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FreeRadical Biology& Medicine, Vol. 12, pp. 63-81, 1992 Printed in the USA. All rights reserved. 0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc Review Article ROUTES OF FORMATION AND TOXIC CONSEQUENCES OF LIPID OXIDATION PRODUCTS IN FOODS STAN KUBOW School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec H9X IC0 (Received 6 June 1991; Revised 26 August 1991; Accepted 26 August 1991) Abstract--Lipid oxidation in foods is initiated by free radical and/or singlet oxygen mechanisms which generate a series of autocatalytic free radical reactions. These autoxidation reactions lead to the breakdown of lipid and to the formation of a wide array of oxidation products. The nature and proportion of these products can vary widely between foods and depend on the composition of the food as well as numerous environmental factors. The toxicological significance of lipid oxidation in foods is complicated by interactions of secondary lipid oxidation products with other food components. These interactions could either form complexes that limit the bioavailability of lipid breakdown products or can lead to the formation of toxic products derived from non-lipid sources. A lack of gross pathological consequences has generally been observed in animals fed oxidized fats. On the other hand, secondary products of lipid autoxidation can be absorbed and may cause an increase in oxidative stress and deleterious changes in lipoprotein and platelet metabolism. The presence of reactive lipid oxidation products in foods needs more systematic research in terms of complexities of food component interactions and the metabolic processing of these compounds. Keywords--Lipid oxidation, Free radical, Toxicity, Atherosclerosis, Mutagenicity, Food, Vitamin E, Oxidative stress are particularly susceptible to oxidation in cooking and frying, beneficial aspects of wide utilization of these fats may be counteracted through formation of toxic oxidation products and increased antioxidant requirements. An exploration of the possible toxic consequences may be particularly relevant at this time since there is increasing evidence of a pathogenic role of endogenous lipid oxidation in a number of chronic and acute disease states2'3 and due to the recent advances in our ability to identify and quantify lipid oxidation products in biological systems via analytical techniques such as gas chromatography-mass spectrometry (GC-MS), high pressure liquid chromatography (HPLC), and nuclear magnetic resonance (NMR). There are a couple of caveats that should be noted regarding our present state of knowledge. First of all, much of our information concerning lipid oxidation processes in foods has derived from the study of individual oils or simple model systems, whereas food is a complex heterogenous material in which lipids exist in environments that are quite different from a single phase model system. The complexity of lipid free radical reactions of foods, especially at higher temperatures, is increased by cross-reactions with other foods components. Such interactions could alter dramatically the spectrum and bioavailability of toxic constituents produced in a food system. Secondly, extrapola- INTRODUCTION The postulated associations between high fat diets and chronic diseases such as heart disease and cancer are well known. In this regard, however, the role of food preparation techniques in chronic disease has not been systematically investigated despite the fact that most of the foods that we consume today have been subjected to various degrees of processing, oxidation, and heat treatment. Convenience and snack foods have risen in popularity, particularly in deep-fat fried foods such as chicken, fish, and French-fried potatoes. These foods absorb substantial amounts of the oils used for deep-frying, l Complex chemical and physical changes occur under these conditions, causing fat deterioration which may reach a point whereby the nutritional value and safety characteristics of the oil may be affected. Since polyunsaturated fatty acids Stan Kubow received his Ph.D. in the department of Nutritional Sciences at University of Guelph at Guelph, Ontario in 1984, and did his postdoctoral work there and at the School of Pharmacy at the University of Toronto. He was appointed to his present position as an Assistant Professor at the School of Dietetics and Human Nutrition at Macdonald Campus of MeGill University in 1987. His laboratory is currently investigating free radical-mediated mechanisms of diabetic- and drug-induced teratogenicity as modulated by diet; mechanisms of alterations of lipoprotein metabolism by dietary fats; and the role of dietary fats on eicosanoid metabolism and fetal development during pregnancy. 63 64 S. K u B o w tions regarding experimental animal toxicity data to the human context are difficult to make, as oxidized fats are typically fed at high levels as the sole source of fat in the diet as opposed to the typical mixed diet of humans. The present review will look at some of the major mechanisms by which lipid oxidation products are generated during the processing of food, and will discuss the potential toxicological relevance of these oxidation products. It should be noted that due to the wide scope of this subject an in-depth review of all of the prevailing issues cannot be made. The reader is referred to a number of excellent recent reviews which give a complementary examination of the topics addressed. 4-21 Moreover, it is possible that important work may have been missed in this coverage as well as in previous reviews. AUTOXIDATION It is generally accepted that the process of lipid oxidation of foods proceeds by way of a free radical mechanism called autoxidation, which is described in terms of initiation, propagation, and termination proc e s s e s . 5-7 This term is a misnomer in that this oxidative process requires a catalyst in biological systems since the triplet state of oxygen forbids direct reaction of molecular oxygen with other biomolecules. 6-7,z2 Activation of molecular oxygen can occur either through a direct input of energy, as in the case of light converting oxygen to singlet oxygen, or by a reduction via some catalyst. Catalysis of lipid oxidation in foods could proceed via both nonenzymatic and enzymatic mechanisms. 6-7'22 Once the reaction has been initiated via a catalyst, however, the process is autocatalytic in the sense that the oxidation products catalyze the reaction and cause an increase in the reaction rate as oxidation proceeds. The following initiation, propagation, and termination reactions characterize the general scheme of autoxidation: Initiation: Propagation: X" + LH ----> L" + XH L" + 02 ----> LOO" LOO" + LH ----> L" + LOOH Termination: LO" + LO" ----> LOO" + LOO" ----> non-radical polymers L" +L" ----> LOO" + L" ----> Autoxidation is initiated by an abstraction of a hydrogen atom from the methylene between a cis dou- ble bond pair of an unsaturated fatty acid (Fig. 1). The abstraction of a hydrogen adjacent to a double bond is favored because of the formation of a stable allylic radical in which the electrons are delocalized over three carbon a t o m s . 6-9'23 In the propagation reaction, the lipid radical formed following initiation will react with molecular oxygen to form a peroxyl radical (Fig. 1). In most food systems where oxygen is present the reaction of the lipid radical with oxygen is very fast, and therefore the concentration of the peroxyl radical is much higher than that of L. 6,7,10 The reaction of a peroxyl radical with another polyunsaturated fatty acid side chain of a lipid molecule yields a lipid hydroperoxide and a lipid radical, thus conserving the number of lipid radicals in the reaction sequence. Due to resonance stabilization of the L. species, the reaction is typically accompanied by shifting of the double bonds resulting in the formation of a mixture of isomeric hydroperoxides usually containing conjugated diene groups (Fig. 1). The newly formed lipid hydroperoxyl radical can itself abstract hydrogen from another fatty acid, thereby propagating the reaction. 7'24 The abstraction of a hydrogen from another unsaturated molecule by the peroxyl radical is the slowest and rate-limiting s t e p . 6 As the propagation step is relatively slow, hydrogen abstraction is selective for the most readily abstractable hydrogen from unsaturated f a t s . 7'11'23 The ease of hydroperoxidation depends on the number of double bonds present. For example, for a series of unsaturated fatty acids ranging from one to five double bonds, the ratio of oxidizabilities increases linearly by the order of 1:2:3:4:5. 2s The length of the propagative cycle of lipid oxidation is thus increased by the degree of lipid unsaturation and limited by the availability of these lipids. The overall reaction has a pyramidal effect in which a relatively few initiating radicals break down many polyunsaturated fatty acids. Termination is the removal of free radicals, either by the combination of two radicals to form a nonradical product or where the propagation reaction is terminated in the presence of a hydrogen or electron donor. 1° The former reactions become more important when the oxygen concentration is low, towards the interior of the fat system. The nature of the initiating reactive intermediate has been difficult to determine, since very low concentrations of the initiating species are usually generated, particularly during the induction period when the extent of oxidation is very low due to the presence of varying amounts of natural or synthetic antioxidants in the fat. 7'23'26 Evidence indicates that lipid oxidation in foods may be initiated by a number of mechanisms including: (a) singlet oxygen; (b) enzymatic Oxidized lipids in foods Catalysts, 1 light -H* I 65 Hydrogen abstraction Molecular rearrangement 0 2 uptake Peroxy radical II ~ II Dimers; polymers; cyclic peroxides; endoperoxide radicals. OI Conjugated diene o 1"" 1 DecomDo$ition Products Lipid hydroperoxide y 0 N~/ ~ (Aldehydes: ketones; hydrocarbons; acids; I / turans; alcohols; O ~ -OH • and epoxides) Cleavage t • Aldehydes; alkyl radicals; Polymers; dimers; keto, hydroxy, and epoxy compounds. II ~ ~ O Alkoxy radical semialdehydes. Fig. 1. A simplified scheme showing products formed from autoxidation of unsaturated lipids. and non-enzymatic generation of partially reduced or free radical oxygen species (i.e., hydrogen peroxide, hydroxyl radical); (c) active oxygen iron complexes (ferryl iron); and (d) thermal- or iron-mediated homolytic cleavage of hydroperoxides. 6-9,~~,~2 Singlet oxygen Generation of hydroperoxides of unsaturated fatty acids can be formed following exposure to light in the presence of oxygen and a photosensitizer which activates the oxygen. There are two types of sensitized photoxidation pathways reported in foods. 7-9'12'27 In the type 1 photosensitization reaction, the triplet photosensitizer absorbs light and the excitation is used to react with an acceptor which then reacts with ground state triplet oxygen to form singlet oxygen. An example of this pathway is illustrated by riboflavin-sensitized photoxidation of unsaturated fatty acid esters. 27 In the type 2 photosensitization mechanism, following reaction with light, the excited photosensitizer reacts with molecular oxygen in the triplet groundstate to form an oxygen molecule of singlet excitedstate IO2. Singlet oxygen is more reactive than triplet oxygen and can react rapidly with carbon-carbon double bonds of unsaturated fatty acids to form hydroperoxides. Another singlet oxygen reaction of potential significance to lipid oxidation is the oxidation of cholesterol to form an allylic hydroperoxide. 5 Photosensitizing substances such as chlorophyll, flavins, retinal, certain heme compounds, and added synthetic color- ings could play a prominent role in the photoxidation of unsaturated lipids in foods.l~'zs Vegetable oils frequently contain chlorophylls or porphyrin compounds which can undergo photosensitization in the presence of visible light. 2s Removal of such compounds in the refinement and bleaching of vegetable oils can effectively reduce deterioration of these oils. zs The importance of singlet oxygen as an active initiator of oxidation of oils is also illustrated by the effect of singlet oxygen quenchers such as/3-carotene and a-tocopherol in decreasing levels of 12-hydroperoxides in soybean esters, s The singlet oxygen reaction involves an "ene" reaction as opposed to the free radical process of hydrogen abstraction from the allylic carbons of unsaturated fatty acids. 6-s Oxygen is inserted at the ends of double bonds with the consequent migration of the double bond to form an allylic hydroperoxide in the trans configuration. The cis double bond shifts to yield hydroperoxides in the trans configuration because of its lower thermal energy state and steric hindrance. 7 The ene reaction with the double bond between carbons 12 and 13 would thus produce the 12 and 13 hydroperoxide isomers. The resulting hydroperoxides would proceed via free radical chain reaction as the main mechanism of oxidative decomposition. 9 Enzyme-induced lipid oxidation Enzyme catalyzed lipid oxidation via lipoxygenase and cytochromes P450 catalysis has been suggested to 66 S. KuBow play a role in the production of hydroperoxides in f o o d s . 7'25'29 Recent reports of lipoxygenase activity in fish gill and skin tissues have suggested that these endogenous enzymes may be a source of initiatory free radicals leading to the production of lipid oxidation compounds in fish. 3° Lipoxygenases initiate the oxidation of polyunsaturated fatty acids via a free radical mechanism to form specific hydroperoxides which can be further oxidized to trihydroxy fatty acid derivatives. 5'7'28 In most tissues, the predominant product contains an oxygenated moiety at the 13-position with smaller amounts of the 9-isomer being forlned. 5,7'25 The breakdown of these unstable hydroperoxides could contribute to subsequent lipid oxidation and further chain reactions in both plant and animal tissues. The improvement of shelf life of fish and frozen stored vegetables following heat treatment has been explained on the basis of the thermal instability of lipoxygenase enzymes. 7,28,3° In addition to lipoxygenases, animal tissues also contain peroxidases and cyclooxygenases which have been implicated to initiate and promote lipid oxidation in a postmortem tissues as these enzymes are active towards unsaturated fatty acid substrates to form hydroperoxides. 7 There is also evidence that lipid oxidation could be initiated via enzymatic generation of hydroxyl radical and singlet oxygen occurring through lipoxygenase, prostaglandin synthase or microsomal oxidase activities. 7 The presence of a membrane fraction capable of forming lipid oxidation products in the presence of NADH or NADPH and iron has suggested a role of these fractions in lipid oxidation of muscle f o o d s . 7'11,30 In mammalian tissues, cytochromes P450, when in the ferric form, have been indicated to catalyze chainbranching reactions very efficiently. 11 The postulated reaction is: CYT P4503+ + LOOH ---> CYT P4503÷ - OH- + LO" Unsaturated fatty acid will bind to cytochromes P450 at the drug binding site which also may be the site where lipid hydroperoxides are decomposed by heme iron.~ 1 The enzymatic lipid oxidation in skeletal muscle microsomes is dependent on the presence of NADH or NADPH and also requires ADP and iron. The maximum rate of this reaction occurs under pH and cofactor concentrations which might be expected to occur in postmortem muscle tissues. 3° Mitochondrial lipid oxidation has also been proposed to play a role in oxidation of lipids in animal tissues. The NADH dehydrogenase of the respiratory chain could initiate lipid oxidation by donating electrons to ADP-chelated Fe 3+ and thereby resulting in free radical generation. I~ An enzymatic lipid oxida- tion system in fish muscle mitochondria has been demonstrated and was found to be similar to that of fish microsomes in terms of co-factor requirements and optimal pH. 3° Activated oxygen-iron interactions Harel and Kanner reported that the interaction of hydrogen peroxide with methemoglobin in muscle tissues can activate methemoglobin to act as an initiator of lipid oxidation in animal muscle foods. 31 The activated heme protein appears to be a porphyrin (P) cation radical, P "+ - Fe 4÷ = 0 which is also a ferryl species. 3~ Initiation of lipid oxidation by this species can occur as following: P'÷ - F e 4÷ = O + RH ----> P - F e 4÷ = O + R" + H ÷ The action of activated myoglobin appears to be more selective than that of the hydroxyl radical since molecules known to interact with the hydroxyl radical did not inhibit lipid peroxidation initiated by activated myoglobin. 5 A reactive species that has been postulated to initiate lipid oxidation during the processing of foods is the hydroxyl radical. 7,32,33 Hydroxyl radical is one of the more potent oxidants due to its capacity to react at high rates with virtually any organic compound. 2 Hydroxyl radical-induced lipid oxidation proceeds by hydrogen abstraction from the methylene between the cis double bond pair of an unsaturated fatty acid. 5 It is generally believed that hydroxyl radical formation could proceed via the Fenton-catalyzed HaberWeiss reaction in which free or chelated forms of iron serve as a catalysts for the reaction between hydrogen peroxide and superoxide anion. 6,11 Metal 0 2 0 2 + O~ ......... "> 0 2 + " O 0 + O H Catalyst The Haber-Weiss reaction is most effectively catalyzed by reduced iron complexes, which can be generated by the reaction of ferric complexes with superoxide anion and by cellular constituents such as ascotbate. 5'1~ This so-called iron-redox cycling catalysis of lipid peroxidation has been suggested to be a driving force for the generation of high concentrations of ferrous ions which can in turn generate hydroxyl radicals. Miller et al. 34 recently proposed an alternative to the traditional Fenton reaction for the role of iron in lipid oxidation reactions in biological systems. These authors suggested that an Fe(II):Fe(III) complex acts as an initiator of lipid oxidation and that formation of this complex was dependent on overall rates of oxidation and reduction of iron not greatly exceeding each other in the surrounding environment. It was felt that Oxidized lipids in foods the proposed model was better reconciled to the apparent requirement of both the ferrous and ferric forms in several model systems of lipid oxidation. Hydroperoxide breakdown A primary mechanism by which initiation could take place is via iron-mediated homolytic cleavage of preformed hydroperoxides which generates organic free radicals. 7'35 Metal catalysts may be coordinated with ligands as complexes, may exist as dimers or higher molecular weight species or complex with hydroperoxides to catalyze autoxidation and decomposition. 28 Alkoxyl radicals are believed to be predominantly formed in the early stages of oxidation by the interaction of the hydroperoxide with the reduced form of the metal via one-electron transfer. 23 LOOH + M "+ + H + ----> LO" + OH- + M ('-1)+ + H20 It has also been suggested that due to the increase in the oxidized form of the metal during the later stages of lipid oxidation this metal could react directly with the hydroperoxide by the mechanism of one-electron transfer to form alkylperoxide lipid radicals. 23 LOOH + M ("+l)+ + HO- ----> LOO" + M "+ + H20 Oils and fats that are poorly handled or stored frequently contain hydroperoxides which can serve as substrates for metal-catalyzed decomposition to form initiatory free radicals. 28A common way that pro-oxidant metal ions enter food is via the water or spices used in food preparation. 3s Additionally, disruption of muscle membranes during grinding, cooking, or deboning breaks up the well organized structure of animal cells to bring together lipids and pro-oxidant catalysts including metal ions. 32 Hydroperoxides can decompose thermally or in the presence of metal catalysts. A major pathway of thermal hydroperoxide decomposition involves a homolytic cleavage of the O-O bond of the hydroperoxide to produce alkoxyl and hydroxyl free radicals (Fig. 1).28 Cleavage of alkoxyl radicals or reaction of these radicals with substrate propagates the chain reaction. The alkoxyl radicals undergo C-C bond scission which can occur on either side of the radical to produce an alkyl radical on one side and a vinyl radical on the other (Fig. 2). 9,24 The alkyl radical can react with a hydrogen radical, hydroxyl radical or molecular oxygen to generate hydrocarbons, alcohols, and hydroperoxides, respectively. The vinyl radical is very reactive and may also react with hydroxyl radical, hydrogen radical, or molecular oxygen to generate aldehydes and olefins. 9 Alkoxyl radicals can also undergo epoxidation or react with each other or with a corresponding alkyl radical to terminate the radical chain 67 by formation of stable products. The recombination of alkoxyl radicals leads to the formation of dialkylperoxides. 23,2s The cleavage reactions explain most of the volatile products such as carbonyls, alcohols, esters and hydrocarbons identified from the decomposition of hydroperoxides of oleate, linoleate and linolenate. 9 On the other hand, numerous speculative mechanisms have been proposed to account for the formation of substituted furans, epoxy aldehydes, ketones, lactones, alkynes, and aromatic compounds. 8a3,24Cleavage of unsaturated aldehydes has been implicated to be the source of many of the volatile compounds that cannot be explained by cleavage of monohydroperoxides such as the formation of lower aldehydes, alcohols, acids, alkyl formates, acrolein, benzene, furans, and hydrocarbons, s,13,24Dihydroperoxides, hydroperoxy cyclic peroxides and other polymeric materials are also potential sources of volatile compounds. 7,s Thermal decomposition of cyclic peroxides from linoleate has been shown to produce most of the same volatile decomposition products as the corresponding monohydroperoxides. 9 Breakdown of cyclic peroxides, however, has been demonstrated to produce some unique oxidation products such as unsaturated methyl ketones. 9 A compound which has been of particular interest regarding lipid oxidation reactions in foods is malondialdehyde. Malondialdehyde has been suggested to be derived from intramolecular cleavage of 5-membered monocyclic peroxides, 36 bicyclic endoperoxides 37 and from decomposition of 1,3-dihydroperoxides9'38; 1,4-dihydroperoxides and monohydroperoxides are believed to be less important precursors of this product. 9'38 More detailed explanations of the postulated stereochemistry involved in the outlined mechanisms can be obtained by some excellent reviews on this subject. 7-9'39 Radicals that do not unimolecularly decompose or bimolecularly react with non-radical substrate react with other radicals by combination or disproportionation to form dimers or intra- and intermolecular cyclizations) 4,23 The probability of collision between alkoxyl or peroxyl radicals to form some of these products increases during the later stages of autoxidation. 23 These nonvolatile products include various cyclic and aromatic monomers, dimers, and polymeric compounds such as hydroperoxyl epoxides, hydroperoxyl cyclic peroxides, and dihydroperoxides. 9 In certain model fatty acid systems such as autoxidized methyl linolenate, monocyclic peroxides can be formed in the same order of magnitude as monohydroperoxides. 9 Radical cyclization may occur when a remote double bond, such as a/3-~,-double bond, is present in the peroxyl radical, which allows for facile 68 S. K u a o w (a) (b) R 2 - CH • CH ; CH * CH 2 I ~ I = O P I R 1 - CH 2 saturated vinyl radical alkenal • 2 2-alkenal R - CH - CH" 2 R 1- C H 2 " • OH/ CH R 2 - CH • CH - C H O aldehyde H, 11 - 1 I - CHO R 2-CH-CH -OH R R - 2 R 2 \.. alpha-olefin - CH " CH 2 R - CH OH 1 2 alcohol CHO alkyl radical R - CH 1 3 hydrocarbon aldehyde Fig. 2. A scheme depicting product formation from homolytic cleavage o f a hydroperoxide of an unsaturated fatty acid. 1,3 cyclization.7' 14Monocyclic peroxides, bicyclic peroxides, and epoxy alcohols are the products derived from radicals formed from cyclization. 7'9 Polymerization reactions are very complex during lipid oxidation and polymeric material is difficult to characterize because of its complex structure.14 Intraand intermolecular combinations of alkoxyl, alkyl, and peroxyl radicals to form dimers and polymers with C-O-C and C-O-O-C cross-links are major reactions in high temperature oxidations? 2 Dimers are a major component of nonvolatile products formed in oxidized and heated fats and increase with the extent of lipid oxidation. 24,4° The temperature of oxidation and the hydroperoxide environment are key factors in determining the role ofhydroperoxides which can act as catalysts or may themselves take place in polymerization reactions.~4 Polymerization can involve mechanisms of both radical and nonradical addition. 23 At temperatures between 60-120°C, decomposition of hydroperoxides can proceed via ester formation with other fatty acids and by homolytic decomposition. 41 These reactions can generate products which react with other fatty acid molecules to form C-O-C bonds and generate dimers, trimers and larger molecular weight products. 4~ Considerable degradation or heating are not prerequisites for dimer formation since Miyashita and co-workers demonstrated the formation ofdimers from methyl linoleate during the initial stages of autoxidation at 30 ¢ after 24 h. 42 They suggested that most of the dimers were formed through peroxide (C-O-O-C) linkages. The compounds formed as a result of thermal oxidation are of special interest, since deep fried fat is continuously or repeatedly used at elevated tempera- tures in the presence of air and moisture. Formation and decomposition of hydroperoxides is markedly increased during deep-fat frying. ~2 The peroxides and hydroperoxides do not survive the heating process and break down into oxy- and cyclic acids as the temperature is raised. 24 In this process, the nonvolatile products which remain in the oil are absorbed into the food and are subsequently ingested. 43 Concentrations of volatile products such as saturated and unsaturated aldehydes, ketones, hydrocarbons, alcohols, acids, and esters are formed much more rapidly during the deep-fat frying process until a balance is reached between the formation of volatiles and their decomposition or evaporation.~2 The removal of the fat through absorption into the food helps to maintain the quality of the fat, and fresh fat is added to replenish the frying b a t h . 43 In establishments in which the fat is only occasionally used for frying and kept hot for long periods of time or cycled through periods of high and low temperatures, polymer formation more readily takes place. After prolonged heating, dimeric and polymeric compounds are formed and can accumulate up to a concentration of 10-20% without the functional properties of the oil becoming noticeably changed. 44 Further, heating of heme-containing foods may increase lipid oxidation by the release of catalysts such as non-heme iron from heme pigments and by disruption of membranes more readily exposing lipid constituents to oxygen and catalysts. 45 Cross-reactions of lipid oxidation products Lipid hydroperoxides and their decomposition products can interact with other biomolecules in Oxidized lipids in foods foods such as pigments, enzymes, proteins, amino acids, membranes and DNA to produce a multitude of other by-products of lipid oxidation.tS The possibility of co-oxidation can increase the complexity of oxidized products) 6 In the process of autoxidation, the peroxyl radicals formed attack the more readily oxidizable substrates due to their relative unreactivity. Alkoxyl radicals, however, are much more reactive and unselective than peroxyl radicals, and as a consequence will react with more slowly oxidizable material such as proteins to increase the complexity of the oxidized products. 16 Lipid radical reaction can cause protein-protein crosslinks; proteinlipid crosslinks, and protein scission. 7'46 A major pathway in lipid-protein interactions is the reaction of primary amines with the carbonyl products of lipid oxidation to form N-heterocyclic compounds including imines, pyridines and pyrroles, t5 Amino sugars can react with malondialdehyde to form pyrroles and other heterocyclic compounds. 47 In the presence of other food components, lipid oxidation reactions are also complicated by the possibility of termination by compounds other than those derived from lipids. The Maillard reaction is of importance in this regard. Several Maillard products which may exert antioxidative actions in foods are produced from the Maillard reaction between reducing sugars and amino acids, peptides, or proteins. 10,47 For example, lipid autoxidation in heated muscle foods continues to increase up to approximately 77°C; beyond this temperature or after prolonged heating, concentrations of lipid autoxidation products decrease due to the formation of significant concentrations of antioxidative Maillard products. 17 Maillard type brown pigments are also formed from interactions of lipid oxidation carbonyl compounds with nitrogenous food constituents such as proteins and amino acids. 48 Antioxidative effects of Maillard products may occur via several mechanisms including metal chelation, reduction of hydroperoxides to non-radical products, and donation of hydrogen atom to break the radical chain. 1°'47 Termination Termination reactions via the combination of two radicals become important when edible oils are heated at elevated temperatures as indicated by formation of polymers in frying oils.rE At 160°C hydroperoxides begin to decompose spontaneously and the radical concentration can become relatively high allowing greater likelihood for radical-radical interactions.l° Autoxidation can also be retarded or inhibited by the presence of low concentrations of chain-break- 69 ing antioxidants that interfere with either chain propagation or initiation.t°,26 The presence of antioxidants in food can retard the development of rancidity via termination reactions. ROO" + A H ----> R O O H + A" A" + ROO" ----> nonradical product A" + A" ----> nonradical product The reactivity of phenolic antioxidants such a-tocopherol allows it to intervene to regenerate the original diene and a phenoxyl radical. The antioxidant free radicals are generally too unreactive to propagate the chain. I° Ascorbate can regenerate the phenol and an ascorbic acid oxidation radical by reaction with the phenoxyl tocopherol radical. 4° At high concentrations, however, tocopherols can act as pro-oxidants in v e g e t a b l e oils. 2s The main limitation of phenolic antioxidants in foods is that they become ineffective during prolonged heating at elevated temperatures. 49 In addition to free radical terminators such as phenolic compounds, a great variety of other substances and conditions can be considered as exerting antioxidant activities other than by converting free radicals to more stable species. 26 For example, free radical production can also be retarded by the presence of chelating agents such as EDTA, citric and phosphoric acids which act to inhibit lipid oxidation by chelating prooxidant metal ions) °,26 A variety of environmental factors, such as packaging material to remove air, redox compounds such as cysteine and ascorbic acid, and physical conditions (temperature, oxygen pressure, moisture) can also act to retard lipid oxidation. 1°'17'26 This variety of factors can cause the nature and proportion of the various products from lipid oxidation to vary widely between different foods and makes the kinetics of oxidation reaction complicated since one cannot account for all the possible interactions. ORAL TOXICITY OF ISOLATED LIPID OXIDATION PRODUCTS Lipid oxidation in foods has received much interest with regards to potential health risks since a number of classes of lipid peroxidation products have been demonstrated to exert toxic effects in studies both at the whole animal and cellular levels. 2-4,5,6,12'23 Lipid oxidation products are known to react with nitrogenous materials in biological systems including amino acids, proteins, bases of phospholipids, and DNA to form brown pigments and fluorescence which have been related to tissue injuries. 5'6'1t'23 A wide array of toxic consequences can be induced by lipid oxidation 70 S. Ktmow in the cell, including destruction of essential membrane and cytosolic enzyme activities, membrane swelling and lysis, mutagenic and carcinogenic activities. 6,11,23 Although some of the adverse effects of lipid oxidation may be relevant to the formation of these products in foods, more systematic research is needed to assess the metabolic and toxicological consequences of ingestion of these compounds. For example, the toxicity of lipid oxidation products formed in situ may have little relevance to the properties of lipid oxidation products taken in orally. In many studies the toxic dose levels of oxidation products animals are exposed to are orders of magnitude higher than would be found in the average diet. Moreover, due to the cascade of reactions in autoxidation, the toxicological relevance of the presence of multitudinal toxic byproducts of lipid oxidation in foods is problematic. Chang and collegues identified a total of 220 compounds formed during deep-fat frying, many of which are potentially toxic. 5° Some approaches to test the toxicity of oxidized fats have either fed the whole oxidized oil or have focused on isolating potentially toxic compounds or fractions via vacuum distillation, column chromatography, or urea adduct formation and fed these fractions to experimental animals. Fatty acid hydroperoxides Although lipid hydroperoxides are very toxic when administered intravenously, oral administration of lipid hydroperoxides is considerably less toxic, most likely due to low absorbability or to some conversion before or during the absorptive processY The presence of a reductase enzyme in the gut as part of a detoxification system has been suggested as lipid alcohol compounds appear in various organs after ingestion of lipid hydroperoxides.18 The role ofglutathione peroxidase in this regard has been suggested by a recent study using everted sacs of rat small intestine and peroxidized methyl linoleate. 52Adding either glutathione or an inhibitor of the glutathione peroxidase system decreased or increased, respectively, the amounts of secondary oxidation products on the contraluminal side. Exogenous glutathione appeared to be taken up and utilized by brush border intracellular reactions to metabolize the peroxides and prevent their transport to the contraluminal side. In contrast to the products generated from autoxidative reactions involving lipid hydroperoxides, such enzymatic action would generate much less reactive hydroxy compounds. Recent work has also indicated that the generation of a 13-hydroxy derivative of linoleic acid formed from peroxidase activity may be coupled to a dehydrogenase enzyme in rat colon thereby resulting in the formation of a 2,4-dienone derivative. 53 The formation of the dienone may be a protective mechanism in that it is much less susceptible to decomposition reactions and thus may further direct the reactivity of the oxidized fatty acid away from the lipid autoxidative cascade. Secondary lipid oxidation products A number of studies have definitively shown that secondary autoxidation products of linoleic acid and other unsaturated fatty acids are absorbed into the circulatory system following oral administration with consequent effects on lipid oxidation products present in body tissues, particularly in the liver. 54'55In one study, following heating of [14C]linoleic acid for seven days at 37°C, the mixture was separated into linoleic acid, the hydroperoxide oflinoleic acid and secondary products. 54 After feeding these various components separately via gavage to Wistar rats, a significantly greater fraction of radioactivity was observed in the livers of rats fed the secondary products than for the other two components. Although all the animals were clinically normal, the feeding of secondary products produced some elevation in serum liver enzymes and a slight hypertrophy indicating a certain amount of liver damage. Studies have also suggested that ingestion of secondary lipid oxidation products can result in lipid oxidation in vivo. Certain carbonyl compounds such as 12-keto oleic acid have a pro-oxidant effect on unsaturated fatty acids in vitro and could conceivably participate in initiation of lipid autoxidation in vivo. 56 Oral administration of autoxidized methyl linoleate hydroperoxide has demonstrated increased tissue chemiluminescence 57 and the presence of lipid oxidation products in various rat organs which were inhibited by administration of chain breaking antioxidants such as tocopherols. 5s The mechanism of chemiluminescence has been explained by a hydroperoxide disproportionation reaction and seems to indicate the generation of short-lived radicals and excited species during lipid oxidation. 59 Singlet oxygen and triplet carbonyl compounds were shown to participate as the major emission species in the chemiluminescence of livers of animals administered with autoxidized oil 6° and in liver homogenates supplemented in vitro with oxidized lipids. 61 In addition, the presence of lipid peroxyl radicals in livers of tocopherol-deficient rats given autoxidized lipid hydroperoxides has been indicated by electron spin resonance spin trapping using the trapping agent, N-tert-butyl-a-4-pyridylnitrone1-oxide. 62 Oxidized lipids in foods It is clear that the toxicity of oxidized oil depends on the amount of secondary oxidation products rather than on the hydroperoxide content. Yoshioka and Kaneda compared the toxicities in mice following autoxidation of methyl linoleate at 60°C for different periods of time. 63 Minimal effects on toxicity were observed in mice fed the hydroperoxides whereas the greatest toxicities occurred while carbonyl values were at their highest. Low molecular weight carbonyl compounds Due to their strong hydrophilic nature the low molecular weight fraction of oxidized oils has been shown to be more easily absorbed and carried to internal organs in the bloodstream and metabolized faster than the polymeric fraction. 5t These characteristics may in turn account for the higher potency of carbonyl compounds among oil autoxidation products in inducing cellular damage and toxicity. Malondialdehyde has received much attention in toxicological and food science literature due to its reported mutagenicity and carcinogenicity. 64'65 Reaction with thiobarbituric acid (TBA) and spectrophotometric determination of the colored compound formed is the most frequently used method to assay malondialdehyde in foods and biological systems. A major difficulty in the use of this test is that the TBA reaction is not specific for malondialdehyde. 39'66 Many other lipid oxidation compounds can form products that can also react with TBA to form colored complexes that co-chromatograph and absorb in the same wavelength as the malondialdehyde-TBA produ c t . 39'66'67 The use of the TBA reagent to determine malondialdehyde in foods has been criticized on the basis that the method can overestimate the amount of malondialdehyde in foods by at least twofold. 66 The term "thiobarbituric acid reactive substances" (TBARS) is more appropriately used in place of"malondialdehyde" in reference to this t e s t . 66 The potential of non-lipid related oxidation products to generate malondialdehyde further limits the usefulness of even direct measurement of this end-product as an indicator of lipid oxidation. 39 Oral toxicity studies administering malondialdehyde as the enol salt in Swiss mice showed a dose dependent increase of total neoplasms and neoplastic lesions in the liver over a 12 month period, 6s although the lowest dose was about ten times what was considered to be the average dietary intake on a body weight basis of the Canadian p o p u l a t i o n . 69 Based on the TBA test, malondialdehyde appears to range in foods from about zero to 10 ppm depending on the extent of oxidative rancidity and the polyunsaturated content pres- 71 ent. 69The food safety concerns regarding the presence of malondialdehyde appears to be mitigated by a recent study which has found that only a negligible fraction of malondialdehyde in several foods of animal origin occurs in the free form. 7° The main portion of malondialdehyde is bound to the lysine residues of food proteins from which it is released in the course of digestion as N-~-(2-propenal)lysine (E-PL). This compound is apparently the product of a reaction between free malondialdehyde formed as a result of oxidative rancidity and the free episolon groups of proteins. 7° During heating, malondialdehyde is released from ~PL and it is probable that this process is involved during the volatilization of malondialdehyde and its decline in content in foods during cooking. 65Intraperitoneal administration of the lysine-bound malondialdehyde to rats does not give rise to free malondialdehyde in the urine but is excreted in rat and human urine in an unchanged form and as its N-a-acetyl derivative. 7° Moreover, ~-PL is extensively metabolized by a route that does not give rise to free malondialdehyde. 6s Since ~-PL does not generate free malondialdehyde endogenously and is non-mutagenic, it is likely therefore that malondialdehyde formed in foods is not of significant toxicological importance. 7° It is noteworthy that aldehydes more toxic than malondialdehyde produced from autoxidation of polyunsaturated fats such as unsaturated aldehydes and 4-hydroxy unsaturated aldehydes can vary considerably in concentration without affecting the TBA reaction. 67 Amongst these aldehydes released by fatty acid peroxides, 4-hydroxyalkenals are of the greatest quantitative and toxic importance. 7~ 4-Hydroxynonenal is of particular relevance since it is a strong electrophilic agent which can react with thiol groups of glutathione and proteins and can inhibit cell division in mammalian cell lines. 7~ This compound has also been shown to inhibit several microsomal and membrane-bound enzymes such as adenylate cyclase and S-adenosylmethione decarboxylase at concentrations of 1-100 #M whereas malondialdehyde has no effect at 5 mM. 67 Such findings further emphasize that the limitations of TBA and malondialdehyde estimations in presenting a completely accurate index of the total cytotoxic potential resulting from both in vitro and in vivo lipid oxidation. It should be noted, however, that apart from malondialdehyde, the bioavailability of these highly reactive short chain aldehydes from the foods in which they are present is not known. Nonvolatile oxidized lipid products It has been demonstrated that nonvolatile oxidized products such as monomers, dimers, and cyclic mono- 72 S. Kuaow mers are absorbed into the body and can be recovered from lymphatic and hepatic lipids. 72 Polymeric material of high molecular weight is not well absorbed and therefore is considerably less toxic than monomeric and dimeric compounds. 12'43 This does not discount the possibility, however, that high molecular weight polymers may induce irritation of the gastrointestinal tract. Oxidized lipids have been treated with urea in order to separate unchanged fatty acids from the oxidized material. The urea-adduct forming fatty acids are composed of a mixture of normal straight chain fatty acids whereas the non-urea-adductable fraction contains predominantly oxidized material. The nonurea-adductable-fraction has been used in feeding studies to demonstrate the toxicity of monomeric, dimeric and polymeric material although other oxidized material is present in these fractions as well. 43 When fed in large amounts, these fractions can be toxic to experimental animals ~2and have been shown to be deleterious to heart cells in c u l t u r e . 73 The amounts of these toxic materials that are normally present following thermal oxidation of oils needs to be considered in any safety evaluation of these oxidation products. Frankel and co-workers in an analysis of cyclic monomer concentrations of commercial frying oils and fats from different restaurants in the U.S. found cyclic monomer concentrations of 0.1% to 0.5%. 74 In a diet containing 15% of oils containing 0.5% cyclic monomer content, approximately 0.086% monomeric material would have been ingested by the rats. 43 This could be considered as a possible health risk since animals fed 0.02 to 0.15% cyclic monomers develop fatty livers, the severity increasing with monomeric concentration. 75 Diffusive fatty degeneration, atrophy, and necrosis have been regarded as the direct injurious effects of cyclic monomers to the liver following intake at these concentrations. Precise information concerning the possible health effects of ingesting low levels of cyclic monomers in humans is not available, although long-term intake of a diet containing 15% of this oil is needed to produce these effects in experimental animals. Chronic consumption of thermally abused, deep-fried oil at the high dietary fat levels of 15% (w/w) is not likely to occur in a typical mixed diet by humans. Dimers of triacylglycerols can contain several functional groups such as epoxides, hydroxyl, and carbonyl groups, and an isolated toxic dimer substance has been isolated from thermally oxidized soybean oil. 19 In short term experiments, rats did not demonstrate toxic symptoms when fed 0.75% non-cyclic dimers. 76 Since the quantities of such compounds in thermally abused fats are far below these concen- trations, 43 the dimeric component of oxidized oils probably pose no significant toxicological problems. The toxicological significance of the presence of these volatile and nonvolatile lipid decomposition materials at the levels present in our foodstuffs is far from clear. In a exhaustive study carried out by Billek, 2° sunflower oil was collected from commercial production of fish fingers at a time of discard. Rats were fed the polar fraction containing most of the oxidation and decomposition products at a concentration of 20% in their diets over a 18 month period. Only mild symptoms were observed in the animals such as growth retardation, enlarged livers and kidneys, and increased serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase indicating some liver damage. An extensive evaluation of other histological and clinical parameters did not show any major abnormalities. The polar fraction contains 90% lipid decomposition products as compared to only 30% in the discarded oil. Moreover, frying oil is usually rejected in most commercial settings by the time significant deterioration occurs in terms of foaming and the development of odor and color defects (at approximately at 30% polar constituents). It is likely, therefore, that human consumption of these products are orders of magnitude lower than was ingested by animals which only demonstrated mild toxic symptoms. In fact, it was calculated that the average daily intake of this polar fraction administered to rats was 10 g/kg body weight whereas average human consumption of frying oil itself is only about 0.1 g/kg body weight. As will be discussed in following sections, however, there may be other safety concerns of oxidized fats based on alterations in metabolic parameters not measured in this investigation. TOXICITY OF WHOLE OXIDIZED OILS Early toxicity studies that carried out feeding trials in which fats were subjected to extreme processing conditions and fed in large amounts to laboratory animals demonstrated severe irritation of the gastrointestinal tract, growth retardation, and death. 19 The conditions used also resulted in the complete destruction of linoleic acid in the oil, and researchers did not usually supplement for the minimum amounts of the essential fatty acid needed. 19 Therefore, some of the severe growth and histological abnormalities could have been attributed to symptoms of essential fatty acid deficiencies. Alternatively, researchers have fed oils and fats oxidized under more realistic cooking practices either in a commercial or laboratory setting as part of a nutritionally balanced diet. Ingestion of these mildly oxi- Oxidized lipids in foods dized oils at 15% of the diet have typically indicated much milder symptoms which included depressant effects on growth or steatorrhea with no change in survival and morphological a p p e a r a n c e . 77-79 A few studies have shown adverse effects which were limited to enlargement of various organs and some histopathological changes. 8°,81 It is likely that many of these effects are secondary to the direct toxic action of the lipid oxidation products themselves. 82When oxidized oils are fed in large quantities to animals, the taste and odor characteristics of the diet may deteriorate with a subsequent decrease in food intake. Growth retardation and other harmful effects may also result from too high an intake of long chain polymers which may cause the fat to be less absorbable and could interfere with the absorption of the other fat-soluble nutrients in the diet. 77,82 Nutrient deficiencies can be induced by the thermal destruction of essential vitamins and fatty acids in the oils or by autoxidation of vitamins in the purified diet fed after the oxidized oil has been mixed in the food. 82 Irritation of intestinal mucosa by peroxides could interfere with nutrient absorption. 19,77.82There also may be decreased protein digestibility and absorption due to cross-linking reactions of secondary lipid oxidation products with proteins. 82 There is some contradictory research on the longterm effects of consuming oxidized fats produced under conditions that mimic commercial and domestic cooking conditions. Some long-term studies have shown that overall mortality rate and tumor incidence did not tend to correlate with extent of heating under either mild or severe oxidation conditions, s3 On the other hand, Kaunitz observed more cardiac fibrotic lesions and hepatic duct lesions in rats fed mildly oxidized oils over their lifespan than in animals fed nonoxidized fats. 84Contrasting results could arise from different experimental approaches; heating conditions (time, temperature, contact with food, and aeration), replenishment of oil, and the antioxidant level in the fat will determine the degree of oxidation. Moreover, the nutritional adequacy of the diet in terms of the level of protein and antioxidants could also determine the degree of toxicity observed. The general lack of serious pathological consequences observed in animal feeding studies of oxidized fats in terms of such general parameters as survival, clinical signs, body and organ weight, growth, and histomorphology of major organs is not surprising. There is a long history of moderate consumption of heated and oxidized fats in humans without obvious adverse effects being observed. The overall lack of gross pathological effects resulting from ingestion of mildly oxidized fats, however, does not obviate the 73 possibility of more subtle deleterious metabolic effects being exerted. Oxidative stress and vitamin E status The feeding of unheated but highly oxidized fish oils at levels of 10-20% in the diet of rat has resulted in a wide spectrum of injurious effects including diarrhea, loss of appetite, growth retardation, cardiomyopathy, hemolytic anemia, and the accumulation of peroxides in adipose tissue. 2~ In particular, the high concentrations of C20-C22 polyunsaturated fatty acids found in certain fish species and fish oils causes such oils to oxidized readily on exposure to air. Although secondary effects are likely to be involved, symptoms such as hemolytic anemia have been reversed by addition of vitamin E to the ration indicating the occurrence of oxidative stress in these fish oil-fed animals. 2~ The possibility of oxidative stress induced by absorption of preformed secondary lipid oxidation products in foods which readily go rancid, such as fish oils, has also been suggested by an immediate and marked increase in urinary malondialdehyde observed in rats after ingestion of a cod liver oil diet. 85A potential risk to humans of exposure to lipid oxidation constituents in fish oils has recently been demonstrated. 86 Ingestion of a commercial cod liver oil product without added antioxidants caused an immediate excretion of malondialdehyde, but remained unchanged in subjects consuming a concentrate of n-3 fatty acids containing dodecyl gallate and vitamin E. The authors suggested that the increased urinary malondialdehyde was likely due to ingestion of preformed malondialdehyde rather than an increased endogenous generation from n-3 fatty acids. This was supported by the finding that subjects receiving the fish oil concentrate with antioxidants over a period of 49-50 days showed no increase in malondialdehyde excretion. In another human s t u d y , a7 the presence of ingested lipid oxidation material in the urine was suggested when TBARS were detected during the first four hours in the urine of humans following a meal consisting of rancid meat fats. The authors indicated that precursor lipid oxidation products rather than malondialdehyde itself were likely measured in the meat and detected in the urine. The red cell membrane has been used as a model for oxidative damage to biomembranes as well as an index of vitamin E status since vitamin E is involved in scavenging free radicals and stabilizing cell membranes by protecting membrane polyunsaturated fatty acids, a8,a9 Red blood cells from vitamin E deficient rats hemolyze faster in the presence of oxidative stress than controls, presumably due to the action of vitamin E acting as a scavenger of lipid peroxyl radi- 74 S. KuBow cals thereby breaking the propagation of the free radical cascade, s9 The occurrence of hemolytic anemia following ingestion of oxidized fish oils may thus indicate an imbalance between the severity of oxidative deterioration of ingested fats and the capacity of the antioxidant system. 21 The ability of primary and secondary lipid oxidation products to hemolyze red blood cells was demonstrated by Yoshioka and Kaneda who observed that 11, 8.7, 4.3, and 0.037 micromolar of methyl linoleate hydroperoxide, alkenal, hydroxylalkenal and hydroperoxyalkenal, respectively, were required to produce 50% hemolysis following incubation of these products with normal red blood cells. 9° Peroxidized fats may accelerate the turnover of vitamin E and consequently increase the requirement for this vitamin 21 such that despite nutritionally adequate amounts of vitamin E, serum and hepatic levels of vitamin E may still be deficient in animals fed mildly oxidized oils in comparison to controls. Rats were fed vitamin E sufficient diets containing 15% of oxidized rapeseed oil which had been used in the frying of fish paste according to standard commercial frying practices. 91 Liver and serum tocopherol levels diminished according to the level of deterioration of the supplied oils. Rats fed oils with increasing degrees of oxidation showed increasing TBARS values in the liver further indicating an increased level of oxidative stress. Alterations in xenobiotic-metabolizing enzymes One of the most common findings of long-term feeding studies of oxidized fats is enlargement of the liver, smooth endoplasmic reticulum proliferation and induction of microsomal cytochromes P450 enzymes. 92'93 This observation is not surprising in light of the hepatomegaly and increase in microsomal enzyme activities frequently seen in animals dosed with xenobiotics. 1~ The chronic feeding of oxidized oil to rats has markedly induced a number of enzymes important in hydroxylation, oxidation and conjugation reactions including cytochromes-P450 content and activities, UDP-glucuronyl transferase and glutathione S-transferase. 93-95 Cyclic monomers which can both induce and act as substrates for cytochromes P450 have been suggested to the component responsible for commonly observed induction of these enzymes by thermally oxidized oils. 94 The observations that the toxicity of ingestion of thermally oxidized oils is much reduced by a concomitant diet higher in protein content has been indicated to be due to a larger induction of hepatic microsomal activities to metabolize lipid oxidation products. 93,96 Ingestion of mildly oxidized oils for eight weeks has been shown to induce cytochrome P450 content as well as various hepatic microsomal enzyme activities such as aniline hydroxylase, aminopyrine N-demethylase and NADPH-cytochrome C-reductase; the level of induction being dependent on the dietary protein level. 93 Not surprisingly, the level of induction of microsomal hydroxylase activities far surpasses that of N-demethylation since lipid oxidation products are not generally abundant in the methyl group. 93 The inhibitory effect of a low protein diet on the inducibility of these enzymes along with the augmented toxicity of oxidized oil products indicate that these enzyme activities may be important in the detoxification of lipid oxidation products. In some rat and guinea pig studies, glutathione peroxidase in the liver, which is known to reduce lipid hydroperoxides to alcohols by linking glutathione reductase, has been observed to be induced in animals receiving orally oxidized oil. 55'6° In contrast, other investigators have not found an increase in liver glutathione peroxidase levels following intake of oxidized oils. 91'97 The absence of supplemented dietary selenium in the former studies is likely responsible for the observed differences between studies. Reddy and Tappel found that that autoxidized oils do not stimulate glutathione peroxidase when the diet is supplemented with selenium. 98 As dietary selenium is an important dietary component for the synthesis ofglutathione, ~ presumably adequate dietary levels of selenium ensured sufficient synthesis of this substrate so that induction of additional enzyme activity was not necessary in the presence of oxidative stress. Further evidence of oxidative stress at the level of the liver has been indicated by reports of increased hepatic glutathione concentrations and hepatic catalase activity following feeding of oxidized oils to rats. 91'97 It has been suggested that the increase in tissue glutathione was a overcompensatory response to oxidative stress induced by the secondary oxidation products absorbed from the oils. 91 The increase in tissue catalase activity has been attributed to an elevation of hydrogen peroxide generation by either enhanced enzyme activities of peroxisomal oxidase or c y t o c h r o m e s P 4 5 0 . 95 A reduction was also seen in the hepatic CuZn superoxide dismutase activity which may represent either an increased turnover of the enzyme or irreversible inactivation by exposure to hydrogen peroxide. 95 Interestingly, investigators have been able to characterize these changes in biochemical criteria related to oxidative stress at levels of intake of mildly oxidized fats that did not show deleterious effects according to clinical, histological or growth parameters. Oxidized lipids in foods These findings tend to support the concept that metabolic harm in terms of oxidative stress may arise by ingestion of mildly oxidized oils in spite of observations of clinical and morphological indices that are generally within the normal range. AN ATHEROGENIC ROLE OF LIPID OXIDATION COMPOUNDS IN FOODS? Some recent reviews have discussed the accumulating evidence which implicates free radical damage via oxidized lipids as a key step in the atherosclerotic process. 99'~°°The potential mechanisms by which oxidized lipids may play an important role in terms of atherosclerotic lesion development will be briefly outlined and will be followed by a presentation of the experimental evidence implicating dietary lipid oxidation products in atherosclerosis. Lipid hydroperoxides have been shown to accelerate the atherosclerotic process in terms of initiation of endothelial injury; the progression phase in which there is accumulation of plaque and the final termination phase of thrombosis. 1°1-1°3 The presence of oxidized lipids in atherosclerotic lesions has been frequently reported 1°3'1°4and a recent study has detected higher levels of lipid peroxides in the serum of patients with cardiovascular disease than in controls, i01 Linoleic acid hydroperoxides can cause irreversible damage to porcine pulmonary artery endothelial cells, ~°5 and injection of this compound into the bloodstream causes marked damage to aortic endothelial cells. 1°2 Also suggestive are studies showing that compounds with antioxidant action markedly reduce atherosclerosis in hypercholesterolemic rabbits. 1°6 Gey has suggested that on a population group basis the correlation between serum cholesterol and cardiovascular disease is improved if serum indices of free radical scavengers are included. 1°7 A key initiatory event in the atherosclerotic process is the formation of foam cells.~°3't°s New evidence has indicated that foam cells can be derived from transformed macrophages which have taken up low density lipoproteins that are loaded with cholesterol esters. 1°9 Uptake of oxidized LDL by macrophages is recognized by a scavenger receptor leading to unregulated increased uptake of cholesterol esters. H° The migration of the lipid-rich transformed macrophage on the surface of the endothelium appears to be the stimulus for the transformation of the macrophage to a reactive cell capable of producing platelet-stimulating factors and other growth promotors which can induce replication of smooth muscle cells and fibroblasts leading to plaque formation.l°S-~10 It is of interest that macrophages take up LDL very slowly and do 75 not change to foam cells unless the LDL has been modified or oxidized, l°s These findings tend to support the concept that a key factor in the atherosclerotic process is an oxidative process to transform cholesterol into a potent stimulus for macrophage uptake. Although it is likely that much of this lipid oxidative process in atherogenesis is mediated endogenously via endothelial cells, monocytes, and macrophages, 1°9 there is a suggestion that oxidative stress induced by environmental factors could also play a role. Some investigators have postulated that free radical mechanisms may account for the positive relationship between environmental factors such as smoking and an increased risk of cardiovascular disease. io3,111Oxidative stress induced by a high chronic intake of dietary lipid oxidation products could also be of significance in this regard. Evidence for a putative role of dietary lipid oxidation products in the atherosclerotic process has come from both animal and human studies. Early animal studies have shown that diets containing polyunsaturated oils heated for 20 min at 215°C were more atherogenic than diets containing unheated oils? 12 The heating effects were related to the polyunsaturated content of the diet as the heating of corn oil caused considerably more atherogenic effects in rabbits than did the heating of olive oil. Higher levels of TBARS have been observed in chylomicrons obtained from humans who had consumed thermally oxidized soybean oil than in chylomicrons from control subjects who had ingested unheated oil. 113 Further studies indicated that the peroxide-rich chylomicrons were taken up more readily by cultured mouse macrophages with over a ten-fold increase in cholesterol esters in these cells than chylomicrons from individuals who had consumed unheated oil. Chylomicrons isolated after ingestion of heated oils were predominantly taken by a macrophages via a scavenger pathway of the acetyl-LDL receptor as opposed to chylomicrons obtained after fresh oil ingestion which were primarily degraded by a ~3-VLDL receptor. This effect of ingested oxidized oils on LDL metabolism is analagous to other studies showing that the generation of TBARS in LDL by endothelial cells cause an increase in the uptake of LDL by macrophages ~°9and suggests that dietary oxidation products could potentially accelerate the accumulation of oxidized lipids in macrophages and monocytes. A possible role of dietary oxidized fats in the thrombotic stage ofatherosclerosis has come from a study of the effects of oxidized fats on prostaglandin synthesis.114 Rats were fed diets containing 10% unsaturated lipids which were heated for ten cycles of nine minutes at 180°C in the presence of potato sticks. Despite 76 s. KuBow the heating of oils, sufficient tocopherol content of the oxidized oil diets was ensured by the addition of vitamin E to the semi-purified diets to produce a ratio of 3.7 mg/g oftocopherol/linoleic acid, which was significantly higher than the nutritionally adequate ratio of 0.6 mg/g. Results showed a doubling of platelet thromboxane A2 production along with a decrease in vascular prostacyclin formation in rats fed the oxidized oil diet, thereby increasing the thromboxane to prostacyclin ratio and consequently, the risk of thrombin formation. Apart from a slight increase in kidney weight, nutritional parameters such as growth, plasma lipids and fatty acid profiles of plasma, liver, and heart lipids were unchanged. Oxidative stress was indicated as supplementation of pharmacological doses of vitamin E (300 mg/kg diet) protected against the changes in the thromboxane and prostacyclin levels. Other studies have shown that the accumulation of lipid peroxides in serum of vitamin E deficient animals was associated with inhibition of prostacyclin synthesis of aortic tissues in vivo 1is and that lipid hydroperoxides inhibit prostacyclin synthetase activity in blood vessels. 116 These findings suggest that dietary-induced oxidative stress could generate an unfavorable thromboxane to prostacyclin ratio. Since platelets participate in the early stages of atheroma these conditions could promote atherosclerosis and thrombogenesis. Although metabolic and epidemiological studies in humans and experimental animals generally tend to support the concept that a higher intake of polyunsaturated fatty acids is beneficial in terms of lipoprotein metabolism and cardiovascular health, there are some new findings that suggest some caution is needed in this approach. Recently, Blankehorn and co-workers carried out the first randomized clinical trial to provide information of effects of dietary fats on the appearance of new coronary lesions in patients who had undergone coronary bypass surgery, i i7 The likelihood of new lesions developing increased significantly with each quartile of consumption of not only saturated fat but also of polyunsaturated (linoleic acid) and monounsaturated (oleic acid) fat. In a recent report of a comparison of cases of atherosclerosis with controls, the ratio of plasma selenium to plasma PUFA levels was negatively correlated with coronary atherosclerosis.118 The authors concluded that high tissue PUFA levels, when insufficiently protected against oxidation, may indicate a higher risk ofatherosclerosis. It is also of interest that ingestion of fish oil by rabbits has been shown to enhance cholesterol-induced atherosclerosis and elevate serum lipid peroxides) t9 Since the lipid peroxide content in the fish oil was not deter- mined, it is not known whether lipid oxidation products in the fish oil played a role in the acceleration of atherosclerosis. As recently reviewed by Smith in this journal, there is also accumulating in vivo and in vitro evidence to indicate that some cholesterol oxidation products are powerful atherogenic agents. 12° Substantial amounts of cholesterol oxidation products have been detected in a variety of foods of animal origin exposed to oxidizing conditions either thermally or through aeration) 21 Several hydroxylated derivatives of cholesterol are potent inhibitors of HMG-CoA reductase in a o r t i c cells. 12°,121 A substantial reduction in cholesterol synthesis may result in cell death due to disruption of membrane function and subsequently lead to lipid infiltration and atherosclerosis. A potential atherogenic role of oxidized cholesterol of dietary origin is further indicated by animal studies which indicate that oxysterols are absorbed from the intestinal tract and are transported in the blood to arterial deposition sites at rates similar to cholesterol) 21 The absorption of oxysterols in human subjects has been studied following intake of a meal of powdered eggs high in cholesterol oxides. 122Increased levels ofoxysterols in chylomicrons and in total plasma was observed. Small amounts of oxidized cholesterol products have been shown to induce arterial injury within 24 h of administration by gavage to rabbits.123 Further, investigations of feeding of low-level of oxysterols to White Carneau pigeons at levels estimated to be an average US dietary intake showed a five-fold increase in coronary atherosclerosis, compared to birds given pure cholesterol. 124 Evidence in humans of the potential atherogenic role of oxidized sterols has come from a recent epidemiological study of Indian immigrants in the U K . 125 The author implicated the consumption of clarified butter (ghee) as having caused the higher than expected mortality from cardiovascular disease in this population, despite the absence of obvious risk factors. Clarified butter contains large amounts of cholesterol oxides (12.3%, w/w, of total sterols) which are not found in fresh butter. It is also of interest to note that the structural similarity of common plant sterols such as B-sitosterol in vegetable oils to cholesterol may also conceivably result in the formation of potentially toxic oxidized products from these plant compounds following air or thermal oxidation. The As. sterols have been demonstrated to form oxidation products in the presence of air that are similar in structure to cholesterol oxidation products) 26 This possibility of toxic atherogenic by-products produced from oxidation of plant sterols has thus far not been investigated. Oxidized lipids in foods MUTAGENICITY AND CARICINOGENICITY Most studies have failed to detect mutagens in frying oils or a carcinogenic effect of feeding trials of normally used frying oils. 12'21'127-129 Although some researchers have reported tumors from animals fed thermally abused fats, the temperatures used in most studies were abnormally high .12'21 The significance of exposure of the digestive tract to a number of lipid oxidation products that are known to act as mutagens, promotors and carcinogenic substances is not clear) 3° The ability of hydroxy and hydroperoxy derivatives of fatty acids to act as mitogens for the colonic mucosa has been suggested as a potential mechanism by which oxidized fatty acids could be involved in the promotion of intestinal tumorigenesis induced by high fat diets, s3 It has been suggested that fats are sources of intermediates for mutagen formation) 31 Oxidation products of linolenic acid and linoleic acids are weakly mutagenic in the "Ames" Salmonella typhimurium assay, which is considered to predict carcinogenic potential accurately.132't 33 Although mutagenic lipid oxidation products such as hydroperoxy cyclic epoxides react with DNA in the presence of metals and ascorbic acid, such compounds have been shown to be formed from highly oxidized esters of linoleate and linolenate at exceptionally high peroxide values ranging from 800 to 3000, whereas the peroxide value of foods are usually less than 10.132'133 Secondary volatile lipid oxidation products such as saturated and unsaturated aldehydes, dicarbonyl compounds and acrolein have also found to be mutagenic) 34 These volatile compounds may pose a more serious risk in terms of inhalation of vapors during cooking rather than ingestion in foods. The risk of lung cancer among women in Shanghai increased with the number of meals cooked by stir-frying or deep-frying, and with the frequency of smokiness and eye irritation from exposure to cooking vapors from rapeseed oil) 35 These effects were assumed to be due to the presence of acrolein which has been detected at concentrations of I. 1-10.3 ppm from the vapor during frying under cooking conditions in which 20 g of potatoes were fried in 100 ml of oil.18 The extracts of condensed volatiles from rapeseed oil heated at 270°C tested positive in tester strain TA98 activated with $9) 36 There was no evidence, however, of mutagenicity in the rapeseed oil itself, heated or unheated. Generally, investigators have not been able to detect mutagens in edible oils and in foods cooked in oils under normal frying conditions using S. typhimurium bioassays. 127-129'137 Only under severe heat- 77 ing and/or oxidizing conditions were appreciable levels of mutagenic activity detected in French-fried potatoes or fish fillets.t37 Taylor et al. have suggested that low levels of mutagenic activity in deep-fried meat compared to pan-fried meat may be related to the presence of antioxidants in the fats and/or reduced access of oxygen to the food that is deep-fried.l as Additionally, Doolittle et al. suggested that the temperature of the bread encased meat does not reach sufficient temperatures to form mutagens) 39 Moreover, the presence of too much fat during heating of foods could lead to decreased mutagen formation, probably the result of dilution of mutagen precursors.~ 39 Based on some recent work by Hageman and coworkers, however, a more specific role of fat in mutagen formation cannot be completely excluded) 4°,~41 Moderate mutagenic activity was detected in the polar fraction of deep-frying samples from restaurants in which the oils were treated according to normal frying practices in which temperatures did not exceed 182°C. 14° Mutagenic activity was highest after 20 hr but was slightly decreased after 40 hr of frying. The authors suggested that mutagenic products found during the early stages of frying may have been diluted or deactivated by other polar compounds or that prolonged heating may result in degradation of mutagenic substances. A positive correlation of TBARS and mutagencity in strain TA97 indicated the involvement of lipid oxidation products.141 The authors, however, suggested that as the mutagenicity of the polar fraction did not increase with increasing levels of dimeric and polymeric materials, mutagenic compounds other than lipid oxidation products were responsible for part of the mutagenic activity of the polar fraction. 14~ It was suggested that mutagenic heterocyclic amines and other pyrolysates may be formed in foods during deep-frying and small amounts may migrate into the frying fat. In this study the consumption by human subjects of potential mutagens formed during the deep-frying of potatoes did not cause an increase in mutagens in the urine. 14°The degree of absorption of these products from the intestine following digestive processes is therefore not clear. The differences in mutagenic activity reported by Hageman et al. may have arisen from differences in frying conditions, fractionation methods or mutagenicity test procedures. Others have found higher mutagenicity in meat samples when butter or margarine was used as a frying fat. 142It was suggested that fat was involved as a more effective heat conductor rather than as a reactant in mutagen formation. The possibility also exists that reactive lipid radicals such as alkoxyl radicals can co-oxidize food com- 78 S. KuBOW ponents other than unsaturated lipids to form mutagenic substances. For example, autoxidized polyunsaturated fatty acids can convert benzo(a)pyrene to mutagenic products that cause sister chromatid exchange in CHV79 cells. 143 The presence of benzo(a)pyrene decreases the formation of TBARS indicating the possible diversion of alkoxyl radicals from formation of endoperoxides as possible precursors of TBARS to a reaction with benzo(a)pyrene.16 Moreover, the formation of potent mutagenic N-heterocyclic compounds may occur in foods from the reaction of primary amines with carbonyl products of lipid oxidation. 15,144 The mode of cooking of foods is also a factor that requires consideration in mutagen formation from fats. A possible source of polyaromatic hydrocarbons in charcoal-broiled foods is the melted fat which drips on the hot coals; the fat is pyrolyzed at the existing temperature and the ascending fumes deposit their polyaromatic hydrocarbons on the foodJ 45 Results show that the production of polyaromatic hydrocarbons in charbroiled meat is dependent on the fat content and closeness of the meat to the source of heat.~45 Although cancer etiology has been related to a high fat diet and some of these lipid oxidation products have mutagenic activity, no realistic assessment of the health hazards of the presence of such products in human foods can presently be made. Better insights into the safety of these products in the human diet may come about through an increased understanding of complexities of food component interactions; increased awareness of metabolic processing of these compounds and improved biomonitoring procedures of risk assessment. CONCLUSIONS The investigation of toxicities of individual dietary lipid oxidation products is complicated by the incomplete knowledge of their interactions within foods. These interactions could result in many of the lipid oxidation products becoming unavailable for absorption and/or subsequent metabolism. On the other hand, the ability of lipid oxidation compounds to cooxidize or react with other food constituents could produce toxic components from non-lipid sources. Although gross pathological effects of ingestion of lipid oxidation products are unlikely in the human feeding situation, more subtle metabolic actions of these compounds on vitamin E status, drug-metabolizing enzymes, platelet activity, lipoprotein metabolism, and mutagenic activity cannot yet be discounted. In general, the presence of reactive lipid oxidation components in foods needs more systematic research in terms of the metabolic effects of these compounds, as well as the associated antioxidant requirements. 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ABBREVIATIONS G C - M S - - g a s chromatography-mass spectrometry H P L C - - h i g h pressure l i q u i d c h r o m a t o g r a p h y NMR--nuclear magnetic resonance ~-PL--N-~-(2-propenal)lysine T B A - - t h i o b a r b i t u r i c acid T B A R S - - t h i o b a r b i t u r i c acid reactive s u b s t a n c e s