addis 1986.pdf

Fd Chem. Toxic. Vol. 24. No. 10/11, pp. 1021-1030, 1986 0278-6915/86 $3.00 +0.00 Printed in Great Britain Pergamon Journals Ltd

OCCURRENCE OF LIPID OXIDATION PRODUCTS IN FOODS

P. B. ADDIS Department of Food Science and Nutrition,

University of Minnesota, St Paul, MN 55108, USA

Abstract--Lipid oxidation products are ubiquitous in foods, although much variation exists in the levels present. Although these levels are generally low, the problem of lipid oxidation severely compromises the quality of some foods and limits the shelf-life of others. Lipid oxidation represents a key barrier in the development of new food products and processes, especially convenience items and processes required to manufacture them. Deleterious changes in foods caused by lipid oxidation include loss of flavour, development of off-flavours, loss of colour, nutrient value and functionality, and the accumulation of compounds which may be detrimental to the health of consumers. All foods that contain lipids are susceptible to oxidation but especially affected are foods which are dehydrated, subjected to high temperatures or cooked and subsequently stored, e.g. dehydrated eggs, cheeses and meats, foods fried in frying oils, and cooked (uncured) meats. Specific examples of compounds which are of health concern include lipid peroxides and the free radicals involved in their formation and propagation, malonaldehyde, and several cholesterol oxidation products. Coronary artery disease (CAD) may be in part caused by the consumption of lipid oxidation products.

Introduction

The study of lipid oxidation has been clearly established as a mature field of scientific investigation. The literature is vast. The chemical reactions involved and the products formed are complex. The effects of lipid oxidation products in the diet on human health, while needing further evaluation, are known to be adverse in some cases. The effects on food technology, including production, processing and distribution, are enor- mous. Taken as a whole, it is difficult to overestimate the monetary impact of lipid oxidation.

Because of the vast scope of lipid oxidation, no at tempt has been made to cover the literature completely and, indeed, some important studies have necessarily been left out. This review will first discuss the health implications of the products of lipid oxidation. Next, the analysis and occurrence of lipid oxidation products will be reviewed. Finally, the need for future research and regulatory action on lipid oxidation will be discussed.

Several reviews and classical research articles are already available and should be consulted by any serious student of lipid oxidation. These include

Published as Paper No. 14900 of the scientific journal series of the Minnesota Agricultural Experiment Station on research conducted under Minnesota Agricultural Ex- periment Station Project No. 18-23.

Abbreviations: CAD = coronary artery disease; GC = gas chromatography; HDL = high density lipoprotein; HMG CoA reductase = 3-hydroxy-3-methylglutaryl coenzyme A reductase; HPLC = high performance liquid chromatography; IR = infra red; LDL = low-density lipoprotein; MS = mass spectrometry, NMR = nuclear magnetic resonance; TBA = thiobarbituric acid; TBARS = thiobarbituric acid reactive substances; TLC = thin-layer chromatography; VLDL = very-low- density lipoproteins.

Addis, Csallany & Kindom (1983), Finocchiaro & Richardson (1983), Kummerow (1979), Logani & Davies (1980), McBrien & Slater (1982), McCay (1985), Parish, Nanduri , Kohl & Taylor (1986), Pear- son, Gray, Wolzak & Horenstein (1983), Sevanian & Hochstein (1985), Simic & Karel (1980), Smith (1981) and Taylor, Peng, Werthessen et al. (1979).

Health implications of lipid oxidation products

Studies on the possible pathological significance of lipid oxidation products have developed in three areas of investigation: lipid peroxides (usually of fatty acids), malonaldehyde and cholesterol oxidation products. Studies are not usually conducted on dietary combinations of these three types of lipid oxidation products, but it must be realized that such mixtures occur in our diets daily.

Lip id perox ides

A series of studies by Yagi and coworkers has established relationships between serum lipid peroxide level and factors that appear to be related to atherosclerosis. Suematsu, Kamada, Abe et al. (1977) studied age-dependent changes in lipid peroxide levels in serum lipoproteins of humans and reported low levels in children and increases with age to 70, followed by declining levels. Hagihara, Nishigaki, Maseki & Yagi (1984) confirmed these findings by demonstrating higher lipid peroxide levels in the low-density lipoprotein (LDL) fraction of serum from old people than from young people. Thio- barbituric acid (TBA) was used to quantify lipid peroxides (Yagi, 1976), and samples were prepared to avoid interference by bilirubin, sialic acid and certain water-soluble substances, all of which react with TBA. In addition, levels of lipid peroxides were adjusted for variations in total lipids in serum so that the differences seen represented true differences in

1021

1022 P.B. ADDIS

Table 1. Serum lipoprotein lipid peroxide levels in normal and diabetic subjects corrected for total lipidst

Lipid peroxides/total lipids$ No. of subjects VLDL LDL HDL

Normal 32 0.71 _+0.30 0.41 _+ 0.11 0.42_+0.18 Diabetic 31 0.72 _ 0.32 0.53 -+ 0.21" 0.74 _ 0.35**

tAdapted from Nishigaki et al. (1981). :~Assayed by method of Yagi (1976). Values are means + SD and those marked with asterisks differ significantly

from the normal value; *P < 0.05; **P <0.01.

absolute quantities. Increases in lipid peroxides in the blood of diabetic patients may be of possible pathological significance (Sato, Hotta, Sakamoto et al. 1979) because plasma lipid peroxide levels in diabetics with angiopathy exceeded significantly the levels in diabetics without angiopathy. Nishigaki, Hagihara, Tsunekawa et al. (1981) noted similar differences between diabetics and controls, even after adjusting for total lipid variations and fractionating the lipoproteins (Table 1).

The primary source of serum lipid peroxides, whether in vivo oxidation or diet, is unknown, but both are probably involved. It is clear that both linoleic acid hydroperoxides and secondary products of linoleic acid oxidation are absorbed into the circulatory system and incorporated into the liver of the rat with detrimental effects, including liver hyper- trophy, increased serum transaminase activities and elevated hepatic lipid peroxide levels (Kanazawa, Kanazawa & Natake, 1985).

The research demonstrating age- and diabetic- association with lipid peroxide levels offers some circumstantial evidence that lipid peroxides in serum could play a role in coronary artery disease (CAD). Absorption studies establish that lipid oxidation products are absorbed from dietary sources into the blood and internal organs and tissues. Recent reports have provided further evidence for the primary in- volvement of lipid peroxides in the pathogenesis of atherosclerosis. Yagi, Ohkawa, Ohishi et al. (1981) showed that intravenous administration of linoleic acid hydroperoxide in the rabbit caused aorta intimal lesions which closely resembled the initial event in atherosclerosis and included the adherence of aggre- gated platelets. Nishigaki, Hagihara, Maseki et al. (1984) noted that LDL uptake by cultured smooth muscle cells was increased by 345 nmol/ml linoleic acid hydroperoxide. Further research on cultured endothelial cells from the human umbilical vein demonstrated that incubation with 1.0 nmol/ml linoleic acid hydroperoxide for 3 hr caused cellular damage, including enlargement and dilatation of the rough- surfaced endoplasmic reticulum and vacuolization (Sasaguri, Nakashima, Morimatsu & Yagi, 1984). A subsequent investigation compared differences in susceptibility to injury by linoleic acid hydroperoxide between endothelial and smooth muscle cells (Sasa- guri, Morimatsu, Kinoshita et al. 1985). The findings demonstrated that smooth muscle cells were more resistant than endothelial cells to attack by linoleic hydroperoxide. Therefore, the incorporation of LDL by smooth muscle cells and their subsequent transformation to foam cells--a putative step in atherogenesis--could be favoured by lipid peroxides.

The foregoing discussion provides interesting and perhaps highly significant data on the origins of CAD. In addition, such findings are consistent with research on the relationship of dietary cholesterol oxides to CAD (to be discussed later). Numerous other systemic toxic effects of lipid peroxides (e.g. hepatotoxicity and growth suppression) are reviewed in many of the excellent reviews cited earlier. The effects of lipid peroxides on cancer are complicated by the effects of antioxidant level and certain paradoxical effects as yet not totally understood (Corn- wall & Morisaki, 1984). It is known that free-radical generators, benzoyl peroxide and hydroperoxy fatty acids, are participants in tumorigenesis. Yet, while antioxidants generally act as inhibitors of tumorigenesis, the number of tumours has increased after antioxidant administration in some cases of chemical carcinogenesis. Cornwall & Morisaki (1984) suggest that these paradoxical effects may be explained thus: cell proliferation is inhibited by lipid peroxidation, and antioxidants would therefore favour tumour growth by reducing the levels of peroxides. Yet tumour initiation is inhibited by antioxidants. An alternative explanation is that low concentrations of antioxidants favour prostaglandin production, promoting cell proliferation, while high levels of antioxidants suppress prostaglandin biosynthesis (Corn- wall & Morisaki, 1984).

Therefore, although much research remains to be done, evidence is increasing that dietary lipid peroxides and antioxidants participate in the development of cancer in humans. Indeed, the recent study of Fujimoto, Neff & Frankel (1984), which demonstrated a strong reaction between deoxyribonucleic acid (DNA) and several primary lipid peroxides, may have provided an insight into a possible mechanism.

Malona ldehyde

There is little doubt that malonaldehyde, a secondary product of lipid oxidation, is toxic to living cells (Addis et al. 1983; Pearson et al. 1983). Malon- aldehyde can be formed in vivo or pre-formed in food products. As a dialdehyde, it can crosslink proteins (Kwon & Brown, 1965), inactivate ribonuclease (Manzel, 1967) and react with DNA, although less intensely than the primary lipid oxidation products (Fujimota et al. 1984). Bird & Draper (1980) noted several untoward effects in skin fibroblast cells incubated in culture with malonaldehyde, including cytoplasmic vacuolization, karyorrhexis, micro- and multi-nucleation, and a marked reduction in protein- synthesizing capacity. Bird, Draper & Basrur (1982) found that malonaldehyde was ten times more active

Lipid oxides: occurrence and toxicology 1023

than acetaldehyde at inducing chromosomal aberra- tions.

Studies suggesting that malonaldehyde may be carcinogenic (Shamberger, Andreone & Willis, 1974) and mutagenic (Mukai & Goldstein, 1976) have been questioned by Marnett & Tuttle (1980), who demonstrated that the mutagenic properties attributed to malonaldehyde in the earlier research may have been caused by an intermediate formed in the reaction used to prepar e malonaldehyde. Moreover, several investigators have criticized the use of TBA reagent to analyse malonaldehyde in food products (Addis et al. 1983). TBA overestimates the amount of malonaldehyde present (Csallany, Guan, Manwaring & Addis, 1984). Therefore, it is difficult at this time to make a realistic assessment of the possible health risk associated with the consumption of foods containing malonaldehyde.

Cholesterol oxidation products

A comprehensive review of cholesterol autoxidation has been published by Smith (1981). More recent reviews covering health implications of cholesterol oxidation in foods include those by Addis et al. (1983), Finocchiaro & Richardson (1983) and Pear- son et al. (1983). A discussion of cholesterol and its oxidation products and human health logically begins with CAD, and indeed numerous studies have been published that implicate autoxidation products of cholesterol in this disease. Data on cancer are extremely contradictory (Addis et al. 1983) with the exception of the mutagenicity of cholesterol ~- and fl-epoxide, discussed by Sevanian (1986). Therefore, the present discussion will be limited to CAD.

Atherogenici ty

Anitschkow (1913) is cited as one of the first to succeed in the induction of atherosclerosis as a specific consequence of feeding cholesterol dissolved in vegetable oil to rabbits (Taylor et al. 1979). Insofar as research into atherosclerosis is concerned, Anits- chkow's method has been frequently repeated as a routine means of provoking experimental atherosclerosis in rabbits. Ostensibly, this resembles human atherosclerosis. Schwenk, Stevens & Allschul (1959) recognized that cholesterol exposed to heat in air was rendered more potent, but considered that purified cholesterol by itself was capable of inducing atherosclerosis. However, investigators conducting experiments on the effects of dietary cholesterol on the circulatory system have often not considered the tendency of cholesterol spontaneously to undergo oxidation in air upon storage. This phenomenon clearly indicates that cholesterol feeding experiments should be evaluated with caution when the purity of dietary cholesterol has not been determined and carefully preserved. Unfortunately, as a rule these precautions are not taken, even nowadays when good analytical procedures are available.

Smith, Matthews, Price et al. (1967) used improved analytical methods such as TLC to find some 30 oxidation products in USP-grade cholesterol, provid- ing decisive evidence for the instability of cholesterol. Suggestions were made that the spontaneous oxidation products of cholesterol, instead of pure cholesterol, might be the actual initiating factor in the

transformation of normal cells into those of the lesion. This scepticism about the role of purified cholesterol in atherosclerosis was strongly supported by earlier studies that demonstrated that endogenously induced hypercholesterolaemia (through a hormonal mechanism) was only minimally atherogenic, whereas a comparable level of hypercholesterolaemia induced by feeding the available commercial cholesterol produced severe atherosclerosis (Chaikoff, Lindsay, Lorenz & Entenman, 1948; Seifter & Baeder, 1956). One would expect that endogenously synthesized cholesterol is protected from autoxidation, probably by in vivo antioxidants in the organism.

Some direct evidence that atherosclerosis could be induced by feeding a cholesterol autoxidation product was reported by MacDougall, Biswas & Cook (1965), who screened a large number of steroids for their cytotoxicity to rabbit aortic cell cultures. In addition, Cook & MacDougall (1968) fed rabbits 30mg cholestanetriol/kg/day for 27-310 days and noted superficial lipid deposition in the aorta, medial fibrosis and calcification.

Imai, Werthessen, Taylor & Lee (1976) were the first to demonstrate angiotoxic effects from contaminants of USP-grade cholesterol. They adminis- tered cholesterol oxide concentrates, made from USP-grade cholesterol, to rabbits by gavage. Within 24 hr of administration of 250 mg/kg body weight, a greater frequency of degenerated aortic cells was observed in the concentrate-treated groups than in those given purified cholesterol. The administration of the concentrate over a 7-wk period at a total of 1 g/kg induced aortic lesions. However, these lesions were distinct from those induced by conventional cholesterol feeding, being characterized by diffuse intimal proliferation of smooth muscle cells and fibrous stroma without foam cells. In contrast to the lesion induction by the concentrate, the rabbits given the purified cholesterol had no induced lesions.

Peng, Taylor, Tham et al. (1978) used a cell cultured technique instead of feeding. Similar results were obtained, in that the concentrate showed re- markable in vitro aortic smooth muscle cell toxicity, while the same dose of purified cholesterol had no effect. The concentrate was separated by TLC into six fractions, including cholesta-3,5-dien-7-one, C-7 cholesterol oxides, 25-hydroxycholesterol and cholestanetriol. The most toxic derivatives included 25-hydroxycholesterol and cholestanetriol, on the basis of the frequency of dying and dead cells from the aortic smooth muscle cell culture. All these observations demonstrated that a compound or compounds present in the current commercially available USP-grade cholesterol, not cholesterol per se, might be responsible for initiating atherosclerosis. Cyto- toxicity of cholesterol oxidation products was confirmed again, together with the absence of cytotoxic effects of purified cholesterol, in a follow-up study of 12 commercially available oxidation products (Peng, Tham, Taylor & Mikkelson, 1979). The degrees of cytotoxicity were graded and measured as the percentage of dying and dead ceils in cell cultures within 24hr. 25-Hydroxycholesterol and cholestanetriol were the most toxic compounds among the oxidized sterols tested, and they significantly de-

1024 P.B. ADDIS

pressed the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), a regulatory enzyme of cholesterol biosynthesis. 25-Hydroxycholesterol was the most potent inhibitor, exerting up to 83% inhibition at a 3-/zg/ml con- centration in culture medium; cholestanetriol was moderately potent. Thus, the sequence of degree of inhibition was not correlated with that of cytotoxicity. Purified cholesterol showed a very minimal effect on the activity of HMG CoA reductase (Bres- low, Lothrop, Spaulding & Kandutsch, 1975; Brown & Goldstein, 1974; Peng e t al. 1979). The discrepancy between the cytotoxicity and enzyme inhibition po- tency of cholestanetriol was discussed by Peng et al. (1979), who speculated that cholestanetriol may also affect the cell membrane. The potent cytotoxicity of cholestanetriol may be due to the stronger polar groups on one end of the molecule and the hydro- phobic group on the other end, which makes it possible for this compound to get into the membrane easily and thereby cause membrane dysfunction.

Imai, Werthessen, Subramanyam et al. (1980) repeated their earlier experiment on the cytotoxicity of the concentrate (Imai et al. 1976) by injecting it into rabbits, instead of using gavage, because the latter route might have led to uncertainties associated with biotransformation, retention, excretion and absorption. The aortae of the rabbits were examined 24 hr after intravenous injection of the same concentrate. The increase in smooth muscle cell death caused by the concentrate confirmed the angiotoxicity of the autoxidation contaminants. Major branches of the pulmonary artery responded to intravenously injected sterols by undergoing grossly visible sequential thickening due to a series of changes ranging from the initial inflammation to subsequent repair by fibromuscular thickening. The concentrate, synthesized cholestanetriol and 25-hydroxycholesterol were equally potent. Neither 7-ketocholesterol nor the epoxide affected the major branches. Neither freshly purified cholesterol nor the vehicle induced changes in the major or minor branches of the pulmonary arteries.

Baranowski, Adams, Bayliss High & Bowyer (1982) investigated the activity of oxidized sterols in promoting tissue inflammation and necrosis and in causing cell death in tissue culture. In mouse fibroblasts and pig vascular smooth muscle, the most marked inhibition of growth was observed with cholestanetriol. The epoxide was found to be more toxic than 25-hydroxycholesterol. When tested sterols were injected subcutaneously into the abdomen of rats, the histological results showed that oxysterols in general promoted a greater inflammatory reaction than did purified cholesterol. Cholestanetriol initially produced the largest lesion but, at a later stage, the 25-hydroxycholesterol implant became the largest.

All of these studies were conducted with unreal- istically high dosages not likely to occur under the normal human dietary practices. Nevertheless, it can- not be denied that these results bear the very significant evidence that it was cholesterol oxides that exerted cytotoxicity and atherosclerotic lesions and not pure cholesterol.

The implication of cholesterol oxides in atherosclerosis is further strengthened by the finding of

compounds identified as 26-hydroxycholesterol and 7-ketocholesterol (Brooks, Harland & Steel, 1966), the esters of 7~t-hydroxy-, 7fl-hydroxy-, 24-hydroxy- and 26-hydroxycholesterol (Brooks, Steel, Gilbert & Harland, 1971), the diesters of 26-hydroxycholesterol (Brooks, Gilbert & Harland, 1972) and 25-hydroxycholesterol (Smith, Teng, Lin & Seitz, 1981) in atheroma from grossly diseased human aortal tissue. Cholesterol ct-epoxide was found in sera of patients with varying degrees of hypercholesterolaemia and atherosclerosis but not in normal volunteers (Gray, Lawrie & Brooks, 1971). According to the obser- vation of McDougall e t al. (1965), 26-hydroxycholesterol was one of the four steroids found to be highly toxic toward organ cultures of rabbit aorta. The quantity of this sterol per unit weight of dry aorta increased with the severity of atherosclerosis in parallel with the increase of cholesterol and total lipids (Brooks et al. 1972). Even though these results may suggest a relationship between some of the cholesterol oxides and atherosclerosis, no definitive explanation for the presence of these products in atherosclerotic tissues or in sera from hyper- cholesterolaemics has yet been forthcoming.

Recently, more convincing information was obtained in support of the hypothesis that some oxidation derivatives of cholesterol are probably the prime cause of atherosclerotic lesions and that cholesterol and its esters have a role that is merely secondary. 25-Hydroxycholesterol was shown to be preferentially incorporated into the atherogenic lipoproteins LDL and VLDL (very-low-density lipoprotein) but to be bound only to a small degree by high-density lipoprotein (HDL), which is anti- atherogenic (Peng, Taylor, Mosbach et al. 1982). Counting of the radioactivity in each lipoprotein fraction 24hr after oral administration of 25-hydroxycholesterol and [~4C]cholesterol to ten squirrel monkeys showed that the majority of labelled 25-hydroxycholesterol was located in the LDL and VLDL (55.1 and 34.7%, respectively) with only 10.2% in the HDL. The distribution of labelled cholesterol in VLDL, LDL and HDL was almost identical to that of unlabelled cholesterol. These results suggest that most 25-hydroxycholesterol is transported to and incorporated in the peripheral tissue, including vascular tissue, by VLDL and LDL in squirrel monkeys. The preferential incorporation of 25-hydroxycholesterol into atherogenic lipoproteins led the authors to speculate that the artery could be injured by the angiotoxicity of 25-hydroxycholesterol derived from dietary sources.

Other cytotoxic effects of cholesterol oxidation derivatives have been noted which are not necessarily related to CAD but are worth mentioning. Higley & Taylor (1984) demonstrated that several cholesterol oxidation products were steatotic and cytotoxic to L cells of mice.

Lipid oxidation products in foods: analysis and occurrence

Numerous studies have established the fact that lipid oxidation products occur in foodstuffs. Un- fortunately, because of the complex nature of such oxidation products and their instability, the pro-


Table 2. Comparison of HPLC and TBA methods for malonaldehyde quantification*

Levels of malonaldehyde (#g/g wet tissue)t determined by:

No. of Sample samples HPLC TBA TBA/HPLC

Beef 9 0.14 _+ 0.085 0.44 + 0.19 3.14 Pork 9 0.11 _+ 0.06 0.39 + 0.12 3.55

*Adapted from Csallany et al. (1984). "t'Means + SD.

pensity of foods to contain large quantities of inter- fering substances, and the lack of specificity of the analytical methods used, the quantification of specific compounds has been difficult. Indeed, it is only recently that reliable methods have been developed for free malonaldehyde and the cholesterol oxides.

Many methods have been used to determine lipid peroxides but TBA is the most widely used and is also used, inappropriately, for quantifying malonaldehyde. Therefore, this discussion begins with a review of the lipid peroxide and malonaidehyde content of foods and the methodology involved, and stresses the need for caution in interpreting published values. Cholesterol oxidation products are discussed separately and, although the methods are quite different, a similar set of cautions applies to them concerning published data on foods.

TBA is the most frequently used method for quantification of lipid peroxides in foods and biological fluids and tissues and, indeed, Yagi (1980) sug- gests that it is useful to apply the same test (TBA) for both types of procedure to permit some degree of standardization. The discovery that TBA was useful as a measure of autoxidation products was made by Kohn & Liversedge (1944). Milk fat was the first food to be analysed by TBA (Dunkley, 1951; Dunkley & Jennings, 1951). Patton & Kurtz (1951) first sug- gested that malonaidehyde was the compound likely to be reacting with TBA to form the coloured complex absorbing at 532 nm. The studies of Sinnhuber & Yu (1985a, b) established the term 'TBA number', defined as ppm malonaldehyde (mg/kg or/~g/g) in a sample of food. Unfortunately, this excellent research has led many to conclude that TBA is a specific measure of malonaldehyde and TBA results are frequently expressed in terms of ppm malonaldehyde. However, the fact that it is erroneous to use TBA as a direct measure of malonaldehyde has been discussed by many authors, including Rethwill (1979), Guan (1982), Csallany et al. (1984), Sevanian & Hochstein (1985) and Fujimoto e t al. (1984). Further complicating matters is the wide variety of TBA tests available (Rethwill, 1979). These problems have stim- ulated the search for alternative methods for mon- itoring lipid oxidation (Sevanian & Hochstein, 1985) and for the direct quantification of free malonaldehyde. Such a method has now been published by Csallany e t al. (1984). High-performance liquid chromatography (HPLC) of free malonaldehyde was conducted on a TSK61000PW column. Rat liver, pork and beef muscle were analysed by the HPLC procedure and the TBA method of Tarladgis, Watts, Younathan & Dugan (1960) and Siu & Draper (1978). Comparison of the HPLC procedure with the TBA method clearly demonstrated a large over-

estimation of malonaldehyde by TBA (Table 2). Therefore, it must be concluded that whereas TBA procedures are very useful for the measurement of some of the numerous lipid oxidation products, it should not be considered as a procedure for malonaldehyde quantification, nor should the results be expressed as 'ppm malonaldehyde'. The term 'thiobarbituric acid reactive substances' or 'TBARS' avoids the inaccuracies inherent in 'ppm malonaldehyde' and should be used in its place.

With the foregoing information as important back- ground material, we can now evaluate the existing literature of lipid peroxides and malonaldehyde in foods.

Virtually every type of food has been reported to contain malonaldehyde (and lipid peroxides). Foods that are of most concern are dehydrated products, cooked (uncured) and stored meats, and animal and vegetable fats used for industrial frying (broasting, deep-fat frying). Shamberger, Shamberger & Willis (1977) surveyed the "malonaldehyde" content of food using TBA and reported levels as high as 39.0ppm (cooked chicken) and 27.0ppm (cooked beef).

On the basis of the work of Csallany et al. (1984) it is extremley doubtful that free malonaldehyde levels approached even 50% of the quoted levels, but the values do indicate the clear presence of primary and secondary lipid oxidation products, some of which are likely to be more toxic than malonaldehyde (Fujimoto e t al. 1984; Nishigaki et al. 1984). A similar survey by Siu & Draper (1978) reported far less "malonaldehyde", presumably because of differences in the methodology used by them and by Shamberger et a/.(1977). The highest value reported by Siu & Draper (1978) was in good agreement with a subsequent publication by Newberg & Concon (1980). Yagi (1980) modified the TBA procedure and analysed foods, reporting the results not as malonaldehyde but as "lipid peroxides, nmol/g". His reported values were as high as 238.3 (fried cuttlefish).

There have been numerous reports of toxicity in animals associated with consumption of oxidized frying oils (Yagi, 1980). Thompson & Aust (1983) noted several deterioration reactions during the heating of soya-bean oil for 100 hr, including the development of toxicologically significant levels of poly- mers. Rhee & Stubbs (1978) noted more rapid deterioration of oils available in health-food stores (no antioxidants) than of conventional oils (with antioxidants).

It is apparent from the foregoing sample of literature on the subject, that levels of both lipid peroxides and malonaldehyde and other secondary oxidation

FoCT. 24/10-11~

1026 P.B. ADDIS

Table 3. Some possible pitfalls in the quantification of cholesterol oxidation products in foods*

Type Example

I. Loss of cholesterol oxides 2. Production of artefacts 3. Loss of 7-ketocholesterol with

purification 4. Mis-identification 5. Insolubility of cholesterol oxide in

non-polar solvent 6. Poor resolution 7. Instability during lengthy TLC 8. Instability during gas chromatography

9. Poor detection by HPLC UV detectors

ct-epoxide, hot saponification 7-ketocholesterol to 3,5-cholestadiene-7-one

Same as (2) No use of MS, IR or N M R

Cholestanetriol insoluble in petroleum ether TLC TLC Production of dehydration products of cholesterol oxides at high column temperatures

- and fl-epoxides of cholesterol and their triol hydrolysis products

*Sources: Tsai (1984) and Park (1985).

products may reach levels high enough to concern researchers.

These results strongly suggest that oxidized frying fats could have deleterious effects on persons con- suming deep-fat-fried foods and they indicate a need for further work in this area.

In spite of the many established toxic effects of cholesterol oxides, their occurrence in the human diet has been successfully studied only recently. There have been technical difficulties with the isolation of cholesterol oxides from food, a much more complicated matrix than air-aged or model system cholesterol. To date, only a limited number of food products has been examined. In most of those studies, some technical questions are left unanswered.

The identification of cholesterol oxides has been based upon the retention index measured by chromatography, in spite of the limitations of such methodology (chromatography was not designed for qualitative analysis). Considering the complex nature of foods and the trace levels of sterols among lipid components, numerous compounds can elute at or around the retention site of the sterol of interest, easily leading to mis-identification. Therefore, cross- confirmation by IR spectroscopy or MS is a necessity. Most early studies did not provide quantitative data, and some studies were conducted without proper precautions against artefact formation during manip- ulation of the samples.

In the light of these common shortfalls, there seems no reliable or authoritative method, among the early reports, for the quantitative determination of cholesterol oxides in food. Methods vary from the simple determination of melting point to state-of-the-art chromatography. Therefore, the studies published should be interpreted with great care. The quantification of trace quantities of sterol oxides from foods that contain much higher levels of cholesterol, triglyceride, phospholipids and other inter- fering chemicals is difficult, and pitfalls abound. A thorough review of methodology has been presented by Park (1985).

In spite of the complex nature of the food matrix, a number of food products have been reported to contain some cholesterol oxides. Two recent reviews listed those foodstuffs (Finocchiaro & Richardson, 1983; Smith, 1981). However, most of the cholesterol oxides found in those foodstuffs appear to be artefacts of human intervention due to intentional oxidative stress, such as irradiation or extended heating.

The lack of proper precautions during the saponification of the samples frequently resulted in the formation of cholesta-3,5-dien-7-one (Tsai, 1984). Moreover, early studies were conducted with less sophisticated analytical tools. Accordingly, early reports should be regarded as the possibility that the oxidation products of cholesterol may occur in food under certain conditions rather than as definitive evidence for their presence in the foodstuffs. An exception to this caution is the recent finding of epimeric epoxides. A brief sampling of meth- odological pitfalls uncovered by Park (1985) and Tsai (1984) in their reviews of the literature is summarized in Table 3.

It therefore appears reasonable to restrict this review to the more recent publications. Summaries of early studies have been published (Park, 1985; Tsai, 1984).

Definitive studies on isolation, identification and quantification of cholesterol ct- and fl-epoxides in powdered eggs were conducted by Tsai and coworkers (Tsai & Hudson, 1984 & 1985; Tsai, Ijichi, Hudson & Meehan, 1980). Briefly, the method involved chloroform-methanol extraction, silicic acid chromatography and GC and HPLC. Qualitative confirmation was accomplished by MS, NMR and IR spectral determination. Tsai & Hudson (1985) published quantitative results obtained on commercial dry egg products. A synopsis of these results appears in Table 4. They demonstrated that a great variation exists among samples, that some very high individual samples were found, that the average of the samples was high and that steam-injected spray dryers cause less cholesterol oxidation than gas-fired. These results do not include data on the six or more other common cholesterol oxides nor on the lipid peroxides and other secondary lipid oxidation products that were probably present. Therefore, the potential for toxicity of powdered eggs may be very significant. It must be

Table 4. Cholesterol ~t- and fl-epoxides in powdered scrambled egg mix*

Mix content (ppm) of cholesterol epoxides:

Egg mix sample ~ + fl ct

A 30 6 B 33 7 C 7 1

*Adapted from Tsai & Hudson (1985).


recognized that cholesterol oxidizes slowly compared to polyunsaturated fatty acids.

Park & Addis (1985a) developed a relatively mild, rapid and accurate HPLC method for cholesterol oxides but were limited by technical difficulties to the C-7 derivatives. Briefly, the method uses a chloroform-methanol extraction, silica-gel chromatography purification and HPLC on a 10pro #-Porasil column. UV detection was used. All positive peaks from food samples were collected, evapo- rated under nitrogen, dissolved in ethyl acetate and subjected to MS. The results showed high levels of the three cholesterol oxidation products (13.8-70.1 ppm) in brain and liver preparations sold at health-food stores, significant levels in french fries obtained at fast-food restaurants (4.1-58.8 ppm) and 1.1 ppm in a pancake mix which contained powdered egg. Raw beef (muscle, liver and brain), fried chicken, cooked hamburger, beef jerky and liver sausage were not found to contain C-7 derivatives.

Because of the limitations of the report by Tsai & Hudson (1985) to the cholesterol epoxides and of their own study to C-7 derivatives, Park & Addis (1985b) developed a method that permits quantification of all cholesterol oxides believed to be of toxicological significance by a single GC injection. Fused-silica capillary columns were evaluated but thermal instability of the diol derivatives of cholesterol necessitated derivatization of the cholesterol oxides to the trimethylsilyl ethers. The derivatized cholesterol oxides were completely resolved on a DB-l column using temperature programming. Flame ionizaton was used as the method of detection (Park & Addis, 1985b). Food was prepared for analysis by adding an internal standard (40/~g 5~t-cholestane), extracting with chloroform-methanol (2:1, v/v), evaporating the solvent and cold- saponifying the dried lipids for 20 hr (23°C). After addition of 10ml distilled water, the non- saponifiables were extracted three times with 10-ml portions of diethyl ether and washed once with 5 ml 0.5 N-KOH and three times with 5 ml distilled water. After drying, the washed non-saponifiables were dissolved in 100pl pyridine, derivatized and injected into the gas chromatograph.

Ryan, Gray & Morton (1981) demonstrated by TLC the development of cholesterol oxides in tallow heated to frying temperatures. Park & Addis (1985a) noted the existence of C-7 oxidized cholesterol in French fries cooked in tallow. Therefore, studies were done by Park & Addis (1986a, b) to apply GC (Park & Addis, 1985b) to quantification of cholesterol oxidation products in tallow heated to temperatures used in fast-food restaurants. All positive chromatographic findings were confirmed by GC-MS. Heating tallow at either 155 or 190°C resulted in the loss of half of the initial content of cholesterol at 250 hr, with samples heated at 190 ° being affected slightly more severely than those at 155 °. After 250 hr at 155 °, 7% of the cholesterol had been converted to 7-ketocholesterol with lesser quantities of 7ct- and 7fl-hydroxy (1.5 and 2.5%, respectively) and ~-epoxide (4%) derivatives of cholesterol (Park & Addis, 1986a), with only 50% of the original cholesterol remaining. A subsequent study confirmed and extended these findings by demonstrating formation of

i.2 and 1.1% of the cholesterol into 7-keto and -epoxide derivatives, respectively, after 70 hr at 135 °

and the delaying of cholesterol degradation by ~t-tocopherol (100ppm) plus ascorbyl palmitate (500 ppm).

Park (1985) also demonstrated that precooked roast beef was resistant to cholesterol oxidation in spite of the well-established propensity of precooked, uncured meats to display muscle membrane phos- pholipid oxidation (warmed-over flavour).

Dry eggnog mix, subjected to fluorescent light exposure, developed increasing levels of 7~- and 7fl-hydroxycholesterol up to 80 days of storage (Herian & Lee, 1985). TLC was used to analyse butter oil and grated cheese for cholesterol oxides (Finocchiaro, Lee & Richardson, 1984). Positive samples were analysed by HPLC and MS. Both bleached butter oils and grated aged cheese displayed levels of cholesterol oxides that were probably significant. However, data are not complete on this question.

Kou & Holmes (1985) developed an HPLC procedure specifically for 25-hydroxycholesterol, probably the most atherogenic cholesterol oxide known. None was detected in lard, cream, fresh egg yolk or spray-dried egg yolk powder. However, other researchers have detected 25-hydroxycholesterol in powdered scrambled egg mix (Missler, Wasilchuk & Merritt, 1985). Pike & Peng (1985) studied the stability of shell egg and liquid yolk lipids during storage for 18 months at 4°C. They noted little in the way of oxidative deterioration. Therefore, it is possible that only in spray-dried products do lipid oxidation products accumulate to a significant degree. Because the egg yolk is an extremely rich source of cholesterol, thorough investigations should be made on the potential for egg yolk oxidation.

It is obvious that the potential for cholesterol oxides to form in foods is very real, although numerous foods are somewhat resistant to this most severe type of autoxidation. Research on the occurrence of cholesterol oxides in foods is in its infancy. Only during the past 4y r have accurate methods been developed. There has not been enough time for a thorough analysis of all cholesterol-containing foods to be conducted. A high priority should be placed on such investigations along with suitable toxicological studies.

Lipid autoxidation, antioxidants and human health

It is apparent from the foregoing discussion that the human food supply includes generous quantities of lipid autoxidation products as well as various antioxidants provided to inhibit them. Attempting to formulate a risk/benefit ratio for antioxidants is a difficult task and will not be attempted here. The purpose of this paper is to assess the risk of not using antioxidants through a preliminary evaluation of the health risk of oxidation products in our food supply. The main focus has been CAD, although numerous other human afflictions can be related to consumption of lipid oxidation products.

With respect to CAD, one must be impressed with the copious data collected on the levels of lipid oxides in our foods, as well as the acute angiotoxicity caused by lipid oxides in animal and cell culture studies.

1028 P. B. ADDIS

Even more impressive is the close agreement of many of these investigations (primarily from the labora- tories of K. Yagi and C. B. Taylor) with the current thinking of pathologists in the field of atherosclerosis research.

An informative recent review of currently accepted models for atherosclerosis has been published by Moore (1985), who outlined the two major con- tending models of atherosclerosis by suggesting that elements of both theories may be true. He states:

"One theory postulates that atherosclerosis is caused by the deposition of lipid in the vessel wall during periods of abnormal elevation of blood lipid levels or in association with disordered metabolism of lipoproteins. The other theory maintains that the disease is essen- tially a response of the vessel wall to injury and although it plays an important part in lesion progression, the deposition of lipid is a secondary phenomenon. Both theories are supported by experimental findings in animals that indicate that lesions, which are morphologically similar to atherosclerotic lesions in humans, can be induced by either a supple- mentation of dietary lipid or by injury. Regardless of experimental setting, the design of the experiments has raised questions regarding the relevance of the results to human atherogenesis."

Injury to arterial endothelial cells, aggregation of platelets and release of platelet-derived growth factor (PDGF) and subsequent proliferation of smooth muscle cells and their conversion to lipid-filled foam cells eventually result in the intimal thickening seen in atherosclerosis. Research from Yagi 's laboratory on lipid peroxides and from Taylor 's laboratory on cholesterol oxides may account for many of the key steps in atherogenesis.

Recent studies reviewed by Majno, Joris & Zand (1985) strongly suggest that a link has been discov- ered between atherosclerosis and inflammation. If true, the studies of Baranowski et al. (1982), showing that oxysterols produce greater inflammation than pure cholesterol, become relevant.

Platelet aggregation is a key factor in CAD. Recent studies have demonstrated that c o - 3 fatty acids from fish oils are very useful in reducing the tendency of platelets to aggregate (Sanders, 1983) as well as in lowering serum triglycerides, cholesterol and even possibly L D L (Illingworth, Harris & Connor, 1984). Fish oils in tablet form are being used by the medical profession to accomplish beneficial changes in serum lipids in patients at risk from CAD. However, by their very nature, 0 9 - 3 fatty acids are very susceptible to autoxidation. Fish oils are also rich in cholesterol. The quality of fish oil preparations varies widely. Recent (unpublished) studies in our laboratory suggest that some of the oils available are of poor quality and that these have significant levels of cholesterol oxidation products. Such fish oils would also be contaminated with fatty acid hydroperoxides and malonaldehyde and could conceivably incur significant losses of the e9 - 3 fatty acids so beneficial to hyperlipidaemic patients.

It is apparent that much research remains to be done to protect the public from levels of lipid oxidation products deleterious to health. Perhaps with more research we will arrive at a level of concern that invokes regulatory action against products contain-

ing significant levels of lipid oxidation products. Given the powerful array of antioxidants and processing techniques available, the appearance of high levels of oxidation products in foods should be a major concern to the food industry and governmental regulatory bodies.

REFERENCES

Addis P. B., Csallany A. S. & Kindom S. E. 0983). Some lipid oxidation products as xenobiotics. In Xenobiotics in Foods and Feeds. A C S Symposium Series No. 234. Edited by J. W. Finley & D. E. Schwass. p. 85. American Chemical Society, Washington DC.

Anitschkow N. (1913). Ober die Ver~inderungen der Kan- inchenaorta bei experimenteller Cholesterinsteatose. Beitr. Path. Anat. Allg. Path. 56, 379.

Baronowski A., Adams C. W. M., Bayliss High O. B. & Bowyer D. B. (1982). Connective tissue responses to oxysterols. Atherosclerosis 41, 255.

Bird R. P. & Draper H. H. (1980). Effect of malonaldehyde and acetaldehyde on cultured mammalian cells: growth, morphology and synthesis of macromolecules. J. Toxic. envir. Hlth 6, 811.

Bird R. P., Draper H. H. & Basrur P. K. (1982). Effect of malonaldehyde and acetaldehyde on cultured mammalian cells. Mutation Res. 101, 273.

Breslow J. L., Lothrop B. A., Spaulding D. R. & Kandutsch A. m. (1975). Cholesterol, 7-ketocholesterol and 25-hydroxycholesterol uptake studies and effect on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in human fibroblasts. Biochim. biophys. Acta 398, 10.

Brooks C. J. W., Gilbert J. D. & Harland W. A. (1972). Oxygenated sterolesters of human atherosclerotic plaque. Protides Biol. Fluids Proc. Colloq. 19, 101.

Brooks C. J. W., Harland W. A. & Steel G. (1966). Squalene, 26-hydroxycholesterol and 7-ketocholesterol in human atheromatous plaques. Biochim. biophys. Acta 125, 620.

Brooks C. J. W., Steel G., Gilbert J. D. & Harland W. A. (1971). Lipids of human atheroma. Part 4. Character- ization of a new group of polar sterol esters from human atherosclerotic plaques. Atherosclerosis 13, 223.

Brown S. & Goldstein J. L. (1974). Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol. J. biol. Chem. 249, 7306.

Chaikoff I. L., Lindsay S., Lorenz F. W. & Entenman C. (1948). Production of atheromatosis in the aorta of the bird by the administration of diethylstilbestrol. J. exp. Med. 88, 373.

Cook R. P. & MacDougall J. D. B. (1968). Experimental atherosclerosis in rabbits after feeding cholestanetriol. Br. J. exp. Path. 49, 265.

Cornwall D. G. & Morisaki N. (1984). Fatty acid paradoxes in control of cell proliferation. In Free Radicals in Biol- ogy. Vol. 6. Edited by W. A. Pryor. p. 130. Academic Press, New York.

Csallany A. S., Guan M. D, Manwaring J. D. & Addis P. B. (1984). Free malonaldehyde determination in tissues by high-performance liquid chromatography. Analyt. Bio- chem. 142, 277.

Dunkley W. L. (1951). Evaluation of the thiobarbituric acid test as a measure of oxidized flavor in milk. Fd Technol. 5, 342.

Dunkley W. L. & Jennings W. G. (1951). A procedure for application of the TBA test to milk. J. Dairy Sci. 34, 1064.

Finocchiaro E. T., Lee K. & Richardson T. (1984). Identification and quantification of cholesterol oxides in grated cheese and bleached butter oil. J. Am. Oil Chem. Soc. 61, 877.

Finocchiaro E. T. & Richardson T. (1983). Sterol oxides in foodstuffs: a review. J. Fd Prot. 46, 917.

Fujimoto K., Neff W. E. & Frankel E. N. (1984). The


reaction of DNA with lipid oxidation products, metals and reducing agents. Biochim. biophys. Acta 795, 100.

Gray M. F., Lawrie T. D. V. & Brooks C. J. W. (1971). Isolation and identification of cholesterol alpha-oxide and other minor sterols in human serum. Lipids 6, 836.

Guan M. D. (1982). The Application of High Performance Lipid Chromatography to the Determination of Free Malondialdehyde in Meat and Other Tissues. MS Thesis, University of Minnesota, Minneapolis.

Hagihara M., Nishigaki I., Maseki M. & Yagi K. (1984). Age-dependent changes in lipid peroxide levels in the lipoprotein fractions of human serum. J. Geront. 39, 269.

Herian A. M. & Lee K. (1985). 7~- and 7fl-Hydroxy- cholesterols formed in dry eggnog mix exposed to fluorescent light. J. Fd Sci. 50, 276.

Higley N. A. & Taylor S. L. (1984). The steatotic and cytotic effects of cholesterol oxides in cultured L cells. Fd Chem. Toxic. 22, 983.

Illingworth D. R., Harris W. S. & Connor W. E. (1984). Inhibition of low density lipoprotein synthesis by dietary co - 3 fatty acids in humans. Atherosclerosb 4, 270.

Imai H., Werthessen N. T., Subramanyam V., LeQuesne P. W., Soloway A. H. & Kanisawa M. (1980). Angio- toxicity of oxygenated sterols and possible precursors. Science, N.Y. 207, 651.

Imai H., Werthessen N. T., Taylor C. B. & Lee K. T. (1976). Angiotoxicity and arteriosclerosis due to contaminants of USP-grade cholesterol. Archs Path. lab. Med. 100, 565.

Kanazawa K., Kanazawa E. & Natake M. (1985). Uptake of secondary autoxidation products of linoleic acid by the rat. Lipids 20, 412.

Kohn H. I. & Liversedge M. (1944). On a new aerobic metabolite whose production by brain is inhibited by apomorphine, emertine, ergotamine, epinephrine, and menadione. J. Pharmac. exp. Ther. 82, 292.

Kou I. L. & Holmes R. P. (1985). The analysis of 25-hydroxycholesterol in plasma and cholesterol- containing foods by high performance liquid chromatography. J. Chromat. 330, 339.

Kummerow F. A. (1979). Nutrition imbalance and angio- toxins as dietary risk factors in coronary heart disease. Am. J. clin. Nutr. 32, 58.

Kwon T. W. & Brown W. D. (1965). Complex formation between malonaldehyde and bovine serum albumin. Fedn Proc. Fedn Am. Socs exp. Biol. 24, 592.

Logani M. K. & Davies R. E. (1980). Lipid oxidation: biological effects and antioxidants--a review. Lipids 15, 485.

McBrien D. C. H. & Slater T. F. (1982). Free Radicals, Lipid Peroxidation and Cancer. Academic Press, New York.

McCay P. B. (1985). Vitamin E: interactions with free radicals and ascorbate. A. Rev. Nutr. 5, 323.

MacDougall J. D. B., Biswas S. & Cook R. P. (1965). The effect of certain C27 steroids on organ cultures of rabbit aorta. Br. J. exp. Path. 46, 549.

Majno G., Joris I. & Zand T. (1985). Atherosclerosis: new horizons. Hum. Path. 16, 3.

Manzel D. B. (1967). Reaction of oxidizing lipid with ribonuclease. Lipids 2, 83.

Marnett L. J. & Tuttle M. A. (1980). Comparison of the mutagenicities of malondialdehyde and the side products formed during its chemical synthesis. Cancer Res. 40, 276.

Missler S. R., Wasilchuk B. A. & Merritt C., Jr (1985). Separation and identification of cholesterol oxidation products in dried egg preparations. J. Fd Sci. 50, 595.

Moore S. (1985). Pathogenesis of atherosclerosis. Metabo- lism 34, 13.

Mukai F. H. & Goldstein B. D. (1976). Mutagenicity of malonaldehyde, a decomposition product of peroxidized polyunsaturated fatty acids. Science, N.Y. 191, 868.

Newburg D. S. & Concon J. M. (1980). Malonaldehyde concentrations in food are affected by cooking conditions. J. Fd Sci. 45, 1681.

Nishigaki 1., Hagihara M., Maseki M., Tomoda Y., Nag- ayama K., Nakashima T. & Yagi K. (1984). Effect of linoleic acid hydroperoxide on uptake of low density lipoprotein by cultured smooth muscle cells from rabbit aorta. Biochem. Int. 8, 501.

Nishigaki I., Hagihara M., Tsunekawa H., Maseki M. & Yagi K. (1981 )..Lipid peroxide levels of serum lipoprotein fractions of diabetic patients. Biochem. Med. 25, 373.

Parish E. J., Nanduri V. B. B., Kohl H. H. & Taylor F. R. (1986). Oxysterols: chemical synthesis, biosynthesis and biological activities. Lipids 21, 27.

Park S. W. (1985). Quantification of Oxidized Cholesterol Derivatives in Foods by Liquid and Gas Chro- matography. PhD. Thesis, University of Minnesota, Min- neapolis.

Park S. W. & Addis P. B. (1985a). HPLC determination of C-7 oxidized cholesterol derivatives in foods. J. Fd Sci. 50, 1437.

Park S. W. & Addis P. B. (1985b). Capillary column gas-liquid chromatographic resolution of oxidized cholesterol derivatives. Analyt. Biochem. 149, 275.

Park S. W. & Addis P. B. (1986a). Identification and quantitative estimation of oxidized cholesterol derivatives in heated tallow. J. agric. Fd Chem. 34, 653.

Park S. W. & Addis P. B. (1986b). Further investigation of oxidized cholesterol derivatives in heated fats. J. Fd Sci. 51, 1380.

Patton S. & Kurtz G. W. (1951). 2-Thiobarbituric acid as a reagent for detecting milk fat oxidation. J. Dairy Sci. 34, 669.

Pearson A. M., Gray J. I., Wolzak A. M. & Horenstein N. A. (1983). Safety implications of oxidized lipids in muscle lbods. Fd Technol. 37, 121.

Peng S. K., Taylor C. B., Mosbach E. H., Hwang W. Y., Hill J. & Mikkelson B. (1982). Distribution of 25-hydroxycholesterol in phase lipoproteins and its role in atherogenesis. Atherosclerosis 41, 395.

Peng S. K., Taylor C. B., Tham P., Werthessen N. T. & Mikkelson B. (1978). Effects of auto-oxidation products from cholesterol on aortic smooth muscle cells. Archs Path. lab. Med. 102, 57.

Peng S. K., Tham P., Taylor C. B. & Mikkelson B. (1979). Cytotoxicity of oxidation derivatives of cholesterol on cultured aortic smooth muscle cells and their effect on cholesterol biosynthesis. Am. J. clin. Nutr. 32, 1033.

Pike O. A. & Peng I. C. (1985). Stability of shell egg and liquid yolk to lipid oxidation. Poult. Sci. 64, 1470.

Rethwill C. E. (1979). Factors which Influence Lipid Ox- idation in Meat and Meat Products. MS Thesis, Univer- sity of Minnesota, Minneapolis.

Rhee K. S. & Stubb A. (1978). Oxidative deterioration in vegetable oils: health-food oils versus conventional oils. J. Fd Prot. 41, 443.

Ryan T. C., Gray J. I. & Morton I. D. (1981). Oxidation of cholesterol in heated tallow. J. Sci. Fd Agric. 32, 305.

Sanders T. A. B. (1983). Dietary fat and platelet function. Clin. Sci. 65, 343.

Sasaguri Y., Morimatsu M., Kinoshita T., Nakashima T., Inagaki T. & Yagi K. (1985). Difference in susceptibility to injury by linoleic acid hydroperoxides between endothelial and smooth muscle cells of arteries. J. appl. Biochem. 7, 70.

Sasaguri Y., Nakashima T., Morimatsu M. & Yagi K. (1984). Injury to cultured endothelial cells from human umbilical cord vein by linoleic acid hydroperoxides. J. appl. Biochem. 6, 144.

Sato Y., Hotta N., Sakamoto N., Matsuoka S., Ohishi N. & Yagi K. (1979). Lipid peroxide levels in plasma of diabetic patients. Biochem. Med. 21, 104.

Schwenk E., Stevens D. F. & AIlschul R. (1959). Experi- ments on cholesterol atherosclerosis in rabbits. Proc. Soc. exp. Biol. Med. 102, 41.

Seifter J. & Baeder D. H. (1956). Occurrence in plasma of

1030 P.B. ADDlS

an extractable lipid mobilizer. Proc. Soc. exp. Biol. Med. 91, 42.

Sevanian A. & Hochstein P. (1985), Mechanisms and con- sequences of lipid peroxidation in biological systems. A. Rev. Nutr. 5, 365.

Sevanian A. & Peterson A. R. (1986). The cytotoxic and mutagenic properties of cholesterol oxidation products. Fd Chem. Toxic. 24, 1103.

Shamberger R. J., Andreone T. L. & Willis C. E. (1974). Antioxidants and cancer. IV. Malonaldehyde has initiating activity as a carcinogen. J. natn. Cancer Inst. 53, 1771.

Shamberger R. J., Shamberger B. A. & Willis C. E. (1977). Malonaldehyde content of food. J. Nutr. 107, 1404.

Simic M. G. & Karel M. (1980). Autoxidation in Food and Biological Systems. Plenum Press, New York.

Sinnhuber R. O. & Yu T. C. (1958a). 2-Thiobarbituric acid measurement of rancidity in fishery products. Fd Technol. 12, 9.

Sinnhuber R. O. & Yu T. C. (1958h). Characterization of the red pigment formed in the 2-thiobarbituric acid determination of oxidative rancidity. Fd Res. 23, 626.

Siu G. M. & Draper H. H. (1978). A survey of the malonaldehyde content of retail meats and fish. J. Fd Sci. 43, 1147.

Smith L. L. (1981). Cholesterol Autoxidation. Plenum Press, New York.

Smith L. L., Matthews W. S., Price V. C., Bachmann R. C. & Reynolds B. (1967). Thin-layer chromatographic exam- ination of cholesterol autoxidation. J. Chromat. 27, 187.

Smith L. L., Teng J. I., Lin Y. Y., Seitz P. K. & McGehee M. F. (1981). Sterol metabolism XLVII. Oxidized cholesterol esters in human tissues. J. SteroidBiochem. 14, 889.

Suematsu T., Kamada T., Abe H., Kikuchi S. & Yagi K. (1977). Serum lipo-peroxide level in patients suffering from liver diseases. Clinica chim. acta 79, 267.

Tarladgis B. G., Watts B. M., Younathan M. T. & Dugan L. (1960). A distillation method for the determination of malonaldehyde in rancid food. J. Am. Oil Chem. Soc. 37, 44.

Taylor C. B., Peng S. K., Werthessen N. T., Tham P. & Lee K. T. (1979). Spontaneously occurring angiotoxic derivatives of cholesterol. Am. J. clin. Nutr. 32, 40.

Thompson L. V. & Aust R. (1983). Lipid changes in french fries and heated oils during commercial deep frying and their nutritional and toxicological implication. Can. Inst. Fd Sci. Technol. 16, 246.

Tsai K. S. (1984). Quantification of Cholesterol Oxidation Products by Capillary Gas Chromatography. MS Thesis, University of Minnesota, Minneapolis.

Tsai L. S. & Hudson C. A. (1984). Cholesterol oxides in commercial dry egg products: Isolation and identification. J. Fd Sci. 49, 1245.

Tsai L. S. & Hudson C. A. (1985). Cholesterol oxides in commercial dry egg products: Quantitation. J. Fd Sci. 50, 229.

Tsai L. S., Ijichi K., Hudson C. A. & Meehan J. J. (1980). A method for the quantitative estimation of cholesterol alpha-oxide in eggs. Lipids 15, 124.

Yagi K. (1976). A simple fluorometric assay for lipid peroxide in blood plasma. Biochem. Med. 15, 212.

Yagi K. (1980). Toxicity of lipid peroxides in processed foods. Biochem. Rev. 50, 42.

Yagi K., Ohkawa H., Ohishi N., Yamashita M. & Nakashima T. (1981). Lesion of aortic intima caused by intravenous administration of linoleic acid hydroperoxide. J. appl. Biochem. 3, 58.

QUESTIONS AND ANSWERS

V. Singh, H o f f m a n - L a Roche: Dr Addis, do you have any data or are there any da ta available to indicate tha t these oxidat ion products of cholesterol are formed in vivo in humans , or are these always available f rom food sources? Secondly, is there any role for an t ioxidants like vi tamin C or E to protect against this?

P. Addis: To answer your first question, there are papers citing the occurrence of these oxidat ion products in biological fluids and serum, and also in m a m m a r y gland aspirates, which were done by Petrakus at the Universi ty of California, San Francisco, However, in many of these cases, one is not sure whether they were dietary or in vivo products. Certainly in the case of in vivo product ion, an t ioxidants could play a role in suppressing oxidat ion products . The problem with in vivo metabol i sm is tha t the levels are so much lower than what we see in foods, tha t it 's difficult to work at such low levels.

addis 1986.pdf

Documents