conventional CBR would contain >0.5 gherving in compound chocolate. In the announcement, the FDA says that the scientific evidence shows that consumption of saturated fat, trans fat, and dietary cholesterol raises LDL (or “bad”) cholesterol levels, thus increasing the risk of coronary heart disease. Therefore, no or low trans alternatives to conventional CBR are in demand. The fats-and-oils industry has been working hard to develop these no and low trans fats not only for the chocolate and compound products but also snack foods and baked goods. Cocoa Butter Substitutes
In the United States, lauric fats, such as fractionated or fully hydrogenated and fractionated palm kernel oil and hydrogenated coconut, are defined as cocoa butter TABLE 26.4
Typical TAG Composition of Soybean Oil-Based Cocoa Butter Replacersa TAG
0%
OTT POT TTT PPO PTT PPT SOT STT
10 6 25 2 20 2 10 10
aO, oleic acid; P, palmitic acid; S, stearic acid; T, trans FA.
K. Yamada e t a / .
648
100 90
80 h
70 4-
5 C 8
4-
60 50
c
P
40
5 30
0
20 Fig. 26.4. Solid fat content of typical cocoa butter
10
0 15
20
30 Temperature ("C) 25
35
40
replacer (CBR): (+), typical palm oil-based CBR; (M), Malaysian cocoa butter.
substitutes (CBS). Typical applications are in compound chocolate coatings or center fillings. These lauric fats have either zero or low trans content. Production Process and Characteristics of CBS. The palm kernel oil (PKO), which is the main raw material, is fractionated and/or hydrogenated. The hard fraction of PKO is called palm kernel stearin, and it is hydrogenated to increase heat resistance. The merits of CBS include the following: elimination of trans fat, elimination of the tempering process, low cost compared with CB, excellent mouth feel compared with CBR, and high oxidative stability. The Chemical and Physical Properties of CBS. CBS are produced from lauric oil, like PKO. Therefore, the major FA are lauric and myristic. The FA composition and SFC of typical CBS are shown Table 26.5 and Figure 26.5. The trans content of hydrogenated palm kernel stearin is zero (
The Effects of CB and CBE on Health The ingredients in chocolate include cocoa liquor, CB, sugar, and emulsifiers such as lecithin. The reason why chocolate has excellent mouth feel and flavor release is due to the quick melt of CB. The m.p. of CB is between 30 and 35"C, which is lower than mouth temperature. Therefore, chocolate melts quickly in the mouth, and we are able to feel the cocoa aroma in the mouth. The m.p. of CB is due to the unique TAGs, SOS,POS, and POP. These symmetric TAGs are ester-bonded stearic and/or palmitic acid at the sn-1 and -3 positions.
Chocolate Fats
649
TABLE 26.5 Major FA Composition of Typical CB Substitutes Lauric
Myristic
Oieic
Major FA
(OIO )
(O h)
(Yo)
Palm kernel stearin Hydrogenated palm kernel stearin
55 55
22 22
0.5
7
Their existence in nature is rare. CBE are composed of the same type of symmetric TAG and have the same FA composition. The only difference between CB and CBE is the ratio of the symmetric TAG combined sn-1,3 saturated FA (SFA) (Tables 26.1and 26.2,Fig. 26.1). On the other hand, CB and CBE are not associated with an increase in the plasma cholesterol level, even though they contain nearly 80% SFA. The lack of adverse effects seems to be related to the difference in chain length and the positional distribution of the SFA in TAG vs. that of animal and other vegetable fats composed of highly SFA. The sn-1,3 stearoyl andor palmitoyl disaturated symmetric TAG characterize the absorption and metabolism of CB and CBE. LDL Lipoprotein Concentrations and the Absorption of CB and CBE Foods containing high levels of SFA have been viewed negatively since the 1990s because they are associated with an increase in the cholesterol level in serum. However, many investigators found that CB does not increase cholesterol although it contains -80% SFA. Chen et al. (2)dosed rats with aqueous emulsions containing CB, PKO or corn oil through surgically inserted duodenal and thoracic duct catheters. The FA composition in collected lymph was reflected in the dietary fat. For the recovery of total absorbed dietary FA in thoracic duct lymph, if corn oil absorption is expressed as loo%, the amount of total FA recovered in the PKO and CB groups was 82.3 and 63.0%, respectively. There was no significant difference in absorption with corn oil or PKO feeding. However, there was a significant reduction ( P < 0.01) in total FA recovered in the lymph after CB feeding. Chen et al. (2) also measured the effect of CB on intestinal absorption of cholesterol in the thoracic duct. The results indicated that CB inhibited the absorption of cholesterol. Questions arose concerning possible effects with human subjects. Several investigators studied this issue to determine the effects on the human body. Denke and Grundy (3) compared serum lipid and lipoprotein concentrations in men with hypercholesterolsterolemia after consumption of four fats and oils: butter fat, beef tallow, CB, and olive oil. The percentages of energy from total SFA were 25.4, 18.8,23.2,and 8%, respectively. The percentages of energy from C,,, C,,, and C,,
650
K. Yamada et a/.
h
8
v
c
c
a
c
E
0
0
5
'c
-
0
$ Fig. 26.5. Solid fat content of typical cocoa butter substitutes.+, hydrogenated palm kernel steari n; palm kernel stearin; I, Malaysian cocoa butter.
+,
Temperature ("C)
were 17.6, 11.2, 10.0, and 6.8%, respectively. Butter fat raised LDL lipoprotein concentrations the most (4.23 f 0.15 mmol/L). Beef-tallow feeding resulted in significantly lower concentrations of LDL (4.03 k 0.18 mmoVL), with CB even lower (3.82 k 0.15 mmoVL). Olive oil resulted in the lowest concentrations (3.62 A 0.18 mmoVL). The rank order for raising LDL lipoprotein concentration is butter fat > beef tallow > CB > olive oil. This rank order matched the rank order of the percentages of energy from C,,, C,,, and C16. Additionally, fecal excretion of FA after adjustment for fecal flow indicated that oleic acid (Clgz1)was 99% absorbed, palmitic acid (C16:o)was 9 6 9 7 % absorbed, and stearic acid was 90-94% absorbed for the three fats containing significant amounts of stearic acid. The lipid-raising effect of CB containing total SFA is less than other fats containing comparable amounts of SFA. Furthermore, Kris-Etherton and Mustad (4) showed the effect of chocolate feeding in humans. They found that a milk chocolate diet did not significantly increase LDL cholesterol concentrations in humans. In general, CB or chocolate does not increase the LDL cholesterol level. This appears to be related to stearic acid metabolism; however, it does not derive from a single metabolism pathway. Emken (5) summarized his study regarding the metabolism of dietary stearic acid in humans. He suggested that the metabolic differences between stearic and palmitic acids are as follows: (i) desaturation of stearic acid is 2.4-fold higher than desaturation of palmitic acid; (ii) plasma total lipid contained 12% more palmitic acid than stearic acid; and (iii) the percentage absorption of stearic acid was 7% less than that of palmitic acid. Although symmetric TAG such as CB and CBE were not examined in that study, the sn-1,3 diSFA of CB and CBE were split into free SFA in the small intestine by lipase.
Chocolate Fats
651
Effect of Positional Distribution on SFA Position in Plasma Cholesterol Concentrations
CB does not significantly increase LDL cholesterol concentrations compared with the high stearic and palmitic acids described above. In the case of stearic acid, the reasons can be explained by the quick desaturation and lower absorption. On the other hand, CB and CBE contain -25% palmitic acid and are composed of symmetric TAG. Several investigators compared symmetric with asymmetric TAG to determine how the sn-2 position works in plasma cholesterol concentrations. Most sn-2 positions in dietary fat are maintained without hydrolysis by lipase until digestion in the small intestine. The effect of positional distribution on SFA in plasma cholesterol is a topic of interest. Fukui et al. (6) examined this effect using synthesized high-purity POP (80.2%) and PPO (84.4%) with a 1,3 specific lipase. Rats were fed a POP or PPO diet with and without cholesterol. Rats fed the PPO had significantly increased plasma total cholesterol concentrations compared with rats fed POP with and without cholesterol, whereas there was no significant difference in weight increase, TAG, and HDL cholesterol (Table 26.6). Kritchevsky et al. (7) demonstrated the same results in rabbits. They were fed not only PPOPOP with cholesterol but also SOS/SSO with cholesterol. Serum lipids and the degree of atherosclerosis were measured. The results suggest that PPO increased the plasma cholesterol concentration and was the most atherogenic fat compared with SOS, SSO, and POP. However, in the case of humans, Zock et al. (8) showed no significant differences between palmitic acid rich in sn-2 position and sn-1 or -3 position in blood lipoprotein concentrations of fasting subjects. They compared palm oil, which has 82 and 18% of palmitic acid at the sn-1 or -3 position and sn-2 position, respectively, with enzymatically modified palm oil, which has 35 and 65%, respectively. With consumption of TABLE 26.6 Concentration of Lipids in Rats Fed Diets Containing 1,3-Dipalmitoyl- 2-oleoyl Glyceride (POP) or 1(3), 2-Dipalmitoyl-3(1)-oleoyl-glycerol-RichOil (PPO) with and Without Cholesterol for 2 wkaIb Dietary group POP+C
PPO+C
POP
PPO SEM Cholesterol
mrnol/L
TG Total cholesterol HDL cholesterol
0.8F
l.lOe
4.32e 1.31
5.2gd 1.12
3.0Id 2.529 1.23
2.72 3.Ogf 1.52
0.22 0.23 0.06
0.01 0.01 NS
ANOVA position C x Pc NS 0.01 NS
NS NS NS
aSource: Reference 6.
values are means, n = 7 rats/group, d-gMeans in a row with different superscripts differ, f'< 0,05; NS, not significant. cC x P =cholesterol x FA position.
652
K. Yarnada e t a / .
the modified palm oil diet, the average lipoprotein concentrations showed a nonsignificant increase for total, HDL, and LDL cholesterol compared with palm oil (Table 26.7). Zock et al. (9) showed that a modified palm oil diet increased palmitic and palmitoleic acids in cholesterol esters at the expense of oleic and linoleic acids. Thus, they suggested that the positional configuration of dietary FA has small but consistent effects on lipid metabolism that persist beyond fat absorption and chylomicron clearance. The effect on health of positional distribution of SFA in plasma cholesterol is not clear. As the above researchers found, it seems that the modification of fats by a sn-1,3 specific enzyme helps to clarify this issue.
The Healthy Fats for Chocolate and the Applications In this section, some of the applications of healthier CBE and CBR are presented. Lower Absorption by Calcium-Fortified CB and CBE
CB and CBE composed of symmetric TAG are split into stearic and palmitic acids and sn-2 oleoyl MAG by pancreatic lipase in the small intestine. However, Denke and Grundy ( 3 ) showed that the absorption of stearic and palmitic acids from CB in humans is 90-94 and 96-9796, respectively. CB is well absorbed in humans. However, it is not known whether the absorption of palmitic and stearic acids is lower with calcium-fortified foods. Fukui et al. (10) investigated the effect of dietary Ca on the absorption ability of POP and CB as symmetric TAG, PPO feeding as asymmetric TAG, corn oil as low SFA content oil, and chocolate made from CB. High-purity POP and PPO were prepared by enzymatic interesterification, and the compositions of the test fats are shown in Table 26.8. Chocolate recipes are shown in Table 26.9 and the compositions of all diets are shown in Table 26.10. The authors showed that the apparent absorption efficiency of fat is lower in rats fed Ca-fortified CB and POP diets than in those fed unfortified CB and Ca-fortified PPO diets. The low apparent absorption efficiency of fat was due to the low absorption efficiency of palmitic and stearic acids (Table 26.11). The fat energy was 10% lower in the Ca-fortified CB diet group than in the unfortified CB diet group and was 18% lower in the Ca-fortified chocolate diet group than in the unfortified corn oil diet group (Table 26.12). Although the apparent absorption efficiency of energy was lower in the unfortified chocolate diet group than in the unfortified corn oil diet group, the apparent absorption efficiencies of palmitic and stearic acids in chocolate were greater than those in CB even with Ca fortification. The apparent absorption efficiency of Ca was lower in rats fed Ca-fortified diets than in the unfortified groups; however, the absolute amount of absorbed Ca did not differ among the groups. The authors suggested that Ca fortification decreased the apparent absorption efficiency of fat with long-chain SFA, particularly at the sn- 1,3 positions of TAG.
Chocolate Fats
653
TABLE 26.7 Serum Lipid and Lipoprotein Cholesterol Concentrations in Subjects Consuming Diets High in Palm Oil and an Enzymatically Modified Palm Oil Analog in Which Palmitic Acid Is in the sn-2 Position Instead of the sn-l,3 Positionsaib
Total cholesterol All Women (n = 37) Men (n = 23) HDL cholesterol Al I Women Men Non-HDL cholesterol A1 I Women Men LDL cholesterol Al I Women Men HDVLDL ratio Al I Women Men
Palm oil diet (mmol/L)
Modified-fat diet (mmol/L)
Change (95% confidence interval)
4.66 i 0.90 4.89 i 0.84 4.31 i 0.89
4.72 i 0.94 4.92 i 0.88 4.41 0.96
0.06(-0.02,0.14) 0.03(-0.09,0.16) 0.10(+0.02,0.18)*
1.60 i 0.33 1.77 i 0.24 1.33 i 0.26
1.79 i 0.31 1.37 i 0.29
0.03(-0.01,0.07) 0.03(-0.04,0.09) 0.04(-0.00,0.08)
* 1.63 * 0.37
*
3.07 i 0.85 3.12 f 0.78 2.98 i 0.94
3.09 0.86 3.13 i 0.79 3.04 i 0.97
0.03(-0.04,0.09) 0.01 (-0.08,O.lO) 0.06(-0.02,0.13)
2.62 f 0.78 2.69 i 0.76 2.51 i 0.81
2.66 i 0.80 2.71 i 0.76 2.59 i 0.86
0.04(-0.03,O.lO) 0.01(-0.08,O.ll) 0.08(+0.00,0.15)*
0.67 0.29 0.70 i 0.20 0.62 i 0.39
0.67 i 0.30 0.70 i 0.1 9 0.63 i 0.41
O.OO(-0.02,0.03) O.OO(-0.04,0.04) O.OO(-0.02,0.03)
0.97 i 0.42 0.92 i 0.33 1.04 i 0.54
0.94 i 0.40 0.91 i 0.38 0.99 i 0.44
*
TC All Women Men
-0.03(-0.08,0.03) -0.02(-0.08,0.05) -0.04(-0.16,0.07)
aSource: Reference 8. bValues are means SD; n = 60. *Significantly different from 0, P < 0.05. Subjects consumed each diet for 3 wk, in different order. To convert values for total, HDL, and LDL cholesterol to mg/dL, multiply by 38.67. To convert values for TAG to mg/dL, multiply by 88.54.
*
In the case of humans, Murata et al. (11) and Shahkhalili et al. (12) investigated this independently. The former group showed that the absorption rate of Ca-fortified chocolate feeding was significantly lowered to 73.2 from 90.1% of unfortified chocolate feeding. The absorption difference between Ca-fortified chocolate and unfortified chocolate was due to the significantly lower absorption rate of longchain SFA ( X I & . Shahkhalili et al. (12) showed that the digestibility of the Casupplemented chocolate feeding was 87% compared with the digestibility of unsupplemented chocolate feeding. In addition, the Ca-supplemented chocolate feeding significantly decreased LDL cholesterol concentration 15% more than unsupplemented chocolate feeding. The scheme of this lower absorption mechanism is shown in Figure 26.6.
K. Yamada e t a / .
654
TABLE 26.8 TAG Composition of Oils in an Investigation on Absorptionatb TAG 1 .o 1.9
POL PLP
MOP 000 PO0
0.5 3.6 18.3
POP PPO PPP
-
so0
3.9
sos PSS
AOS
sss OthersC
-
1.3
2.2 0.9
-
-
-
37.2 0.6 0.7 25.7 0.9 1.9 0.5 3.1
5.2 0.2
4.3 0.7
SLS
A00
0.3
84.8
OPO
POS PPS
0.1 0.1
0.1 0.3 0.5 0.7 5.3 80.2
-
-
-
-
-
4.2
5.2
aSource: Reference 10. See Table 26.6 for other abbreviations. bAbbreviations used for acyl chains in the TAG: M = C,:, (rnyristic), P = C,:, C,8:2 (linoleic), S = CI8:, (stearic), A = C,:, (arachidic). Cothersare unknown.
(palmitic), 0 = C,:,
(oleic), L =
TABLE 26.9
Composition of Chocolate in an Investigation of Absorptiona Choco Component Cocoa massc Whole-milk powderC Sugar Calcium powderd CaCO, Cocoa butter Lecithin Vanillin Fat ( O h ) Calcium (mg/g)
(Oh
1
16.0 16.0 48.0
20.0 0.4 0.03 32.9 0.1
Choco + Cab
Choco + Capb
(YO)
(Oh)
16.0 16.0 45.1
-
16.0 16.0 44.9 3.1
2.9 20.0 0.4 0.03 32.9 11.7
20.0 0.4 0.03 32.9 11.7
-
aSource: Reference 10. bCa carbonate and Ca powder contain 40 and 38.4% Ca, respectively. Change of chocolate composition by Ca addition was adjusted by sugar. ‘The fat content of cocoa mass and whole-milk powder was 8.8 and 4.1%, respectively, and that of chocolate was 32.9%. Ca is calcium carbonate. CaP is calcium powder from bone powder.
TABLE 26.1 0
Composition of Diets in Experiments on Absorption” Experiment 1 Components Casein m-Methionine a-Cornstarch Sucrose Experimental oilc Chocolated Cellulose powder Mineral mixturee Vitamin mixturee Choline bitartrate Calcium carbonate Protein (%) Fat (%) Calcium (mg/g) Energy (kcal/g)
Experiment 2
CB
CB + Ca
POP + Ca
PPO + Ca
Corn
Corn + Ca
Chocolate
20.0 0.3 15.0 45.0 10.0 -
20.0
20.0 0.3 15.0 44.1 1 10.0
20.0 0.3 15.0 44.1 1 10.0
20.0 0.3 15.0 45.0 10.0
20.0 0.3 15.0 44.1 1 10.0
20.0 0.3 15.0 24.6 -
-
-
-
-
-
-
5.0 3.5 1 .o 0.2 0.89
5.0 3.5 1.o 0.2 -
5.0 3.5 1 .o 0.2 0.89
30.4 5.0 3.5 1 .o 0.2
30.4 5.0 3.5 1.o 0.2 -
30.4 5.0 3.5 1 .o 0.2 -
17.0 10.0 8.1 4.58
17.0 10.0 4.6 4.49
17.0 10.0 8.1 4.48
20.9 10.0 8.1 4.67
20.9 10.0 8.1 4.68
0.3 15.0 44.1 1 10.0 -
5.0 3.5 1 .o 0.2 -
5.0 3.5 1 .o 0.89
5.0 3.5 1.o 0.2 0.89
17.0 10.0 4.6 4.65
17.0 10.0 8.1 4.62
17.0 10.0 8.1 4.58
0.2
Choco + Cab
-
20.9 10.0 8.1 4.69
aSource:Reference 10. See Tables 26.1 and 26.6 for other abbreviations. %a is calcium cabonate. cap is calcium powder from bone powder. (PPO) and corn oil. ‘Txperimental oils are CB,1,3-dipalmitoyl-2oleoyl glycerol (POP), 1(3),2dipalmitoyl-3~1lmyl2-gIycerol dCa carbonateand Ca powder contain 40 and 38.4% Ca, respectively. Change of chocolate composition by Ca addition was adjusted by sugar. eAIN-76.
20.0 0.3 15.0 24.6
Choco + Capb 20.0 0.3 15.0 24.6
3 0 c, 0
2 ic;
P G:
TABLE 26.1 1 Apparent Absorption Efficiency of Food, Protein, Fat, Ash, and FAdpb FA
Experiment 1 CB CB + Ca POP + Ca PPO + Ca Experiment 2 Corn Com + Ca Choco Choco+Ca Choco + CaP
(6) (6) (6) (6)
90.77 f 0.1 6a 88.33 f 0.2Sb 88.26 f 0.1 7b 90.30 f 0.25a
92.62 f 0.03a 91.67 f 0.34b 91.53 f 0.56b 93.07 f 0.40a
75.25 f 0.78b 64.58 f 1.7gC 62.80 f 1.27c 82.14 f 1.34a
66.75 f 0.41 a 49.74 f 1.37b 50.71 f 0.88b 52.53 f 0.84b
63.5 f 0.3b 50.7 f 2.8= 41.4 f 0.8d 77.6 f 0.3a
(6) (6) (6) (6) (6)
93.20 f 0.20a 91.91 f 0.27b 89.89 f 0.26= 87.87 f 0.34d 87.77 f 0.34d
96.20 f 0.08a 94.51 f 0.36a,b 93.35 f 0.30b 92.55 f 0.53b*C 92.13 f 0.37c
96.49 f 0.37a 94.99 f 0.91 a 84.72 f 0.60b 79.82 f 0.72c 75.91 f 1.55d
71.63 f 0.96a 69.54 f 1.1 l a 7 2 . 1 6 i 1.42a 62.39 f 1.12b 56.92 f 1 .0gc
92.5 f O.ga 87.7 f 2.1 79.4 f 0.8c 72.2 f 0.8d 63.3 f 2.4e
52.9 f 0.Y 39.9 f 3 9
-
75.5 f 1.la 67.8 f O.gb 54.8 f 2.7=
aSourcer Reference 10. See Tables 26.1 and 26.6 for other abbreviations. bValues are means f SE. Means in a column with different superscripts within an experiment differ, P < 0.05 (Duncan's Multiple Range Test).
98.5 f 0.1 a 98.0 f 0.2a 89.2 f 0.4b 86.7 f 0 . F 98.1 f 0.3a 94.8 f 0.5c 97.7 f 0.3a 95.9 f 0.2b 95.8 f 0.4b
P
'' P
e
Chocolate Fats
657
TABLE 26.12
Energy of Lipid Ingested Food, Feces, and Absorbed Food and Apparent Absorption Efficiency of Energya,b
Group
(n)
ingested food (kcalid)
Experiment 1 CB CB + Ca POP + Ca PPO + Ca
(6) (6) (6) (6)
17.8 0.5b 17.6 f 0.5b 78.5 f 0.4a 16.7 0.3b
Experiment 2 Corn Corn + Ca Choco Choco + Ca Choco+CaP
(6) (6) (6) (6) (6)
16.7 0.3b 17.0 f 0.5a,b 18.3*0.7a,b 18.7 f 0.8a 17.9*0.6a,b
*
* *
Feces (kcal/d)
* *
0.2b 6.0 f 0.3a 6.6 0.3a 3.0 0.3c
4.2
*
Absorption of fat food (kcalid)
Absorption rate of fat foodC
13.6 i 0.5a 11.6 k 0.7b 11.9 f 0.3b 13.7 0.3a
76.5 0.8b 65.5 f 2.6c 64.3 f 1 .2c 82.1 1.6a
*
16.2 f 0.3a 16.1 f 0.5a 15.5*0.6a 15.0 f 0.6a,b 4.3~k0.3~ 13.6i~0.6~ 0.6 ~t0.lc 0.8 0.2c 2.8*0.2b 3.8 0.2a
* *
* *
96.6 f 0.4a 95.0 O.ga 84.5+0.6b 80.0 0.7c 76.1 +1.5d
* *
aSource:Reference 10. See Tables 26.1 and 26.6 for other abbreviations. balues in Experiment 1 are means i SE. Values in Experiment 2 are means. Means in a column with different superscripts within an experiment differ, P < 0.05 (Duncan’s Multiple Range Test). =Absorbed lipid energy/ingested food energy.
Increase in Stearic and Palmitic Acids in Ca-Fortified Chocolate
According to Table 26.1 1, the key point of Ca-fortified chocolate is to increase the stearic and palmitic acids in CB to reduce the absorption of FA as much as possible. However, there is a limitation to the amount that stearic and palmitic acids can be increased by using only CB. Healthier Cocoa Butter Improver to Increase Stearic Acid for Chocolate Coating Application. Healthier CBI can help to increase stearic acid. Table 26.13 shows the major FA content of Healthier CBI. Healthier CBI is one of Fuji’s CBI, which contains 56% stearic acid. Healthier CBI provides chocolate with not only an increase in stearic acid but also improved heat and bloom resistance. The solid fat content is shown in Figure 26.7. Palm Mid-Fraction to Increase Palmitic Acid for Center Filling Applications. The PMF can help to increase palmitic acid. PMF contains 56% palmitic acid and -70% sn-1,3 disaturate sn-2 oleoyl TAG. A typical recipe is shown in Table 26.14. PMF requires at least 9% powdered CaP to reduce the absorption of SFA. Additionally, PMF has a sharp melting profile. Therefore, it has superior flavor release and mouth feel. When PMF is applied for a center filling compound containing fresh flavor such as fruit or mint flavor, the flavors will be enhanced in the mouth. The solid fat content is shown in Figure 26.8.
--K. Yamada eta/.
658
s
ss
0
ss
0
s
0
CaCaCa
+
Dietary Food: Three Molecules of SOS TAG + 3 Molecules of Calcium
-------
4-
-OH
OH
0
0
s s
OH
c
OH OH
0
OH
s s
s s
SIX Molecules of FFA
Three Molecules of MAG
+
Ca2+ Ca2+ Ca*+ Three Molecules of Calcium
1
Bile Acid
s s s s s s \ / \ / \ /
Micellea
Ca
Ca
Ca
1
i
Insoluble in Water
Excretion with Feces
Absorption
Fig. 26.6. The excretion image scheme of Ca-fortified sn-l,3 distearoyl sn-2 oleoyl acyl glycerol TAG. S, stearic acid; P, palmitic acid.
SLS Fat: High Linoleic and Stearic Acid Nontempering Fat
Linoleic acid is one of the essential FA, and its intake can reduce LDL cholesterol concentration. In addition, linoleic acid is one of the sources for synthesis of arachidonic acid in humans. However, typical CB and CBR contain 4%of this TABLE 26.1 3 Stearic Acid Composition of Healthier Cocoa Butter Improver (CBI) and Blend Oil with Brazilian CBa Blend ratio (YO) Malaysian CB Healthier CBI Stearic acid Palmitic acid 3 e e Table 26.1 or other abbreviations.
100
34 26
85 15 37 23
70 30 41 19
100 56 4
Chocolate Fats
659
100
90 h
80 70
4-
c c
$
60
850
c
40 '0
=
CE
30 20 10 0
15
20
25
30
35
40
Temperature ("C)
Fig. 26.7. Solid fat content of healthier CB improver (0);(o), Malaysian CB.
essential FA. From the viewpoint of cholesterol, linoleic acid seems to be a healthier FA. Safflower oil is rich in linoleic acid, but it is a liquid oil and cannot be used for chocolate applications. If stearic acid, which does not affect LDL cholesterol, can be combined with safflower oil, the safflower oil becomes a solid fat and the triacylglycerol will be sn-1,3 stearoyl sn-2linoleyl (SLS) TAG (13-15). This SLS fat can be produced by using sn-1,3-specific enzymatic interesterification. The process is shown in Figure 26.9. Safflower oil is used as the highlinoleic source and is interesterified enzymatically. The major TAG of this fat are SLS and sn-1 stearoyl sn-2,3linoleyl glycerol (SLL). The typical linoleic and stearic acids contents are 36 and 49%, respectively. This is considerably higher than CB and typical CBR (Table 26.15). The SFC of the SLS fat is shown in Figure 26.10in a comparison with CB or typical CBR. The SLS fat is softer than the others and is suitable for center filling applications. In addition to its health benefits, the SLS fat has rather unique physiTABLE 26.1 4 Compound Recipe of Palm Mid-Fraction for Center Fillinga Ingredient
(Oh
)
Palm mid-fraction Lactose Whole-milk powder Strawberry powder Strawberry flavor CaP
40 10 20 3 0.3 0.3
Sugar
23.1
5 e e Table 26.9 or other abbreviations.
K. Yarnada eta/.
660
100
90 80 c
70
a c
60
C C
8
c c
m
50 40
30 20 10
0 15
25
20
30
35
40
Temperature ("C)
Fig. 26.8. SFC of palm mid-fraction (0); (O), Malaysian cocoa butter
cal properties. SLS fat is nontempering but also has plasticity. That is, when SLS fat is blended into a chocolate compound recipe, the chocolate compound becomes a plastic, moldable chocolate. This means that the chocolate compound made of SLS fat can be changed easily to any shape. An example is shown in Table 26.16 and Figure 26.1 1. The compound chocolate made from SLS fat can be molded into shapes such as a sheet, roll, and leaf without cracking. The mechanism is explained as follows. The most stable crystal form of SLS is the y-form. The y-form of SLS crystals gathers and aggregates. Liquid oil such as SLL is held in the crystal aggregates, thereby allowing the SLS fat to provide plasticity (Fig. 26.12). No Trans, No Lauric, and No Tempering Fat for
Center Filling Applications CBR generally has a high trans FA content, which increases the LDL concentration level. Therefore, it is important to minimize intake as much as possible. The
EL+$-
lnteresterification
srrl,3 Specific Lipase
EK EF
+ L,s
Safflower Oil
7. Ei EL Distillation
L, s Fig. 26.9. The process scheme of sn-I ,3 stearoyl sn-2 linoleyl glycerol
(SLS)TAG.
Chocolate Fats
661
TABLE 26.1 5 Major FA Composition of SLS Fat, CB, and Typical CBRa FA composition SLS fat Malaysian CB Typical CBR
Stearic
Palmitic
Linoleic
(Old
(YO)
(YO)
49
5 26 37
36 4 4
34 6
Table 26.1 or other abbreviation.
-5
90 80 70
.I-
c
$
60
C
850
c
d!
40
30 v)
20 10
0 15
20
25 30 Temperature ("C)
35
40
Fig. 26.10. Solid fat con(01, tent of SLS fat (0); Malaysian cocoa butter; (W), typical cocoa butter replacer.
Fuji Oil Company (16) developed a non-trans, nonlauric, nontempering fat for center filling application. This fat system is a combination of POP and PPO made from triple-fractionated PMF and a polyglycerol FA ester. The typical production process is shown in Figure 26.13, and SFC is shown in Figure 26.14. This fat has a sharp melting profile. Therefore, it has excellent mouth feel and flavor release in the mouth. Typical compound recipe and bloom test results are shown in Tables 26.17 and 26.18, respectively. This fat has excellent bloom resistance. TABLE 26.1 6 Typical Compound Chocolate Recipe for SLS Fata Ingredient
YO ~~
Cocoa mass Skim-milk powder Sugar SLS fat Lecithin Table 26.1 or other abbreviation.
13 12 49
26 0.4
K. Yamada e t a / .
662
Plastic Chocolate made from SLS fat
SLS gamma form crystal
Liquid
SOS beta-2 form crystal
A Fig. 26.12. Image of SLS y-form crystal matrix, compared with sn-1,3 distearoyl sn-2 oleoyl acyl glycerol (SOS) crystal. (A) SLS y-form crystals gather and aggregate and hold liquid oil during crystal growth. Therefore, the compound chocolate can have plasticity without oil migration. (B)SOS p-2 form grows as needle crystals in liquid oil and cannot hold liquid oil. Therefore, oil migration occurs. See Figure 26.9 for other abbreviation.
Non-Trans, Nonlauric, Nontempering Fat Palm MidFraction
High Melting Point Fraction Low Melting Point
Solid Fat
Polyglycerol FA Ester
Chocolate Fats
663
100
90 h
80 70
c
S
a 6 0
c
S
850
c
$ 4 0 0
cz-
30 20
Fig. 26.14. Solid fat content of non-trans, nonlauric, nontemperingfat (+); (MI, transtype center fi IIing fat.
10
0 15
20
30
25
40
35
Temperature ("C)
In summary, consumers are beginning to examine their eating habits seriously and they must be allowed to have options available to enjoy healthier foods. With new laws going into effect such as the FDA's requirement for trans fats labeling, TABLE 26.1 7 Typical Recipe of No trans Fat for Center Filling Ingredient Cocoa mass Cocoa powder Whole-milk powder Skim-milk powder Sugar Fat Vani IIin Lecithin
10 2 15 5 40 28 0.02 0.4
TABLE 26.1 8 Bloom Resistance TestaJb CyclesC
Non-trans fat Typical trans fat
5
10
15
20
25
-
-
-
-
i
-
i
*
T h e chocolate recipe was same as that in Table 26.1 1. b-, no bloom; i,loss of gloss; +, bloom.
The bloom resistance test was performed under cycle temperatures of 18-30.5"Ud.
30
+ +
664
K. Yamada eta/.
the government is taking steps to address the rising increase in obesity and cardiovascular disease. A few examples of “healthier” fats were presented in this paper, and the food industry, including the fats and oils industry, is working hard to develop new and interesting products that have good functionality and taste but also offer some positive health benefits. References 1. 21 Code of Federal Regulations, Part 101 (http:l/www.fda.gov/oclinitiativesltransfat/). 2. Chen, IS., S. Subramaniam,G.V. Vahouny, A.A. Casssidy, I. Ikeda, and D. Kritchevsky,A Comparison of the Digestion and Absorption of Cocoa Butter and Palm Kernel Oil and Their Effects on CholesterolAbsorption,Lipids 24: 1568-1569 (1989). 3. Denke, M.A., and S.M. Grundy, Effects of Fats High in Stearic Acid on Lipid and Lipoprotein Concentrations in Men, Am J. Clin. Nutr. 54: 1036-1040 (1991). 4. Kris-Etherton, P.M., and V.A. Mustad, Chocolate Feeding Studies: A Novel Approach for Evaluating the Plasma Lipid Effects of Stearic Acid, Am. J . Clin Nutr. 60: 1029s-1036s (1994). 5. Emken, E.A., Metabolism of Dietary Stearic Acids Relative to Other Fatty Acids in Human Subjects, Am. J. Clin. Nutr. 60: 1023s-1028s (1994). 6. Fukui. K., K. Taniguchi, S. Nagaoka, and Y. Hashimoto, Absorption and Metabolism of Lipids in Rats Depend on Fatty Acid Isomeric Position, J. Nutr. 126: 225-231 (1996). 7 . Kritchevsky, D., S.A. Tepper, S.C. Chen, G.W. Meijer, and R.M. Krauss, Cholesterol Vehicle in Experimental Atherosclerosis. 23. Effects of Specific Synthetic Triglycerides, Lipids 35: 621-625 (2000). 8. Zock, P.L., J.H. de Vries, N.J. de Fouw, and M.B. Katan, Positional Distribution of Fatty Acids in Dietary Triglycerides: Effects on Fasting Blood Lipoprotein Concentrations in Humans, Am. J. Clin. Nutr. 61: 48-55 (1995). 9. Zock, P.L., J. Gerritsen, and M.B. Katan, Partial Conservation of the sn-2 Position of Dietary Triglycerides in Fasting Plasma Lipids in Humans, Eur. J. Clin. Znvestig. 26: 141-150 (1996). 10. Fukui, K., K. Taniguchi, S. Nagaoka, T. Yamamoto, and Y. Hashimoto, Effect of Dietary Calcium on the Absorption of Triglycerides Esterified at 1,2 and 1,3 Positions of Glycerol with Long Chain SaturatedFatty Acids in Rats, Nutr. Rex. 15: 1005-1018 (1995). 11. Murata, T., T. Kuno, M. Hozumi, H. Tamai, M. Takagi, T. Kamiwaki and Y. Itou, Study on Fat Absorption Effect of Calcium Derived from Egg Shell Fortified Chocolate Feeding in Human Subjects, J. Jpn. SOC.Nutr. Food Sci. 51: 165-171 (1998). 12. Shahkhalili, Y., C. Mursel, I. Meirim, E. Duruz, S. Guinchard, and C. Cavadini, Calcium Supplementation of Chocolate: Effect on Cocoa Butter Digestibility and Blood Lipids in Humans, Am. J. Clin Nutr. 73: 246-252 (2001). 13. Yamaguchi, K., T. Nishimoto, Y. Ebihara, H. Matsunami, and S. Fujita, JP Patent 2,513,104 (1996). 14. Yamaguchi, K., T. Nishimoto, Y. Ebihara, H. Matsunami, S. Fujita, and A. Kakuhara, U.S. Patent 5,271,950 (1993). 15. Okumura, Y., U.S. Patent 5,279,846 (1994). 16. Okada, T., K. Yamada, and A. Nago, U.S. Patent 6,258,398 (2001).
Chapter 2 7
Margarine and Baking Fats Vijai K.S. Shukla International Food Science Centre NS, Soenderskowej 7, DK-8520 Lystrup, Denmark
Introduction Margarine was created by a Frenchman, Hippolyte, from Provence, France, in response to a request from the Emperor Louis Napoleon 111 for the production of a satisfactory substitute for butter. There was a pressing need to find a cheaper butter alternative with which to feed a growing population. Noticing its pearly sheen, MegbMouri&scoined the name “Margarine” for his invention, taken from the Greek word “Margarites” meaning pearl. Commercial production was initiated in the 1870s by the Dutch company Jurgens. The popularity of margarine soon grew on a worldwide scale as it became recognized as a valuable and economical food product. Although it has been available for over a century, it was not the preferred table spread in the United States. In 1930, per capita consumption of margarine was only 2.6 lb (vs. 17.6 lb of butter). Today, per capita consumption of margarine is 8.3 lb, whereas butter consumption is down to -4.2 lb. At that time, margarine was produced from physically fractionated tallow, skim milk, salt, and other components. Cooling of the margarine emulsion was provided by adding cold water which was drained off after solidification. Since then, developments in oil refining technology and fat modification techniques have allowed the development of a wide range of fats with different functional properties for margarine and other types of food products. Definitions
It is difficult to define precisely what margarine and baking fats are due to the large diversification of products on the market. The definitions used here are as follows: Margarine: A spreadable partially crystalline water-containing product with fat-like properties. Baking fats: Plastic fat products used for baking and cooking. Also called shortenings.
In summary, when a baking fat has 80434% fat and the rest water, it is called margarine, whereas a fat used in baking which is 100%fat is called shortening. 665
666
V.K.S. Shukla
Some of functions of fat in margarine applications are as follows (1): (i) spreadability; (ii) texture/hardness; (iii) crystal network; (iv) emulsion stability (with added emulsifiers); (v) stability against oil separation in the end-product; and (vi) organoleptic appeal. The functions of fat in bakery applications are as follows (2): (i) shortening power and lubricity; (ii) batteddough aeration (with added emulsifiers); (iii) emulsifying properties (with added emulsifiers); (iv) provision of an impervious layer; (v) improvement in keeping properties; and (vi) provision of flavor. This chapter will deal only with the general principles for making margarine and baking fats. No specific types of margarine and baking fats will be discussed. For more details see References 1,3, and 4. Fat Types
Today, the animal fats used in the past have been replaced extensively by vegetable oil products due to the worldwide expansion of oilseed agriculture. Due to a lower content of crystalline fat, the vegetable oils used are often hardened to a certain extent by chemical or physical means. The two main types of oils and fats used are of vegetable and animal origin. The former include the lauric oils (palm kernel oil and coconut oil), palm oil and its fractions (palm olein and palm stearin), olive, peanut and rapeseed oils, and oils rich in essential fatty acid (FA) such as soybean, cotton, corn, and sunflower oils. The latter include animal fats (lard and tallow) and marine oils or fish oils Most margarine types have a melting point (m.p.) between 28 and 42°C. If unmodified oils are to be used, the only choices are palm, palm kernel, and coconut oils as well as fats from land animals. Unmodified marine oils cannot be used because of their high unsaturation and semi-instantaneous reversion of taste. Minor amounts of health food oils may be incorporated into special dietary margarines. Such products may be used for nutritional supplementation in cases of digestive disorders. Examples of this are margarines prepared with medium-chainlength triglycerides based on caprylic and capric acids (9,natural highly unsaturated n-3 fish oils (6), and y-linolenic acid-containing oils such as evening primrose oil (7). World production and consumption of oils and fats are illustrated in Figure 27.1. The use of various oils for margarine and shortenings over a period of years is depicted in Figure 27.2. Modifications If one wishes to extend the number of raw materials to obtain higher flexibility in the composition of the fat phase and to obtain functionalities not possible with unmodified oils, different types of modification can be used. To improve their functional properties, the oils and fats used may be hydrogenated, fractionated, or interesterified as shown in Figure 27.3. In general, oils and fats should be well refined. This means they should have been subjected to a sequence of treatments:
Margarine and Baking Fats
667
Animal Nutrition Fig. 27.1. World production and consumption of oils and fats. degumming (for oils rich in lecithins), neutralization, bleaching, and deodorization. Refining and deodorization should result in discolored oils with a neutral taste. Coloring agents and flavor may be added to obtain desirable visual and organoleptic characteristics. ~
~
+ Corn -et-
Cottonseed
--A-
Soybean
~+ Animal Fat
V.K.S. Shukla
668
Beef tall ow
n
Low temperature
a a a
Separation of oil
Filtered
Allowed to crystallize
Crystallized beef fat
Wrapped in filter
clothes and pressed
Liquid fraction obtained = oleo oil ar HYDROGENATION m~ R
INTERESTERIFICATION
FRACTIONATION Fig. 27.3. Different types of oil modification.
Hydrogenation. Many vegetable oils contain a high proportion of unsaturated FA residues and are quite soft or even liquid at room temperature. This restricts their application in food. Certain amounts of higher melting fat, particularly those melting close to body temperature, and also of appropriate polymorphic form (discussed later), are necessary for margarine and baking fats (8). Unsaturation of triacylglycerol oils can be reduced by hydrogenation. The hydrogenation reaction is the addition of hydrogen to the double bonds or of the unsaturated FA residues in the presence of a metal catalyst. Under certain conditions, hydrogenation leads to isomerization of the normal cis-unsaturation of the double bond to trans-unsaturation. Both types of reactions are used for the manufacture of margarine and baking fats. During the hydrogenation process, numerous addition and isomerization reactions take place simultaneously. The reaction conditions, including the type of catalyst, may accelerate some of these reactions over others. The terms “nonselec-
Margarine and Baking Fats
669
tive,” “selective,” or “highly selective” hydrogenation are used to characterize such different types of reactions. Saturation of the double bonds of oils by hydrogenation gives rise to an increase in m.p. But trans formation during hydrogenation of oils also results in a considerably increased m.p. The oxidative stability is greatly increased in high-trans fats. Hightrans margarines were once very popular, but in recent years there has been a trend to reduce the amount of trans FA in edible oils and fats for health reasons. Generally, hydrogenation of oils for baking fats is done under less selective conditions than for margarine fats. Table 27.1 illustrates the difference selectivity can make in the hydrogenation of soybean and cottonseed oils. The solid fat content (SFC) profile of the selectively hydrogenated oil is much steeper than that of the oil hydrogenated to the same iodine value (IV) under extremely nonselective conditions. Thus, for solids content, the nonselective oil is more suitable for a shortening with a wide plastic range. Fractionation. Because oils and fats are mixtures of high-, medium-, and lowmelting triacylglycerols, a fractionation process can be used to separate oils into two or more acylglycerol mixtures, which can have significantly different physical properties from the feed material. During fractionation, the high-melting acylglycerols can be crystallized from the melt and separated. The crystals will form a higher-melting fraction (stearin) relative to the original oil, whereas the liquid phase will be lower melting (olein). There are three well-established processes: (i) detergent fractionation, (ii) solvent fractionation, and (iii) dry fractionation. Solvent fractionation is an expensive process and is used mainly in the manufacture of high-quality confectionery fats (9). In some situations, flavor problems may arise due to solvent residues. TABLE 27.1 Characteristics of Soybean Oil and Cottonseed O i l Hydrogenated Selectively and Nonselectivelya ~
Soybean oil Selective
~~~
Cottonseed oil
Nonselective
Analysis
Selective
Nonselective
(Old ~~
Trans fatty acids Iodine value Solid fat content at 10°C at 21 “C at 26°C at 33°C at 38°C aSource:Reference 3,
38.9 80
33.3 79
36.5 78
35.5 75
26 12 6 1 0
35 26 24 16 11
28 15 10 1 0
39 30 26
16 -
670
V.K.S. Shukla
In the dry fractionation process, liquid oils or melted fats are cooled and the resulting crystalline mass is separated from the remaining liquid. The process is most effective when the crystals to be isolated are large and easily separated by filtration or centrifugation. The dry fractionation process is employed most frequently when the desired product does not require the efficient separation of triglycerides with closely related solubility properties. In this process, both crystallization and crystal separation take place in the absence of additives. Crystal separation is generally achieved through the use of vacuum or high-pressure filters. Dry fractionation is relatively simple in terms of equipment, but in reality, it is difficult to conduct and generally the separations are difficult to carry out. For that reason, it is limited to a few products such as palm, palm kernel, and some hydrogenated oils. The dry fractionation process has a better reputation (greener technology) than solvent fractionation. Interesterification. During interesterification, the glyceride structure of the oils is modified whereby molecular rearrangement of FA on glycerol takes place without modification of FA composition. Interesterification results in the modification of physical properties of the interesterified product such as m.p., crystalline characteristics, SFC, and plasticity. After interesterification of an unblended oil, the m.p. often go up. Interesterification of blends of very unsaturated oils with very solid fats may result in a lowering of the m.p. to <37"C, which makes the product consumable. It is possible to prepare soft zero trans margarines without hydrogenation by interesterification alone, but is unlikely that it will be possible in the future even with advanced technology to make satisfactory stick margarine in this way. The limited amount of natural saturated fatty acids (SFA; mainly stearic and palmitic acids) would appear to exclude this possibility. Combined Modifications. Hydrogenation, fractionation, and interesterification are often combined to obtain desirable product characteristics. Some examples are described. Margarines high in polyunsaturates and containing low- or zero-trans FA can be formulated with nonhydrogenated oils such as soybean, corn, or sunflower oils with fats that are completely hydrogenated (10). Palm oil and lauric fats, such as coconut and palm kernel oil, are rich in natural SFA. However, because of eutectic formation and steep melting profiles, satisfactory stick margarine oil blends cannot be achieved with these oils as the sole raw materials without some additional modification. Interesterification of a fully hydrogenated mixture of palm and palm kernel oil yields hardstocks that can be used in very high polyunsaturated soft and stick margarines capable of being processed using standard equipment (1l ,12). Another approach to low-trans margarines makes use of a combination of fractionation and interesterification. It is possible to make soft low-trans margarines from a 100% liquid oil such as sunflower oil in this way (13). Soft and stick margarines may be prepared with an unhydrogenated hard fraction consisting
Margarine and Baking Fats
671
of an interesterified mixture of coconut, palm oil, and palm stearine (14). There are many more combinations or possibilities for producing margarine oil blends. Frequently, oil formulation may be dictated by economics and availability. Fat Crystallization
Edible fats consist of suspensions of various amounts of crystals in a liquid oil. The crystals are composed of solidified triglycerides. The properties of fats, such as hardness, work softening, and plastic behavior, are highly dependent on how these crystals interact by reason of their number, size and shape, and the type of bonding force between them. In general, the crystallization of fats is assumed to consist of a nucleation and a growth phase, and both of these processes can exhibit a maximum value with respect to temperature. The crystallization process is highlighted by polymorphism, the formation of mixed crystals, and the dimension of the crystals. The heterogenous type of nucleation of fat crystallization predominates in commercial fats due to the presence of trace impurities such as high-melting glycerides and other compounds. In bulk fat systems, degrees of supercooling will be relatively low, but in the globular fat state of dairy cream, it is likely that degrees of supercooling will be greater due to the relative isolation of impurities within individual globules with the capability of seeding crystallization, The nucleation rate in fat globules increases with a decrease in temperature within the normal range of physical ripening temperatures for cream. Oils and fats can solidify in more than one crystalline form, and these differ from each other in terms of melting temperature, density, heat of fusion and crystallization, or refractive index, for example. The a crystals are the least stable form, have the lowest m.p., and occur when melts are crystallized rapidly. Although each form has minor variants, the three main forms are a,p’, and p in ascending order of stability. The forward transitions between a and p take place with the liberation of heat and are not reversible. The various polymorphic forms are distinguished by X-ray powder spectroscopy, which reflects the arrangement of the molecules within each lattice structure although other techniques, such as differential scanning calorimetry, can also be used. Fat blends for margarine and shortenings usually crystallize in the a form within the units of a continuous process line; under the influence of an increase in temperature and agitation, they rapidly transform into a p’ form. Further transition to the p forms is much less likely because these require very close packing of the triglycerides, and the complex nature of many margarine blends (in terms of difference in length and shape of the FA chains) makes such close packing difficult even after a considerable period of time. Simpler blends, however, can crystallize in the p form. The presence of molecules other than triglycerides, such as diglycerides, can inhibit the creation of more closely packed molecular arrangements, and thus act as
672
V.K.S. Shukla
@’phase stabilizers. Such an effect was noted in low-erucic rapeseed oil blends (15 ) . The process of the formation of solid phases in fats is described by solubility curves, and each polymorphic form has a different curve depicting the relation between temperature and fraction of solute in the soluble/insoluble state. Each triglyceride has its own polymorphic and melting behaviors. However, in a mixture of triglycerides, they do not behave independently but take on a totally new character in terms of crystallization behavior. The systems are so complex that it is easier, in the case of fat blends, to describe them in terms of their different phases; thus, the physical properties of a fat can be discussed in terms of its phase behavior. In a fat blend at a given temperature, there will always be a liquid phase and a solid phase and the solid phase can have several components, which can change with temperatures and composition. A phase diagram can be used to show how blended fats interact, for example, to produce minima points (eutectic behavior) or maxima points (solid solutions). When the fats are compatible, a horizontal line results on the iso-solids line at different compositions. Fat Crystal Size. The dimensions of fat crystals are related to the manner of their formation. Rapid cooling below the solubility temperature of the a-form gives a large number of imperfect crystals, whereas slow cooling at higher temperature results in larger crystals. a-Crystals transform to more stable forms above the transition point but tend to retain their original size and crystal habit. The shape of the crystals can be needle-like, but they are often thin platelets of some tenths of a micrometer. Triglyceride molecules tend to add on during the growth phase in the form of successive layers. Interesterificationusually results in a decrease in crystal size. The tempering of fats can increase the mean crystal size, especially if the fats contain significant amounts of symmetrical triglycerides. Crystals that are small and do not melt quickly on the palate can give a rather slow melting sensation in margarines. Small crystals having a large surface area, which may not be smooth, can retain a large amount of liquid oil in the network, and the close approach of such crystals to each other tends to result in a tough strong lattice structure. The a crystal form, the least stable and lowest melting polymorph, is initially formed during the rapid chilling conditions used in margarine manufacture; however, it transforms quickly to the @’state. The @’structure of margarine consists of a very fine network that, because of its great surface area, is capable of immobilizing a large amount of liquid oil and aqueous-phase droplets. Most margarine fats are stable in the @’state, but if the fat blend has strong @ tendencies, it may, under certain storage conditions, transform to the @ crystalline state. This is usually accompanied by the development of a coarse, sandy texture consisting of large crystals. In severe cases, this transformation also may result in exudation of liquid oil from the product and partial coalescence of the aqueous phase, which increases the microbiological susceptibility. In general, the more diverse the triglyceride structure of the highest melting portion of the fat, the lower the @-formingtendencies. Therefore, fats such as sun-
Margarine and Baking Fats
673
flower, safflower, and low-erucic rapeseed (canola) are most likely to undergo this transformation because the palmitic acid content is very low; when they are hydrogenated, the solid component consists of a series of closely related homologs. In baking fats, p’ stable fats are also preferable because they have the best effect in stabilizing air in the dough. The dimension of the crystals plays a vital role in defining various properties of margarine such as the following: spreadability, formation of crystals, oil exudation, elasticity, m.p. of the crystals, melting, quantity of crystals (in wt%), mouth feel, and type of bonding. For table margarine, 1518% of the crystals have dimensions <0.3 pm; 70% are <1.O pm, and 50% of the crystals fall below 0.6 pm. Fat Crystal Networks. The three-dimensional crystal network in plastic fats gives rise to both viscoelastic behavior and a yield value. The intercrystal forces that exist in the fat crystal matrix can be described as being of two types. Primary bonds are very strong and are formed by the crystallization of glycerides between two adjacent crystals. Once broken they are not easily reformed; in this sense, they are irreversible. However, weaker secondary bonds of association are formed within seconds (16) by van der Waals-type bonding between crystals. Such bonds are relatively easily broken; thus, they are weak, can be reformed by reflocculation, and are reversible. In practice, a range of such primary and secondary forces likely exists in a typical network. Individual crystals can be organized in fat-continuous spreads into various structures. Crystals dispersed in an interstitial fashion between aqueous phase droplets contribute to product rheology . When clustered and aggregated around water droplets they can stabilize the emulsion by forming protective “shells.”
Solid Fat Content It is important to know the extent to which a fat or fat mixture crystallizes at the temperatures of practical interest and the extent of crystallization at various temperatures. The modern method for measuring the SFC is based on the difference in molecular mobility in liquid and solid triglycerides (15); because the solid-to-liquid ratio varies with temperature, a temperature profile of the solids content of the blend can be obtained. The technique used is low-resolution pulsed nuclear magnetic resonance (NMR) spectroscopy (17,18). Formulation of Oil Blend Recipes
Blends that are optimum in terms of physical and performance properties and cost can be calculated using certain mathematical models (19). These calculations, which are based on measurements taken on a large number of available components, assume that each component yields a linear contribution to physical properties such as the solids content of the blend. More advanced models use triglyceride classification and FA composition from which the solid content can be generated mathematically (20).
V.K.S. Shukla
674
As more knowledge is gained concerning mutual solubility relations and the mutual influence of triglyceride classes on melting behavior, even more precise models may be developed. In addition, the effects of the technology of crystallization and particularly the application of scraped-surface heat exchangers, which are now widely used to chill and crystallize fat-based products, must be evaluated. Establishing the correct blend for the manufacture of margarine and baking fats requires close consultation with the user or customer, and an understanding of the process by the manufacturer. Usually, knowledge of the FA composition and triglyceride structure of the blend is enough to ensure correct functionality and crystallization behavior. A typical formulation of the oil blend may have the following profile: palm oil, 46%; palm olein (IV = 55-58), 16%; soybean oil, 30%; hydrogenated soybean oil (m.p. = 35”C),8%. The FA compositions of various products in Europe are shown in Table 27.2. This clearly reveals that in Europe there is predominance of palm oil and palm kernel oil (presence of high proportions of C,, and C12)in the formulations. The oils contain negligible quantities of trans-FA. Hydrogenated oils are absent, and substantial amounts of 18:l and 18:2 are present. Thus, blends of palm and palm kernel oils with sunflower and soybean oils were used extensively in developing these oils. The FA composition of North American products is illustrated in Table 27.3. These results show high percentages of trans-FA in active use in partially hydrogenated oils. Thus, blends of partially hydrogenated soybean oils, sunflower oils, corn oils, and pure liquid oils of the same family were utilized to develop products in North America. Several possible compositions are described in Figure 27.4. Zero Trans/f ow-Trans Feedstocks. We developed a series of feedstocks based upon the interesterification process. The analytical constants of these fats are depicted in Figures 27.5 and 27.6 and Table 27.4. These feedstock products help in the design of a newer set of margarines and spreads with low or no trans values. Attempts were made to reduce trans levels employing newer catalytic hydrogenation techniques (21). These technologies are yet to be developed and perfected to gain the best advantage for producing low-trans margarines or shortenings. TABLE 27.2 Fatty Acid Composition by Gas Chromatography of Various Margarines from Europe ‘8
‘10
‘12
‘14
‘16
‘18
‘18:lt
‘181
‘18:2
0.3
45.9 40.7 20.6 35.0 22.0
0
38.9
12.8 9.9 54.5 11.1 31.2 11.5
‘18:3
(Old
D1 D2
D3 D4 D5 D6
0.3 0 0.1 0.4 0.5 1.5
0.4 0 0.1 0.5 0.5 1.4
4.6 0.2 2.3 6.2 8.7 12.6
1.9 0.8 0.9 2.9
21.7 35.9 7.7 29.5
5.1 8.5 9.7 9.7
3.2 5.6
9.9 19.2
20.6 3.4
0.2
0.3 0.3 0.3
4.6 2.0 0.7 2.8 1.1 4.5
Margarine and Baking Fats
675
TABLE 27.3 Fatty Acid Composition of Various Margarines from North America Cl,
1‘,
‘18
‘18:lt
‘18:l
‘18:2
47 36.7 30.8 31.7 47.3 30.6
34.5 41.1 44.3 44.1 32.0 44.7
‘,8:3
(Old
A1 A2 A3 A4 A5 A6
0.0 0.0 0.1 0.1 0.1
0.0
10.8 11.9 11.5 10.7 12.6 11.7
6.1 5.9 7.3 7.2 6.5 7.0
14.6
7.9 5.3 6.1 14.5 5.4
1.6 4.4 6.1 6.3 1.5 6.1
Table Margarine Compared with pastry margarine, table margarine is softer in consistency and is formulated with a lower solid-to-liquid ratio. It spreads with ease even when it is just out of the freezer. The “refrigerated” type is used in temperate countries and “nonrefrigerated” margarine is found in tropical and subtropical countries. In tropical areas, relatively higher SFC is important to avoid oil exudation and oil separation at higher temperatures. Some desirable characteristics of table margarine are discussed below. Rapid Melting in the Mouth. Table margarine is an emulsion of water in oil in which the droplets of water are separated by a network of fat crystals. A close-knit and distinct network exists around the water droplets, with the crystal agglomerates having few points of contact. The structure of the emulsion can be (i) fine, a stable emulsion with delayed melting in the palate or (ii) coarse, a less stable structure in which the emulsion breaks up more quickly and the flavor and aroma are better developed. A coarse structure gives a more pronounced sensation of freshness compared with a fine-structured emulsion. Palm oil and palm kernel olein are suitable choices; 30% of palm kernel oil develops some kind of brittleness. Broad Range of Plasticity. Plasticity is incorporated by the kneading of the crystallized mix in the last cooling chambers or in the complector system. The mix is subject to weak secondary Van der Waal’s forces of attraction, and an irreversible primary liaison occurs due to the precipitation of fat crystals during the formation of weaker liaisons. The primary and secondary forces are broken under a uniform deformative force. With the withdrawal of the force, the secondary liaison is reestablished and the initial hardness (Hi) is superseded by the new hardness (Hf). The ratio (Hi - Hf/Hi) x 100 = P, is referred to as the plasticity P at t”C. When P, = 30- 40% , the product is sticky in nature; at P, = 50-60%, the product has good
plasticity; at P, = 70430% , the product is mildly brittle in consistency; and at P, > 80% ,the product is brittle.
676
V.K.S. Shukla
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677
Bakery Margarine. The characteristics of this margarine include: (i) it contributes softness and richness, which enhance the flavor and mouth feel; (ii) it provides the desired grain and texture qualities; (iii) it enhances aeration for leveling and volume; (iv) it prevents adhesion by providing lubrication; (v) it affects moisture retention, which gives the baked product a longer shelf-life; (vi) it provides a high flavor level; (vii) it is generally stronger and has to be more heat-stable; (viii) it improves the buttery flavor in baked products. Commercially available flavors contain butyric acid and/or lactones. Bakery margarines are formulated with higher salt levels, e.g., -3% in contrast to 1.5-2% in consumer products. Functional Margarines. To counteract the high fat content in food in Western Europe, low-energy spreads were developed in the late 1960s. Recent formulations have concentrated on other functional aspects of table spreads. A low-calorie spread can be designed as follows: (i) with only water and salt in the aqueous phase; (ii) with hydrocolloids in the aqueous phase; (iii) with protein and as an oilin-water emulsion with phase reversion in the tube chiller; or (iv) by other methods that involve usage of a stabilizer and protein, and a water-in-oil emulsion. Lowcalorie spreads typically contain -40% fat and 20%. Modern research showed that margarines enriched with plant sterols are capable of lowering the P-carotene concentration in the blood plasma. A daily intake of 0.8-3.2 g of plant sterols via spreads consisting primarily of soybean oils decreased blood cholesterol by 5-7%. Low-density lipoprotein (LDL) cholesterol is reduced by 7-lo%, but high-density lipoprotein (HDL) cholesterol remained unaffected. Plasma levels of P-carotene are 6 9 % (as a percentage of total plasma lipids) at sterol consumption levels of 0.8-3.2 g. Margarines with a low trans content have a fully hydrogenated hardstock component (16-24 carbon chain) and fats containing low-molecular-weight FA such as propionic and butyric acids. Margarine with such oil blends have excellent rheological properties and very low oil exudation characteristics. Specifications. In the oils and fats business, well-documented tests determine
whether the parameters usually included in the specifications for a margarine blend or baking fat are met. Classical tests for free fatty acid (FFA) content, peroxide value, SFC, and color are well known and are often supplemented by FA composition and accelerated stability tests such as the Rancimat test. Finally, the flavor of the oil must be judged by an expert panel to ensure that it is bland. In addition to those listed, specifications also sometimes include limits on heavy metals and microorganism content. The oil used in margarines and shortenings should be of the highest quality, be as bland as possible, and freshly deodorized. Transportation specifications are often made for issues such as truck delivery, loading temperatures, whether the oil must be nitrogen-blanketed, and temperature on arrival. Processing. The vast majority of margarines and baking fats are manufactured on scraped-surface heat exchangers; although these exchangers vary in design, the basic
V.K.S. Shukla
678 - _. .- __
100
- _____
- -_--
90
80
70
-+-Danmag 20
60 50
-c- Danmag 30
40
30
-A-
Danfat 33
-Q--
Danfat 40
20 10
0 15
20
25 30 Temperature ("C)
40
35
Fig. 27.5. Melting profile of IFSC interesterified basestock fats. NMR, nuclear magnetic resonance; SFC, solid fat content.
60
-
- - --
.- -.
_. .
-
-
. _ _ _
-
-1
j 50 40 I
30
20
10 0
I
I
I
I
I
I
1
Fig. 27.6. Melting profile of IFSC non-trans margarine fats. NMR, nuclear magnetic resonance; SFC, solid fat content.
Margarine and Baking Fats
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principles apply to all. The heat exchanger is made of two concentric tubes; in the annular space thus created, a compressible refrigerant is circulated. The inner tube has a heated shaft that mns the length of the tube on which are mounted floating scraper blades. As the shaft rotates, these blades scrape the internal surface of the tube. In the process, the liquid emulsion or fat blend is pumped along the tube at a fixed speed, and the rotating blades remove the chilled product from the walls. This constant renewal of the cooling surface and the turbulence created leads to supercooling, the initiation of crystal nuclei, and hence crystallization. The supercooled and partially crystallized product can then be pumped to a worker unit where crystallization is completed and the heat of the crystallization released. Systems of much greater complexity are available to obtain the texture and plasticity of a widening range of blend types and to meet greater specificity according to the requirements of the user. Factors such as shaft rotation speed, scraper blade design, size of the annular space, and the size and location of the worker units all can affect the final texture of the product. The Water Phase. The water phase of margarine usually contains optionally cultured milk components, which provide flavor to the product. In low-fat products, hydrocolloids such as gelatins, alginates, or carrageenan are added to improve product rheology .
Structure of Margarines. Traditionally, fat-continuous type of spreads have water droplets in the range of 2 4 pm (margarine) to 4-80 pm (low-fat spreads). However, with the move toward even lower-fat spreads, water-continuous types are being marketed. High internal phase emulsion technology indicates that fat-continuous emulsions with as little continuous phase as 5% are possible, so that emulsions of this type with 10-20% are certainly feasible. Commercially, however, they may offer characteristics different from those of higher-fat spreads, and their stabilization systems are possibly unique. With highly structured aqueous phases, a true emulsion system may not be necessary. Bicontinuous phase systems are also possible. Clearly, such systems will exhibit both water- and fat-continuous characteristics as indeed some butter was shown to do. Margarine contains fat-soluble and water-soluble components. The former include emulsifiers, coloring agents, aromas, and vitamins. Emulsifiers. The structural composition of emulsifiers includes both lipophilic and hydrophilic parts with the remainder dissolved in both the aquatic and lipid phase as a homogeneous union of the two phases. The emulsifiers can be monoglycerides of FA or of SFA (C14-C22) such as monopalmitate and monostearate. The monoglycerides of FA with C < 12 have a bitter taste. Emulsifiers are added at a level of 0.054.2% for 90/95% distilled monoglycerides and 0.34.5% in the case of commercial diglycerides. They act as regulators in fermented doughs for pastry margarine and slow down the conversion of starch to sugar in the dough by sequestering the starch amylase.
m W
0
TABLE 27.4 Analytical Constants of Various Non-Trans Industrial Margarine Fats Parameter Melting point ("C) Total saturated ('YO) Monounsaturated (Yo) Polyunsaturated (%) Trans fatty acids
A
C
D
E
F
C
H
I
J
K
44.0 68 30 5.3 1.4
49.2 86.9 11 1.9 0.4
50.1 90 9.8 1.2 1.6
47.3 88.4 9.9 1.7 0.5
45.9 89.8 8.5 1.6 0.3
43.0 67 27 5 0.6
40.0 74 22 5 0.4
47.0 99
0.4
45 87 10 2 0.6
41 67 27 6 0.3
-
Pulsed Nuclear Magnetic Resonance (NMR) Values of Various Non- Trans Industrial Margarine Fats
NMR
A
C
D
E
F
C
H
I
J
K
10°C 20°C 30°C 40°C
80.9 60.0 33.7 10.4
95.4 86.7 68.5 32.5
96.3 85.9 64.1 31.6
95.7 91.6 75.0 37.6
96.2 90.4 71.5 30.9
84.1 63 .O 37.0 13.0
84.0 63.2 33.1 6.3
96.0 92.1 72.3 25.2
94.2 88.2 63.1 24.1
76.1 54.0 25.0 5.2
< ? i
F
$
Margarine and Baking Fats
681
Lecithin. Lecithins, which are generally extracted from soybean oil, contain high amounts of F A . The do not have a neutral taste and are added to a level <0.5%. Modified lecithins include the following: hydrolyzed lecithin, which is hydrolyzed by enzymatic hydrolysis (pancreatic phosphatase) or by chemical hydrolysis (ammonia or soda); lecithin fractionated by acetone/alcohol; commercial lecithin fractionated to obtain lecithin enriched with phosphatidylethanolamine. Enzymatically hydrolyzed lecithin is contained at a level of 0.1-0.2%. Coloring Agents. Color may be added by the incorporation of red palm oil or 0carotene at 5 and 8 ppm, respectively, and is a direct function of the content of the coloring agent. The carotenoids are natural products consisting of isopropene carbides and oxygenated by-products thereof. By convention, the carotenoid content is expressed in terms of p-carotene. Aromas. The addition of artificial flavoring agents is prohibited. Natural compounds such as diacetyl or butanedione 2,3 possess a strong quinoid odor and are used at a level of 2-4 ppm. There are aroma mixes with &lactones as the primary ingredient; such mixes are partly soluble in both the oil and aqueous phases. Vitamins. The addition of 20-30 IU vitamin A/g of the products is common. In countries with insufficient exposure to sunlight, incorporation of 2-3 IU of vitamin D is often practiced. The components that are soluble in the aqueous phase of margarine include milk, salt, pH regulators, and antioxidants. Milk. In some cases, milk is added after maturation. The incorporation of aroma is prohibited when other flavors are added. Milk protein also plays the role of stabilizer for oil-in-water emulsions; however, the addition of greater than usual amounts might lead to phase reversal. Salt. Salt conveys the salty taste and is bacteriostatic. It must be of food grade, i.e., essentially free of all kinds of Fe, or Mg compounds bcause these metal ions have a propensity to catalyze the peroxidation of lipids even in trace amounts, The salt used is anhydrous and weakly alkaline.
pH Regulators. The pH of the aqueous phase is generally 4.0-5.5.The Na, K, or Ca salts of citric or lactic acid are generally used with a maximum dosage of 1 gkg margarine. Antioxidants. These include the natural antioxidants, the tocopherols, and the synthetic antioxidants tert-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, and octyl gallates. The shift from butter to margarine and total spread during the last decade is vividly illustrated in Figure 27.7.
V.K.S. Shukia
682 25
20
--t. Margarine
e 15
0
f
B
i -c- Butter
10
&Total
Spread
5
0 1910
1920
1930
1940
1950
1960
1970
1980
1990
ZOO0
Year Fig. 27.7. Consumption of table spreads during the 20th century.
Future Aspects
Changing nutritional attitudes and the ever-increasing demand for qualitative excellence have put pressure on the oils and fats processors and food manufacturers to continue to do research and develop new products and systems. Margarine and bread manufacturers are well placed to take advantage of those fats created by current and novel methods of modification. Changes in basic fats have also been achieved by plant biology. The use of these techniques, combined with developments in processing equipment to achieve specific rheological characteristics, means that the processor has the opportunity to tailor products to the customer’s requirements. The publicity given to the relation between the so-called saturated fats and high cholesterol contents has led to an increased use of margarines and spreads high in polyunsaturated fatty acids (PUFA). Recipes are now available for the use of such high liquid oil blends in bakery products. The continuous demand for the use of fats high in PUFA could popularize liquid and slurry shortenings in which the baked products’ quality will rely heavily on the presence of emulsifiers (22). Margarine is a good food system in which to introduce health oil components. In the future, there may be a market for margarines containing FA that modify the ratios of prostaglandin syntheses such as highly unsaturated n-3 acids or y-linolenic acid. Good sources of such acids are fish oils (6) and evening primrose oil (7), respectively.
Margarine and Baking Fats
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The demands for higher quality, improvements in nutritional standards, and greater naturalness are major challenges for all sectors of the oils and fats industry, and they can be met only with increases in our understanding of the material that is handled and developments in processing techniques.
References 1. Moran, D.P.J., Fats in Spreadable Products, in Fats in Foods Products, edited by D.P.J. Moran and K.K. Rajah, Blackie Academic & Professional, Glasgow, 1994, pp. 155-21 1. 2 . O’Brien, R.D., Fats and Oils: Formulating and Processing for Applications, 2nd ed., CRC Press, New York, 2003, pp. 305-336. 3. Chrysam, M.M., Table Spreads and Shortenings, in Bailey’s Industrial Oil and Fat Products, edited by T.H. Applewhite, 4th ed., John Wiley & Sons, New York, 1985, Vol. 3, pp. 41-126. 4. Podmore, J., Fats in Bakery and Kitchen Products, in Fats in Foods Products, edited by D.P.J. Moran and K.K. Rajah, Blackie Academic & Professional, Glasgow, 1994, pp. 213-253. 5. Weiske, T., and H.U. Menz, Medium-Chain Triglycerides Fat Stock for Margarine, Fette Seifen Anstrichm. 74: 133-136 (1972). 6. Stansby, M.E., Marine-Derived Fatty Acids or Fish Oils as Raw Material for Fatty Acids Manufacture, J . Am. Oil Chern. SOC.56: 793A-796A (1979). 7. Hudson, B.J.F., Evening Primrose (Oenothera Spp.) Oil and Seed, J . Am. Oil Chem. SOC. 61: 540-543 (1984). 8. DeMan, L., J.M. DeMan, and B. Blackman, Polymorphic Behavior of Some Fully Hydrogenated Oils and Their Mixtures with Liquid Oil, J . Am. Oil Chem. SOC. 66: 1777-1780 (1989). 9. Deffense, E., Fractionation of Palm Oil, J . Am. Oil Chem. SOC.62: 376-380 (1985). 10. List, G.R., E.A. Emken, W.F. Kwolek, T.D. Simpson, and H.J. Dutton, “Zero trans” Margarines: Preparation, Structure, and Properties of Interesterified Soybean Oil-Soy Trisaturate Blends, J . Am. Oil Chem. SOC.54: 408-413 (1977). 11. Ward, J. (to Nabisco Brands),U.S. Patent 4,341,812 (1982). 12. Ward, J. (to Nabisco Brands), US.Patent 4,341,813 (1982). 13. Sreenivasan, B. (to Lever Brothers), US.Patent 3,859,447 (1975). 14. Fondu, M., and M. Willens, (to Lever Brothers), US.Patent 3,634,100 (1972). 15. Anonymous, IUPAC Method 2.150, in Standard Methods for the Analysis of Oils, Fats and Derivatives, edited by C. Paquot and A. Hautfenne, Blackwell Scientific Publications, Oxford, 1987, pp. 59-70. 16. Haighton, A.J., The Measurement of Hardness of Margarine and Fats with Cone Penetrometers, J . Am. Oil Chem. SOC.36: 345-348 (1959). 17. Shukla, V.K.S., Studies on the Crystallization Behavior of the Cocoa Butter Equivalents by Pulsed Nuclear Magnetic Resonance-Part I, Fette Seifen Anstrichrn. 85: 467-471 (1983). 18. Gunstone, F.D.G., and V.K.S. Shukla, NMR of Lipids, in Annual Reports on NMR Spectroscopy, edited by G. Webb, P.S. Belton, and M J . McCarthy, Vol. 31, Academic Press,London, 1995,pp. 219-236.
19. Haighton, A.J.,Blending, Chilling, and Tempering of Margarines and Shortenings, 1.Am. Oil Chem. SOC.53: 397-399 (1976).
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20. Wieske, T., British Patent 1,479,287(1977). 21. Mangnus, F., and A. Beers, Hydrogenation of Oils at Reduced TFA Content, Oils Fats Int. 20: 33-35 (2004). 22. Hartnett, D.I. and W.G. Thalheimer, Use of Oil in Baked Products-Part II: Sweet Goods and Cakes, J . Am. Oil Chem. SOC.56: 948-952 (1979).
Chapter 28
Nutritional Characteristics of Diacylglycerol Oil and Its Health Benefits Noboru Matsuo Kao Corporation, Health Care Products Research Laboratories, Tokyo, Japan
Introduction Obesity is a serious problem that is increasing not only in industrialized countries but also in urban areas of developing countries (1). The World Health Organization (WHO) reported that currently >1 billion adults are overweight and at least 300 million of these are clinically obese in 2000, worldwide. In the United States, the proportion of overweight and obese adults (BMI 225 kg/m2 and 230 kg/m2, respectively) reached 58% of the total population as of 2001 (2). In Japan, the National Nutrition Survey conducted in 2001 revealed that >30% of men 240 yr old are obese (BMI 225 kg/m2 is regarded as obese by the Japan Society for the Study of Obesity). This prevalence of obesity is a dramatic increase from 1990. On the other hand, BMI in women in 2001 was equal to or lower for all generations except for women in their seventies, compared with 1999 (3). Many investigators and surveys have noted the importance of preventing the accumulation of body fat that is associated with lifestyle-related diseases. Clinical studies suggest that weight loss in the range of 5 1 0 % of initial weight can confer a significant reduction in obesity-related disorders such as heart disease, diabetes mellitus, and hypertension (4). Although energy restriction and the limitation of total and saturated fat intake may be the primary measures for the treatment of obesity, these are difficult to achieve. Therefore, numerous studies on dietary fats that influence the accumulation of body fat have been conducted. During the last two decades, fat substitutes such as starch, cellulose, protein, pectin, and dextrose to obtain the same texture as fat or fats with low or no energy value were developed (5). Although modification of the fatty acid (FA) composition of triacylglycerol (TAG) is one approach, we have focused on the structure of acylglycerols. Dietary diacylglycerol (DAG) is a natural component of various edible oils and is used in foods as an emulsifier. We studied the nutritional characteristics of dietary DAG in comparison with TAG and found that DAG, particularly in the 1,3-isofom, has metabolic characteristics distinct from those of TAG that may produce beneficial effects with regard to the prevention and management of postprandial lipemia and
obesity. A cooking oil product containing 280% (w/w)DAG was approved as a “Food for Specified Health Use” by the Ministry of Public Health and Welfare of 685
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Japan. It is on the market in Japan and in the United States. The beneficial effects of DAG oil consumption in humans were summarized recently and its mechanisms were discussed (6,7). In this review, the nutritional benefits of dietary DAG observed in clinical trials under various conditions and the current state of the mechanistic studies using animal models will be outlined with data including recent findings. Structure, Energy Value, and Absorption Coefticient of DAG
DAG is a component of many edible oils with varying contents depending on their origin. Table 28.1 shows the content of DAG and other acylglycerols in edible oils of various origins (8,9). Edible oils generally contain DAG up to -lo%, with the relative content depending on the origin and the condition of the oil material. DAG has two isoforms, 1,2 (or 2,3)-diacyl-sn-glycerol (1,2-DAG) and 1,3-diacyl-snglycerol (1,3-DAG). Figure 28.1 illustrates the structures of DAG in comparison with TAG. DAG has been recognized as a metabolic intermediate after ingestion of TAG. This metabolic intermediate is in the isoform of 1,2-DAG. DAG contained in edible oil is partly converted to 1,3-DAG by migration of the acyl group during the manufacturing process under elevated temperatures or during storage (10,ll). The amount of the 1,3-isoform in equilibrium is intrinsic to the FA in the molecule of DAG (10). At equilibrium, the amount of 1,3-dipalmitoyl-sn-glycerol is 56% (w/w) (1 1). In edible oils with a common FA composition, -70% (w/w) of DAG exists as 1,3-DAG. Use of a reactor of immobilized lipase with 1,3-regioselectivity made it possible to produce a large quantity of oil high in 1,3-DAG content (12). 1,3-DAG-rich oil was prepared by esterifying glycerol with FA from natural vegetable oils. Taguchi et al. (13) determined the energy values of DAG oil by calculations and by measurements using bomb calorimetry. The combustion heat of cooking oil containing 87% DAG was 38.9 kJ/g and that of the TAG oil with the same FA composition was 39.6 kJ/g. The difference (2%) is considered to be negligible in practical intake of oils because it will produce only 0.1% difference in total daily energy intake. Furthermore, the apparent digestibility or the absorption coefficients of the DAG and TAG oils determined in rats, a good model with which to predict TABLE 28.1 Contents of Acylglycerols in Edible Oils of Various Originsa ~~~~~
Soybean
Cottonseed
Palm
-
0.2 9.5 87.0 3.3
5.8 93.1 1.1
Corn
Safflower
Olive
Rapeseed
-
0.2 5.5 93.3 2.3
0.1 0.8 96.8 2.3
(wt%) Monoacylglycerol Diacylglycerol Triacylglycerol Others
aSource: References 8.9.
1 .o 97.9 1,l
2.8 95.8 1.4
2.1 96.0 1.9
Diacylglycerol Oil
Triacylglycerol
FbOCOR, FHOCOR, CH,OCOR,
687
Dlacylglycerols
YH,OCO& $XIOCOR, CH,OH 1(3),2-Dlacylglycerol
FH,OCOR, FHOH CH,OCOR, 1,3-Diecylglycerol
Fig. 28.1. Schematic structures of TAG and DAG.
digestibility of oils and fats in humans, were similar (96.3%) (13). These results indicate that the physiologic differences between DAG and TAG ingestion observed in humans and animals are most likely caused by the different metabolic characteristics of these oils. Digestion and Absorption of DAG Compared with TAG
The digestion and absorption processes of DAG were investigated in an experiment tracing time-course changes in the lipid composition after perfusion of triolein and diolein (1,2- : 1,3-diolein = 3:7) in the portion of the small intestine of rats that contains the opening of the common bile duct (14). The rate of absorption as a whole did not differ when either oil was infused. In the diolein perfusion, monoolein and oleic acid were produced as diolein decomposed; unlike in the triolein perfusion, at 60 min after initiation of perfusion, 65% of monoolein was l(3)monoolein. Kondo et al. (15) reported that when DAG was infused intraduodenally as an emulsion, TAG was digested to 1,2-DAG, 2-MAG and FFA, whereas 1,3DAG was digested to l(3)-MAG, and FFA. Thus, the production of l(3)-MAG rather than 2-MAG seems to be a unique characteristic of DAG metabolism. Focusing on the resynthesis of TAG in the mucosal cells further clarifies the characteristics of DAG metabolism. The resynthetic pathways of triglyceride’ (TG) in the small intestinal epithelial cells include the 2-MAG pathway and the glycerol-3-phosphate pathway, with the former considered the main pathway (16). 2-MAG is a good substrate for the reactions in the 2-MAG pathway, whereas the reactivity of l(3)-MAG is lower (17). Free glycerol is a substrate of the glycerophosphate pathway, but the reaction rate is slower than that in the 2-MAG pathway, and its contribution to TG resynthesis is small (16). Although it has not been
‘The term “triglyceride” (TG) is used according to the conventional terminology for TAG resynthesized and circulated in the body.
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quantitatively concluded what ratio of 1(3)-monooleinproduced from DAG is directly absorbed, the amount of 2-MAG produced is smaller after ingestion of DAG than after ingestion of TAG. Thus, the resynthesis rate of TG in the small intestinal epithelium may be lower with the ingestion of DAG. Murata et al. (18) reported that the release rate of resynthesized TG into the intestinal lymph was lower after administration of a DAG emulsion than after a TAG emulsion. In their experiment, there were no differences in the total FA composition of chylomicron-TG between the rats administered the TAG and DAG emulsions. Considerable differences between the two groups of rats were observed in the FA composition at the 2-position of TG. Thus, intragastric infusion of DAG consisting mainly of 1,3-DAG compared with TAG not only altered the rate of lipid transport by lymph chylomicrons but also altered the structure of the TG moiety in the rats. These results support the altered pathway of TG resynthesis after DAG and TAG ingestion. Incorporation of 14C-labeledlinoleic acid into TG was significantly retarded in the intestinal mucosa of rats into which a DAG oil emulsion had been infused compared with TAG oil emulsion (15). These results indicated that the contribution of the 2-MAG and glycerol-3-phosphate pathways might differ between rats given these different acylglycerols . Figure 28.2 illustrates the speculation about metabolic pathways of DAG vs. TAG according to the results obtained to date. Another beneficial effect besides the nutritional effect was that the stomach-emptying time was reduced by using DAG oil compared with TAG oil in scrambled eggs. The subjects ingested scrambledsgs cooked with or without test oils. The stomachemptying time was monitored by using technetium-labeled albumin as a probe and quantification by scintigram. Stomach-emptying time for scrambled eggs cooked with DAG oil was less than that for eggs cooked with control TAG oil (19). Improved Postprandial lipemia After Ingestion of DAG in Humans
TAG in the serum is a source of fat accumulated in the body, and postprandial hyperlipidemia is a risk factor for cardiovascular disease. A reduction in postprandial hypertriglyceridemia is one of the nutritional characteristics of DAG. Figure 28.3 shows changes in serum TG concentrations after a single ingestion of lipid emulsion in healthy men (20). The two test oils had almost the same FA composition. We showed in another experiment that the magnitude of the postprandial increase in chylomicron-TG concentration after ingestion of DAG was cut in half compared with TAG ingestion (21). The human studies described above were conducted using either a fat emulsion as an experimental food or DAG oil products under strictly controlled dietary intake conditions. Therefore, it would be of interest to investigate changes in TG levels in the serum or body weight after the ingestion of DAG or TAG that had been incorporated into ordinary meals. The postprandial response to DAG vs. TAG ingestion was examined using a mayonnaise-type solidified dressing prepared from DAG oil (22) or a typical
N.Matsuo
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250 200 n
?s
Y
Q)
P 150
c
0
100 I
50
0
2
4
6
Time after fat loading (h)
8
Fig. 28.3. Differential effects of DAG and TAG on postprandial serum TC concentration. Serum TG was measured after a single ingestion of a lipid emulsion (30 g/m2) in healthy men. The percentage change from the initial value f: SEM (n = 6) is shown. Asterisks indicate significant difference between treatments: *f < 0.05; **f< 0.01. Source: Reference 20.
breakfast into which a mayonnaise-type DAG (10 g DAG oi1/60 kg) was incorporated (23). These studies were double-blind, placebo-controlled, crossover studies in which healthy volunteers were recruited (mean BMI, 23-25 kg/m2). The control was regular mayonnaise prepared with TAG with a FA composition similar to that of DAG. Increments in the serum triglyceride levels over baseline values were significantly smaller in the DAG ingestion group than in the TAG group. Differences were more pronounced for changes in chylomicron-TG and remnant-like particle (RLP)-TG levels. Among several mechanisms that may be involved in the decreased response of chylomicron-TG to the dietary DAG, it is unlikely that the impaired absorption of the dietary fat from the small intestine reduced postprandial chylomicron-TG responses. Dietary DAG had the same digestibility coefficient as TAG with a similar FA composition (13). Because the calculated amount of FA released from the DAG oil (913 mg/g) was lower than that of the TAG oil (956 mg/g), the theoretical amounts of FA ingested differed between the emulsions. However, the differences in serum and chylomicron-TG concentrations observed these studies were far greater than those expected from the small difference in the amounts of FA ingested. Murata et al. (18) showed that the lymphatic transport of chylomicron-TG caused by the DAG oil was slower than that induced by TAG oil using rats infused intragastrically with the emulsions. We speculate that a common mechanism is underlying these observations because FA contents had been adjusted to be equal in this animal study.
Diacylglycerol O i l
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Repeated DAG vs. TAG Consumption Reduces Body Fat in Humans
We conducted a long-term comparative study of DAG and TAG oils in 38 healthy Japanese men (24). The men ingested 10 g of test oil with a total of 43 g of fat intake in the daily diet for a period of 16 wk in a double-blind, parallel study. Total energy intake was not restricted. As shown in Table 28.2, significantly greater decreases were observed in the DAG group than in the TAG group in body weight, visceral fat, subcutaneous fat, and waist circumference. Abdominal fat was accessed by the fat area of the abdominal cross-section image of a CT scan. This Japanese study was expanded by using men and women who were categorized as obese or overweight. The Chicago Center for Clinical Research (Chicago, IL) conducted a double-blind, parallel study in which 131 overweight or obese men and women ingested DAG or TAG diets for 24 wk (25). In that study, 15% of energy intake was ingested as the test oil under a mild hypoenergetic condition established by subtracting 500-800 kcal from the energy requirement calculated for the body weight, activity level, and age. The decreases in body weight and body fat were significantly greater in the DAG group than in the TAG group (Fig. 28.4). Long-term studies on ad libitum consumption of DAG oil in adults were conducted. These include a randomized, double-blind, controlled, parallel trial in which 312 adults were randomly assigned to receive either a treatment (DAG oil, n = 155) or placebo (TAG oil, n = 157) product (26). The subjects were in overall good health with a BMI 225 kg/m2 and/or a fasting serum TG level 2 150 mg/dL. They consumed either DAG oil or TAG oil for 1 yr without any dietary restrictions and maintained a constant level of physical activity. BMI ( P = 0.002) and skin-fold TABLE 28.2 Changes i n Anthropometric Values and Body Composition
of the Subjectsatb
DAG group
Wk Body weight (kg) Waist circumference (cm) Visceral fat area (crn2) Subcutaneous fat area (cm2)
0 16 0 16 0 16 0 16
TAG group Change
ChangeC
72.1 i 1.8 69.5 i 1 .7#nd -2.6 85.0 i I .4 80.6 1.3## -4.4 79 i 7.0*e 63 i 7.0” -6
*
148+ii 126 i 10”
* 0.3** * 0.6*
68.1 i 1.3 67.0 i 1.5# -1 .I 82.0 i 1.o
* 10.4
79.5 i 1.2#’ -2.5 i 0.6 56 6.0 2.0***f 51 i 6.0 -5.0 3.0 126 10 -22 i 3.0** iiaki3 -8.ort4.0
*
aSource: Reference 24. values are means i SEM (n = 19/group). Value at wk 16 - value at wk 0. dDifferent from wk 0 value by Student’s t-test: ‘ P < 0.05, *‘P< 0.01, eDifferent from TAG diet group by Student’s t-test for paired value: * P < 0.05, ‘Different from TAG diet group by analysis of covariance: * P < 0.05.
*
*
**P<0.01.
*
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692
0
‘treatment
1
= 0.024
Pvisit4 . 0 0 1 1
0
4
8
12
16
20
24
Time (wk)
B
-o-0-
-10.
DAG Oil TAG Oil
= 0.011 Pvlsit 4.001
‘treatmen*
-12
i
0
12
24
Time (wk)
*
Fig. 28.4. Panel A: The percentage change (mean SEM) in body weight from baseline among subjects assigned to the DAG oil or TAG oil group. P-values represent results of repeated-measures ANOVA. Panel B: The percentage change (mean SEM) in fat mass from baseline among subjects assigned to DAC oil or TAG oil groups. Pvalues represent results of repeated measures ANOVA. Source: Reference 25.
*
thickness ( P = 0.028) decreased significantly in the DAG group compared with the TAG group. In conclusion, long-term consumption of DAG oil, when it was simply replaced with conventional TAG cooking oil, may modestly but significantly
Diacylglycerol Oil
693
reduce body weight and prevent weight gain compared with TAG oil consumption, even at a similar energy intake. Thus, DAG oil may be beneficial for maintaining a healthy body weight. In addition to these parallel, controlled studies, a 2-yr monadic study (n = 60) (27) and a 1-yr monadic study (n = 114) (28) were also conducted. Similar efficacies were noted in both trials. In the 2-yr study, the number of risk factors for subjects who initially had >3 categories of risk factors for the metabolic syndrome was significantly decreased. No serious adverse effects with respect to hematology or serum chemistry were noted. These results, which were obtained using practical meals, indicate that the effects of DAG oil in preventing body fat accumulation are closely associated with the metabolic differences between DAG and TAG.
Antiobesity Effects of DAG in Animal Studies Animal models were used to further confirm the effects of DAG consumption and to elucidate its mechanism. Watanabe et al. (14) investigated the effect of DAG feeding on body fat and body weight in male SD rats. Although the percentage of body fat was significantly less in the group fed 10% DAG food at 3 and 4 wk of feeding compared with the group fed TAG food, no difference in body weight occurred between the diet groups during the feeding period. Another experiment conducted by Soni et al. (29) revealed no compound-related effects on body weights in male and female SD rats fed either 5.3% DAG or TAG diet for up to 77 wk. On the other hand, Meng et al. (30) recently reported that body weight and fat mass were decreased significantly in SD rats fed a 20% DAG diet compared with the corresponding TAG diet. Sugimoto et al. investigated the efficacy of DAG or TAG oil on body fat accumulation in male Wistar rats (31) and female Wistar rats (32). There were no significant differences in body fat accumulation between the DAG and TAG diet groups. Thus, although some argument remains concerning the efficacy of DAG feeding in rats, most of the studies indicated a trend for less bodyfat accumulation after high DAG diet feeding. When C57BL/6J mice (obesity- and diabetes-prone model mice) were fed a high-fat (30%, w/w) and high-sucrose (13%, w/w) diet for 5 mon, body weight and body fat (white adipose tissue) mass increased compared with a control low-fat diet. Insulin and leptin (peptide product of the OB gene, which is associated with obesity in mice as well as in humans) concentrations also increased in these mice. Substituting DAG for TAG prevented the increases in body weight and fat accumulation associated with the high-fat and high-sucrose diet (Fig. 28.5) (33). Increases in insulin and leptin concentrations were also prevented by replacing TAG with DAG. Therefore, the mice consuming the DAG diet maintained smaller fat stores, suggesting that DAG consumption produced an increase in energy expenditure. Although the mechanism for the suppression of body fat accumulation by a DAG diet has not been fully elucidated, it may be due to increased P-oxidation in
694
N. Matsuo
n
en
W
Fig. 28.5. Effects of diets on (A) body weight and (B) body fat accumulation in C57BL/6J mice. Control (Cont.), standard control diet containing 5% TAG; High TAG, TAG diet containing 30% TAG + 13% sucrose; High DAG, DAG diet containing 30% DAG + 13% sucrose (n = 5/group; values are means r SD). WAT, white adipose tissue mass after 22 wk. Source: Reference 33.
the liver induced by a DAG diet. When food containing -10% DAG was given to SD rats for 2-3 wk, enzyme activity associated with FA synthesis in the liver decreased and that for P-oxidation of FA increased (34). Meng et al. (30) reported that hepatic acyl-CoA carnitine acyltransferase was stimulated and hepatic acylCoA DAG acyltransferase activity was decreased after 8 wk of 20% DAG diet feeding, suggesting that dietary DAG affects FA metabolism in the liver by stimulating P-oxidation and repressing anabolism of TAG. In addition, experiments with a high-fat-induced obesity model mice (C57BL/6J), a high-DAG diet, compared with a high-TAG diet, increased hepatic acyl-CoA oxidase activity and mRNA for acyl-CoA synthase, suggesting a higher capacity for hepatic lipid oxidation (33). Using the same animal model, they showed that within the first 10 d (before the onset of obesity), DAG consumption stimulated P-oxidation and lipid metabolismrelated gene expression including acyl-CoA oxidase, medium-chain acyl-CoA dehydrogenase, and uncoupling protein-2 in the small intestine but not in the liver, skeletal muscle, or brown adipose tissue, suggesting the predominant contribution of intestinal lipid metabolism on the effects of DAG (35). Beneficial Effects of DAG Consumption in Pathological Conditions
Type 2 diabetes, abnormal lipid metabolism, hypertension, hyperuricemidgout, arteriosclerotic diseases, and fatty liver are complications of obesity. The importance of diet therapy as a treatment for these lifestyle diseases is widely recog-
Diacylglycerol Oil
695
nized. DAG oil was tested in pathological states such as dialysis patients and type 2 diabetes patients. Using type 2 diabetic patients, a randomized, single-blind, controlled parallel trial was conducted. In that study, the influence of long-term ingestion of DAG on blood lipids in type 2 diabetics with hypertriglyceridemia was examined. The serum TG levels in these patients were persistently increased despite continuous nutritional counseling (for 14 mon on average) at the outpatient clinic (36). The subjects were 16 patients with diabetes and a mean BMI of 26.3 f 2.9 kg/m2. The baseline serum TG, total serum cholesterol, and HDL cholesterol concentrations did not differ significantly between the DAG and control groups. Changes in these variables during the test period were not significant in the control TAG group. The serum TG levels in the DAG group, in contrast, were significantly decreased (from 2.51 f 0.75 to 1.52 f 0.28 mmol/L, mean f SD, P < 0.01) after 3 mon of treatment to the levels significantly ( P < 0.05) lower than those of the control group (1.52 f 0.28 vs. 3.59 1.70 mmol/L). There were no significant changes in blood sugar levels during the study period in either group. In the DAG group, the glycohemoglobin (HbA,,) level decreased significantly from 6.41 f 1.15 to 5.79 f 0.85% (9.7% reduction, P < 0.05). The normal range of HbA,, established by the Japan Diabetes Society is 4.3-5.8%. No significant changes in HbA,, levels were observed in the control group (6.88 f 0.53 to 6.65 f 0.73%).
*
Further Application of DAG to Serum Cholesterol-Lowering Food
In addition to obesity, hypercholesterolemia is another health concern because it was shown to be associated with various lifestyle-related diseases. Clinical investigations revealed that phytosterol administration in humans reduces plasma total cholesterol and LDL cholesterol levels. Meguro et al. (37) found that the DAG oil is a good solvent for phytosterols compared with TAG oil. They conducted a study to investigate the difference in serum cholesterol-lowering activities between phytosterols dissolved in DAG oil and those dispersed in TAG oil. Administration of 500 mg/d of phytosterols dissolved in the DAG oil for 2 wk significantly reduced the total and LDL cholesterol levels from 5.57 to 5.31 mmol/L (4.7% reduction) and 3.69 to 3.39 mmol/L (7.6% reduction), respectively, whereas the same amount of phytosterols dispersed in the TAG oil had no significant effect. DAG oil containing phytosterols has been on the market in Japan since 2001 as a “Food for Specified Health Use” as approved by the Ministry of Health, Labor, and Welfare.
Conclusions Cooking-oil product containing DAG at 2 80% (w/w) was approved as a “Food for Specified Health Use” by the Ministry of Health, Labor, and Welfare of Japan. The approved claims vs. conventional oil are that there is a smaller elevation in postprandial blood concentration of TG and the oil is less likely to become body fat.
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N.Matsuo
These effects are likely caused by metabolic differences in the oils after their absorption into mucosal cells rather than by a difference in the bioavailability of the oils. Although a more detailed analysis of the digestion, absorption, and metabolic processes is warranted, consumption of DAG oil was shown to improve lipid metabolism in various states. Clinical studies are in progress to further confirm the efficacy and reliability of DAG oil consumed under various conditions.
Summary DAG oil is an edible oil containing ;r 80% (w/w) DAG with natural FA, -70% of which is the 1,3-DAG isoform. The energy value of the DAG oil is practically the same as that of ordinary oil containing TAG. In contrast to TAG, the main digestive product of DAG is l(or 3)-monoacylglycerol, which is poorly reesterified into TG in the small intestinal mucosa. The magnitude of postprandial elevations of TG content in chylomicrons is markedly smaller after DAG ingestion compared with TAG with a similar FA composition. DAG consumption increases hepatic andor enzyme activities for P-oxidation and increases oxygen consumption compared with TAG. It was shown that long-term DAG consumption prevents the accumulation of body fat and body weight and reduces risk factors for lifestyle-related diseases. References 1. World Health Organization (2003) Controlling the Global Obesity Epidemic, see WHO official web site: http://www.who.int/nut/obs.htm. 2. Mokdad, A.H., E.S. Ford, B.A. Bowman, W.H. Dietz, F. Vinicor, V.S. Bales, and J.S. Marks, Prevalence of Obesity, Diabetes, and Obesity-Related Health Risk Factors, 2001, J . Am. Med. Assoc. 289: 76-79 (2003). 3. Ministry of Health, Labor and Welfare, Japan, in The National Nutrition Survey in Japan, 2001, edited by Research Association of Health and Nutrition Information, Daiichi-Shuppan, Tokyo, 2003, p. 58. 4. Bray, G.A., D.H. Ryan, and D.W. Harsha, Diet, Weight Loss, and Cardiovascular Disease Prevention, Curr. Treat. Options Cardiovasc.Med.: 259-269 (2003). 5. Wylie-Rosett, J., Fat Substitutes and Health: An Advisory from the Nutrition Committee of the American Heart Association, Circulation 105: 2800-2804 (2002). 6. Tada, N., and H. Yoshida, Diacylglycerol on Lipid Metabolism, Curr. Opin. Lipidol. 14: 29-33 (2003). 7. Tada, N., Physiological Actions of Diacylglycerol Outcome, Curr. Opin. Clin. Nutr. Metab. Care 7: 145-149 (2004). 8. Abdel-Nabey, A.A., Y . Shehata, M.H. Ragab, and J.B. Rossell, Glycerides of Cottonseed Oils from Egyptian and Other Varieties, Riv. Ital. Sostanze Grasse 69: 443-447 (1992). 9. D’Alonzo, R.P., W.J. Kozarek, and R.L. Wade, Glyceride Composition of Processed Fats and Oils as Determined by Glass Capillary Gas Chromatography. J. Am. Oil Chem. SOC.59: 292-295 (1982). 10. Crossley, A., I.P. Freeman, J.F. Hudson, and J.H. Pierce, Acyl Migration in Diglycerides, J. Chem. SOC.:760-764 (1959).
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11. Kodali, D.R., A. Tercyak, D.A. Fahey, and D.M. Small, Acyl Migration in 1,2Dipalmitoyl-sn-glycerol,Chem. Phys. Lipids 52: 163-170 (1990). 12. Watanabe, T., M. Shimizu, M. Sugiura, M. Sato, J . Kohori, N. Yamada, and K. Nakanishi, Optimization of Reaction Conditions for the Production of DAG Using Immobilized 1,3-Regiospecific Lipase Lipozyme RM IM, J . Am. Oil Chem. SOC. 80: 1201-1 207 (2003). 13. Taguchi, H., T. Nagao, H. Watanabe, K. Onizawa, N. Matsuo, I. Tokimitsu, and H. Itakura, Energy Value and Digestibility of Dietary Oil Containing Mainly 1,3-Diacylglycerol Are Similar to Those of Triacylglycerol, Lipids 36: 379-382 (2001). 14. Watanabe, H., K. Onizawa, H. Taguchi, M. Kobori, H. Chiba, S. Naito, N. Matsuo, T. Yasukawa, M. Hattori, and H. Shimasaki,Nutritional Characterization of Diacylglycerols in Rats, J . Jpn. Oil Chem. Sci. 46: 301-307 (1997). 15. Kondo, H., T. Hase, T. Murase, and I. Tokimitsu, Digestion and Assimilation Features of Dietary DAG in the Rat Small Intestine, Lipids 38: 25-30 (2003). 16. Friedman, H.I., and B. Nylund, Intestinal Fat Digestion, Absorption, and Transport, Am. J . Clin. Nutr. 33: 1108-1139 (1980). 17. Lehner, R., A. Kuksis, and Y. Itabashi, Stereospecificity of Monoacylglycerol and Diacylglycerol Acyltransferases from Rat Intestine as Determined by Chiral Phase High-Performance Liquid Chromatography, Lipids 28: 29-34 (1993). 18. Murata, M., K. Hara, and T. Ide, Alteration by Diacylglycerols of the Transport and Fatty Acid Composition of Lymph Chylomicrons in Rats, Biosci. Biotechnol. Biochem. 58: 1416-1419 (1994). 19. Yasunaga, K., Y. Seo, Y. Katsuragi, N. Oriuchi, H. Ootake, K. Endo, and T. Yasukawa, Gastric Emptying Rate of Dietary Diacylglycerol, 54th Annual Meeting of the Japanese Society of Nutrition and Food Science, Matsuyama, May 12-14 (2000). 20. Tada, N., H. Watanabe, N. Matsuo, I. Tokimitsu, and M. Okazaki, Dynamics of Postprandial Remnant-Like Lipoprotein Particles in Serum After Loading of Diacylglycerols, Clin. Chim. Actu 311: 109-117 (2001). 21. Taguchi, H., H. Watanabe, K. Onizawa, T. Nagao, N. Gotoh, T. Yasukawa, R. Tsushima, H. Shimasaki, and H. Itakura, Double-Blind Controlled Study on the Effects of Dietary Diacylglycerol on Postprandial Serum and Chylomicron Triacylglycerol Responses in Healthy Humans, J . Am. Coll. Nutr. 19: 789-796 (2000). 22. Takei, A., T. Toi, H. Takahashi, Y. Takeda, J. Moriwaki, H. Takase, and Y. Katsuragi, Effects of Diacylglycerol-Containing Mayonnaise on Lipid-Metabolism and Body Fat in Humans, J . Nutr. Food 4: 89-101 (2001). 23. Tomonobu, K., T. Hase, and I. Tokimitsu, Subsequent Blood Lipid Variance in Subjects Consuming Diacylglycerol Meal, 25th Annual Meeting of Japan Society of Clinical Nutrition, Yokohama, Oct. 3-5 (2003). 24. Nagao, T., H. Watanabe, N. Goto, K. Onizawa, H. Taguchi, N. Matsuo, T. Yasukawa, R. Tsushima, H. Shimasaki, and H. Itakura, Dietary Diacylglycerol Suppresses Accumulation of Body Fat Compared to Triacylglycerol in Men in a Double-Blind Controlled Trial, J . [email protected]: 792-797 (2000). 25. Maki, K.C., M.H. Davidson, R. Tsushima, N. Matsuo, I. Tokimitsu, D.M. Umporowicz, M.R. Dicklin, G.S.Foster, K.A. Ingram, B.D. Anderson, S.D. Frost, and M. Bell, Consumption of Diacylglycerol Oil as Part of a Reduced-Energy Diet Enhances Loss of Body Weight and Fat in Comparison with Consumption of a Triacylglycerol Control Oil, Am. J. Clin. Nutr. 76: 1230-1236 (2002).
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26. Koyama, W., H. Kawashima, Y. Wakagi, K. Mori, H. Takase, and T. Yamaguchi, Long-Term Effects of Diacylglycerol Used Ad Libitum as Cooking Oil in Home, 24th Annual Meeting of Japan Society for the Study of Obesity, Makuhari, Chiba, Nov. 13-14 (2003). 27. Otsuki, K., K. Mori, H. Takase, and Y. Katsuragi, Two Years, Long-Term Effects of Dietary Diacylglycerols on the Risk of the Metabolic Syndrome, J . Jpn. Human Dry Dock 19: 29-32 (2004). 28. Katsuragi, Y., T. Toi, and T. Yasukawa, Effects of Dietary Diacylglycerols on Obesity and Hyperlipidemia, J . Jpn. Human Dry Dock. 14: 258-262 (1999). 29. Soni, M.G., H. Kimura, and G.A. Burdock, Chronic Study of Diacylglycerol Oil in Rats, Food Chem. Tonicol. 39: 317-329 (2001). 30. Meng, X., D. Zou, Z. Shi, Z. Duan, and Z. Mao, Dietary Diacylglycerol Prevents HighFat Diet-Induced Lipid Accumulation in Rat Liver and Abdominal Adipose Tissue, Lipids 39: 37-41 (2004). 31. Sugimoto, T., T. Kimura, H. Fukuda, and N. Iritani, Comparisons of Glucose and Lipid Metabolism in Rats Fed Diacylglycerol and Triacylglycerol Oils, J . Nutr. S c i . Vitaminol. 49: 47-55 (2003). 32. Sugimoto, T., H. Fukuda, T. Kimura, and N. Iritani, Dietary Diacylglycerol-Rich Oil Stimulation of Glucose Intolerance in Genetically Obese Rats, J . Nutr. Sci. Vitaminol. 49: 139-144 (2003). 33. Murase, T., T. Mizuno, T. Omachi, K. Onizawa, Y. Komine, H. Kondo, T. Hase, and I. Tokimitsu, Dietary Diacylglycerol Suppresses High Fat and High Sucrose Diet-induced Body Fat Accumulation in C57BL/6J Mice, J . Lipid. Res. 42: 372-378 (2001). 34. Murata, M., T. Ide, and K. Hara, Reciprocal Responses to Dietary Diacylglycerol of Hepatic Enzymes of Fatty Acid Synthesis and Oxidation in the Rat, Br. J . Nutr. 77: 107-121 (1997). 35. Murase, T., M. Aoki, T. Wakisaka, T. Hase, and I. Tokimitsu, Anti-obesity Effect of Dietary Diacylglycerol n C57B6/6J Mice: Dietary Diacylglycerol Stimulates Intestinal Lipid Metabolism, J . Lipid Res. 43: 1312-1319 (2002). 36. Yamamoto, K., H. Asakawa, K. Tokunaga, H. Watanabe, N. Matsuo, I. Tokimitsu, and N. Yagi, Long-Term Ingestion of Dietary Diacylglycerol Lowers Serum Triacylglycerol in Type I1 Diabetic Patients with Hypertriglyceridemia, J . Nutr. 131: 3204-3207 (2001). 37. Meguro, S., K. Higashi, T. Hase, Y. Honda, A. Otsuka, I. Tokimitsu, and H. Itakura, Solubilization of Phytosterols in Diacylglycerol Versus Triacylglycerol Improves the Serum Cholesterol-Lowering Effect, Eur. J . Clin. Nutr. 55: 513-517 (2001).
Chapter 2 9
Plant Stanol Ester as a Cholesterol-Lowering Ingredient of Benecol@Foods Pia Salo, Anu Hopia, Jar; Ekblom, Ritva Lahtinen, and Paivi Laakso Raisio Benecol Limited, Finland
Introduction Dietary plant sterols and stanols have a serum cholesterol-lowering effect when dietary intake is sufficient, -2 g of sterol or stanol equivalenud. The regular daily intake of sterols or stanols is recommended as one low density lipoprotein (LDL) cholesterol-lowering therapeutic dietary option in those individuals who want to lower their serum cholesterol levels by lifestyle means (1). During the last few years, sterol- and stanol-enriched functional foods increased in popularity in the international food market. Plant stanol ester (stanol ester)-enriched functional foods were the pioneers of these food types. Stanol ester is a patented cholesterol-lowering ingredient of Raisio Benecol Ltd., and the active ingredient of Benecol@ foods. The first Benecol food, stanol ester-enriched margarine, was first marketed in November 1995 in Finland. Today Raisio Benecol Ltd. and McNeil Nutritionals market stanol ester internationally to food companies worldwide. Stanol ester is one of the forms of plant sterols existing in nature (see Chapter 15). This chapter introduces the properties of stanol ester as an ingredient in cholesterol-lowering functional foods. The first part of the chapter describes the chemistry and technology of stanol esters in different food types. The second part of the chapter discusses their role as part of a heart-healthy diet. Occurrence of Plant Stanols in the Diet
Plant stanols are present in our daily diet in small amounts (2); the main sources are whole-grain foods, mainly wheat and rye (Table 29.1). The daily intake of stanols in the average Western diet is -60 mg/d, whereas the intake of plant sterols is -150-300 mg/d, and that of cholesterol is 500-800 mg/d. The relatively low natural levels of stanols in the diet are insufficient to have a significant effect on serum cholesterol levels. Chemistry of Plant Stanol Esters
Plant stanols are hydrogenated derivatives of plant sterols. Figure 29.1 shows the molecular structures of cholesterol and the most abundant plant sterols and plant 699
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TABLE 29.1 Dietary Sources of Plant Sterols and S t a n o P Sterols
Source
Cholesterol Plant sterols Plant stanols
Egg yolk, liver, crustaceans Vegetable oils, corn, bean Grains (wheat, rye, corn)
Daily intake (mg)
300-400 150-300 2 0-5 0
aSource:Reference 2 .
stanols. The main dietary plant sterols, sitosterol and campesterol, differ in structure from cholesterol by an ethyl and methyl group, respectively, at C-24 in the side chain (Fig. 29.1). Sitostanol and campestanol are the corresponding stanols in which the sterol backbone is fully saturated. In different plant stanols, the molecular structure differs in the side chain of the stanol backbone only. Saturation of sterols to the corresponding stanols changes their technological properties as well as behavior in the digestive track, compared with unsaturated sterol derivatives. Stanol esters of Benecol foods are FA esters of plant stanols. The stanol part of the molecule is sitostanol or campestanol, whereas the FA residue originates from different vegetable oils. As an example, Figure 29.1 shows the structure of sitostanyl oleate, which is the major stanol ester component when stanols are esterified with low erucic acid rapeseed oil (LEAR) FA. Production of Stanol Esters
Plant stanol esters are produced from natural plant sterols and vegetable oils. Plant sterols are derived either from vegetable sources or from tall oil. The main commercial vegetable source of sterols is soy, but corn and rapeseed are also potential sources. Typically, vegetable sterols are separated as a by-product during the vegetable oil refining process at the steam distillation step. The sterols are then refined from the recovered deodorizer distillate (3). Another source of plant sterols is tall oil, which is a by-product of the wood pulp industry formed when coniferous woods are digested under alkaline conditions (4). The production scheme of stanol esters is described in Figure 29.2. Free sterols are first saturated by hydrogen in the presence of a catalyst. After hydrogenation, the catalyst is removed, and free stanols are crystallized from the reaction mixture. Crystallization serves also as a purification step. The composition of the stanol produced depends on the sterol source used. A typical sitostano1:campestanol ratio in a vegetable oil-based stanol is 70:30 and 90:lO in tall oil-based stanol. Free stanols are further esterified with vegetable oil FA esters resulting in plant stanol esters. This part of the manufacturing process consists of dissolving stanols into FA esters, the esterification reaction, and the postprocessing steps. The same basic process principles are followed as in the interesterification of edible oil, i.e., the reaction is conducted at elevated temperature and vacuum in the presence of a catalyst. Once the reaction is completed, postprocessing takes place. It consists
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#
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Campestanol
Cholestanol
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Sitostanol
& & &
HO
#
HO
Cholesterol
HO
Sitosterol
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Stigmasterol
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-
c-0
6
Sitostanyl oleate
Fig. 29.1, Chemical structures of selected sterols and stanols.
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L PLANT STEROLS
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PLANT STANOLS
~~
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VEGETABLE OIL FATTY ACID ESTERS ESTERTFICATION PLANT STANOL
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of steps generally used in edible oil and fat processing, e.g., washing to inactivate the catalyst, bleaching, and deodorization. The final stanol ester product is cooled, packed into containers, and shipped to the customers after passing quality assurance tests. Physical Properties of Stanol Esters
Stanol ester is a fat-like product with a waxy texture; the ester is a creamy white color in solid form. Molten stanol ester is a viscous clear liquid with a bright yellow color. Odor and taste are bland. Stanol ester is insoluble in water and soluble in fat. The viscosity of stanol ester is higher than that of the triglyceride oil with the same FA composition. For example, stanol ester with LEAR-FA has a viscosity of 153 CPat 60°C (by Brookfield RVDV-I1 viscometer, SC4-18 spindle) and 65 CP at 80°C, whereas the viscosity of low erucic acid rapeseed (LEAR)-oil is 21.4 CPat 60°C (5). The physical properties of stanol esters can be tailored by changing the FA composition. This is achieved by selecting an FA source that contributes the targeted melting profile for the stanol ester. The carbon chain length and the degree of
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unsaturation of the FA affect the melting point of the stanol ester. The composition of stanols, i.e., the ratio of sitostanol to campestanol, does not significantly affect the physical properties of stanol esters. In different technological applications of Benecol foods, the FA part is selected so that the melting properties, texture, and other sensory characteristics of the stanol ester closely resemble the corresponding properties of the fat that is being replaced. The physical properties of stanol ester are of great importance, especially in food applications containing insignificant amounts of fats. Depending on the food matrix, the required melting range of stanol esters varies from liquid to solid at ambient temperature. Typical FA that are useable in stanol esters are derived from liquid vegetable oils, such as rapeseed oil, sunflower oil, soybean oil, olive oil, corn oil, or mixtures of vegetable oils. By using these FA, the melting point of stanol ester by dropping point (DP) measurement varies typically between 35 and 50°C. This melting range is suitable for texturizing purposes for many food applications such as spreads and dairy products. As an example, stanol ester produced from LEAR-FA has a DP of 4143°C. Figure 29.3 presents the solid fat contents (SFC-%) of LEAR-stanol ester, partially hydrogenated soybean oil, and an interesterified palm stearidcoconut oil blend. LEAR-stanol ester has a melting profile such that it can replace traditionally used hard fat fractions. Moreover, LEAR-
90 80 n
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70
60
e
50
u*
40
I
30
2 0 rn
20 10
,. 10
15
20
25
30
35
40
45
Temperature ("C) Fig. 29.3. Solid fat content (%) of low erucic acid rapeseed oil (LEAR)-stanol ester [dropping point (DP) 42'C], hydrogenated soybean oil (DP 42°C) and interesterified palm stearinkoconut oil blend (DP 42OC) at different temperatures.
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stanol ester provides the product with a nutritionally more beneficial FA composition. LEAR-stanol ester is rich in n-3 FA and contains a low amount of saturated fatty acids (SFA), whereas traditional hard fats contain high amounts of saturated and/or trans-unsaturated FA. Stanol esters can be tailored also to be in liquid form at ambient temperature by selecting an FA composition containing a high amount of PUFA and a very small amount of SFA, typically 4%of the total FA. Oxidative and Processing Stability of Stanol Ester
Stanol ester is a stable molecule under major food-processing conditions. The ester bond is resistant to hydrolytic conditions of foods such as presence of water under typical pH ranges of all food types. Furthermore, the saturated nature of the stanol moiety improves the oxidative stability of stanol ester compared with more unsaturated lipids. The major deterioration in the quality of fats and oils is caused by oxidation, resulting in unacceptable sensory properties and shortening of the product’s shelflife. Oxidation is also the major cause of quality deterioration and off-flavors in stanol esters. The unsaturated FA moieties are the most likely parts of the stanol ester molecules to become oxidized, whereas the stanol moieties, without any sites of unsaturation, are very stable. Oxidation of the FA part of the stanol ester is comparable to that occurring in vegetable oils and fats. Plant sterols are expected to be oxidized in a way similar to cholesterol (6). Few data are available on the oxidation stability of plant sterols, but the area is currently one of intensive research. The major oxidation products of plant sterols and stanols are 7-hydroxysterols, 5,6-epoxysterols, 7-ketosterols, and triols. In general, the stanols are much less prone to oxidation than sterols due to the lack of unsaturation sites in the molecule. For example, a recent study shows that only 0.15% of the 1% sitostanol enrichment in rapeseed oil was oxidized during a thermooxidation study conducted for 2 h at 180°C (7). By comparison, 3.6% of the natural content of campesterol and sitosterol (7.3 mg/g) in rapeseed oil was oxidized during 6 h of treatment at 180°C (8). Fully comparable studies on the oxidative stability of sterols and stanols are not yet available in the literature. In general, oxidized plant sterols are expected to be present in plant sterol-containing foods in the same quantities as oxidized cholesterol in cholesterol-containing foods (pg/g levels) (6). The amount of oxidized stanols in stanol-containing foods is much lower due to the lack of double bonds in the stanol structure. The stability of stanol esters is measured using the same methods as those applied for stability studies of oils and fats. Most often, the properties measured are peroxide value (PV), FFA, and sensory quality. The PV is the measure of primary oxidation products of FA origin, hydroperoxides, whereas the FFA level indicates the degree of hydrolysis occurring during storage. Sensory properties are the most important quality attributes affecting the acceptance of the product. Figure 29.4 presents the stability of an LEAR stanol ester, rapeseed oil, and an interesterified palm stearinlcoconut oil vegetable fat blend as a function of PV vs.
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705
, .....
Vegetable fat blend
/' /
/
/
Rapeseed oil ///
Stanol ester
/
/
/
/
/
/
/
0
2
4
6 Time (d)
8
10
12
Fig. 29.4. Stability of low erucic acid rapeseed oil (LEAR)-stanol ester, rapeseed oil, and interesterified palm stearinkoconut oil vegetable fat blend during an accelerated oxidation trial carried out at 60°C.
time during an accelerated oxidation test carried out at 60°C (9). Stanol ester is clearly more stable than rapeseed oil and behaves quite similarly to the palm stearin/coconut vegetable fat blend, which represents a relatively saturated fat. During long-term stability trials under normal storage conditions, stanol esters proved to be very stable. For example, during 12 mon of storage at refrigerator temperature (4-8"C), a slight increase in PV occurs after 10 mon and practically no change in the sensory quality of the stanol ester (Fig. 29.5). To summarize, stanol esters are very stable under normal storage and food preparation conditions because they are more resistant to oxidation than the most common vegetable oils. Using stanol esters in food applications instead of conventional fats does not decrease the shelf-life of the end-product. As is the case for all fats and oils, stanol esters should be protected from heat, air, and light to avoid oxidation. If longterm storage is required, stanol esters are typically stored in solid form at refrigeration temperatures. Furthermore, antioxidants in general use can also be added to stanol ester products as they are to other oils or fats to minimize oxidation. Benecop Food Applications
Benecol foods are targeted to those who want to lower their serum cholesterol levels by dietary means. In every Benecol-branded food, there is a sufficient amount
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-
-
0
3
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Fig. 29.5. Stability of stanol ester at normal storage conditions (4-8OC) as a function of peroxide value (PV) and sensory quality. The sensory quality is presented as the mean value of the taste evaluations of at least 5 trained panelists and the taste scores from 1 to 5 are used: 5 represents an excellent product quality and 1 a product with strong off-flavor and after-taste. Scores 3-5 represent acceptable sensory quality.
of plant stanol ester as an ingredient to provide the cholesterol-lowering effect. This effect is demonstrated when the product is consumed at normal dietary levels. Benecol foods are also tailored to have a heart-healthy nutritional profile such as low fat content and a nutritionally desirable FA profile. At their best, Benecol foods provide consumers dietary choices by which they can promote heart health. When developing new Benecol foods, the target is to provide consumers with healthy choices for different consumer circumstances and needs. It is important to find healthy food forms in which an adequately high level of the active ingredient can be incorporated into the particular food. Furthermore, it should be convenient for the consumers to know the stanol ester content and be able to estimate hisker daily intake of stanol. Local eating habits have to be considered because the sterol-enriched foods should be staple foods, making it easy to incorporate them into every meal. Nutritionally balanced FA compositions, lower fat content, or lower energy content compared with typical foods in the same category would help consumers identify the stanol-enriched foods as heart-healthy choices. Benecol margarine and low-fat spread are the most commonly known Benecol foods. Although stanol ester is fat by its physical nature, it can also be added into many low-fat foods. Benecol food applications vary from high-fat margarines to dairy and cereal foods in which the stanol ester is the sole fat source in the product, Table 29.2 lists the different Benecol food applications that are currently available on the mar-
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TABLE 29.2 Benecol Foods Available o n the Market (Spring 2004) Product
Country
Butter milk Cereal bar Chews Cream cheese type spread Softgel Liquid rapeseed oil product Margarine Milk Pasta Spread
Finland Ireland, UK United States Belgium, Finland, Ireland, UK United States Finland Belgium, Finland, Poland, The Netherlands Spain, United Arab Emirates Finland Argentina, Belgium, Finland, France, Germany, Greece, Ireland, The Netherlands, Sweden, UK, United States Belgium, Finland, France, Ireland, United Arab Emirates, UK Austria, Belgium, Finland, Germany, Ireland, The Netherlands, Portugal, Spain, Switzerland, UK
Yogurt Yogurt drink
ket in Spring 2004. Clinical tests were conducted for each new type of Benecol food to ensure that the cholesterol-lowering efficacy of stanol ester is not lost when the product platform is changed. Adding Stanol Esters to Different Food Forms
The same basic procedures for handling and manufacturing are used for stanol esters as are used for other edible fats. For most of the end-product applications, stanol esters have to be melted and mixed to homogeneity before use. The melting procedures and equipment are the same as those used for edible fats. The melting temperature must be set -15-20°C above the measured melting point to obtain a totally liquid and clear stanol ester. This is due to the fact that stanol ester is always a mixture of different FA esters. The palmitic and stearic acid esters of stanol, in particular, require higher temperatures to be dissolved into the main bulk. All foods that contain edible oils and fats can be considered as candidates for stanol ester enrichment, as well as products in which stabilizers or emulsifiers exist naturally or are added to achieve specific properties. Stanol esters can also be used in foods containing insignificant amounts of fats. Stanol esters should be added and mixed into the end-product formulation in the same way as the fat they replace. Basically the same process equipment can be used for manufacturing a stanol ester-enriched product as is used for the base product. Their higher viscosity compared with normally used oils and fats must be taken into account when adding stanol esters into a food matrix. Effective mixing may be required to achieve a homogenous mixture or emulsion.
In fat-containing foods, stanol esters normally replace a similar amount of the regular fat blend used. When emulsion-based products such as mayonnaise, salad
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dressings, margarine, and spreads are produced, it is recommended that molten stanol ester first be mixed with the fat blend and other fat-soluble ingredients before mixing with the water phase. Stanol esters can also be added as solids, for example, as a powder or in ground form, together with other dry ingredients. Stanol esters with different FA profiles can be selected for different food types. The type of stanol ester is chosen according to its texturizing properties to provide properties that correspond to those of the saturated fat that it is replacing in the food application in question. A good example is LEAR-stanol ester, which is a versatile product for most typical fat-containing food applications. Stanol esters rich in PUFA are liquid at ambient temperature and can be used for liquid or coldprocessed food applications, salad dressings, mayonnaise, and vegetable oils.
Stanol Esters in Spreads and Dressings The first commercial food application for stanol ester was the Benecol margarine launched in Finland in 1995. Since then, margarines with fat contents from 60 to 65% were introduced in the market in Belgium, Ireland, The Netherlands, Poland, the UK, and the United States. Benecol@light spreads containing 30-55% fat are sold in Argentina, Belgium, Finland, France, Germany, Greece, Ireland, The Netherlands, Sweden, the UK, and the United States. An olive oil margarine (Belgium) and liquid margarine (Finland) enriched with stanol ester are also available. Spreads other than margarines include light cream cheese-type vegetable oilbased spreads that are available in Belgium, Finland, Ireland, and the UK. In traditional spreads, the hard stock is made from naturally occumng hard fats, such as tropical oils and animal fats, or fats are prepared by either partially or fully hydrogenating liquid oils with or without subsequent interesterification with liquid oils. Hard-fat fractions are also obtained by fractionation procedures and are used as such or are subjected to further modification processes such as interesterification. Stanol esters replace a part of the hard stock in spread making. Molten stanol ester is added to the fat phase, and the process continues with conventional emulsion preparation, cooling crystallization, and packing. In applications utilizing direct crystallization, it is important to first totally melt the entire fat blend including the stanol esters. Replacing traditional saturated and trans-unsaturated hard fat with stanol esters results in a 45% reduction in SFA content of the spread, but only 5 and 10% reduction in monounsaturated FA and PUFA contents, respectively (Fig. 29.6). Particularly in low-fat spreads, the structurizing effect of stanol esters can be utilized to even fully replace hard fat that contains nutritionally undesired saturated and trans-FA, for example, in the fat blends of spreads (10). The energy content of stanol ester-enriched spreads is typically lower than that of the corresponding conventional spreads. This is because the stanol part, which represents 60% of the stanol ester molecule, is unabsorbable and is not a source of energy in the product. Thus, stanol ester-enriched spreads and margarines have a lower energy content than their conventional counterparts (lo).
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100 %
80% 60% 40%
20% 0%
Light spread (40%)
Benecol* light spread (32%)
Benecol** light spread (32%)
Fig. 29.6. The amounts of SFA and unsaturated FA of different kind of spreads. *Benecol light spread made of stanol ester [dropping point (DP) 42"CI; **Benecol light spread made of stanol ester (DP 45OC).
One of the nutritional benefits of Benecol spreads is that the content of unsaturated FA can be increased in significant amounts in the product. The cholesterollowering effect of Benecol spreads is an additive effect of the stanol esters and unsaturated FA in the product (see below). Salad dressings and mayonnaise containing stanol ester are also available in the market. Salad dressings are sold as such, but mayonnaise can also be added into meal salads such as potato salad. As fat-based emulsions, salad dressings and mayonnaise form a good product platform for Benecol foods. Stanol esters can almost entirely replace the oil phase in this type of product, enabling production of very low fat foods. Stanol Esters in low-Fat Foods Dairy and Nondairy Foods. Dairy foods are gaining popularity among cholesterol-lowering foods. During 2003, different types of dairy applications of plant stanol-enriched Benecol foods were launched in 12 European countries (see Table 29.2). The first sterol-enriched dairy food applications were yogurts enriched with plant stanol ester in the UK in 1999. Although yogurt remains a common product, yogurt drinks have been the most popular version of new dairy Benecol foods in recent years.
Dairy foods have several benefits as plant stanol ester-enriched foods. Several low-fat dairy products are considered to be a natural part of a healthy diet among a
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large number of consumers. Many dairy foods are available as single servings into which it is easy to incorporate either half or all of the daily recommended amount of stanol esters. Because many dairy products are oil-in-water emulsions, stanol esters are added into the product before the homogenization step similar to what occurs when milk fat is replaced by vegetable oils in dairy products (Fig. 29.7). Plant stanol esters give dairy products a creamy texture with a very low fat content. Furthermore, because the FA of stanol ester are derived from vegetable oils, the FA profile of the product is high in unsaturated FA and very low in saturated fats. Different soy- and cereal-based nondairy alternatives are also potential platforms for Benecol foods. Especially in the United States, Asia, and Central Europe, soy- and cereal-based nondairy foods are gaining popularity as heart-healthy dietary choices. Plant stanol esters can be added to these food forms using a technology similar to that used in the dairy industry. Stanol esters have an additional benefit in soy and cereal food applications because they are able to mask some of the off-flavors of these foods. For example, unsweetened soy beverages and fermented soy products often have an unpleasant “beany” taste. A commonly used method to mask off-flavors is to add sugar, other sweetening agents, or flavors. Stanol esters can mask bitterness. When they are added, less sugar or other sweetener is required to obtain a pleasant taste and mouth feel with soy products. Cereal Foods and Pasta. Cereal foods, especially those rich in soluble fiber, are widely acknowledged as part of a heart-healthy diet. Thus, different types of cereal foods are also potential Benecol applications. The first cereal-based Benecol food was dry pasta first marketed in Finland at the beginning of 2003. Stanol ester was incorporated into pasta dough with only minor modifications and alterations to traditional production facilities. The stanol ester content of uncooked and cooked pastas is the
Water phase (+ stabilizer)
1
Stanol ester (+ emulsifier)
PasteurizationKJHT
1 Fig. 29.7. Adding stanol
Packaging
ester to dairy products. UHT, ultra-high temperature.
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same per net weight. Furthermore, no reduction of active ingredients was noticed during cooking. The sensory properties of Benecol pasta are comparable to those of conventional pasta. The addition of plant stanol ester even improves the “a1 dente” texture and mouth feel, i.e., the feeling of the surface of the pasta product (11). In addition, the cholesterol-lowering effects continued to be present in this type of stanol esterenriched product (see below). Stanol ester can also be added to breakfast cereals, such as muesli. Portion-packaged products in particular would be ideal vehicle for incorporating the cholesterollowering feature into the daily diet. As in spreads and margarines,stanol esters replace part or all of the hard stock of the fat in the granulated muesli products. Thus, the SFA content is lowered. The sensory properties of stanol ester-enriched cereal products are comparable to or better than those of their conventional counterparts. Stanol esters provide the desirable crispy texture of these products without the need to add extra fat to the product. Similar to other stanol ester-enriched products, the total energy intake is lower, and the FA composition is nutritionally more favorable than in conventional breakfast cereals. Beverages and Other Low-Fat Applications. Beverages are a convenient way for consumers to obtain effective amounts of stanol esters from the diet. Although stanol esters typically have been associated with fatty foods, they can easily be incorporated into beverages and other low-fat or fat-free foods. However, currently only a few sterol- or stanol ester-enriched drinks and low-fat food applications other than dairy products are on the market. Cloudy or juice-based beverages can be produced with conventional production facilities including homogenization. The product can have an acceptable, stable structure through the addition of commonly used amounts of emulsifiers and stabilizers, or even without those, if the product contains proteins with good emulsifying capacity. Protein-containing beverages, such as milk or cereal protein-based smoothies as well as coffee or cacao drinks, are examples of potential stanol ester-enriched drinks. The amount of additives, such as emulsifiers, can be kept low to obtain a stable, acceptable taste and appearance. Stanol esters provide a creamy texture to beverages and may also smooth excess acidity of fruit juices without the addition of sweetener. Stanol ester-enriched beverages can also be produced by using a premix or dried powder as a form of stanol ester to be added to the drink base at the production plant. Consumers are becoming increasingly demanding concerning how functional food products fit in with their existing lifestyles. Demand for on-the-go functional food products is also increasing. Because drinks are a growing segment in the functional food market, it is expected that more of these types of products will appear on the market in the future. Stanol Esters and Heart Health
The prevalence of cardiovascular disease (CVD) , i .e., coronary heart disease (CHD), stroke, and peripheral arterial disease, is increasing as populations age and
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unhealthy lifestyles are adopted. Three risk factors in particular, i.e., elevated cholesterol, smoking, and high blood pressure, or combinations of these, are responsible for >75% of all CVD worldwide. Of these three, elevated cholesterol carries the greatest attributable risk for CHD; in other words, eliminating this risk factor would result in the greatest decrease in CHD incidence. In addition to medical therapies, dietary tools to control the risk factors of such diseases are vigorously being developed. Plant stanols are added to foods to aid in controlling serum cholesterol levels and, ultimately, reducing the risk of heart disease. Plant stanols are present in the everyday diet in small amounts. When enriched in foods, plant stanols effectively reduce the absorption of all sterols from the digestive tract. Consequently, they reduce serum levels of cholesterol and of plant sterols, both of which are implicated as risk factors for CVD. Reducing elevated serum cholesterol and especially the LDL cholesterol level is the single most important thing to do to hamper atherosclerosis, the process underlying CVD. Dietary intervention is always the cornerstone of therapy for dyslipidemia according to all national and international recommendations and guidelines, even when cholesterol-lowering drug therapies have been initiated (1,12). Importantly, diet may have beneficial cardiovascular consequences beyond its effects on lipid concentrations, such as antithrombotic effects and improved endothelial function. History of Plant Stanol Research
Early studies in the 1950s showed that plant-derived sterols reduced serum cholesterol levels and prevented the formation of atheroma (13). However, the marked increases in plant sterol levels in plasma raised safety concerns especially in the light of the newly described disease, sitosterolemia. Sitosterolemia is a rare, recessively inherited disorder characterized by excessive absorption, reduced clearance, and high plasma levels of plant sterols, resulting in premature atherosclerosis (14,15). Some years later, however, new interest in this field of research was generated when Sugano et al. (16) reported that p-sitostanol was not absorbed and had a greater hypocholesterolemic effect than p-sitosterol in rats. Similar results were obtained later with rabbits (17). In addition, sitostanol depressed the formation of aortic atheroma more than sitosterol. These results were also verified recently by Ntanios et al. (18-20), who showed that the cholesterol-lowering effect of plant sterol blends depended on the amount of sitostanol in the blends. The first description of the use of plant stanols to lower plasma cholesterol in humans was by Heinemann et al. (21). In a small uncontrolled study, they showed that administration of capsules of sitostanol dispersed in oil at a dose of 1.5 g/d lowered LDL cholesterol by 15%. Similar intakes of sitosterol and sitostanol that were infused directly into the small intestine decreased cholesterol absorption by 50 and 85%, respectively. Becker et al. (22) showed that LDL cholesterol decreased in children suffering from familial hypercholesterolemia by 33% with 1.5 g sitostanoYd compared with 20% for 6 g sitosterol/d, suggesting higher efficacy
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for sitostanol than for sitosterol in lowering LDL cholesterol.The negative outcome of free sitostanol administered in capsules was useful in drawing attention to the importance of the physical state of plant sterols and stanols in determining their efficacy (23). Mechanism of Action
Stanols reduce serum sterol levels by inhibiting the absorption of sterols from the small intestine. Importantly, however, stanols themselves are only minimally absorbed. Recent evidence indicates that the inhibition of absorption may be a multistep process (Fig. 29.8). In the first step, stanols replace sterols from micelles. In vitro and in vivo studies showed that plant sterols and stanols compete with and displace cholesterol from the micelles, the form from which cholesterol absorption occurs. Ikeda et al. (24) showed that administration of sitosterol and sitostanol displaced cholesterol from micelles by 24 and 53%, respectively. Later, this was shown also by Nissinen et al. (25). It was shown recently that plant stanols as FA esters consumed with a meal were as effective in one daily dose as when the same amount was divided over three doses in the day (26). This discovery indicated that there may be a second step in the absorption inhibition process involving a mechanism within the enterocytes. Studies using cell cultures and animal models showed that stanols and sterols are taken up by the enterocytes (27-30). Interestingly, stanols were shown to be potent inducers of ATPbinding cassette transporter A1 (ABCAI) expression (3 l), which indicates that within the enterocyte, plant stanols increase the ABCAl -mediated cholesterol efflux back into the intestinal lumen (Fig. 29.8). With decreased cholesterol absorption from the intestine, the liver increases both the synthesis of cholesterol from its precursors and the uptake of cholesterol by increasing the expression of LDL receptors. This further reduces the amount of cholesterol circulating in the bloodstream (22,32,33). Because the incorporation of stanols into the micelles is the key step for the mechanism, the optimal effect is achieved when plant sterols and stanols are ingested as part of a meal, preferentially consumed as food. Reduction in Dietary and Biliary Cholesterol
Of the total cholesterol in the digestive tract (1 0-1.5 g/d) -33% is derived from the diet and the remainder from bile. Studies using the dual-isotope method showed that the ingestion of plant stanols reduces cholesterol absorption efficiency between 50 and 80% (34,35). This decrease in absorption applies to both dietary and biliary cholesterol, which explains the effect of plant stanols even during low-cholesterol diets and statin medication when the endogenous synthesis of cholesterol is strongly depressed (32,36,37) (Fig. 29.9).
Poor Absorption of Plant Sterols. Compared with cholesterol, plant sterols are poorly absorbed, which suggests that the enterocytes differentiate between their
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Fig. 29.8. The proposed two-step mechanism of action of stanols. In the small intestine, stanol ester is hydrolyzed by pancreatic esterase. Free stanols have a greater affinity for micelles than cholesterol and therefore displace cholesterol from them (24). Within the enterocyte, sitostanol upregulates the ATP-binding cassette transporter Al (ABCAI) transporter, which i s responsible for pumping more cholesterol out from the enterocyte and back into the intestinal lumen (31).
molecular structures (35,3842). Absorption of plant stanols is even lower than the absorption of sterols. The reason for this difference is still not clear. However, plant sterols and stanols are poorly esterified by the intestinal enzyme acyl-CoA cholesterol acyltransferase (ACAT) (43). Because esterification is needed for effective incorporation into chylomicrons ,the absence of an efficient esterification mechanism may explain in part the poor absorption of plant sterols from the enterocyte into the lymph. Recently, studies in mice revealed that all sterols are rapidly taken up by the intestinal mucosa but are resecreted back into the intestinal lumen depending on the side chain length and the saturation of the A5 double bond. Thus, the resecre-
Stanot Ester-Enriched Functional Foods
Absorption of dietary cholesterol
71 5
Absorption of biliary cholesterol
Fig. 29.9. ingestion of 2.6 g/d plant stanols in FA ester form reduced total cholesterol absorption by 44% (from 7.1 7 mg/kg body weight during the control margarine period to 4.03 mg/kg body weight during the stanol ester margarine period). Absorption rates of dietary and biliary cholesterol were reduced by 43.7% (from 1.35 to 0.76 mg/kg body weight) and 37.4% (from 5.22 to 3.27 m o o d y weight), respectively (32).
tion into the intestinal lumen may be the most important limiting step in the absorption. Difference in the side chain length of cholesterol by a methyl (campesterol) or ethyl group (sitosterol) produces marked differences in absorbability and hepatic elimination. In humans, the absorption rate of plant sterols also depends on the side chain length and the saturation of the As double bond. Furthermore, hepatic clearance of sitosterol is faster than that of campesterol, and the clearance of campesterol is higher than that of cholesterol. These data indicate that in humans, the ABC G5/8 transporters are also regulating absorption and hepatic secretion of the different sterols (44). Clinical Trials with Plant Stanols
A combination of results from a number of clinical trials shows that with mean consumption of 2 g esterified stanol/d, serum levels of cholesterol decrease by 10-15%
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(49, which calculates to a reduction in CVD risk of -25% (46). Importantly, the stanol-specific cholesterol-lowering effect is in addition to the effects of other dietary cholesterol-lowering options. Therefore, numerous international expert boards have recommended the inclusion of stanol ester as a dietary option to reduce cholesterol levels in addition to the more conventional dietary means (lJ2). Sustained Effect in Long-Term Use
The full effect of stanol esters on serum total and LDL cholesterol levels is obtainable within 1-2 wk (47-49). For clinical benefit, however, it is more important that the cholesterol-lowering effect be sustained. Most of the clinical studies on stanols and sterols were unfortunately of short duration only. The publication of results from the landmark study on the effect of 52-wk long consumption of stanol ester preceded the launch of the first commercial stanol ester food product, Benecol margarine in 1995 (Fig. 29.10). The North Karelia stanol ester study involved 153 moderately hypercholesterolemic subjects who were randomly assigned to replace 24 g/d of their usual dietary fat intake with rapeseed oil margarine with or without 6 mon
12 mon
0.9
-1.1
-24-
-6-
Benecol
8
m Control spread
-8
-12 -1 4 -16
4
-10.4
i
j
Fig. 29.10. The 12-mon, randomized, double-blind study showed that substituting sitostanol-ester margarine for part of the daily fat intake in subjects with mild hypercholesterolemia was effective in lowering serum total cholesterol and LDL cholesterol by 10 and 14%, respectively. There was no difference between the daily doses of 1.8 and 2.6 g of stanol in LDL-lowering effect (50).
Stanol Ester-Enriched Functional Foods
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sitostanol ester (50). The intervention started with a dose of 2.6 g sitostanol/d and was continued as such for 6 mo. At 6 mo, half of the subjects in the treatment group reduced their intake of stanol to 1.8 g and the other half continued with 2.6 g. In those who continued to receive 2.6 g, LDL cholesterol decreased at the end of the year by 14% from baseline or by 13% compared with those consuming placebo margarine. No effect was seen between the two doses studied. These results are in striking contrast to those found in a similar study with sterol ester; during 52 wk of consumption of 1.6 g of sterol ester-enriched spread, total and LDL cholesterol levels were reduced by only 4 and 6%, respectively (51). The only study to date that has evaluated the difference in long-term efficacy between stanol and sterol esters in a head-to-head trial showed that during mon 1 of stanol and sterol consumption, the effect was equal with respect to LDL lowering. However, after 2 mon, stanol efficacy remained constant, whereas the sterol effect was attenuated with no difference from baseline in total or LDL cholesterol (52). Because this study showed differences between the stanol and sterol groups with respect to bile acid metabolism, it was hypothesized that the higher absorption of sterols may downregulate bile acid synthesis and thus reduce the long-term efficacy. Similar results were obtained in diabetics in whom sterol ester spread reduced cholesterol levels during the first weeks of consumption, whereas after 8 wk, the effect was diminished (53). Clearly, more studies are warranted to address the question of long-term efficacy. Formulation of Stanols
The physical form of stanols poses restrictions to the food matrix used to transport them into the digestive system. Solubilization in the emulsified fat phase of the food digest is a prerequisite for plant stanols to be incorporated into the micelles. Free plant sterols and stanols form highly stable crystals that may require several days or even weeks to dissolve in bile salt solutions (5435). Direct dissolution of free sterols and stanols in fat is not very efficient because the solubility in triglyceride is only -1-2%. However, after esterification with FA, the solubility increases to 10-20% (56,57). The esterified sterols and stanols are fat-soluble and can mix directly and homogeneously with the fat phase of the food digest. In contrast, the efficacy of free sterols and stanols is highly dependent on the extent to which they are first solubilized in the emulsified fat phase of the food digest in the stomach. In fact, LDL lowering with low doses of sterols or stanols was shown in studies in which plant sterols were first dissolved in vegetable oil (50), egg fat (57), emulsified with triglyceride monooleate (58) or lecithin (55), or finely micronized and mixed with fatty foods (59,60). Indeed, data from clinical studies with free sterols and stanols show that their efficacy is reduced when they are dispersed in proteinrich, low-fat food matrices such as low-fat yogurts, marmalade, and bread rather than, for example, fat-based spreads (61-64). Thus, with free sterols and stanols, key conditions include the presence of fat in the food matrix and the way in which the free sterol or stanol is dispersed within that fat phase.
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To show the efficacy of esterified stanol also in low-fat food forms, new product forms were tested in several randomized intervention trials. One low-fat vehicle that was tested is yogurt. Total and LDL cholesterol were reduced by 9 and 14%, respectively, in subjects consuming stanol ester vs. placebo low-fat (
With esterified stanols, combining the data from the clinical studies shows that optimal LDL lowering is achieved at an intake of -2 g/d, and increasing the dose does not further reduce serum LDL levels (45).The minimum amount of stanols required to produce a significant cholesterol-lowering effect is -0.8-1 .O g/d. However, most of the studies focused on higher intake levels, -2-3 g/d of plant stanols in their esterified, fat-soluble forms. In a dose-response study, daily intake of 0.8, 1.6,2.3, and 3.0 g reduced serum LDL cholesterol after 2 wk of stanol ester margarine consumption with 10.5, 11.2, 17.4, and 17.4%, respectively (Fig. 29.1 1). Furthermore, Cater and Grundy (unpublished results) showed that stanol ester margarines delivering 2, 3, and 4 g plant stanols for 6 wk each reduced serum LDL cholesterol levels by 12, 13, and 14%, respectively. Effects of Consumption Frequency
Most of the studies on stanol consumption evaluated the effects of using stanol divided in two or more doses. Plat and Mensink (26) showed, however, that daily intake for 4 wk of 2.5 g of esterified plant stanols once at lunch was as effective in lowering serum LDL cholesterol concentrations as when the dose was divided over three meals (0.42 g at breakfast, 0.84 g at lunch, and 1.25 g at dinner, proportional to the dietary cholesterol intake) (Fig. 29.12). Also, when plant stanol ester was incorporated into pasta or meat products that were eaten once a day, LDL cholesterol levels were reduced by 10-1 1% in two placebo-controlled, randomized trials (unpublished results).
Stanol Ester-Enriched Functional Foods
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Fig. 29.11. This study determined the dose-response relationship for serum cholesterol with different doses of plant stanol ester in hypercholesterolemic subjects and showed that significant reduction of serum total and LDL cholesterol concentrations was reached with a 1.6-g stanol dose, and increasing the dose from 2.4 to 3.2 g did not provide a clinically important additional effect (89).
€ffecfs of Background Diet
The common feature of all international recommendations to reduce CVD risk is an emphasis on the interaction of multiple risk factors as increasing the risk for CVD and an emphasis on the full implementation of appropriate lifestyle changes as the foundation of treatment. Diet provides several tools for cholesterol lowering, and it was shown that by combining different dietary components, LDL reductions similar to those achieved with statins can be achieved (65). Current dietary guidelines aiming at heart health include as key elements of the diet decreases in saturated fat and cholesterol intake and increases in soluble fiber and plant stanol or sterol intake. Stanols add to the cholesterol-lowering effect of other recommended dietary changes. In other words, stanol has an additional cholesterol-lowering effect even after dietary changes such as increasing fiber intake and reducing saturated fat and cholesterol intake (6668) (Fig. 29.13). In addition, stanol ester proved to be effective with the consumption of both high- (50,69) and low-fat (66,67,70) diets and
diets with high and low PUFA to SFA ratios, irrespective of dietary cholesterol intake. Similarly, the effect seems to hold for many diverse food cultures. Although
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Once per day
Three times per day
Fig. 29.12. To lower LDL cholesterol concentrations, it is not necessary to consume products rich in plant stanol ester at each meal. In a Dutch study, consumption of 2.5 g plant stanols at lunch resulted in a similar LDL cholesterol-lowering efficacy compared with consumption of 2.5 g plant stanols divided over the three meals (-0. 29 mmol/L compared with the control period for the 1 time/d diet and -0.31 mmol/L for the 3 times/d period) (26).
most of the studies were done in Europe and the United States, similar results were obtained in Japan (71,72). Source and Composition of Stanols
Ikeda et al. (73) assumed that the cholesterol-lowering action is greatest for the least absorbable plant sterols. Because the absorbability of sitostanol is lower than that of campestanol, a greater hypocholesterolemic effect might be expected after consumption of plant stanol ester mixtures in which the amount of sitostanol is higher than that of campestanol. Stanol esters derived from wood sources such as tall oil contain more sitostanol and less campestanol than those derived from vegetable oil sources. However, the results of three separate studies showed that the composition of stanol esters would appear to be irrelevant to their efficacy because no significant difference in the LDL-lowering effect of sitostanol ester-rich vs. campestanol ester-rich mixtures was found (67,74).
Stanol Ester-Enriched Functional Foods
7;
-1 2%
-12%
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Usual diet and stanol ester
Low-safa diet
1;
Low-safa diet and stanol ester
Fig. 29.13. Stanol adds to the cholesterol-lowering effect of a recommended dietary regimen. The combined use of the stanol ester margarine and the strictly controlled low-saturated fat (safa) diet reduced serum total and LDL cholesterol by 15 and 19%, respectively. These reductions were significantly greater than those in patients consuming a controlled low-safa diet and a standard, low-fat margarine (-8 and -12%, respectively) or those in the reference group consuming a normal diet but with stanol ester margarine (-9 and -1 2%, respectively) (66). Simultaneous Cholesterol-Lo wering Medication
There are several options for medical therapy to lower serum cholesterol levels (75). Statins (3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors) are the most widely used cholesterol-lowering medications to date. They reduce serum cholesterol concentration by inhibiting the synthesis of cholesterol in the liver. Resins (e .g ,, cholestyramine) bind bile acids in the intestine, thereby interrupting the enterohepatic circulation of bile acids and increasing the conversion of cholesterol into bile acids in the liver. The newest cholesterol-reducing drug is ezetimibe, which blocks cholesterol absorption at the intestinal brush border (76). Statins and stanol act additively in a clinical setting because they have different cholesterol-lowering mechanisms. Blair et al. (36) showed that in patients who were taking statin medication for hypercholesterolemia, the addition of stanol ester margarine in the diet reduced serum LDL cholesterol by an additional 17% compared with the control group who also benefited from a 10% reduction in LDL due to the beneficial FA composition of the margarine (Fig. 29.14). Gylling et al. (32) also showed that adding stanol ester margarine to the diet of women taking statin medication led to a further reduction in serum LDL cholesterol levels of 20% in a 12-wk study.
To investigate the cholesterol-lowering efficacy of a triple therapy combining bile acid malabsorption with the inhibition of cholesterol synthesis and absorption,
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Fig. 29.14. Stanol adds to the cholesterol-lowering effect of statins. In this study, daily consumption of a stanol ester spread effectively reduced elevated total and LDL cholesterol levels in participants on a stable regimen of a statin. Plant stanol ester spread reduced total cholesterol at 8 wk by 12% compared with a 5% reduction in the placebo (= regular vegetable-oil margarine) group and a 17% in LDL cholesterol compared with a 7 % reduction in the placebo group (36).
Gylling and Miettinen (77) added a low-dose of simvastatin (20 mg/d) for 3 mo, and then dietary plant stanol ester margarine (2.25 g stanoldd) for 8 wk; finally, 8 g cholestyramine/d was added for another 8 wk. They showed that simvastatin lowered LDL-cholesterol by 39%, and the additional stanol ester margarine by a further 13%. In total, the triple treatment led to 67% reduction in LDL cholesterol from baseline. In patients who had undergone cardiac transplantation, Vorlat et al. (37) showed that the statin dose could be reduced in two thirds of those patients who added stanol or sterol ester margarine to their daily diet. Dual Effect of Stanol and the Importance of Lowering Cholesterol and Plant Sterol Levels
Elevated plant sterol concentrations were implicated as an independent risk factor for CHD. Two recently discovered ABC transporters, ABCGS and ABCG8, play an important role in the regulation of intestinal plant sterol absorption by resecreting already absorbed plant sterols out of the enterocytes back into the intestinal lumen, Mutations in these transporter proteins lead to the rare heritable disease of sitosterolemia, which is characterized by severely elevated serum plant sterol concentrations, normal to moderately increased serum cholesterol concentrations, and a high risk of developing CHD at a very young age. It was shown recently that polymorphisms in the ABCGS and ABCGS genes contribute to the variation in
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serum plant sterol levels in healthy, nonsitosterolemic individuals. Furthermore, according to several epidemiologic evaluations, the risk of developing heart disease seems to be increased even at more “normal” levels of plant sterols (78-80). Statins were shown to increase serum plant sterol concentrations (8 1$2). Therefore, patients should probably not be treated by statins only but by a combination of interventions focused simultaneously on improving the serum lipoprotein profile and lowering serum plant sterols. Recently, Plat et al. showed that a certain genotype of the ABCG8 is characterized by higher serum plant sterol concentrations when examined cross sectionally; these subjects are also responsive to interventions that affect serum plant sterol concentrations, i.e., the addition of plant stanol to the diet of these subjects significantly reduced plant sterol concentrations. Safety of Stanol Ester
Extensive safety evaluation studies in humans and animals showed that plant stanol esters are safe to use, well-tolerated, and without adverse effects. Furthermore, plant stanols are absorbed only minimally. Thus, because of the lack of bioavailability after the ingestion of stanol ester, adverse systemic effects of plant stanols are highly unlikely. Plant stanol esters have obtained Generally Recognized As Safe status (GRAS) in the United States. They were also evaluated by food authorities in several EU countries before their introduction to the market in each country. In addition, food authorities in Europe evaluated the use of stanols and sterols in foods. Their recommendation is that a daily intake should range between 1 and 3 g phytosterols and stanols (83). As a consequence of the reduced absorption of cholesterol, the absorption of fat-soluble components other than cholesterol, such as vitamins and antioxidants, might also be reduced. Like cholesterol, carotenoids and tocopherols are transported by lipoproteins. Because the number of LDL particles decreases in the circulation after consumption of plant sterols or stanols, plasma concentrations of carotenoids and tocopherols also decrease. For this reason, these antioxidants are often standardized to plasma lipid concentrations. The results from randomized, placebo-controlled trials concerning the effects of plant sterols or stanols on fatsoluble vitamins and antioxidants were summarized recently (45). Significant reductions were seen in clinical trials for hydrocarbon carotenoids; part of the reduction is likely due to reduced absorption and part to the reduced concentration in blood of the carrier, LDL. After correction for cholesterol levels, only the reduction in p-carotene level remained. Importantly, however, the levels of carotenoids and tocopherols were still within the normal ranges. Furthermore, clinical trials showed that adherence to dietary recommendations, including consumption of vegetables and fruit, prevented the decrease in carotenoids (84,85). Plasma concentrations of retinol (vitamin A), 25-hydroxy-vitamin D, and vitamin K are unaffected by dietary plant sterols and stanols.
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Concluding Remarks Considerable progress in the primary and secondary prevention of CVD has occurred in the past 30 years. Risk factors are identified and treated in those not yet ill (primary prevention) and among those with established CVD to prevent recurrent events (secondary prevention). As understanding of the mechanisms of atherosclerotic disease evolved, recommendations for therapeutic interventions were revised with more patients identified as candidates for lipid-lowering therapy and the establishment of more stringent lipid goals. Statin trials showed that lowering cholesterol levels is beneficial at any age and from any previous levels and across all population groups (86,87). Although the development of risk-factor thresholds (e.g., hypercholesterolemia defined as total cholesterol >5 mmol/L or 200 mg/dL) has helped patients and clinicians focus on treatment objectives, such cut-off points have obscured the continuum of risk, i.e ., average population levels are inappropriately assumed to be desirable. Because atherosclerosis is a lifelong process, it is important to target not only those with high-cholesterol levels but also those who are borderline high and to focus on prevention. Dietary intervention studies support the concept that restriction of SFA and cholesterol and increasing the intake of fiber and essential FA, especially n-3 FA, reduce CHD risk. However, compliance with dietary recommendations remains a major problem (88). In short-term dietary trials, cholesterol reductions of 15-20% were achieved, but long-term follow-up has mainly seen reductions of only -5%. Incorporating plant stanols into the diet offers a sustained LDL-lowering effect to complement the favorable changes in the diet. Because the incidence of CHD and its treatment costs are growing constantly, the search for effective preventive measures such as dietary and lifestyle tools to lower the risk factors for CHD is becoming increasingly important. For this purpose, food products with stanol ester offer an efficient, tasty, and convenient solution to be combined with a healthy diet. References 1. Expert Panel, Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel 111), J . Am. Med. Assoc. 285: 2486-2497 (2001). 2. Piironen, V., D.G. Lindsay, T.A. Miettinen, J. Toivo, and A.-M. Lampi, Plant Sterols: Biosynthesis, Biological Function and Their Importance to Human Nutrition, J . Sci. Food Agric. 80: 939-966 (2000). 3. Clark, J.P., Tocopherols and Sterols from Soybeans, Lipid Technol. 111-117 (1996). 4. Salo, P., I. Wester, and A . Hopia, Phytosterols, in Lipids for Functional Foods and Nutraceuticals, edited by F.D. Gunstone, The Oily Press, Bridgewater, UK, 2003, pp. 183-224. 5. Noureddini, H., B.C. Teoh, and L.D. Clements, Viscosities of Vegetable Oils and Fatty Acids, J . Am. Oil Chem. SOC. 69: 1189-1 191 (1992).
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6. Dutta, P.C., Determination of Phytosterol Oxidation Products in Foods and Biological Samples, in Cholesterol and Phytosterol Oxidation Products: Analysis, Occurrence, and Biological Effects, edited by F. Guardiola, P.C. Dutta, R. Codony, and G.P. Savage, AOCS Press, Champaign, IL, 2002, pp. 319-334. 7. Soupas, L., L. Juntunen, S. Saynajoki, A.-M. Lampi, and V. Piironen, GC-MS Methods for Characterization and Quantification of Sitostanol Oxidation Products, J . Am. Oil Chem. SOC.81: 135-141 (2004). 8. Lampi, A.-M., L. Juntunen, J. Toivo, and V . Piironen, Determination of ThermoOxidation Products of Plant Sterols, J . Chromatogr. B 777: 83-92 (2002). 9. AOCS Cg 5-97: Oven Storage Test for Accelerated Aging of Oils, in OfJicial Methods and Recommended Practices of the AOCS, 5th edn., AOCS Press, Champaign, IL, 1998. 10. Wester, I., U S . Patent Application WO 9819556 (1998). 11. Aalto, T., P. Alho-Lehto, and J. Ekblom, U.S.Patent Application WO 200365822 (2003). 12. Harmonized Guidelines on Prevention of Atherosclerotic Cardiovascular Diseases, International Atherosclerosis Society, 2003. 13. Pollak, O.J., Reduction of Blood Cholesterol in Man, Circulation 7: 702-706 (1953). 14. Salen, G., I. Horak, M. Rothkopf, J.L. Cohen, J. Speck, G.S. Tint, V. Shore, B. Dayal, T. Chen, and S. Shefer, Lethal Atherosclerosis Associated with Abnormal Plasma and Tissue Sterol Composition in Sitosterolemia with Xanthomatosis, J . Lipid Res. 26: 1126-1133 (1985). 15. Hidaka, H., T. Nakamura, T. Aoki, H. Kojima, Y. Nakajima, K. Kosugi, I. Hatanaka, M. Harada, M. Kobayashi, A. Tamura, T. Fujii, and Y. Shigita, Increased Plasma Plant Sterol Levels in Heterozygotes with Sitosterolemia and Xanthomatosis, J . Lipid Res. 31: 881-888 (1990). 16. Sugano, M., F. Kamo, I. Ikeda, and H. Morioka, Lipid-Lowering Activity of Phytostanols in Rats, Atherosclerosis 24: 301-309 (1976). 17. Ikeda, I., A. Kawasaki, K. Samezima, and M. Sugano, AntihypercholesterolemicActivity of Beta-Sitostanol in Rabbits, J . Nutr. Sci. Vitaminol. 27: 243-251 (1981). 18. Ntanios, F.Y., and P.J. Jones, Effects of Variable Dietary Sitostanol Concentrations on Plasma Lipid Profile and Phytosterol Metabolism in Hamsters, Biochim. Biophys. Acta 1390: 237-244 (1998). 19. Ntanios, F.Y., D.E. MacDougall, and P.J. Jones, Gender Effects of Tall Oil Versus Soybean Phytosterols as Cholesterol-Lowering Agents in Hamsters, Can. J . Physiol. Pharmacol. 76: 780-787 (1998). 20. Ntanios, F.Y., P J . Jones, and J J . Frohlich, Dietary Sitostanol Reduces Plaque Formation but Not Lecithin Cholesterol Acyl Transferase Activity in Rabbits, Atherosclerosis 138: 101-110 (1998). 21. Heinemann, T., 0. Leiss, and K. von Bergmann, Effect of Low-Dose Sitostanol on Serum Cholesterol in Patients with Hypercholesterolemia, Atherosclerosis 61: 219-223 (1986). 22. Becker, M., D. Staab, and K. von Bergmann, Treatment of Severe Familial Hypercholesterolemia in Childhood with Sitosterol and Sitostanol, J . Pediatr. 122: 292-296 (1993). 23. Denke, M.A., Lack of Efficacy of Low-Dose Sitostanol Therapy as an Adjunct to a Cholesterol-Lowering Diet in Men with Moderate Hypercholesterolemia, Am. J . Clin. Nutr. 61: 392-396 (1995).
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24. Ikeda, I., Y. Tanabe, and M. Sugano, Effects of Sitosterol and Sitostanol on Micellar Solubility of Cholesterol, J. Nutr. Sci. Vitaminol. 35: 361-369 (1989). 25. Nissinen, M., H. Gylling, M. Vuoristo, and T.A. Miettinen, Micellar Distribution of Cholesterol and Phytosterols After Duodenal Plant Stanol Ester Infusion, Am. J. Physiol. 282: G1009-Gl015 (2002). 26. Plat, J., and R.P. Mensink, Effects on Serum Lipids, Lipoproteins and Fat Soluble Antioxidant Concentrations of Consumption Frequency of Margarines and Shortenings Enriched with Plant Stanol Esters, Eur. J. Clin. Nutr. 54: 671-676 (2000). 27. Field, F.J., E. Born, and S.N. Mathur, Effect of Micellar Beta-Sitosterol on Cholesterol Metabolism in CaCo-2 Cells, J. Lipid Res. 38: 348-360 (1997). 28. Bhattacharyya, A.K., and L.A. Lopez, Absorbability of Plant Sterols and Their Distribution in Rabbit Tissues, Biochim. Biophys. Acta 574: 146-153 (1979). 29. Sanders, D.J., H.J. Minter, D. Howes, and P.A. Hepburn, The Safety Evaluation of Phytosterol Esters. Part 6. The Comparative Absorption and Tissue Distribution of Phytosterols in the Rat, Food Chem. Toxicol. 38: 485-491 (2000). 30. Igel, M., U. Giesa, D. Lutjohann, and K. von Bergmann, Comparison of the Intestinal Uptake of Cholesterol, Plant Sterols, and Stanols in Mice, J. Lipid Res. M200393MJLR200 (2002). 31. Plat, J., and R.P. Mensink, Increased Intestinal ABCAl Expression Contributes to the Decrease in Cholesterol Absorption After Plant Stanol Consumption, FASEB J. 16: 1248-1 253 (2002). 32. Gylling, H., R. Radhakrishnan, and T.A. Miettinen, Reduction of Serum Cholesterol in Postmenopausal Women with Previous Myocardial Infarction and Cholesterol Malabsorption Induced by Dietary Sitostanol Ester Margarine: Women and Dietary Sitostanol, Circulation 96: 42264231 (1997). 33. Jones, P.J., M. Raeini-Sarjaz, F.Y. Ntanios, C.A. Vanstone, J.Y. Feng, and W.E. Parsons, Modulation of Plasma Lipid Levels and Cholesterol Kinetics by Phytosterol Versus Phytostanol Esters, J. Lipid Res. 41: 697-705 (2000). 34. Lutjohann, D., I. Bjorkhem, U.F. Beil, and K. von Bergmann, Sterol Absorption and Sterol Balance in Phytosterolemia Evaluated by Deuterium-Labeled Sterols: Effect of Sitostanol Treatment, J. Lipid Res. 36: 1763-1773 (1995). 35. Ostlund, R.E., Jr., J.B. McGill, C.M. Zeng, D.F. Covey, J. Steams, W.F. Stenson, and C.A. Spilburg, Gastrointestinal Absorption and Plasma Kinetics of Soy Delta 5 Phytosterols and Phytostanols in Humans, Am. J. Physiol. 282: E911-E916 (2002). 36. Blair, S.N., D.M. Capuzzi, S.O. Gottlieb, T. Nguyen, J.M. Morgan, and N.B. Cater, Incremental Reduction of Serum Total Cholesterol and Low-Density Lipoprotein Cholesterol with the Addition of Plant Stanol Ester-Containing Spread to Statin Therapy, Am. J. Cardiol. 86: 46-52 (2000). 37. Vorlat, A., V. Conraads, and C. Vrints, Regular Use of Margarine Containing Stanol Esters Reduces Total and LDL Cholesterol After Cardiac Transplantation and Allows Reduction of Statins, J. Heart Lung Transpl. 21: 99-100. (2002). 38. Bosner, M.S., L.G. Lange, W.F. Stenson, and R.E. Ostlund, Percent Cholesterol Absorption in Normal Women and Men Quantified with Dual Stable Isotopic Tracers and Negative Ion Mass Spectrometry, J. Lipid Res. 40: 302-308 (1999). 39. Lutjohann, D., I. Bjorkhem, U.F. Beil, and K. von Bergmann, Sterol Absorption and Sterol Balance in Phytosterolemia Evaluated by Deuterium-Labeled Sterols: Effect of Sitostanol Treatment, J . Lipid Res. 36: 1763-1773 (1995).
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40. Salen, G., E.H. Ahrens, and S.M. Grundy, Metabolism of Beta-Sitosterol in Man, J . Clin. lnvestig. 49: 952-967 (1970). 41. Salen, G., G.S. Tint, S . Shefer, V. Shore, and L. Nguyen, Increased Sitosterol Absorption Is Offset by Rapid Elimination to Prevent Accumulation in Heterozygotes with Sitosterolemia, Arterioscler. Thromb. Vasc. Biol. 12: 563-568 (1992). 42. Salen, G., G. Xu, G.S. Tint, A.K. Batta, and S. Shefer, Hyperabsorption and Retention of Campestanol in a Sitosterolemic Homozygote: Comparison with Her Mother and Three Control Subjects,J. Lipid Res. 41: 1883-1889 (2000). 43. Field, F.J., and S .N. Mathur, Beta-Sitosterol: Esterification by Intestinal Acylcoenzyme A:Cholesterol Acyltransferase (ACAT) and Its Effect on Cholesterol Esterification, 1. Lipid Res. 24: 409417 (1983). 44. Igel, M., U. Giesa, D. Lutjohann, and K. von Bergmann, Comparison of the Intestinal Uptake of Cholesterol, Plant Sterols, and Stanols in Mice, J . Lipid Res. 44: 533-538 (2003). 45. Katan, M.B., S.M. Grundy, P. Jones, M. Law, T. Miettinen, and R. Paoletti for the Stresa Workshop Participants, Efficacy and Safety of Plant Stanols and Sterols in the Management of Blood Cholesterol Levels, Mayo Clin. Proc. 78: 965-978 (2003). 46. Law, M., Plant Sterol and Stanol Margarines and Health, Br. Med. J . 320: 861-864 (2000). 47. Hallikainen, M., E . Sarkkinen, I . Wester, and M . Uusitupa, Short-Term LDL Cholesterol-Lowering Efficacy of Plant Stanol Esters, BMC Cardiovasc. Disord. 2: 14 (2002). 48. Miettinen, T.A., M. Vuoristo, M. Nissinen, H.J. Jarvinen, and H. Gylling, Serum, Biliary, and Fecal Cholesterol and Plant Sterols in Colectomized Patients Before and During Consumption of Stanol Ester Margarine, Am. J . Clin. Nutr. 71: 1095-1102 (2000). 49. Mensink, R.P., S. Ebbing, M. Lindhout, J. Plat, and M.M. van Heugten, Effects of Plant Stanol Esters Supplied in Low-Fat Yoghurt on Serum Lipids and Lipoproteins, NonCholesterol Sterols and Fat Soluble Antioxidant Concentrations, Atherosclerosis 160: 205-213 (2002). 50. Miettinen, T.A., P. Puska, H. Gylling, H. Vanhanen, and E. Vartiainen, Reduction of Serum Cholesterol with Sitostanol-Ester Margarine in a Mildly Hypercholesterolemic Population, N . Engl. J . Med. 333: 1308-1312 (1995). 51. Hendriks, H.F., E.J. Brink, G.W. Meijer, H.M. Princen, and F.Y. Ntanios, Safety of Long-Term Consumption of Plant Sterol Esters-Enriched Spread, Eur. J . Clin. Nutr. 57: 681-692 (2003). 52. O’Neill, F.H., Effects of Dietary Plant Sterols and Stanols on Cholesterol Metabolism in Humans, Ph.D. Thesis, 2003. 53. Lee, Y.M., B. Haastert, W. Scherbaum, and H. Hauner, A Phytosterol-Enriched Spread Improves the Lipid Profile of Subjects with Type 2 Diabetes Mellitus: A Randomized Controlled Trial Under Free-Living Conditions, Eur. J . Nutr. 42: 111-1 17 (2003). 54. Armstrong, M.J., and M.C. Carey, Thermodynamic and Molecular Determinants of Sterol Solubilities in Bile Salt Micelles, J . Lipid Res. 28: 1144-1 155 (1987). 55. Ostlund, R.E., C.A. Spilburg, and W.F. Stenson, Sitostanol Administered in Lecithin Micelles Potently Reduces Cholesterol Absorption in Humans, Am. J . Clin. Nutr. 70: 826-831 (1999).
56. Mattson, F.H., R.A. Volpenhein, and B.A. Erickson, Effect of Plant Sterol Esters on the Absorption of Dietary Cholesterol, J . Nutr. 107: 1139-1 146 (1977).
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57. Mattson, F.H., S.M. Grundy, and J.R. Crouse, Optimizing the Effect of Plant Sterols on Cholesterol Absorption in Man,Am. J . Clin. Nutr. 35: 697-700 (1982). 5 8 . Grundy, S.M., and H.Y. Mok, Determination of Cholesterol Absorption in Man by Intestinal Perfusion, J . Lipid Res. 18: 263-271 (1977). 59. Jones, P.J., F.Y. Ntanios, M. Raeini-Sarjaz, and C.A. Vanstone, Cholesterol-Lowering Efficacy of a Sitostanol-Containing Phytosterol Mixture with a Prudent Diet in Hyperlipidemic Men, Am. J . Clin. Nutr. 69: 1144-1 150 (1999). 60. Christiansen, L.I., P.L. Lahteenmaki, M.R. Mannelin, T.E. Seppanen-Laakso, R.V. Hiltunen, and J.K. Yliruusi, Cholesterol-Lowering Effect of Spreads Enriched with Microcrystalline Plant Sterols in Hypercholesterolemic Subjects, Eur. J . Nutr. 40: 66-73 (2001). 61. Nestel, P., M. Cehun, S. Pomeroy, M. Abbey, and G. Weldon, Cholesterol-Lowering Effects of Plant Sterol Esters and Non-Esterified Stanols in Margarine, Butter and LowFat Foods, Eur. J . Clin. Nutr. 55: 1084-1090 (2001). 62. Volpe, R., L. Niittynen, R. Korpela, C. Sirtori, A. Bucci, N. Fraone, and F. Pazzucconi, Effects of Yoghurt Enriched with Plant Sterols on Serum Lipids in Patients with Moderate Hypercholesterolaemia, Br. J . Nutr. 86: 233-239 (2001). 63. Tikkanen, M.J., P. Hogstrom, J. Tuomilehto, S . Keinanen-Kiukaanniemi, J. Sundvall, and H . Karppanen, Effect of a Diet Based on Low-Fat Foods Enriched with Nonesterified Plant Sterols and Mineral Nutrients on Serum Cholesterol, Am. J . Cardiol. 88: 1157-1 162 (2001). 64. Clifton, P.M., M . Noakes, D. Sullivan, N. Erichsen, D. Ross, G . Annison, A . Fassoulakis, M. Cehun, and P. Nestel, Cholesterol-Lowering Effects of Plant Sterol Esters Differ in Milk, Yoghurt, Bread and Cereal, Eur. J . Clin. Nutr. 58: 503-509 (2004). 65. Jenkins, D.J., C.W. Kendall, A. Marchie, D. Faulkner, E. Vidgen, K.G. Lapsley, E.A. Trautwein, T.L. Parker, R.G. Josse, L.A. Leiter, and P.W. Connelly, The Effect of Combining Plant Sterols, Soy Protein, Viscous Fibers, and Almonds in Treating Hypercholesterolemia, Metabolism 52: 1478-1483 (2003). 66. Andersson, A., B. Karlstrom, R. Mohsen, and B. Vessby, Cholesterol-Lowering Effects of a Stanol Ester-Containing Low-Fat Margarine Used in Conjunction with a Strict Lipid-Lowering Diet, Eur. Heart J . Suppl. 1 : S80-S90 (1999). 67. Hallikainen, M.A., and M.I. Uusitupa, Effects of 2 Low-Fat Stanol Ester-Containing Margarines on Serum Cholesterol Concentrations as Part of a Low-Fat Diet in Hypercholesterolemic Subjects, Am. J . Clin. Nutr. 69: 403410 (1999). 68. Tammi, A., T. Ronnemaa, H. Gylling, L. Rask-Nissila, J. Viikari, J. Tuominen, K. Pulkki, and 0. Simell, Plant Stanol Ester Margarine Lowers Serum Total and Low-Density Lipoprotein Cholesterol Concentrations of Healthy Children: The STRIP Project. Special Turku Coronary Risk Factors Intervention Project, J . Pediatr. 136: 503-510 (2000). 69. Vanhanen, H.T., S. Blomqvist, C. Ehnholm, M. Hyvonen, M. Jauhiainen, I. Torstila, and T.A. Miettinen, Serum Cholesterol, Cholesterol Precursors, and Plant Sterols in Hypercholesterolemic Subjects with Different ApoE Phenotypes During Dietary Sitostanol Ester Treatment, J . Lipid Res. 34: 1535-1544 (1993). 70. Hallikainen, M.A., E.S. Sarkkinen, H. Gylling, A.T. Erkkila, and M.I. Uusitupa, Comparison of the Effects of Plant Sterol Ester and Plant Stanol Ester-Enriched Margarines in Lowering Serum Cholesterol Concentrations in Hypercholesterolaemic Subjects on a Low-Fat Diet, Eur. J. Clin. Nutr. 54: 715-725 (2000).
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71. Homma, Y., T. Ishikawa, M. Tateno, A. Mitaniyama, and M. Sugano, Cholesterol and Apolipoprotein Lowering Effect of Plant Stanol Ester in Healthy Japanese Men and Women. A Randomized, Placebo Controlled Study, Nippon Eiyo Shokuryo Gakkaishi 53: 155-162 (2000). 72. Ishiwata, K., Y . Homma, T . Ishikawa, H. Nakamura, and S . Handa, Influence of Apolipoprotein E Phenotype on Metabolism of Lipids and Apolipoproteins After Plant Stanol Ester Ingestion in Japanese Subjects, Nutrition 18: 561-565 (2002). 73. Ikeda, I., K. Tanaka, M. Sugano, G.V. Vahouny, and L.L. Gallo, Inhibition of Cholesterol Absorption in Rats by Plant Sterols, J . Lipid Res. 29: 1573-1582 (1988). 74. Plat, J., and R.P. Mensink, Vegetable Oil Based Versus Wood Based Stanol Ester Mixtures: Effects on Serum Lipids and Hemostatic Factors in Non-Hypercholesterolemic Subjects, Atherosclerosis 148: 101-1 12 (2000). 75. Knopp, R.H., Drug Treatment of Lipid Disorders, N . Engl. J . Med. 341: 498-51 1 (1999). 76. Davis, H.R., K.K. Pula, K.B. Alton, R.E. Burrier, and R.W. Watkins, The Synergistic Hypocholesterolemic Activity of the Potent Cholesterol Absorption Inhibitor, Ezetimibe, in Combination with 3-Hydroxy-3-methylglutarylCoenzyme A Reductase Inhibitors in Dogs, Metabolism 50: 1234-1241 (2001). 77. Gylling, H., and T.A. Miettinen, LDL Cholesterol Lowering by Bile Acid Malabsorption During Inhibited Synthesis and Absorption of Cholesterol in Hypercholesterolemic Coronary Subjects, Nutr. Metab. Cardiovasc.Dis. 12: 19-23 (2002). 78. Glueck, C.J., J. Speirs, T. Tracy, P. Streicher, E. Illig, and J. Vandegrift, Relationships of Serum Plant Sterols (Phytosterols) and Cholesterol in 595 Hypercholesterolemic Subjects, and Familial Aggregation of Phytosterols, Cholesterol, and Premature Coronary Heart Disease in Hyperphytosterolemic Probands and Their First-Degree Relatives, Metabolism 40: 842-848 (199 1). 79. Sudhop, T., B.M. Gottwald, and K. von Bergmann, Serum Plant Sterols as a Potential Risk Factor for Coronary Heart Disease, Metabolism 51: 1519-1521 (2002). 80. Assmann, G., P. Cullen, J.R. Erbey, D.R. Ramey, F. Kannenberg, and H. Schulte, Elevation in Plasma Sitosterol Concentration Is Associated with an Increased Risk for Coronary Events in the PROCAM Study, Circulation 108 (Suppl.IV-730): 3300 (2003). 81. Miettinen, T.A., T.E. Strandberg, and H. Gylling, Noncholesterol Sterols and Cholesterol Lowering by Long-Term Simvastatin Treatment in Coronary Patients: Relation to Basal Serum Cholestano1,Arterioscler. Thromb. Vasc. Biol. 20: 1340-1346 (2000). 82. Gylling, H., and T.A. Miettinen, Baseline Intestinal Absorption and Synthesis of Cholesterol Regulate Its Response to Hypolipidaemic Treatments in Coronary Patients, Atherosclerosis 160: 477-48 1 (2002). 83. European Commission, Scientific Committee on Foods, General View on the Long-Term Effects of the Intake of Elevated Levels of Phytosterols from Multiple Dietary Sources, with Particular Attention to the Effects on &Carotene, 26 September 2002. 84. Hallikainen, M.A., E.S. Sarkkinen, and M.I. Uusitupa, Effects of Low-Fat Stanol Ester Enriched Margarines on Concentrations of Serum Carotenoids in Subjects with Elevated Serum Cholesterol Concentrations, Eur. J . Clin. Nutr. 53: 966-969 (1999). 85. Noakes, M., P. Clifton, F. Ntanios, W. Shrapnel, I. Record, and J. McInerney, An Increase in Dietary Carotenoids When Consuming Plant Sterols or Stanols Is Effective in Maintaining Plasma Carotenoid Concentrations, Am. J . Clin. Nutr. 75: 79-86 (2002). 86. MRClBHF Heart Protection Study of Cholesterol Lowering with Simvastatin in 20,536 High-Risk Individuals: A Randomised Placebo-ControlledTrial, Lancet 360: 7-22 (2002).
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87. Cannon, C.P., E. Braunwald, C.H. McCabe, D.J. Rader, J.L. Rouleau, R. Belder, S.V. Joyal, K.A. Hill, M.A. Pfeffer, and A.M. Skene, for the Pravastatin or Atorvastatin Evaluation and Infection Therapy -Thrombolysis in Myocardial Infarction 22 Investigators, Comparison of Intensive and Moderate Lipid Lowering with Statins after Acute Coronary Syndromes, N . Engl. J . Med. 350: 1495-1504 (2004). 88. Tang, J.L., J.M. Armitage, T. Lancaster, C.A. Silagy, G.H. Fowler, and H.A.W. Neil, Systematic Review of Dietary Intervention Trials to Lower Blood Total Cholesterol in Free-Living Subjects Commentary: Dietary Change, Cholesterol Reduction, and the Public Health-What Does Meta-Analysis Add?, Br. Med. J . 316: 1213-1220 (1998). 89. Hallikainen, M.A., E.S. Sarkkinen, and M.I. Uusitupa, Plant Stanol Esters Affect Serum Cholesterol Concentrations of Hypercholesterolemic Men and Women in a DoseDependent Manner, J . Nutr. 130: 767-776 (2000).
Chapter 30
Palm Oil, Its Fractions, and Components Oi-Ming Lai Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Introduction Palm, soybean, rapeseed, and sunflower oils comprise 70% of the world’s edible vegetable oil supply. Among these four oils, palm oil has a very high annual growth rate, primarily because it has the highest productivity at 5 tons oilhectare compared with 0.30 for soybean oil, 0.37 for rapeseed oil, and 0.42 for sunflower oil (1). Currently, Malaysia is the largest producer and exporter of palm oil. In 2002, Malaysia produced 11.9 million metric tons (MMT) of crude palm oil and 1.5 MMT of crude palm kernel oil (PKO) (2).The commitment of the government in ensuring a steady supply of the oils as well as the added advantage of tocotrienols and carotenes in the oils led to the exportation of 10.6 and 0.7 MMT of processed palm oil and palm kernel oil, respectively (Table 30.1). Malaysia now produces more palm oil than all of the other countries in the world combined; the oil is consumed in >150 countries.
Palm Oil and Its Fractions Palm oil is obtained from the mesocarp of the fruit of the oil palm species Elaeis guineensis. Each fruit is made up of a hard kernel (seed) inside a shell (endocarp), which is surrounded by the fleshy mesocarp. The mesocarp produces 49% palm oil, whereas the kernel yields -50% PKO (3). These oils differ greatly in their composiTABLE 30.1 World Production and Exports of Palm Oil (in metric tons)a 2001
Major producers Malaysia (palm kernel oil) Indonesia Papua New Guinea Others Total
2002
Production
Exports
Production
Exports
11,804 (1532) 7950 330 3940 24,024
10,625 (669) 4940 326 1690 17,581
11,909 (1472) 8850 3 04 3904 24,967
10,886 (698) 6040 330 1,710 18,966
aSource: Malaysian Palm Oil Board statistics (1).
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tion. Palm oil contains 44.1% palmitic acid (16:O) and 39.0% oleic acids (18:l) ( 4 3 , whereas PKO contains -48.7% lauric acids (12:O) and is more saturated than palm oil (6). Because palm oil is made up of a mixture of triacylglycerols (TAG) with a broad range of melting points, it can be separated using fractional crystallization into solid and liquid fractions, known as palm stearin and palm olein, respectively. Figure 30.1 shows the different fractions and products derived from the oil palm fruit. Palm stearin consists of high amounts of C,, [mainly tripalmitin (PPP)] and C,, and C,, TAG (7). The physical characteristics of palm stearin differ from those of palm oil and palm olein; it is also available in a wider range of melting points and iodine values (IV). Stearin samples obtained through detergent separation are much harder than those obtained using partial vacuum filtration. This makes palm stearin a very useful source of fully natural hard fat for products such as pastry margarine, shortenings, or vegetable ghee (vanaspati), and it allows the manufacturer a wider choice of solid fats to suit the properties and performance of the final food product. Palm olein is traded as a major oil in its own right. Palm olein is fully liquid in warm climates, has a narrower range of glycerides, and blends well with any seed oil. It contains large amounts of C,, [mainly dioleopalmitin (POO) and palmito-oleolinolein (PLO)] and C,, [mainly triolein (OOO)] TAG (7). There is a high demand for a premium palm olein with higher IV and lower cloud point. This olein, known as superolein, has a maximum IV of 60 and a maximum slip melting point ( S M P ) of 19°C. Palm oil also contains high amounts of dipalmitoolein (POP) which is used extensively in the production of cocoa butter equivalents (CBE). A product known as the palm mid-fraction (PMF) can be obtained from the refractionation of olein from the fxststage fractionation process of palm oil. Thus, PMF enriched in C,, TAG is the secondstage stearin produced from this two-stage fractionation process (8). Although originating from the same tree source as palm oil, PKO resembles coconut oil in its composition and characteristics. The major TAG are C,, and C,, with no others >lo% (3). The oil is semisolid at ambient temperatures <28"C and has a sharp melting profile that is suitable for applications in the confectionary industry. Like palm oil, PKO can undergo fractionation to yield palm kernel stearin, which is a valuable component in the production of sharp-melting lauric-based cocoa butter substitutes (CBS). These lauric stearins have a pronounced cooling effect in the mouth and are often used as filling fats in confectionary as well as in toffees and coatings. Fractionation enriches palm kernel stearin with high amounts of lauric and myristic acids, whereas the olein contains more of the lower-melting short chains such as caprylic and capric acids, as well as the unsaturated oleic and linoleic acids (9). Minor Components in Palm Oil
Minor components such as tocopherols, tocotrienols , carotenoids, phosphatides , sterols, triterpenic and aliphatic alcohols comprise <1% of an oil's constituents. Some of these, e.g., tocopherols, tocotrienols (vitamin E), and carotenoids, are nutritionally beneficial,
Fresh Fruit Bunches
Kernels
Crude Palm Oil Refining
b
Crushing a d Extraction
1
RBD Palm Olein
RBD Palm Oil
+I
RBD Palm Stearin
Crude Palm Kernel Oil Crude Palm Kernel Meal
I
1
Margarines Shortenings Frying Fats Vanaspati Ice Cream
Margarines Shortenings
Frying Oils Cooking Oils Shortening Margarines
Refining
9
30 4
2 nd Fractionation
Superolein
I
Palm Mid-Fraction
4 Cooking Oil Premium
I +
Hard Stearin
Blending
a
'
RBD P Im Kemel Olein
Cocoa Butter Equivalent
Coffee Whitener, Coating Fats, Filled Milk
1 -
- -4
. Margarines
\
alm Kernel Stearin A o n k c t i o n e r y Fat
v
Hydrogenation Hydrogenated Palm Kernel Olein/Stearin
Fig. 30.1. Fractionsand products derived from oil palm fruit. RBD, refined, bleached, and deodorized.
W u
W
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Vitamin E. “Vitamin E ’ is the collective term describing bioactivities of both tocopherols and tocotrienols derivatives. Tocotrienols are fat-soluble vitamins related to the family of tocopherols. Crude palm oil is a rich source of vitamin E (600-1000 ppm) with most of the vitamin E occurring as tocotrienols (10). However, refining processes can lead to a partial loss of the vitamin E in palm oil. Deodorization produces the greatest loss, whereas during hydrogenation, little or no vitamin E is destroyed (1 1). During deodorization, the temperature and vacuum produce a distillation effect on the tocopherol fraction, which increases its concentration in the distillates. Deodorization distillates are currently an important commercial source of vitamin E (12). In 1994, Ong et u1. (13) reported that palm fatty acid distillate can contain as much as 0.9% by weight of a mixture of palm oil tocopherols and tocotrienols. In refined, bleached, and deodorized (RBD) palm oil, palm olein, and palm stearin, almost 69,72, and 76% of the original vitamin E present in the crude oils is retained, respectively (10). Refined palm oil contains -350-450 ppm of vitamin E in the form of tocopherol (30%) and tocotrienol(70%) isomers. Accurate quantitation of vitamin E in foods requires the conversion of each homolog to RRR-a-tocopherol equivalents (a-TE) (14): 1 a-TE activity is the activity of 1 mg of RRR-a-tocopherol (15). As such, oils with the highest a-TE levels contain a-tocopherol as the primary homolog. Palm oil is the only edible oil consumed in quantities that provides tocotrienols. Other sources of tocotrienols include cereal oils (wheat, barley, and rice bran), but their low dietary intake limits their relevance as vitamin E sources. In the Western diet, cereals and legumes represent the other sources of tocotrienols (12). The intake of antioxidant nutrients such as vitamin E was found to have beneficial effects in the prevention of various cancers, cataracts, and cardiovascular diseases (16). Tocotrienols also exhibit hypocholesterolemic activity. In 1991, Qureshi et ul. (17) and Tan et al. (18) conducted human studies with a tocotrienolrich fraction (Palmvitee) from palm oil. Both studies revealed lower levels of LDL cholesterol. Several studies also reported on the anticancer properties of tocotrienols (19,20). Sundram et al. (21) suggested that crude palm oil, which contains tocotrienols and carotenoids in higher amounts, is more effective than refined palm oil in increasing the tumor latency period in 7,12-dimethylbenz(a)anthracene (DMBA)-treated rats. When the vitamin E content in palm oil was removed, significantly more tumors were apparent (22).
Carotenoids. Carotenoids derived their name from carrots because carrots contain one of the most widespread plant pigments. It is a naturally occurring fat-soluble pigment that gives red, orange, or yellow color to fruits and vegetables. The amount of carotenoids found in vegetable oils is generally in the low range of
Palm Oil
735
p-Carotenes have provitamin A activity and convert to vitamin A in vivo. In Africa, crude palm oil is used as a source of vitamin A because the carotenoids in the commercially refined palm oil are destroyed during refining. Consequently, Choo et al. (25,26) developed a method using phosphoric acid degumming, followed by bleaching earth treatment, to produce a deodorized and deacidified red palm oil that retains S O % of the original carotenoids. Elsewhere, in India, red palm oil was used in dietary intervention studies to evaluate its role in preventing vitamin A deficiency among the poor. Children aged 5-10 yr old with keratomalacia were given an emulsion containing 0.6 mL of red palm oil for 15 d. Outcomes in the treated group compared well with the results from children treated with cod liver oil (27). p-Carotene is also a good singlet oxygen quencher, thus playing an important role in preventing photooxidation damage. Together with vitamins E and C, these micronutrients minimize the effects of free radical damage. fi-Carotene was also reported to have anticarcinogenic properties because it improves cell-cell communication (23). Additionally, palm oil carotenoids were approved by the F A 0 to be used as a natural food colorant (23).
Food Applications of Palm Oil, Its Fractions and Components The use of palm oil in food goes back 5000 yr. At present, 90% of palm oil and its products is used for edible purposes; the remaining 10% is used in nonedible applications, mainly in the oleochemical and soap industries. Figure 29.1 shows a chart of the different fractions obtainable from the oil palm fruit and the products that are derived from the different fractions. Palm olein, the liquid fraction of palm oil, is used widely as the main cooking oil in Malaysian households. Its unique properties such as longer frying life and reduced tendency for foaming and polymerization make it a better frying oil than corn or soybean oils (28). For industrial frying of instant noodles (29) or snack foods, palm oil is very suitable due to its stability. Palm oil’s stability is due to its composition, which includes minor amounts of unstable linolenic acid and a moderate amount (10-12%) of the more stable linoleic acid. The presence of tocopherol (380-890 ppm) also acts as a natural antioxidant (30). In the preparation of margarines and shortenings, the fat blend must have certain plasticity. This often means that liquid oils have to be hydrogenated or hardened to the right consistency before use. Hydrogenation produces trans fatty acids (TFA), and there is an increasing awareness of their detrimental effects on human health. TFA were implicated in raising blood serum cholesterol levels in humans (31), and this has provided an impetus to produce trans-free products. Palm oil, which is semisolid at ambient temperature, is thus a good natural hardstock for use in the preparation of shortenings,margarines, and vanaspati. The palm kernel fractions, which have different compositions and properties, can be used either on their own or in combination with other vegetable oils for the production of varied edible products such as those described here.
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Margarines and Spreads
Margarine was originally developed as a butter substitute by the French chemist, Hippolyte Mbge-Mouriez in 1869 (32). The first margarines were made from animal fat, but today, most margarines are made using vegetable oils. Margarine is defined by the Codex Alimentarius (33) as a liquid or plastic emulsion, fortified with vitamin A and consisting of not <80% fats and not >16% water. Thus, it is a water-in-oil emulsion. There are three main types of margarine that are formulated for different purposes, i.e., (i) table margarine in tub and packet form, (ii) industrial or bakery margarine, and (iii) pastry margarine. Tub margarine has fairly low solids at low temperatures, making it immediately spreadable when taken directly from the refrigerator. Packet table margarines, on the other hand, resemble butter in consistency in having higher solids at low temperatures; they are spreadable at ambient temperatures. Industrial margarine must be spreadable at usage temperature and must possess a wide plastic range for optimum functionality, whereas pastry margarine has a high solid content to give a flaky texture to the end product (34). Today, we can find a wide range of margarines with different physical properties to suit a specific consumer requirement such as stick margarine, regular stick margarine, whipped soft margarine, premium soft margarine, polyunsaturated margarine, high monounsaturated margarine, pourable margarine, and different varieties of lowenergy or diet margarine. The introduction of spreads is the result of the high consumer demand for lower-energy foods. By avoiding the use of the term “margarine” or “butter,” spreads do not fall under the existing legal definition and may have a lower fat composition. Such spreads can be classified as (i) reduced energy spreads, which usually contain 50-70% fat, and (ii) dietary spreads, which have 6 5 0 % fat. Table Margarine. Palm olein was found to be suitable as the liquid component in margarine blends, whereas -10-15% palm stearin or hardened palm oil can be used as the solid component (35). Margarines containing palm oil are also able to impart an extended plastic range with good working properties. Its tendency to crystallize in the p’ form produces a good textured margarine (34). A good margarine base stock can also be made using interesterified palm kernel olein and palm stearin or palm stearin with other liquid vegetable oils. Palm kernel olein’s quick crystallization properties give good creaming properties, and its high content of short-chain fatty acid (FA) gives a cool sensation when it melts in the mouth. Kurashige et al. (36) reported on the improved fluidity of blended oils (palm oil and canola oil) by enzymatic reaction and found the results to be better than that produced using soybean oil. Lai et al. (37-39) reported on the use of the lipase interesterification method to produce trans-free fat blends suitable for use as table margarines. Similarly, Cho and deMan (40) reported on the use of interesterified palm stearid canola blends for margarine production. A new palm-based pourable or squeezable margarine that differed from the conventional margarine was developed by using liquid oils such as palm olein as the main ingredient (41).
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IndustriaV5akery and Puff Pastry Margarines. Industriabakery margarines should have a higher solid fat content (SFC) than table margarines. In such instances, palm stearin, which contains higher solids and has a higher melting point, is used to a greater extent in the formulations. In 1996,Noor Lida et al. (42) reported on the different ratios of palm stearin, palm oil, and PKO blends in the preparation of pastry margarine. Blends with high amounts of palm stearin (30% of palm stearin with IV 32 or >50% palm stearin with IV 40) had cooling characteristics similar to those of commercial pastry margarines. This finding agreed with that reported by Young (43), who incorporated 60%palm stearin in his formulation. Reduced Energy Spreads. The first low-fat spread was introduced in 1968 in Europe and generally contained 40-80% fat. Unlike table margarine formulations, liquid oils are preferred in the formulation of reduced energy spreads because fats that are too hard can cause the product to be unstable. For this reason, palm olein is often used in the formulation. Interesterifying palm oil with other shorter-chain FA oils such as PKO is another option used to improve the melting properties and accelerate the meltdown of the fat in the mouth. Shortenings
Shortenings were originally used to “shorten” or tenderize baked products, hence its name. It was initially formulated to replace lard, which was in short supply. Today, the materials that make up the shortenings have changed from natural fats to blends of oils with hard fats or hydrogenated liquid oils (44). Depending on their application, shortenings can be divided into frying shortenings, baking shortenings, household shortenings, and icing or filling shortenings (45). Each of these shortenings appears in a solid (plastic), semisolid, pourable, or liquid form at room temperature. The melting points of shortenings are usually in the range of 34-44°C. Shortenings with a higher melting point provide better support to the cake batter in the early stages of baking. However, when the melting point is too high, it affects the mouth-feel and creaming takes longer to achieve. Shortenings from the United States are usually made from hydrogenated soybean oil, beef fat, and cottonseed oil, whereas in Europe (UK, Germany, and The Netherlands), they are mostly based on palm oil, hydrogenated rapeseed oil, and hydrogenated fish oils (3). Palm oil, beef fat, and fish oil are preferred as ingredients because of their tendency to promote p’ crystals. However, only palm oil is vegetable based. The physicochemical characteristics of experimental shortenings based on various palm oil products were reported by Nor Aini et al. (46). These authors reported higher SFC for palm oil-cottonseed oil blends compared with blends containing palm oil/soybean oil and palm oil/low-erucic acid rapeseed oil (LEAR) blends. Interesterification of palm oil followed by blending with anhydrous milk fat (AMF), also known as butter fat, also improved the SFC profile of the shortenings (47). Additions of AMF were found to produce shortenings that were more plastic and spreadable. In their work, Chu et aZ. (48) reported on experimental shortenings
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made from a lipase-catalyzed palm stearidpalm kernel olein blend at 1:l (w/w ratio), which had physical properties similar to the domestic commercial samples. Shortenings for cream fillings should be able to incorporate air during efficient mixing. Nor Aini et al. (46) reported that a high-palmitic acid content was good for aeration of fadsugar mixtures and indicated that palm stearidcottonseed oil at a 3:2 ratio was the best for that purpose. For applications in cream fillings and baking, interesterified palm olein proved to be the best formulation. The tendency of palm oil to promote 6’ crystals is essential for optimum baking and creaming performance of a shortening. However, compared with other oils, the development of an equilibrium SFC in palm oil is slow, thus promoting a phenomenon known as posthardening, which is a disadvantage in certain palm oil-based applications. This disadvantage can be overcome, however, by employing an interesterification process or by adding butterfat to palm oil, which reduces the SFC at lower temperatures, thus producing shortenings with improved plasticity. Products derived from the use of such shortenings are found to be finer and softer, and have a better flavor (49). Palm oil shortenings were also used to replace chicken fat or raw beef fat in the production of burgers. Not only were the cholesterol contents greatly reduced, but the shortenings blended well with the meat and their ingredients. However, when used in emulsion meat products such as sausages and frankfurters, palm oil shortenings require special properties to enhance their emulsification with the meat proteins because incomplete emulsificationwill cause a substantial loss of fat during and after cooking (50). Vanaspati
Vanaspati, or vegetable ghee, is the Indian term for hydrogenated vegetable fat used as a substitute for butterfat (ghee) in cooking. It is widely used in India, Pakistan, and eastern Mediterranean countries. In Greece, it is known as voutyros and in Turkey, Algeria, and Morocco, it is known as smin (3). In India and Pakistan, consumers prefer a product with a granular texture, whereas in Iraq and Iran, a smooth texture is preferred. As a result of urban population growth, the demand and price for vanaspati have increased. Cheaper hydrogenated vegetable oils are currently being used as an alternative and are widely accepted. In the early stages of the industry, only single hydrogenated oils were used. Later, blends of hydrogenated vegetable oils were mixed with animal fats. Currently, palm oil, soybean, rapeseed, and cottonseed are the most commonly used oils in the formulation of vanaspati. In India, vanaspati was reported to contain between 5 and 20% palm oil, whereas in Pakistan, up to 50% palm oil was reported in the formulation (51).
The granular or grainy texture of the vanaspati is an important quality criterion in India and Pakistan. In India, the vanaspati is grainy, firm, dry and crumbly, whereas in Pakistan, the granules are dispersed in some liquid oil. In most countries, hydrogenation is the common process used. This process produces the undesirable TFA, thus raising the need to replace the hydrogenated fats with natural
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hard fats such as palm oil, which has physical characteristics very similar to those of vanaspati, and does not require hydrogenation to achieve this effect. Palm oil will produce the desired granular texture after interesterification. Other studies (5253) showed that a trans-free vanaspati formulation can be produced by simply blending palm stearin with other liquid oils such as soybean, rapeseed, and sunflower. When interesterification was used, the amount of palm stearin incorporated into the formulation can be increased. Interesterification causes a reduction in the slip melting point (SMP) of all of the blends as well as the SFC at all temperatures. The products produced using interesterification were also softer. Ternary blends of palm oi1:palm stearin:palm oleidpalm kernel olein can also be formulated to have characteristics similar to those of the hydrogenated vanaspati. Cooking and Frying Oils/Fats
Palm olein is the main source of oil used in Malaysian households and catering establishments, whereas in temperate European countries, sunflower, peanut, or winterized cottonseed oils are highly regarded. In the United States, canola, soybean, and sunflower are the most common oils. For a good cooking oil, the quality criteria include clarity and the oxidative stability of the oil. Price also plays a major role in determining the oil used in less developed countries. In hot tropical countries, palm olein with a maximum cloud point of 10°C is the preferred choice among consumers. Palm olein can also be easily blended with other oils such as peanut and sesame oils, which are highly favored by certain segments of the consumer market, especially the Chinese. Cooking oils with an addition of 5% peanut oil or 3 and 2% sesame oil into palm olein is well accepted by the Chinese. Compared with all the other vegetable oils, such as cottonseed, corn, peanut, olive, sesame, sunflower, and soybean, palm olein had the longest induction period of 4044°C at IOO'C, indicating that it is stable against oxidation (54).Also, when palm olein was added to the other oils, it increased the induction time (54). At low temperatures, palm olein crystallizes and becomes cloudy. Although this does not mean a deterioration of oil quality, consumers prefer clear-looking oils. The double fractionated, superolein with a minimum IV of 60, has a low cloud point (maximum of 6°C) and therefore remains clear even in temperate or cold climates. In the EU countries, three major companies have introduced blends of superolein, sunflower oil, and peanut oil for their retail market (3). Frying is a widely used cooking method in the industry. It is rapid, more efficient, and produces foods that look and taste better, and have a longer shelf-life. With the increase in the production of convenient fast food or snack foods, deep frying has become an important method of cooking. In normal shallow frying at home or small restaurants, any fat including polyunsaturated oils can be used because the oils are not reused. For commercial deep-fat frying, there are many attributes that an oil/fat should have, including resistance to gumming and oxidation, low foaming and darkening
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rates, low FFA rise and smoking, low melting point, and a nutritionally good FA composition (3). Solid fat such as tallow and hydrogenated soybean, canola, or sunflower oil is often used as frying fat in commercial establishments to reduce oxidation and polymerization of the oils, and to extend the shelf-life of the fried products (55). These liquid oils cannot be used for commercial frying in their unhydrogenated state. A way to resolve this problem without the introduction of TFA would be to blend the vegetable oils with palm olein, which would give an effect similar to that of partial hydrogenation in lowering the linoleic and linolenic acid contents (35). A series of frying experiments with potato chips/French fries fried with different vegetable oils was conducted by Razali and Badri (56). In their experiment, the potato chips were intermittently fried for 4 min at intervals of 30 min at 180 f 5°C in several vegetable oils for 8 h/d over five consecutive days. At the end of each day, the oils were filtered and replenished with fresh oils at a level 4 0 % . At the end of the experiment, sunflower and soybean oils had more polar compounds than palm oil and palm olein. For certain products such as potato crisps or chips, palm olein performed better than palm oil. This is because these products absorb a large amount of fat (-40%) and are eaten cold. With a melting point of 22"C, palm olein gives a better mouth-feel and improved product gloss; because it is fluid, it allows immediate start-up after overnight and weekend stops. Palm olein has become the standard snack frying oil in the industry in the UK and The Netherlands (3). For fried nuts, palm olein gives a good gloss. Palm oil is equally good in all aspects except gloss but it has better saltholding capacity. The instant noodle industry is a big business. In Japan alone, total production in 1998 was 5.2 billion packs, equivalent to 42 packdperson per year (3). The advantages of instant noodles include its taste, convenience, and low cost. In West Europe, palm oil or palm olein is virtually the standard oil used in preparation of instant noodles. In Asia (Japan and Korea), lard and beef fats were once used because of their availability and low price (3). However, these animal fats oxidize rapidly and are unhealthy. Palm oil and palm olein are fast gaining ground in this industry. Red Palm OiI/Olein
Crude palm oil is reddish in color due to the high content of p-carotenes in the oil. It is a very nutritious oil because it also contains high amounts of vitamin E. In the crude form, this unrefined oil is suitable for use in the preparation of curry and chili paste, satay gravy, and chutney (57) because its deep red color enhances the color of the products making them more appealing. The development of the refined red palm oil/olein has the potential to combat vitamin A deficiency in the world. This was shown through the treatment of schoolchildren in India and lactating mothers in Honduras and in Guatemala (58-60). This oil has garnered wide interest and acceptance by the health conscious especially among the Japanese. One tablespoon of red palm olein each day will provide a child's daily requirement of vitamin E and p-carotenes (35). However, red palm oil/olein is not suitable for use as a frying oil due to the breakdown of carotene at high temperatures.
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SpecialtyKonfectionary Fats
Specialty or confectionary fats can be categorized into CBE, CBS or hard butters, coating fats for cakes, biscuits, sugar and frozen confectionaries, toffee fats, and cream filling fats (35). Among the important characteristics of a specialty fat are sharp melting, bland flavor, good crystallization stability, and no waxy sensation on the palate. Cocoa butter, although it has excellent eating qualities, is subject to bloom; it is difficult to temper and melts in hot climates. In addition, its escalating price and inconsistent supply have encouraged many confectioners to search for newer alternatives. Palm oil and palm kernel oil are suitable raw materials. Cocoa butter is the only commercially available fat that has a high saturated acid content with SOS-, POS-, and POP-type TAG. This simple chemical composition of cocoa butter imparts the sharp melting behavior much appreciated by consumers. Palm oil contains a high amount of POP- and SOS-type TAG, which gives the sharp melting characteristic in the mouth. The lauric-type oils such as PKO and coconut oil are also ideal raw materials.
CBE. CBE are specialty fats composed of the same symmetrical unsaturated TAG of C,, and C,, FA as cocoa butter. They are fully compatible with cocoa butter and can be mixed in proportions used in the production of chocolate. In many countries, for a product to be labeled “chocolate,” the use of vegetable fat (CBE) is allowed up to a maximum of 5% in total chocolate formulation or 15% in UK, Denmark, and Ireland (61). In the manufacture of chocolate, all CBE are tempered just like cocoa butter. PMF is the best known source of POP, and generally CBE are formulated with PMF blended with vegetable fats rich in SOS and POS such as illipe and shea fats. In Western Europe, Coberine and Illexao are well-known CBE, whereas in Malaysia, Chocomate is a popular high-quality brand. All of these CBE are made with PMF as their major component. However, in the United States and certain EU countries, only cocoa butter and milk fat may be used in chocolate formulations. The compatibility of CBE and cocoa butter can be affected by the addition of milk fat and its fractions for which eutectic interactions were reported (62). CBS. CBS are fats with physical properties similar to those of cocoa butter but with a different TAG composition; they can therefore be mixed with cocoa butter in very limited amounts. Thus, the degree of compatibility and their melting characteristics will determine the quality and price of the CBS. A good-quality CBS is hard at ambient temperature and gives the product a good snap and resistance to fat bloom. Products made from CBS are called substitute chocolate or coatings and they do not require tempering. CBS can be divided into the lauric-type CBS and nonlauric CBS. The predominant TAG in the lauric-type CBS are the saturated TAG of lauric (C,,) and myristic acids (C,,). Palm kernel oil and coconut oil are the two major sources of laurictype CBS. These lauric oils can be blended, fractionated, hydrogenated, or interesterified. These fats have the lowest compatibility with cocoa butter and when
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used in substitute chocolate and coating formulations, all of the cocoa mass must be replaced with cocoa powder. The use of milk fat should also be minimal, and skimmed milk powder is normally used instead. PKO is a good chocolate coating fat for ice cream and deep frozen confections because the coating formed is hard, elastic, and not brittle (35). Hydrogenated PKO and hydrogenated palm kernel olein are suitable for use for toffee formulations. Nonlauric CBS are made from liquid oils such as soybean, peanut, and cottonseed that have been hydrogenated. Sometimes, these fat are called cocoa butter extenders (CBEx) or cocoa butter replacers (CBR). They are usually used as compound coatings for chocolate-flavored baking chips and biscuits and wafers. Coatings made with them have a long shelf-life for bloom and are resistant to soapy flavor. Organoleptically , they have poor flavor release and mouth-feel. The major advantage to their use is their low cost. Interesterification of PMF with stearic acid or tristearin using a 1,3-specific lipase will also result in the formation of more complex mixtures of TAG, which contain 40-50% cocoa butter-like TAG. Transesterification of palm oil with stearic acid will also increase the stearic acid content of the oil and increase its melting point. This can be critical in the chocolate confectionary industry in providing a sharp melting sensation in the region of the body temperature. Confectionary/Cream Fillings. Cream fillings, used in cakes, wafers, or biscuits are usually made from hydrogenated palm kernel olein, which sets and melts rapidly in the mouth. Confectionary fillings are made from either lauric fat or trans hardened fat because both these fats give the stable p’ crystals. However, lauric fats are not compatible with cocoa butter and can cause bloom formation (63), whereas trans hardened fat gives a waxy mouth-feel (61).
Nondairy Products Ice Cream. Ice cream is an oil-in-water emulsion made up of milk solids. Due to the rising cost of milk fat, dairy ice creams are expensive. In the US., only milk fat is allowed to be used in ice cream making, whereas in most European countries, vegetable fats have replaced milk fats. For a suitable vegetable fat for ice cream, the SFC at 0°C should be high, with melting points of up to 25-35°C. Lauric-type fats such as coconut oil and PKO with their sharp melting profile are suitable ingredients for ice cream. However, in the UK and elsewhere in Europe, palm oil or hydrogenated palm oil and mixtures thereof are more commonly used because they offer a better compromise between price and quality. For a lower-cost ice cream, 10-15% palm stearin can be added into palm oil mixtures. Palm-based ice cream is considered to be healthier than that based on dairy fat, which was shown to elevate blood cholesterol levels. A study by Liew et al. (64) showed that mycelium-bound lipase could be used to transesterify PKO and AMF mixtures at a 70:30 ratio to produce a fat suitable for use in ice cream formulation. Additionally, ice cream powder,
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a new product, can also be conveniently reconstituted to meet sudden high demands. The new product is made up of palm oil, skim milk powder, corn syrup solids, sugar, protein, and small amounts of emulsifier, stabilizers, flavors, and optional colorings. This dry powder is hygienic, and can be easily packed and transported (65). Filled Milk. Milk powder is expensive, has a short shelf-life, and many people in Asia and Africa cannot tolerate it. Others may avoid it for health reasons (e.g., cholesterol) or to avoid eating animal fats. For these reasons and more, it has become more appealing to use skimmed milk powder or sodium caseinate and reconstitute it with vegetable fat. These products are known as “filled milk.” Three types of filled milk are available in Malaysia, i.e., sweetened condensed, evaporated, and powdered. Partially hydrogenated PKO or palm oil is a natural choice. Palm oil is cheaper, whereas the lauric oils give a better mouth-feel. Nondairy CreamerdCoffee Whiteners. Coffee whiteners are dry powders similar to filled milks used as substitutes for cream, evaporated milk, or fresh milk in coffee, tea, or cocoa drinks. These creamers contain higher fat levels than filled milk to impart richness and whitening power to the drink. A whitener also imparts body to the drink when added; it contains special additives to make it easily wetted and to prevent it from caking. The creamer should be resistant to oxidation and flavor reversion. Fats with a melting point of 35-37°C and high SFC at storage temperature are preferred. Lauric oils are usually considered the best. Hydrogenated palm oil is a good second choice (3). Whipping Creams. Dairy cream with 380400 g k g (fresh weight) butterfat is considered to be the best when the performance of whipping cream is evaluated (35). The peaks obtained are softer and the cream more stable. The criteria for a whipping cream fat include a semisolid structure at 5°C and a good meltdown in the mouth. Hydrogenated PKO is a suitable fat to be used in making whipping cream or whipped toppings (66). An interesterified blend of palm stearin and hydrogenated PKO at a 66:34 ratio produced a good whipping cream in terms of its whipping performance, stability, and mouth-feel(67). Palrn-Based Processed Cheese. In 1985, Narimatsu et al. (68) patented the method for making cheese-like food by adding rapeseed oil or mixtures of rapeseed oil with other oils such as palm oil as the fat component. For processed cheese, young or mature cheese (natural cheese) is blended in the presence of water, emulsifying salts, and coloring matter before being heated and agitated to form a homogenous mixture. Different fractions of palm-based oil consisting of 30% palm oil and 70% palm kernel olein can be blended into the formulation (69). The final moisture content of the block processed cheese should be -45%. Palrn-Based Yogurt. Yogurt is a product of lactic acid fermentation of milk or milk products. There are two types of yogurts, the set type, which exists as a strong
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jelly-like gel, and the stirred or soft-type yogurt, which exists as a highly viscous semifluid. The production of yogurt involves heating the homogenized milk at 85-95”C, followed by cooling before inoculation of the starter cultures such as Lactobacillus bulgaricus and Streptococcus thermophilus. The mixture is then incubated at 42OC for a few hours until the titratable acidity reaches 0.85-0.95% and the pH is 4.0-4.5.For palm-based yogurt, 30% palm oil and 70% palm kernel olein are blended along with the skimmed milk powder. Palm blends with the range of 2-5% can be used for palm-based yogurt production. Spray-Dried Products
lnfant Formula. Palmitic acid is an important nutrient source for infants when 5040% of their dietary energy comes from human milk. The FA composition of breast milk is influenced by the mother’s diet. However, palmitic acid (20-30%) is the main FA present with 70% of the FA located at the sn-2 position of the TAG. This is important due to the increased absorption of palmitic acid in the 2-monoacylglycerol form rather than in free acid form. Palm olein when blended or interesterified with other vegetable oils is suitable for use in infant food formulations. Low-melting palm olein has 10-15% of its palmitic acid in the sn-2 position of the glycerol molecule, and this will contribute to the high digestibility of the product. Palm-Based Coconut Milk Powder. Coconut milk, locally known as “suntan,” is widely used in Asian food recipes and it is obtained by pressing the coconut flesh to obtain the liquid milk. The coconut milk can be spray-dried to obtain a dry powder form that can be handled easily during transportation and storage. However, concerns exist due to the high levels of saturated FA (C12and C14)in coconut milk, a possible cause of hypercholesterolemia. As such, palm oil was used to replace coconut milk fat because it contains only 0.1-1.0% C, and C, FA. The nutrient composition of the palm-based coconut milk powder is similar to that of the commercial coconut milk powder with 60.5% fat, 29.7% carbohydrate, 7.35% protein, 1.82% moisture, and 0.65% ash (70). Other additives include anticaking agents and artificial flavors.
Dry Soup Mixes. Beef fat is the traditional fat used for dry soup mixes. In recent years, however, meat fats were replaced by the healthier vegetable fats. Hydrogenated palm oil is often used in this case because it has good stability and high solids at room temperature. Fats that are high melting are used because soups are almost often eaten hot (except for some very uncommon types). The fat in this case is spray-dried into a free-flowing powder form and encapsulated in sodium caseinate. Other Uses
Salad Oil. The most common oils used in salad dressing include sunflower, soybean, corn, and canola. Palm oil and peanut oil are now being introduced because
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they break the emulsion at refrigerated temperatures (3). Superolein is also very promising as a salad oil for use in the manufacture of mayonnaise and salad dressing, The basic recipe in making a palm-based salad dressing includes palm olein (IV 60-67), egg yolk, vinegar, starch, sugar, salt, mustard, and water. Mono- and Diacylglycerols. Monoacylglycerols (MAG) and diacylglycerols (DAG) are surface-active agents (emulsifiers) that act by reducing the surface tension of the product. They are used in large amounts to improve the performance of shortenings and margarines during baking and other edible applications. MAG and DAG are produced during a process known as glycerolysis in which fats with glycerols are reacted at high temperatures of -250°C in the presence of catalysts such as sodium ethylate or sodium hydroxide. Partial glycerides (MAG and DAG) as high as 4045% can be obtained through the process, and they can be concentrated to >90% by molecular distillation. Lipases can also be used in the manufacture of MAG emulsifiers from palm oil (7 1,72). The reaction conditions for lipase glycerolysis are mild and have no deleterious side reactions, thus producing a “purer” and more “natural” product compared with that produced during chemical reactions. At the high temperatures used for chemical reactions, dark-colored products with an undesirable flavor due to the polymerization of unsaturated FA are produced together with the desired products.
Vitamin Supplements. Research showed that vitamin E functions as an antioxidant that protects the unsaturated FA of lipid foods against oxidative deterioration. Vitamin E has also been reported to quench free radicals produced in our bodies and protect us against diseases such as cardiovascular, cancer, cataract, and diabetes. Tocotrienols from palm oil were also shown to have anticholesterol and anticarcinogenic effects. Thus, palm oil can be used as a source of vitamin supplements in the form of an emulsion. This is based on 70% red palm olein which contains natural provitamin A and vitamin E (35).
Conclusions Palm oil and its fractions have proved to be very versatile oils, finding applications in almost all areas in which fat is used in edible foods. Although the applications are diversified, the majority of the commercialized processes used involve either simple blending or chemical interesterification methods. Although enzymatic reactions have the advantage of being FA or position specific, the process is expensive when scaled up. The instability of the enzymes is also another limitation that adds to the high cost. Chemical interesterification, on the other hand, is a true and tested method, easier to scale up, and less expensive. Unfortunately, the chemical catalysts used lack the specificity to produce certain structured lipids with specific FA composition and distribution. The high temperature used can also cause damage to the lipids. Through greater consumer preference for “all natural,” “trans-free” products, as well as awareness on issues such as religious considerations on animal-based materials,
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bovine encephalopathy (BSE) issues, use of sustainable and “green” technology, biodegradable by-products or wastes, ecologically safe methods, and environmentally friendly techniques, I foresee a day when large-scale commercial enzymatic interesterification production will become a reality. Until then, the production of structured lipids such as medium-chain TAG (MCT) using palm oil and its fraction, the improvement of the nutritional value of palm oil by incorporating essential FA such as eicosapentaenoic acid and docosahexaenoic acid using lipases will continue to remain a research and development effort, published in scientific journals. Traditional industrial companies have to be more receptive toward this new area of research, which in the long run will produce a new niche market for “healthful lipids.” References 1. Berger, K.G., Palm Oil, in Strucrured and Modfied Lipids, edited by F.D. Gunstone, Marcel Dekker, New York, 2001, p. 119. 2. Ahmad, S., Malaysia: The Hub for Plant-Based Oleochemicals, inform 14: 604606 (2003). 3. Pantzaris, T.P., Pocketbook of Palm Oil Uses, 5th edn., Malaysian Palm Oil Board, 2000, pp. 1-32. 4. Siew, S.L., C.L. Chong, Y.A. Tan, T.S. Tang, and C.H. Oh, Identity Characteristics of Malaysian Palm Oil Products, Elaeis 4: 79-85 (1992). 5. Siew, S.L., T.S. Tang, C.H. Oh, C.L. Chong, and Y.A. Tan, Identity Characteristics of Malaysian Palm Oil Products: Fatty Acids and Triglyceride Compositions and Solid Fat Content, Elaeis 5: 38-45 (1993). 6. Tang, T.S., C.L. Chong, and M.S.A. Yusoff, Malaysian Palm Kernel Stearin, Palm Kernel Olein and Their Fractionated Products, PORZM Technology No. 16: 1-19 (1995). 7. Tan, B.K., and F.C.H. Oh, Malaysian Palm Oil Chemical and Physical Characteristics, PORIMTechnology No. 3: 1-5 (1981). 8. Wong, S., The Business of Specialty Fats Versus Cocoa Butter, Malays. Oil Sci. Technol. (MOST) 2: 44-49 (1993). 9. Tang, T.S., Composition and Properties of Palm Oil Products, in Advances in Oil Palm Research, edited by Y. Basiron, B.S. Jalani, and K.W. Chan, Malaysian Palm Oil Board, 2000, Vol. 2, pp. 845-894. 10. Sundram, K., and N. Chandrasekharan, Nutritional Properties of Palm Oil and Its Components, in Advances in Oil Palm Research, edited by Y. Basiron, B.S. Jalani, and K.W. Chan, Malaysian Palm Oil Board, 2000, Vol. 2, pp. 1061-1 101. 11. McLaughlin, P.J., and J.C. Weihrauch, Vitamin E Content of Foods, J . Am. Diet. Assoc. 75: 647-655 (1979). 12. Eitenmiller, R.R., Vitamin E Content of fats and Oils-Nutritional Implications, Food Technol. 51: 78-81 (1997). 13. Ong, A.S.H., W.S.S. Chee, and Y.M. Choo, Carotenoids and Tocols from Palm Oil, Transcript of a paper presented at the 85th AOCS Annual Meeting and Expo, Atlanta, 1994. 14. Eitenmiller, R.R., and W.O. Landen, Jr., Vitamins, in Analyzing Food for Nutrition Labeling and Hazardous Contaminants, edited by I.J. Jeon and W.G. Ikins, Marcel Dekker, New York, 1995. 15. National Research Council, Recommended Dietary Allowances, 10th edn., National Academy Press, Washington, 1989.
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16. Diplock, A.T., Antioxidants and Disease Prevention, Mol. Aspects Med. 15: 293-376 (1994). 17. Qureshi, A.A., N. Qureshi, J.J.K. Wright, S . Shen, G. Kramer, A. Gabor, Y.H. Chong, G. DeWitt, A.S.H. Ong, D. Peterson, and B.A. Bradlow, Lowering of Serum Cholesterol in Hypercholesterolemic Humans by Tocotrienols (Palmvitee), Am. J . Clin. Nutr. 53:
1021s-1026s (1991). 18.Tan, D.T.S., H.T. Khor, W.H.S. Low, A. Ali, and A. Gapor, The Effect of Palm Oil Vitamin E Concentrate on the Serum and Lipoprotein Lipids in Humans, Am. J . Clin. Nutr. 53: 1027s-1030s (1991). 19. Gould, M.N., J.D. Haag, W.S. Kennan, M.A. Tanner, and C.E. Elson, A Comparison of Tocopherol and Tocotrienol for the Chemo-Prevention of Chemically Induced Rat Mammary Tumors,Am. J . Clin. Nutr. 53: 1068s-1070s (1991). 20. Kato, A,, M. Yamaoka, A. Tanaka, K. Komiyama, and I. Umezawa, Physiological Effect of Tocotrienol, J . Jpn. Oil Chem. SOC.34: 375-381 (1985). 21. Sundram, K., H.T. Khor, A.S.H. Ong, and R. Pathmanathan, Effect of Different Palm Oils on Mammary Carcinogenesis in Female Rats Induced by 7,12-dimethylbenz(a)anthracene, Cancer Res. 49: 1447-1451 (1989). 22. Nesaretnam, K., H.T. Khor, J. Ganeson, Y.H. Chong, K. Sundram, and A. Gapor, The Effect of Vitamin E Tocotrienols from Palm Oil on Chemically Induced Mammary Carcinogenesis in Female Rats, Nutr. Res. 12: 63-75 (1992). 23. Choo, Y.M., Specialty Products: Carotenoids, in Advances in Oil Palm Research, edited by Y. Basiron, B.S. Jalani, and K.W. Chan, Malaysian Palm Oil Board, 2000,Vol. 2,pp.
10361060. 24.Tan, B., Novel Aspects of Palm Oil Carotenoid Analytical Biochemistry, in international Oil PalmlPalm Oil Conferences: Technical Progress and Prospects, Palm Oil Research Institute of Malaysia, Bangi, 1987,pp. 370-376. 25. Choo, Y.M., and A.S.H. Ong, Refining of Edible Oil, Australian Patent PI 7267/88(1988). 26. Choo, Y.M., A.N. Ma, S.C. Yap, C.K. Ooi, and Y. Basiron, Production and Applications of Deacidified and Deodorized Red Palm Oil, Palm Oil Developments No. 19: 30-34 (1993). 27. Aykroyd, WR., and R.E. Wright, Red Palm Oil in the Treatment of Human Keratomalacia, Indian J . Med. Res. 25: 7-10 (1937). 28. Augustine, M.A., L.K. Heng, and I. Nor Aini, Evaluation of Potato Crisps Fried in Market Samples of Palm Olein, Corn Oil and Soya Oil, Pertanika 11: 393-398 (1988). 29. Mashashi, S.,T. Yoshikazu, and S . Masanori, Quality of Fried Food with Palm Oil, J . Am. Oil Chem. SOC.62: 449454 (1985). 30.Gapor, A.M.T., Content of Vitamin E in Palm Oil and Its Antioxidant Activity, Palm Oil Developments No. 12: 25-27 (1990). 31. Mensink, R.P., and M.B. Katan, Effect of Dietary trans Fatty Acids on High-Density and Low-Density Lipoprotein Cholesterol Levels in Healthy Subjects, N. Engl. J . Med. 323:
439-445 (1990). 32. Andersen, A.J.C., and P.N. Williams, Margarine, Pergamon Press, Oxford, 1965,p. 1. 33. Codex Alimentarius, Codex Stan 32-1981,Codex Standardfor Margarine 8: 97 (1992). 34. Rasid, M., M. Jaais, M.S.A. Yusoff, and B.A. Elias, PORIM’s Experiments on Low Trans-Margarine, Palm Oil Technical Bulletin, Nov. 1996,pp. 9-13. 35. Nor Aini, I., and M.S.A. Yusoff, Food Uses of Palm and Palm Kernel Oils, in Advances in Oil Palm Research, edited by Y. Basiron, B.S. Jalani, and K.W. Chan, Malaysian Palm Oil Board, 2000,Vol. 2,pp. 968-1035.
748
0.-M. Lai
36. Kurashige, J., N. Matsuzaki, and H. Takahashi, Enzymatic Modification of CanolaPalm Oil Mixtures: Effects on the Fluidity of the Mixture, J . Am. Oil Chem. SOC.70: 849-852 (1993). 37. Lai, O.M., H.M. Ghazali, and C.L. Chong, Enzymatic Transesterified Palm StearinSunflower Oil Blends in the Preparation of Table Margarine Formulation, Food Chem. 64: 83-88 (1999). 38. Lai, O.M., H.M. Ghazali, and C.L. Chong, Physical Properties of Pseudomonas and Rhizomucor miehei Lipase-Catalyzed Transesterified Blends of Palm Stearin: Palm Kernel Olein, J . Am. Oil. Chem. SOC.75: 9.53-959 (1998). 39. Lai, O.M., H.M. Ghazali, and C.L. Chong, Effect of Enzymatic Transesterification on the Melting Points of Palm Stearin-Sunflower Oil Mixtures, J . Am. Oil. Chem. SOC. 75: 881-886 (1998). 40. Cho, F., and J.M. deMan, Physical Properties and Composition of Low trans CanolaPalm Blends Modified by Continuous Enzymatic Interesterification, Elaeis 6: 39-48 (1994). 41. Miskandar, M.S., and M.S.A. Yusoff, Palm Oil Pourable Margarine, PORIM Information Series No. 70: 1-4 (1998). 42. Noor Lida, H.M.D., A.R. Ali, and I. Mahadhir, Blending of Palm Oil, Palm Stearin and Palm Kernel Oil in the Preparation of Table and Pastry Margarine, Intl. J . Food Sci. Nutr. 47: 71-74 (1996). 43. Young, F.V.K., Palm Kernel and Coconut Oils: Analytical Characteristics, Process Technology and Uses, J . Am. Oil Chem. SOC.60: 374-379 (1983). 44. O’Brien, R.D., Shortening: Types and Formulations, in Bailey’s Industrial Oil and Fat Products, 5th edn., edited by Y.H. Hui, John Wiley and Sons, New York, 1996, Vol. 3, pp. 161-192. 45. Metzroth, D.J., Shortening: Science and Technology, in Bailey’s Industrial Oil and Fat Products, 5th edn., edited by Y.H. Hui, John Wiley and Sons, New York, 1996, Vol. 3, pp. 115-160. 46. Nor Aini, I., K.G. Berger, and A.S.H. Ong, Evaluation of Shortenings Based on Various Palm Oil Products, J . Sci. FoodAgric. 46: 481-493 (1989). 47. Nor Aini, I., M.S. Embong, A. Aminah, R. Mohd. Ali, and C.H. Che Maimon, Physical Characteristics of Shortenings Based on Modified Palm Oil, Milkfat and Low Melting Milkfat Fractions, Fat Sci. Technol. 97: 2.53-360 (1995). 48. Chu, B.S., H.M. Ghazali, O.M. Lai, Y.B. Che Man, S. Yusoff, S.B. Tee, and M.S.A. Yusoff, Comparison of Lipase-Transesterified Blend with Some Commercial Solid Frying Shortenings in Malaysia, J . Am. Oil Chem. SOC. 78: 1213-1219 (2001). 49. Nor Aini, I., Utilization of Palm Oil and Milkfat in Shortening Formulations for Madeira Cake and Short Dough Biscuits, Ph.D. Thesis, Universiti Kebangsaan Malaysia, Selangor, Malaysia, 1991, pp. 1-239. 50. Osman, A., and Wan Sulaiman, Malaysian Palm Oil Shortenings as Chicken Fat and Chicken Skin Substitute in the Production of Chicken Pattie Burger, PORIM Viva No. 0055/98,1998, pp. 1-20. 51. Kheiri, M.S.A., and F.C.H. Oh, Formulation of Vegetable GheeNanaspati, in Palm Oil Product Technology in the Eighties, edited by E. Pushparajah and M. Rajadurai, Incorporated Society of Planters, Kuala Lumpur, 1983, pp. 449-474. 52. Nor Aini, I., L. Lina, M.S.A. Yusoff, and Arif Simeh, Palm Oil-Based trans Free Vanaspati, Proc. of the 1997 PORIM Technology Transfer Seminar, 1997, pp. 28-39. 53. Nor Aini, I., M.S.A. Yusoff, and Arif Simeh, Palm-Based trans Free Vanaspati, PORIM Information Series No. 66: 1-4 (1997).
Palm Oil
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54. Teah, Y.K., Improvements in the Frying Quality of Vegetable Oils by Blending with Palm Olein, Palm Oil Developments No. 8: 1-4 (1988). 5 5 . deMan, L., J.M., deMan, and B. Blackman, Physical and Textural Characteristics of Some North American Shortenings, J . Am. Oil Chem. SOC.68: 63-69 (1991). 56. Razali, I., and M. Badri, Oil Absorption Polymer and Polar Compounds Formation During Deep-Fat Frying of French Fries in Vegetable Oils, in Proc. 1993, PORIM Inst. Palm Oil Congress-Update and Vision (Chem. & Technol.), edited by B. Yusof, A. Salmiah, Y.M. Chow, I. Nor Aini, T.S. Tang, W.L. L o n g , and J. Maycock, Palm Oil Research Institute of Malaysia, Bangi, Malaysia, 1995, pp. 80-89. 57. Nor Aini, I., The Use of Palm Oil in Food Preparation, Palmsearch Circular No. 2 , 1990, pp. 87-92). 58. Manorama, R., M. Sarita, R. Kavita, and C. Rukmini, Red Palm Oil for Combating Vitamin A Deficiency, in Proc. PORIM Nutr. Conf., 1996, p. 122. 59. Canfield, L.M., Y. Liu, and R. de Kaminsky, Supplementation of Mothers with Red Palm Oil Increases Infant Vitamin A Status, in Proc. PORIMNuh. Conf., 1996 p. 138. 60. International Vitamin A Consultative Group XVII Meeting, Guatemala, September 1996, Nutr. Rev. 3: 6. 61. Wong, S., Specialty Fats Versus Cocoa Butter, Atlanto Sdn. Bhd., Subang, Malaysia, 1 9 9 1 , ~101-317. ~. 62. Sabariah, S., A.R. Mohd. Ali, and C.L. Chong, Physical Properties of Malaysian Cocoa Butter as Affected by Addition of Milkfat and Cocoa Butter Equivalent, Intl. J . Food Sci. Nutr. 49: 211-218 (1998). 63. Laustern, K., The Nature of Fat Bloom in Molded Compound Coatings, Manuf. Confect. 5: 137-144 (1991). 64.Liew, M.Y.B., H.M. Ghazali, K. Long, O.M. Lai, and A.M. Yazid, Physical Properties of Palm Kernel Olein-Anhydrous Milkfat Mixtures Transesterified Using Mycelium-Bound Lipase from Rhizomucor miehei, Food Chem. 72:447454 (2001). 65. Wan Rosnani, I., and M.S.A, Yusoff, Ice Cream Mix Powder, PORIM Information Series NO.54: 1-2 (1996). 66. Towler, C., Cream Products for the Consumer, N Z . J . Dairy Sci. Technol. 17: 191-202 (1982). 67. Nesaretnam, K., N. Robertson, B. Yusof, and C.S. Macphie, Application of Hydrogenated Palm Kernel Oil and Palm Stearin in Whipping Cream, J . Sci. Food Agric. 61: 401-407 (1993). 68. Narimatsu, H., K. Sakamoto, T. Edayoshi, and H. Kobota, Method of Making CheeseLike Food, U S . Patent 4,560,560 (1985). 69. Karimah, A., M. Yazid, and M.S.A. Yusof, Production of Palm-Based Processed Cheese, PORIM Information Series No. 52: 1-2 (1996). 70. Zaida, Z., M.S.A. Yusof, H.M.D. Noor Lida, 0. Muhammad Nor, and A.S. Burhanuddin, Production and Characterization of Palm-Based Santan Powder, PORIM Information Series No. 65: 1-2 (1997). 71. Kawahara, Y., Progress in Fats and Oils Food Technology, INFORM4: 663-667 (1993). 72. Krishnamurthy, R.E., Biotechnology Offers Route to New Fats and Oils, inform 3: 457458 (1992).
INDEX
Index Terms
Links
A Acute respiratory distress syndrome (ARDS), γ-linolenic acid administration and
307
Agrobacterium tumefaciens
508
Alcon process
54
Allergens in GM foods
37
514
149
671
α polymorph Amino acids as antioxidant synergists and metal chelators
292
Analytical measurement of crystal growth
150
of lipids, hyphenated techniques for
78
of trans FA
20
Antioxidants
574
in beverage plants
286
carotenes as
280
from cereals and pulses
288
in edible oils
275
essential oils as
290
in herbal plants
287
mechanisms of action
273
from oilseeds
283
metal chelators as
290
578
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Antioxidants (Cont.) phenolic compounds as
281
580
phytosterols as
280
349
from spices
285
synergists of
290
581
tocopherols as
275
575
from vegetables and fruits
289
xanthophylls as
280
Apoptotic cell death induced by γ-linolenic acid
313
Appetizer (brand of shortening)
593
Arachidonic acid role of, in cardiovascular health ARASCO
®
6
65
222
231 593
Arrythmias, cardiac
371
Arthritis
236
Ascorbic acid
291
574
Astaxanthin, purification of
406
407
Asthma
236
Atherosclerosis
173
Atmospheric pressure chemical ionization (APCI)
351
177
316
82
Atopic dermatitis
316
Attalea colenda
536
Australia regulation of GM foods
36
regulation of trans FA
23
Autacoids
205
Autocatalytic oxidation
549
Autoxidation
558
mechanisms of Avrami index
213
560 159
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
B Baby foods
16
17
677
737
Bacillus thuringiensis. See Bt Bakery margarines Baking fats. See Shortenings Barley (Hordeum sativum L.)
289
Beans (Phaseolus vulgaris L.)
289
Beany flavor of soy
567
Becel Pro-Activ
TM
(Unilever)
Bell peppers (Capsicum annuum L,)
350 289
®
699
®
Benefat (Cultor Food Science)
601
Benzoic acid series
281
BetapolTM (Loders Croklaan)
411
597
β/β′ polymorph
149
671
of palm oil
157
Beverages, low fat
711
Biohydrogenation
2
Benecol (Raisio Benecol Ltd.)
of linoleic acid Biologically active components Biosafety
622
250 164 34
Bipolar disorder, role of n-3 FA in
375
Black currant (Ribes nigrum)
302
Black tea
286
Bleaching of vegetable oil
705
617
61
Blood pressure. See Hypertension Bohenin
593
Bone health
235
601
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Borage (Borago offcinalis)
302
Borage oil
323
617
Brain. See Nervous system Breast cancer, γ-linolenic acid administration and Breast milk
310 16
distribution of acyl moieties in
624
glycolipids in
615
lipid content of
607
phospholipids in
615
triacylglycerols in
613
Bt (Bacillus thuringiensis),
38
18
Buckwheat (Fagopyrum esculentum L.)
288
Butter
162
592
illipe
163
594
shea
186
529
321
597
cocoa. See Cocoa butter
Butyrivibrio fibrisolvens,
594
250
C Cachexia
379
California laurel (Umbellularia califomica Nutt.) Canada, regulations on trans FA
536 6
19
Cancer eicosapentaenoic acid and
379
γ-linolenic acid and
308
and the n-6:n-3 FA ratio
233
and phytosterols
349
This page has been reformatted by Knovel to provide easier navigation.
607
Index Terms
Links
Canola oil, FA composition of
4
Canola (genetically modified)
34
536
Capreninm (Proctor & Gamble)
593
600
Carbohydrates
179
®
Cardiovascular/coronary heart disease carbohydrate intake, influence of
179
cholesterol intake, influence of
177
and depression
376
fish consumption, cardioprotective effect of
229
237
362
and eicosapentaenoic acid, health studies of
364
and highly unsaturated FA
209
and metabolism of dietary fats
204
mono-and polyunsaturated FA intake, influence of
173
and the n-6:n-3 FA ratio
233
and palmitoleic acid
533
plant stanols, influence of
711
role of γ-linolenic acid in
314
saturated FA intake, influence of
172
trans FA implicated in
1
Carrot (Daucus carota L.)
289
Carotenes and carotenoids
577
as antioxidants
578
as oxidants
280
in palm oil
734
patents
434
547
190
483
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Cartagena Protocol on Biosafety
40
Catalysts, precious metal
70
Catechins Caustic refining of vegetable oils Cell proliferation inhibited by γ-linolenic acid
282
287
59 313
Cereals antioxidants in
288
Benecol
710
Cheese, processed
743
Chelating agents
549
573
as antioxidants
290
574
Cherries (Cerasus spp.)
290
Chlorophylls as prooxidants
573
Chocolate products
162
physiologic effects of Chocomate
351
594
601
647 741
Cholesterol absorption of, lowered by phytosterols
344
dietary
177
effects of individual SFA on
183
effect of γ-linolenic acid on
315
in plants
337
predictive equations, relating FA to
180
182
total plasma
171
178
and trans FA, clinical trials on
191
Cholesteryl ester transfer protein (CETP)
173
Cinnamic acid series
281
Citric acid
290
344
368
574
This page has been reformatted by Knovel to provide easier navigation.
641
Index Terms
Links
Clove (Eugenia caryophyllata Thunb.)
286
Coatings, lipid
595
Coberine
741
Cocoa (Theobroma cacao)
529
Cocoa butter
162
594
641
741
529
594
641
741
composition/characteristics of
641
crystalline properties of
146
physiologic effects of
647
substitutes and equivalents
163
411
642
741
Coconut milk Codex Alimentarius Commission
744 22
36
Coffee whiteners
743
Confectionary products
159
594
Conjugated linoleic acid (CLA)
162
249
effects of, in humans
254
natural sources of
250
purification of
399
Continuous loop, shallow bed extractor
55
Controlled powder formation (CPF) process Cooking oils
118 51
Copper-catalyzed lipid oxidation
572
Corn (Zea mays L.)
288
genetically modified
515
Cotton (Gossypium hirsutum)
514
genetically modified
514
Cottonseed oil, hydrogenated
669
739
515
This page has been reformatted by Knovel to provide easier navigation.
641
Index Terms CO2-based processes
Links 99
Coumarins
282
Creosote (Lurrea tridentate),
283
Crypthecodinium cohnii
594
619
Crystallization of fats
146
159
659
661
302
304
363
analytical techniques to study
150
essential FA
164
sterols
165
Cuphea spp.
536
Cyclooxygenase (COX), metabolism of FA by
224
Cytellin®
349
Cytochrome P450, metabolism of FA by
224
Cytokines
373
363
D Dairy products
11
Deep bed extractor
55
Degumming
54
Delivery systems, lipid-based Denmark, regulation of trans FA Deodorization of vegetable oils recovery of material from distillate
14
159
58
118 2
23
62 402
Depression and cardiovascular health
376
role of n-3 FA in
375
Depressive disorders
236
This page has been reformatted by Knovel to provide easier navigation.
671
Index Terms
Links
Dermatitis
316
Δ6 desaturase
301
Δ desaturase
250
Desaturase hypothesis for CLA production
251
9
Desolventizing
56
CO2-based
103
Detergent fractionation
669
DHA. See Docosahexaeonic acid DHASCO®
594
619
Diabetes, γ-linolenic acid supplementation and Diacylglycerols
319 685
nutritional characteristics of
687
patents
433
Dietary intake of fats
527
cholesterol
177
and conversion of food energy
211
isoenergetic substitutions
171
lipid digestion and transport
205
and mental health
375
monounsaturated FA (MUFA)
176
n-3 FA
381
n-6 FA
361
phytosterols
344
polyunsaturated FA (PUFA)
173
role in cardiovascular health
204
saturated FA
172
446
178
183
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Dihomo-γ-linolenic acid
301
Docosahexaenoic acid (DHA)
163
210
221
Droplet, emulsion
548
550
553
Dry fractionation
669
7
51
222
E Echium (Echium plantagineum L.)
302
Eczema
316
Edible oils
6
See also Vegetable oils antioxidants in
275
autoxidation of
560
copper and iron content
572
genetically engineered
52
1
564
oxidative stability of
569
tocopherol contents of
575
O2 oxidation of
Eggs
15
Eicosanoids
213
223
224
Eicosapentaenoic acid (EPA)
163
221
222
antiarrhythmic effect of
371
and cardiovascular health, studies of
364
immunoregulatory action of
373
metabolic pathways for
363
and neuropsychological disorders
375
Electrocatalytic hydrogenation
66
Electrospray (ES) ionization
82
364
This page has been reformatted by Knovel to provide easier navigation.
362
Index Terms Emulsions
Links 548
oxidation of lipids in
549
protein stabilized
553
surfactant micelles in
552
®
Enova oil (Kao/Archer Daniels Midland)
351
Enrichment of lipid products
111
Enteral feeding, lipid products for
597
596
Environmental safety, of genetically modified organisms Enzyme-catalyzed reactions Enzyme inactivation
38 105
153
395
419
54
EPA. See Eicosapentaenoic acid Essential FA (EFA) competition with nonessential FA
163
222
204
207
Essential oils as antioxidants
290
Esterification
153
lipase catalyzed
395
of phytosterols
349
selective
398
Ethylene diamine tetraacetic acid (FDTA) European Union, regulation of trans FA
549 23
Evening primrose (Oenothera biennis spp.)
284
Evening primrose oil
323
Excess food energy
211
Expellers, mechanical
574
302
617
57
Expert Consultation (Joint WHOFAO) on Diet, Nutrition and the Prevention of Chronic Disease on Foods Derived from Biotechnology
10
18
35
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Extraction of oil
54
“green processes” Extrusion-expanding of oilseeds
99 53
F Falling film layer crystallization
155
Fat crystals
672
See also Crystallization of fats Fat-soluble vitamins, analysis of
88
Ferulic acid patents
435
Filled milk
743
488
Fish consumption and cardiovascular health
229
237
362
FHOWHO Expert Consultation. See Expert Consultation (Joint WHOFAO) Flavonoids
282
Flavr Savr tomato (Calgene) Flax (Linum usitatissirnum L.) Flora Pro-Activ
TM
(Unilever)
Food coatings
37 278 350 595
Food labeling. See Labeling regulations Food Linked Agro-Industrial Research (FLAIR) Programme Food products, fats and oils in
38 145
Food safety, of genetically modified products
35
Fourier transform infrared spectroscopy
89
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Fractionation
154
669
dry
154
669
of FA
134
of milk fat
160
of mono- and polyunsaturated FA
166
of oils and fats
137
of palm oil
157
solvent
155
669
supercritical fluid-based
105
111
Free FA (FFA), removal of
114
133
Fruits, antioxidants in
289
Frying oils
739
Functional foods
121
margarines
677
phytosterols in
345
Functional lipids
351
592
G Gallic acid
281
Garlic (Allium sativum L.)
289
Gas chromatography Gastric cancer and γ-linolenic acid-rich diet Genetically modified crops
79 311 33
508
genetic transformation methods
508
517
for high-laurate oils
535
for high-oleate oils
530
for high-stearate oils
528
public acceptance and education
41
regulation of
40
518
safety of
34
518
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Ghee
738
Ginger (Zingiber officinale Roscoe)
286
Gingko biloba
288
Gingseng (Panax spp.)
288
Glioma, γ-linolenic acid administration and
310
Glycolipids, in human milk fat
615
GoodFry oil
278
593
“Good nutrition”
204
207
Grapes (Vitis vinifera)
290
antioxidants in
287
“Green solvents”
216
99
Green tea
286
Growth, crystalline
146
152
See also Crystallization of fats G77 developing countries
40
H Herbal extracts
121
Herbal plants, antioxidants in
287
Hexanes in oil extraction
54
High-carbohydrate diets
179
High-density lipoprotein (HDL) cholesterol
171
animal studies of metabolism of
189
effect of EPA supplementation on
368
effect of γ-linolenic acid on
315
effects of individual SFA on
183
predictive equations, relating FA to
180
and trans FA, clinical trials on
191
182
This page has been reformatted by Knovel to provide easier navigation.
Index Terms Highly unsaturated FA (HUFA) role in metabolism and health
Links 207 207
High-laurate oils
535
High-oleate oils
530
High-stability oils (HSO)
167
High-stearate oils
528
Horizontal bed extractor
56
Human milk
16
distribution of acyl moieties in
624
glycolipids in
615
lipid content of
607
phospholipids in
615
triacylglycerols in
613
18
321
597
56
152
668
105
110
Hybridization of genetically modified and wild species Hydrogenation biohydrogenation
38 3 2
new processes
65
supercritical fluid-based
73
Hydrolysis of triglycerides
56
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
178
Hypertension, effect of EPA on
367
Hyphenated techniques Fourier transform infrared spectroscopy
89
gas chromatography
79
liquid chromatography
82
mass spectrometry
78
nuclear magnetic resonance (NMR) spectroscopy
91
This page has been reformatted by Knovel to provide easier navigation.
607
Index Terms
Links
I Ice cream
742
Illexao
741
Illipe (Shorea stenoptera) fat
163
Immune response
372
effect of CLA on
260
effect of eicosapentaenoic acid on
373
effect of γ-linolenic acid on
304
594
Industrial margarine. See Bakery margarine Infant formulas
15
17
210
191
236
153
670
553
321
See also Human milk lipid composition of
625
sources of lipids for
616
Inflammatory response
372
effect of EPA on
373
effect of γ-linolenic acid on
304
Institute of Medicine, National Academy of Sciences Insulin sensitivity, effect of CLA on Interesterification for cocoa butter equivalents
18 260 57 643
Interfacial membrane, of emulsion droplet
548
550
Interferon
304
373
Interleukin
304
373
Iron-catalyzed lipid oxidation
549
551
2
57
Isomeric forms of FA Isotope-ratio monitoring
572
80
This page has been reformatted by Knovel to provide easier navigation.
744
Index Terms
Links
J Juniper extracts
286
K KIM-2 software
217
Kokum (Garcinia indica) butter
529
594
L Labeling requirements for genetically modified foods for trans FA Lauric acid
Laurical
1
16
18
535
influence of, on cholesterol levels TM
43
(MonsantoCalgene)
183 537
Laurus nobilis
536
Layer crystallization
155
LDL receptor. See RLDL Lecithin
350
Lentil (Lens culinaris)
289
Leukotrienes
224
Licorice (Glycyrrhizza glabra L.)
286
Lignans
278
Linoleic acid conjugated.See CLA metabolism of Linoleic acid
304
283
580
581
4 222 222
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Linolenic acid
4
α-Linolenic acid
4
γ-Linolenic acid
301
and acute respiratory distress syndrome
307
and arthritis
306
and cancer
308
and cardiovascular health
314
and diabetes
319
metabolism of
303
physiologic effects of
304
purification of
399
and skin health
316
sources of
302
Linseed oil, antioxidants in
277
Lipase-catalyzed reactions
105
Lipases, role in lipid metabolism
205
Lipid-based delivery systems
118
Lipid content, alteration of
105
163
222
153
395
419
304
363
Lipid-lowering diets. See Dietary intake of fats Lipid oxidation products, analysis of Lipoprotein (a) [Lp(a)]
88 193
195
224
302
Lipoxygenase (LOX), metabolism of fatty acids by Liquid chromatography
82
Liver cancer, γ-linolenic acid administration and
311
Living modified organisms
41
Lorenzo’s oil
596
This page has been reformatted by Knovel to provide easier navigation.
Index Terms Low-density lipoprotein (LDL) cholesterol
Links 171
effect of γ-linolenic acid on
315
effects of individual SFA on
183
metabolism of. See rLDL activity oxidative stress from
207
predictive equations, relating FA to
180
and trans FA, clinical trials on
191
Low-energy fats
182
600
M Macadamia nut oil
534
Maize (Zea mays)
515
genetically modified Mangosteen (Garcinia mangostana)
37
515
529
Manic-depressive illness, role of n-3 fatty acids in Marc Margarines
375 56 6
8
156
592
736 bakery
677
737
Benecol
706
708
definition of
665
736
FA composition of
674
675
functional
677
low-trans
670
processing and physical properties of
677
table
675
736
10
153
zero-trans
676
670
This page has been reformatted by Knovel to provide easier navigation.
665
Index Terms
Links
Mass spectrometry
78
Mayonnaise
51
Meat
15
Mechanical pressing for oil extraction
57
60
Mège-Mouriès, Hippolyte
665
736
Melting-point profile of a fat
151
Mental health and the n-6: n-3 FA ratio
236
Mercosur regulations on trans FA
22
Metabolic role of oils
50
709
Metabolism of oils eicosapentaenoic acid pathways
363
role in cardiovascular health
204
Metastasis of cancers, inhibition of, by γ-linolenic acid Mevalonate pathway Miami Group Micellar solubilization Milk
312 341 40 552 11
filled human Milk fat
14
743 16
18
321
160
crystalline properties of
147
modification of
160
Molecular distillation. See Short-path distillation Monoacylglycerol patents
343
458
Monounsaturated FA (MUFA)
166
527
consumption of
176
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Mortierella alpina
594
Mortierella isabellina
617
Mucor circinneloides
617
TM
Multi-Bene
351
Mustard (Sinapis alba L.)
284
Myristic acid, influence of, on cholesterol levels
184
619
N n-3 FA
174
adverse affects of
230
antiarrhythmic effect of
371
beneficial effects of
229
consequences of metabolism of
205
dietary recommendations for
236
excessive dietary supply of
208
hypotriacylgylcerolemic effects of
175
immunoregulatory action of
373
and neuropsychological disorders
375
physiologic role of
362
inverse relation to n-6 FA levels
224
n-6 FA
174
adverse affects of
230
consequences of metabolism of
205
dietary dominance of
361
dietary recom mendations for
236
222
222
228
excessive dietary supply of, inverse relation to n-3 FA levels
224
and oxidative stress
175
This page has been reformatted by Knovel to provide easier navigation.
Index Terms n-6:n-3 FA ratio
Links 225
relation to health and disease
232
of vegetable oils
238
n-9 FA
176
222
(NCEP)
171
174
Natural extracts
121
Neobee (Stepan Company)
601
Nervous system, role of n-3 FA in
362
National Cholesterol Education Program
Neutral lipids, analysis of
83
New Zealand, regulation of GM foods
36
regulation of trans FA
23
Nightshade (Solanum spp.)
338
“No longer equivalent” genetically modified foods Nondairy creamers
43 743
Non-insulin dependent diabetes mellitus and the n-6:n-3 FA ratio
235
Nuclear magnetic resonance spectroscopy
91
Nucleation
147
152
Nutraceuticals
121
597
Nutrient content claims
20
Nutritional effects of diacylglycerol oils
687
of milk fats
161
Nutritional imbalances
216
Nutrition education
216
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
O 1
O2. See Singlet oxygen
3
O2.See Triplet oxygen
Oat (Avena sativa L.) Octadecenoic acid Oil extraction
288 3
14
54
“green processes”
99
Oil palms (Elaeis spp.)
516
Oilseeds antioxidants from
283
cleaning and drying of
52
oil extraction from
54
structure and composition of
49
Oleic acid Oleins of palm oil
4
530
154 157
Olive (Olea europaea L.)
278
Olive oil, antioxidants in
278
732
581
ω-FA. See n-FA Onion (Allium cepa L.)
289
Oolong tea
286
Orange (Citrus sinensis)
290
Oregano (Origanum vulgare L.)
285
Oryzanol
279
339
435
486
patents Oxidation
558
autocatalytic
549
autoxidation
560
563
decomposition products of
561
566
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Oxidation (Cont.) effect of surfactant micelles on oxidative stability
552
of lipids
273
role of metals in
572
sensitizers
564
stability of lipids against
547
of triglycerides Oxidative stress
56 175
and hydrolysis of plasma VLDL
207
and n-6 FA
174
Oxygen
558
570
P Palladium-catalyzed hydrogenation
70
Palmitic acid (16:0), influence of, on cholesterol levels Palmitoleic acid
185 528
531
containing oils
531
dietary intake of
528
Palm oil/palm kernel oil
156
576
crystalline properties of
149
157
food applications of
735
594
731
Pancreatic cancer, γ-linolenic acid administration and
311
Parenteral feeding, lipid products for
597
Partially hydrogenated fats Particle formation technology
3 118
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Pasta, Benecol
710
Peanuts (Arachis hypogaea L,)
284
Pea, yellow (Pisum sativum L.)
289
Phenolic compounds as antioxidants
281
580
54
58
Phosphatides, removal of Phospholipids analytical techniques for
84
as antioxidant synergists
291
in human milk fat
615
as prooxidants
573
Phosphoric acid
573
574
Physiologic action of milk fats
161
of n-3 FA
362
Phytosterols and phytostanols, patents
434
Phytosterols
165
468
as antioxidants
280
biosynthesis of
341
and cancers
349
commercial formulations and products
349
concentration of, in foods
345
function of
339
metabolism of
343
344
natural occurrence of
335
343
and prostate health
349
Pine bark (Pinus pinaster)
349
351
288
Plant sterols. See Phytosterols Plastic fats. See Individual types Platelet-activating factor (PAF)-mimics
212
This page has been reformatted by Knovel to provide easier navigation.
Index Terms Platelet aggregation
Links 315
Platinum-catalyzed hydrogenation
70
Polar lipids, analysis of
84
Polymerization of triglycerides
57
369
Polymorph
149
671
Polymorphism, crystal
148
151
of cocoa butter substitutes Polyunsaturated FA (PUFA)
163 166
in human milk
609
increasing consumption of
173
in infant formulas
616
patents
434
structure and metabolism of
222
Potato (Solanum tuberosum L.) genetically modified
671
527
461
289 41
Poultry
15
Precious metal-catalyst hydrogenation
70
Preterm infants. See also Human milk Infant formulas Processed foods
11
Propane-based solvation
103
Prostate health
349
Protein-stabilized emulsions
553
Prunes (Prunus domestica)
290
Pulses, antioxidants in
288
Purification of FA.
395
12
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
Q Qualea grandiflora
536
R Rama Pro-ActivTM (Unilever)
350
Rape (Brassica napus)
510
genetically modified
39
Rapeseed oil
284
Reactive oxygen species (ROS)
211
creation of platelet-activating factor-mimics by
740
Reduced-calorie fats
600
Reducol (Forbes Medi-Tech) Refining of vegetable oils
510
212
Red palm oil/olein ®
284
350
351
58
Retina. See Visual system Rheumatoid arthritis, γ-linolenic acid administration and
306
Rice bran oil, antioxidants in
279
Rice (Oryza sativa L.)
279
Risk assessment and management
35
rLDL activity animal studies of
188
regulation of, effect of fat intake on
171
172
285
286
Rosemary Rotacel extractor
55
Rotary extractor
55
TM
Roundup Ready
soybeans
Rumen bacteria Ruthenium-catalyzed hydrogenation
175
44 250 70
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
S Sage (Salvia officinalis L.)
285
Sal (Shorea robusta)
529
594
Salad oils
51
709
®
593
Salatrim (Nabisco Foods Group) Saponification of triglycerides
56
Saturated FA (SFA), consumption of
172
Schizochytrium sp.
619
Schizophrenia, role of n-3 FA in
375
Screw presses
745
57
Sesame oil, antioxidants in
277
Sesame (sesarnum indicum L.)
277
Sesamin
580
Sesaminol
278
580
582
Sesamol
278
580
582
Shea (Butyrospemum parkii) fat
186
529
594
51
156
592
665
618
620
Shortenings
580
definition of
665
Short-path distillation
127
Silver-ion liquid chromatography
413
Single-cell oils
593
617
Singlet oxygen, O2
558
564
Skin health
316
Sodium dodecyl sulfate
536
Solexol process
103
1
Solid-polymer electrolyte reactor
68
This page has been reformatted by Knovel to provide easier navigation.
737
Index Terms Solvent-based extraction “green”
Links 54 99
of polar solutes
100
toxicity reduction
103
Solvent fractionation
669
Solvent-free enzymatic reaction systems
420
Soup mixes
744
South Africa, regulation of trans FA,
23
Soybean (Glycine max spp.)
284
genetically modified
34
36
38
44
513 Soybean oil, hydrogenated Sphingolipids
669 84
Spices, antioxidants in
285
Spreads
156
737
706
708
Benecol Squash (genetically modified) Stanols
37 165
and cardiovascular health
711
foods enriched with
699
history of research
712
physiologic effect of
713
Statins
721
Stearic acid
528
influence on cholesterol levels Stearins of palm oil Step 1 and Step 2 diets (NCEP)
699
707
186 154 157
732
171
174
This page has been reformatted by Knovel to provide easier navigation.
Index Terms Sterols
Links 165
See also Cholesterol; Phytosterols possessing antioxidant activity purification of
280 402
Structured lipids
591
Structured triacylglycerols
622
Substantial equivalence of GM and unmodified crops
35
Summer rape (Brassica rapa L.)
284
Summer savory (Satureja hortensis L.)
286
Sunflower (Heianthus annuus L.)
284
SUN-TGA25
®
Supercritical fluid-based processes
619 99
coupled with particle formation and delivery methods
118
desolventizing
103
fractionation
105
111
73
105
110
73
105
110
hydrogenation Supercritical fluid-state hydrogenation Supersaturated solution
147
Suspension crystallization
155
Symmetrical triacylglycerols
411
chromatographic separation of
412
412
in cocoa butter and cocoa butter equivalents enzymatic synthesis of Synergists of antioxidants
641 419 290
581
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
T Table margarine ®
675
Take Control (Unilever)
350
Tea beverages, antioxidants from
286
736
Term infants. See also Human milk; Infant formulas Thrombosis
369
Thromboxanes
224
Thyme (Thymus vulgaris L.)
286
230
304
575
582
315
Tocopherols as antioxidants
275
chemical structure
276
in palm oil
734
purification of
402
vitamin E activity of
277
Tocotrienols as antioxidants
275
chemical structure
276
in palm oil
734
Tomato (Lycopersicum esculentum Mill.)
289
Tota1:HDL cholesterol ratio
171
Transesterification, lipase catalyzed
395
Transferrin
549
575
339
Trans FA analytical measurement of
20
cardiovascular health, effects on
1
chemical structures
2
in cocoa butter substitutes food content of
65
190
593
17
18
19
646 6
This page has been reformatted by Knovel to provide easier navigation.
369
Index Terms
Links
Trans FA (Cont.) in human milk
16
isomeric forms of
2
origin and sources of
2
reducing levels of, via hydrogenation reducing levels of, via interesterification
18
65 156
Trans free margarines. See Zero-trans margarines Transgenic organisms. See Genetically modified crops Triacylglycerols (TAG) analysis of
83
84
crystalline properties of
146
in human milk
613
in infant formulas
621
patents
433
437
plasma
171
315
predictive equations, relating FA to
180
182
structured
622
symmetrical
411
Triglycerides, chemical reactions in 3
368
57
Triplet oxygen, O2
558
Trolox
276
Tumor necrosis factor α
236
Turmeric (Curcuma domestica L.)
286
560
373
TXA, platelet aggregation agent. See Thromboxanes
This page has been reformatted by Knovel to provide easier navigation.
Index Terms
Links
U Ubiquinone patents
435
Undercooling
147
United Kingdom, regulation of trans FA
23
United States, regulation of trans FA
22
491
V Vaccenic acid (llt-18:1) Vanaspati
4
14
162
738
Vegetable oils. See also Edible oils antioxidants in.
275
bleaching of
61
caustic refining of
59
chemical reactions in
56
composition and uses of
50
CLA produced from
253
copper and iron content of
572
deodorization of
62
402
FA content of
181
509
genetically engineered
526
n-6:n-3 ratios of
238
production of
48
refining of
58
specifications for
58
tocopherol contents of
575
Vegetables, antioxidants in
289
Very low-density lipoprotein (VLDL)
172
52
63
180
207
This page has been reformatted by Knovel to provide easier navigation.
Index Terms Visual system, role of n-3 FA in Vitamin E
Links 362 88
activity of tocopherols
277
in palm oil
734
patents
435
485
Vitamins, fat-soluble vitamin A
89
vitamin E
88
W WHOFAO Expert Consultation. See Expert Consultation (Joint WHOFAO) Whipping creams
743
Wine, antioxidants in
287
Winter rape (Brassica napus L.)
284
X Xanthophylls as oxidants
280
Y Yogurt, palm-based
744
Z Zero-trans margarines and oils
10
153
538
593
This page has been reformatted by Knovel to provide easier navigation.
