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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:533-538.)
© 1996 American Heart Association, Inc.

Articles

Oxidized Lipids in the Diet Accelerate the Development of Fatty Streaks in Cholesterol-Fed Rabbits

Ilona Staprãns; Joseph H. Rapp; Xian-Mang Pan; David A. Hardman; Kenneth R. Feingold

From the Department of Veterans Affairs Medical Center (I.S., J.H.R., K.R.F.) and the Departments of Surgery (I.S., J.H.R., X.-M.P.), Medicine (K.R.F.), and Cardiovascular Research Institute (J.H.R., D.A.H.), University of California, San Francisco.

Correspondence to Ilona Staprãns, PhD, Lipid Research Laboratory (151L), Veterans Administration Medical Center, San Francisco, CA 94121.

	Abstract

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Abstract Studies have indicated that oxidized lipoproteins may play a role in atherosclerosis. We have recently demonstrated that the levels of oxidized lipoproteins in the circulation can be directly correlated to the quantity of oxidized lipids in the diet. The present study tested the hypothesis that dietary oxidized lipids accelerate the development of atherosclerosis. For 12 to 14 weeks, 36 male New Zealand White rabbits were fed a low-cholesterol (0.25%) diet containing either 5% unoxidized corn oil (control diet) or 5% oxidized corn oil (oxidized-lipid diet). Serum cholesterol levels increased to a similar extent in both groups, with the majority of the cholesterol in the ß-migrating very low density lipoprotein (ß-VLDL) fraction. ß-VLDL from control animals contained 3.86±0.57 versus 9.07±2.14 nmol conjugated dienes per µmol cholesterol (P<.05) in rabbits fed the oxidized-lipid diet. No difference in oxidized lipid levels was detected in LDL. Most important, feeding a diet rich in oxidized lipid resulted in a 100% increase in fatty streak lesions in the aorta. Additionally, rabbits that were fed the oxidized-lipid diet had a >100% increase in total cholesterol in the pulmonary artery that was primarily due to an increase in cholesteryl ester. Oxidized lipids are frequently present in the typical US diet, and our results suggest that consumption of these foods may be an important risk factor for atherosclerosis.

Key Words: ß-VLDL • lipid peroxides • corn oil • polyunsaturated fat • oxidized diet

	Introduction

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The etiology of atherosclerosis is complex and multifactorial, but recent studies have indicated that oxidized lipoproteins may play a key role. Oxidized LDL displays a number of atherogenic properties in a variety of cell types in vitro, including stimulation of monocyte chemotactic factor production, potentiation of monocyte–endothelial cell adhesion, the ability to stimulate and inhibit cytokine expression, accelerated deposition of lipid in macrophages, and cytotoxicity in endothelial cells.^{1 2} Moreover, several lines of evidence have demonstrated the in vivo occurrence of oxidized LDL, as atherosclerotic lesions have been shown to contain oxidized LDL–like particles and lipid peroxidation products.^{3 4 5 6} Additionally, it has been shown that antioxidants in the diet slow the progression of atherosclerosis in animal models.^{7 8 9 10} Together these studies suggest that oxidation may play an important role in the pathogenesis of atherosclerosis. However, the site where and the mechanism whereby lipoproteins are oxidized have not been resolved, and it is not clear whether oxidized LDL forms locally in the artery wall, as suggested by several investigators^{1 2} and/or is sequestered in the atherosclerotic lesion after uptake of circulating oxidized LDL. In addition to the atherogenicity of LDL in animal models,^{7 8} the potential role of ß-VLDL in the atherogenic process has been described by Rosenfeld et al.^{11 12} They have shown that fatty streak formation is similar in both Watanabe Heritable Hyperlipidemic rabbits, in which cholesterol is primarily in LDL, and in cholesterol-fed rabbits, in which cholesterol is in ß-VLDL.

Recent studies in our laboratory have suggested that oxidized lipids in the diet may play a significant role in lipoprotein oxidation in vivo. In rats the levels of oxidized lipids in chylomicrons that have been isolated from mesenteric lymph and in the serum VLDL+LDL fraction can be directly correlated with the quantity of oxidized lipids in the diet.^{13 14} Similarly in humans, consumption of diets that are high in oxidized lipids also results in increases in oxidized lipids in the postprandial serum chylomicron fraction.^{15 16} These results suggest that oxidized lipids in the diet may be major contributors to the levels of oxidized lipids in intestinally derived lipoproteins and endogenous lipoprotein particles such as VLDL and LDL.

Given the evident importance of dietary regulation of oxidized lipoprotein concentrations in the circulation and the role of oxidized lipoproteins in atherosclerosis, we have postulated that dietary oxidized lipids may be atherogenic. The present study tests the hypothesis that oxidized lipids in the diet accelerate the development of atherosclerosis.

	Methods

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Animal Model
Thirty-six male New Zealand White rabbits (10 to 11 weeks old; 2 kg initial weight) were divided into two equal groups. During the 12- to 14-week experimental period, 6 rabbits died because they refused their food and 2 did not develop hypercholesterolemia during the cholesterol-feeding period. Rabbits were individually housed in stainless steel cages at the San Francisco Veterans Affairs Animal Housing facility, which is accredited by the American Association for Accreditation of Animal Laboratory Care. All procedures were reviewed and approved by the Institutional Animal Care Subcommittee.

Diets and Experimental Protocol
One group of rabbits (control group) was fed a low-fat, low–vitamin E synthetic diet (Bioserv) containing 0.25% cholesterol by weight in dry food to which 5% natural corn oil was added. This diet contained negligible amounts of lipid peroxide. The other group (oxidized-diet group) was fed the same diet except that the corn oil was depleted of vitamin E (ICN Biochemicals) and was oxidized by heating for 2 hours at 100°C. To equalize the vitamin E content in both diets, it was added to the oxidized oil before feeding to yield the same concentration as in natural corn oil (0.14 mg/mL oil). Oxidized oil contained 0.180 µmol conjugated dienes per milligram of oil and 0.350 µmol peroxide per milligram of oil. Thus, the oxidized diet contained 17.5 µmol lipid peroxide and 7 µg vitamin E per gram of food. Because the peroxide value depends on the reference standard and the conditions of the assay, fatty acid degradation due to oxidation of the oxidized oil was examined by gas-liquid chromatography. We found a 3% fatty acid loss after the oil had been heated. On the basis of this value, a rabbit that consumed 80 g food (5% total fatty acids, 3% of which were oxidized) had an intake of 400 µmol oxidized fatty acids per day. Thus, in our experiments the oil was oxidized to contain levels of oxidized fatty acids similar to those in foods from restaurants, institutions, and home cooking.^{17 18 19 20 21} Additionally, care was taken to maintain low levels of peroxides in the dietary oils, because a high lipid peroxide intake may be toxic to rabbits.^{22 23}

To acclimate the rabbits to the synthetic diet, during the first week 30% (wt/wt) of regular laboratory rabbit chow (No. 5326, Purina) was added to the synthetic diet. During the second week, the concentration of chow was reduced to 15%. During the third and subsequent weeks, rabbits were fed the synthetic diet only. The amounts of the aforementioned diets were adjusted so that all animals consumed all of their daily food allotment (80 g/d) and therefore had a constant food intake. Rabbit weight was monitored for the duration of the experiment, and both the control and oxidized-diet groups had a similar weight gain during the experiment. We measured serum cholesterol levels every 2 weeks after the animals had fasted overnight to determine the cholesterol exposure for each rabbit. Because the severity of arterial fatty streak lesions depends on the serum cholesterol concentration,^{9 10} cholesterol exposure was calculated as the AUC (MacDraft and Cricket Graph software) of the serum cholesterol concentration–versus-time graph (12 to 14 weeks) as described^{9 10} and is given in millimoles per liter times days. Serum TG levels in rabbits were measured at the beginning and end of the experiment.

Serum Lipid Analysis
At 12 to 14 weeks, the rabbits were euthanized (200 mg/kg pentobarbital IV) in pairs (one control rabbit for each rabbit that had been fed the oxidized-lipid diet) after an overnight fast and their blood collected. Lipoproteins were isolated by sequential ultracentrifugation²⁴ in the density range 1.006 to 1.019 (ß-VLDL), 1.019 to 1.063 (LDL), and 1.063 to 1.225 (HDL) g/mL. All serum and lipoprotein samples included 1 µmol/L EDTA and 100 µg/mL gentamicin sulfate. EDTA concentrations were low to have minimal interference on the conjugated diene measurements. However, in a trial experiment in which EDTA concentrations of 1 µmol/L and 50 µmol/L were compared, there was no difference in oxidation level in the lipoprotein fractions. Cholesterol was measured in serum and lipoprotein fractions to determine the cholesterol distribution among lipoproteins.

Assessment of Oxidation
Lipid oxidation in serum lipoproteins and dietary oils was measured by determining the conjugated diene content by the method of Corongiu et al.²⁵ In brief, serum lipoproteins were extracted by the method of Dole,²⁶ and conjugated dienes were measured by second-derivative UV spectroscopy in a Perkin-Elmer 555 spectrophotometer. This method was chosen because it is more sensitive than direct colorimetric peroxide and thiobarbituric acid–reactive substance measurements, which are not sufficiently sensitive to detect oxidized lipids in rabbit serum lipoproteins. Lipid peroxides were measured by a direct peroxide assay that uses a color reaction with a methylene blue derivative²⁷ as described previously.^{13 15} Cumene hydroxide was used as a standard in this assay. Fatty acid loss due to oxidation was measured by gas-liquid chromatography.²⁸ Serum ß-VLDL and LDL fractions were also examined for susceptibility to copper oxidation by the procedure of Esterbauer et al.²⁹ Freshly isolated ß-VLDL (0.5 mg TG per milliliter of incubation mixture) was incubated with CuSO₄ (final concentration, 0.01 mmol/L) at 37°C, and conjugated dienes were measured every 30 minutes for 4 hours as described previously.¹⁵ For determination of LDL susceptibility to oxidation (0.3 mg TG per milliliter of incubation mixture), the final CuSO₄ concentration was 0.005 mmol/L. In experiments with LDL the copper concentration was lower because in our experience LDL is more easily oxidized. For determination of oxidation in the dietary oils, solutions of 5 mg oil per milliliter of isopropanol were used.

Morphological Examination of Atherosclerotic Lesions
At the end of the experiment the rabbits were anesthetized with ketamine and xylazine (35 and 10 mg/kg body weight, respectively). The chests were opened, and the rabbits were bled by cardiac puncture and then killed with an overdose of pentobarbital (200 mg/kg body weight). After laparotomy the aortas and pulmonary arteries were removed. The aorta was dissected from the aortic valve to the iliac bifurcation and as much adventitia as possible was removed to prevent errors during Sudan IV staining of the vessel. The aorta was opened longitudinally and pinned flat on a Styrofoam surface. After overnight fixation in 10% formalin (buffered Formalde-Fresh, Fisher Scientific Co), the aorta was rinsed in 70% ethanol for 10 minutes and then stained with 0.5% Sudan IV in 35% ethanol–50% acetone for 20 minutes. Destaining was carried out for 20 minutes in 80% ethanol. Lipid deposition in the aorta was determined by morphological assessment of the percentage of lesion-covered aorta as visualized by Sudan IV staining of the region between the aortic root and bifurcation. Fatty streak lesions on enlarged photographs were traced on a digital tablet (Kurta IS/ADB, Inmac Inc), and lesion areas were measured using MacDraft software on a Macintosh computer.

Biochemical Analysis of the Artery Wall
Because all aortas were fixed and stained for fatty streak lesion determination, they were not suitable for chemical analysis; therefore, pulmonary arteries were used for cholesterol, CE, and oxidized cholesterol analyses to determine the effect of oxidized dietary fat on arterial lipid deposition. It has been shown that fatty streak lesions in the pulmonary artery develop similarly to those in the aorta.³⁰ Pulmonary arteries were washed in ice-cold saline, blotted dry between sheets of filter paper, and weighed. Cholesterol and CE contents in the arteries were measured after they were homogenized in phosphate buffer and the lipid was extracted from the homogenates as described by Folch et al³¹ ; quantitative gas-liquid chromatography was performed as described by Rapp et al.²⁸ The samples were analyzed for free cholesterol before and after saponification, and the calculated difference represents the cholesteryl ester concentration. Cholesterol and 7ß-hydroxycholesterol (Steraloids Inc) were used as standards for free- and oxidized-cholesterol determinations. Oxidized cholesterol eluted from the column at 17 minutes and free cholesterol at 14 minutes.

Analytical Methods
Total serum cholesterol (kit No. 352-20, Sigma Chemical Co) and TGs (kit No. 339-20, Sigma) were determined by enzymatic assay as specified by the manufacturer. Vitamin E concentration in dietary oils was measured by high-performance liquid chromatography.³² Unless stated otherwise, all results are expressed as mean±SEM. Student's t test was used to test for the significance between means, which was set at P<.05. A multiple regression model was used to determine the two best-fit equations to represent lesion percent after oxidized and unoxidized oils were given in the diet and to determine the degree of statistical significance between their slopes. An indicator variable was used in the regression model for the type of oil (oxidized=1; unoxidized=0).³³ The Statistica 4.1 application for the Macintosh (StatSoft Inc) was used to perform the computations.

	Results

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Serum Cholesterol and TG Levels
As expected, serum cholesterol levels increased in both diet groups (control and oxidized-lipid diets). After initiation of either diet, both of which contained 0.25% cholesterol, serum cholesterol concentrations were markedly increased at 2 weeks and reached maximum levels at 4 weeks. At the end of the experiment (12 to 14 weeks), mean serum cholesterol level was 22.22±4.12 mmol/L for the control group and 19.55±2.58 mmol/L for the oxidized-diet group (NS). Fig 1 shows the cholesterol distribution among lipoprotein particles, and as described in previous studies,³⁴ >50% of the serum cholesterol was in the ß-VLDL fraction (d<1.019 g/mL). The serum cholesterol exposure (AUC of the cholesterol level–versus-time graph) was 2345.12±481.96 mmol/Lxdays for the control group and 2013.63±260.96 mmol/Lxdays for the oxidized-diet group (NS). Thus, cholesterol exposure and distribution among lipoproteins were similar for rabbits fed either the control or oxidized-lipid diet.

View larger version (34K): [in this window] [in a new window]   Figure 1. Bar graph showing serum cholesterol levels in rabbits (n=14) and serum cholesterol distribution (n=8) among lipoproteins at the end of the experiment (12-14 weeks). Lipoproteins were isolated by sequential ultracentrifugation in the density range 1.006-1.019 (ß-VLDL), 1.019-1.063 (LDL), and 1.063-1.225 (HDL) g/mL. Rabbits were fed either a control or oxidized-lipid diet. Values are expressed as mean±SEM in mmol/L. NS for all lipoprotein groups.

At the end of the experiment, the serum TG concentrations in the control and oxidized-diet groups were 0.63±0.14 and 0.74±0.11 mmol/L, respectively. At the start of the experiment, the mean TG concentration in all rabbits was 0.83±0.13 mmol/L. Thus, there was no difference in serum TG concentrations between the control and the oxidized-diet group.

Serum Lipoprotein Oxidation
Freshly isolated ß-VLDL and LDL were examined for the presence of oxidized lipid by measuring conjugated dienes.²⁵ ß-VLDL from rabbits that were fed the control diet contained 3.86±0.57 versus 9.07±2.14 nmol conjugated dienes per micromole cholesterol in rabbits that were fed the oxidized diet (P<.05; Fig 2). Thus, feeding rabbits an oxidized lipid–enriched diet in conjunction with cholesterol results in conjugated diene levels in the ß-VLDL fraction that are 2.35 times higher than controls. No difference in oxidized lipid levels was detected in the LDL fractions.

View larger version (34K): [in this window] [in a new window]   Figure 2. Bar graph shows lipid oxidation in the ß-VLDL (d=1.006-1.019 g/mL) and LDL (d=1.019-1.063 g/mL) fractions measured as amount of conjugated dienes. Rabbits were fed either the control or oxidized-lipid diet and serum samples were obtained at the end of the experiment. Data are expressed as mean±SEM in nmol conjugated dienes per µmol cholesterol. *P<.05 for dienes compared in ß-VLDL fractions. n=7 for each group.

When ß-VLDL fractions were subjected to copper oxidation,²⁹ we found no difference in the oxidation lag times of lipoproteins that were isolated from control rabbits versus those fed the oxidized diet (165 versus 156 minutes). There was also no significant difference in the susceptibility of LDL fractions to copper oxidation between those from the control and those from the oxidized-diet group (38 versus 43 minutes). (The time values represent an average of two separate determinations.) Thus, the increase in oxidized lipids in the ß-VLDL fraction did not alter the susceptibility of these lipoproteins to additional copper oxidation.

Effect of Oxidized Dietary Fat on Fatty Streak Formation
Fig 3 is a scatterplot of aortic lesion area versus cholesterol exposure (the AUC of serum cholesterol as a function of time) for rabbits that were fed either the control or oxidized-lipid diet. The data show an increase in fatty streak lesions in the group that was fed the oxidized-lipid diet. Because a linear fit by the least-squares method resulted in intercepts not significantly different from 0, the data could be fitted with a straight line through 0. The slope of the line for the oxidized-lipid diet group increased by 100% when compared with that of the control group, and the difference between the slopes was highly significant (P<.02). Thus, a diet rich in oxidized lipid results in an 100% increase in aortic fatty streak lesions for a similar cholesterol exposure. There was a large range in the severity of fatty streak lesions within not only the experimental but also the control group. This phenomenon has been shown in previous rabbit studies^{9 10} and has been discussed by Stender et al.³⁵

View larger version (16K): [in this window] [in a new window]   Figure 3. Scatterplot showing the dependence of lesion area on cholesterol exposure for rabbits fed either the control () or oxidized-lipid (o) diet. Cholesterol exposure was calculated as the AUC of serum cholesterol level (expressed as mmol cholesterol/Lxdays) vs time. n=14 for each group.

Chemical Analysis of Pulmonary Arteries
To further evaluate the extent of atherosclerosis, pulmonary arteries from both dietary groups were analyzed for TC and CE deposition (Fig 4). Cholesteryl ester contents were 4.2 times higher in the experimental group than in rabbits that were fed the control diet, but TC levels increased by only 100%. Free cholesterol content in the pulmonary artery was only modestly increased in the oxidized-lipid diet group (55% increase; P=.056). When the TC content in the pulmonary artery was adjusted for serum cholesterol exposure, the mean cholesterol content in control arteries was 5.30±0.85 nmol cholesterol per gram of tissue per unit of cholesterol exposure (mmol cholesterol/Lxdays), whereas in arteries from the oxidized diet–fed animals, cholesterol deposition was 12.65±2.39 nmol cholesterol per gram of tissue per unit of cholesterol exposure (P<.05). Additionally, in 40% of rabbits that were fed the oxidized-lipid diet, a small amount (0.26±0.05 nmol/g tissue) of 7ß-hydroxycholesterol (oxidized cholesterol) was detected. In controls, 7ß-hydroxycholesterol was not detected in any animal.

View larger version (39K): [in this window] [in a new window]   Figure 4. Bar graphs showing cholesterol and CE deposition in rabbit pulmonary arteries after rabbits were fed either the control or oxidized-lipid diet for 12-14 weeks. Data are expressed as mean±SEM in µmol cholesterol per g arterial tissue (wet weight) *P<.02, **P<.05. n=8 for each group.

	Discussion

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In recent years lipoprotein oxidation has been implicated in the pathogenesis of atherosclerosis.^{1 2} It has been hypothesized that lipoproteins are oxidized in the vessel wall, but it is also possible that oxidized lipoproteins may be transported into lesions from the circulation. Previous studies from our laboratory^{13 14 15} and others^{16 36} have demonstrated that one source of such oxidized lipid in the circulation is oxidized dietary lipid. Given the evidence that oxidized lipoproteins are atherogenic and that oxidized dietary lipids influence the concentration of oxidized lipoproteins in the circulation, in this study we tested the hypothesis that dietary oxidized fat would accelerate the progression of atherosclerosis in rabbits that were fed a low-cholesterol diet.

As expected, we found a substantial portion of the serum cholesterol in the ß-VLDL fraction. Numerous studies have shown that ß-VLDL can induce foam cell formation.^{37 38 39} Moreover, consumption of the oxidized-lipid diet resulted in a small but significant increase in the quantity of oxidized lipids in serum ß-VLDL. ß-VLDL from rabbits that were fed the control diet contained 3.86±0.57 nmol conjugated dienes per micromole of cholesterol, whereas ß-VLDL from rabbits that were fed the oxidized-lipid diet contained 9.07±2.14 nmol conjugated dienes per micromole of cholesterol (P<.05). However, no differences were found in the susceptibility to copper oxidation of ß-VLDL isolated from oxidized diet–fed and control diet–fed rabbits. This finding conflicts with our previous observation in humans, whose chylomicron fractions had an increased susceptibility to copper oxidation after ingestion of an oxidized-lipid diet.¹⁵ The amount of preexisting oxidized lipid necessary to increase the susceptibility of ß-VLDL to copper oxidation has not been established and, therefore, it is possible that in rabbits the increase in oxidation level in ß-VLDL was not high enough to enhance oxidation susceptibility. In human studies, we found that oxidation levels in the chylomicron fraction increased from 10.99±1.05 to 52.63±6.40 nmol conjugated dienes per micromole of TG after consumption of an oxidized-lipid diet.¹⁵ It is also possible that in ß-VLDL, preexisting peroxides do not enhance susceptibility to copper oxidation.

At present we have no explanation for the origin of oxidized lipids in the sera of rabbits that were fed the control diet, although this finding is similar to our previous observation in rat sera¹³ that contained oxidized lipids in the LDL+VLDL fraction after the rats had been fed a fat-free sucrose diet for 2 weeks. This finding strongly suggests that endogenous oxidation occurs and therefore that oxidized dietary lipids are not the only source of oxidized serum lipoproteins.

The major finding of the present study is that consumption of a diet rich in oxidized lipid results in an increase in fatty streak lesions in the aorta and a >100% increase in TC in the pulmonary artery, primarily due to an increase in CEs. Moreover, small amounts of oxidized cholesterol were found in the pulmonary arteries at the end of the oxidized-lipid diet period. Because the synthetic diet contained trace amounts of 7ß-hydroxycholesterol, it could have originated from the diet and been detected in the arteries of rabbits that were fed the oxidized-lipid diet due to increased deposition of cholesterol. Another explanation for the increases in oxidized cholesterol in the pulmonary arteries may be that the oxidized fatty acids in dietary oil facilitate cholesterol auto-oxidation. It has been shown by Sevanian and McLeod⁴⁰ that in human hypercholesterolemic plasma, the presence of peroxidized unsaturated fatty acids results in the increased auto-oxidation of cholesterol.

The effects of oxidized dietary fat on the atherosclerotic process have been described in several animal species. In 1965 Kaunitz et al⁴¹ fed cottonseed oil to rats for 2 years and found increased atherosclerosis in the coronary arteries when the oil was heated before feeding. Kritchevsky and Tepper⁴² reported that in cholesterol-fed rabbits, heated dietary polyunsaturated fat (corn oil) was more atherogenic than unheated corn oil. In contrast, there was no increase in atherogenicity if the heated oil was monounsaturated (olive oil). At the time of those studies, the role of oxidation in atherosclerosis was not yet appreciated and measurement of the extent of oxidation in dietary oils was not performed. However, in conjunction with our experiments it seems reasonable to speculate that during heating, polyunsaturated fats become oxidized, which leads to increases in oxidized serum lipoproteins and subsequent atherosclerosis.

At present, the mechanism whereby oxidized dietary lipid increases atherogenesis remains to be elucidated. However, it has been shown that oxidized ß-VLDL is degraded by macrophages at an accelerated rate when compared with unoxidized ß-VLDL^{43 44} and that oxidized ß-VLDL in turn results in increased lipid accumulation in smooth muscle cells in culture.^{45 46} Thus, oxidized ß-VLDL may exhibit increased atherogenic properties compared with its unoxidized form. It has also been suggested that preformed lipid peroxides in oxidized polyunsaturated fatty acids in ß-VLDL may accelerate auto-oxidation of other lipids,^{40 47} such as cholesterol and phospholipid, resulting in increased atherogenicity of the oxidized ß-VLDL particle.^{48 49}

Our results and those of others suggest that foods that contain oxidized lipids may be important risk factors for atherosclerosis. Owing to the popularity of fried foods and the widespread fast-food industry, the typical US diet contains large quantities of oxidized fat. Frankel et al¹⁷ examined the relative percentages of oxidation products in frying oils used in fast foods and found that most of them contained oxidized lipids; french-fried potatoes, in fact, contain as much as 8.2% oxidized material. Alexander¹⁸ has shown that the oils used in many restaurants are kept at 180°C for extended periods, which results in the formation of oxidized lipids. Thompson et al¹⁹ have also shown that the oils used for deep frying in restaurants, institutions, and the armed services contain high quantities of oxidized lipids and that during commercial preparation of french-fried potatoes, at least 15% of the polyunsaturated oil becomes oxidized.²⁰ Thus, the amount of oxidized fat consumed per medium serving of french-fried potatoes that contain a total of 15 g fat is 8000 µmol oxidized fatty acids, whereas the rabbits in our experiments consumed only 400 µmol oxidized fatty acids per day. Finally, Yagi et al²¹ measured peroxides in 30 kinds of food and found that even such foods as crackers and frozen shrimp contained relatively high concentrations of oxidized lipids (320 nmol/g). The daily intake of such oxidized lipids would depend on the amounts and types of food consumed. Thus, there is abundant evidence that oxidized lipids are frequently present in the typical US diet. Because humans can also absorb oxidized fat,¹⁵ oxidized dietary oils may also promote atherogenesis in humans.

In summary, the present study indicates that dietary oxidized lipids accelerate fatty streak lesion formation in rabbits. If further studies demonstrate that oxidized lipids are atherogenic in humans, dietary recommendations will need to be altered to reduce the intake of oxidized lipid.

	Selected Abbreviations and Acronyms

 

AUC = area under the curve CE(s) = cholesteryl ester(s) GLC = gas-liquid chromatography TC = total cholesterol TG(s) = triglyceride(s) ß-VLDL = ß-migrating VLDL

	Acknowledgments

 
This work was supported by the American Diabetes Association, the Medical Research Service, the Veterans Affairs Administration, and the Pacific Vascular Research Foundation. The excellent technical assistance of Agnes Frank is appreciated. Dr Kem A. Rogers is gratefully acknowledged for helpful discussions. We also thank Dr Vojtech Licko for carrying out the statistical analyses.

Received June 23, 1995; accepted January 4, 1996.

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