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Institute of Nutritional Sciences, Martin-Luther-University of Halle-Wittenberg, Emil-Abderhalden-Straße 26, D-06108 Halle/Saale, Germany
2To whom correspondence should be addressed. E-mail: eder{at}landw.uni-halle.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol equivalents/kg were used. In Experiment 2, diets with fresh or oxidized fat, heated at a temperature of 55°C, containing 25 mg -tocopherol equivalents/kg, were used. In Experiment 1, rats fed all types of oxidized fats had higher concentrations of HDL cholesterol and lower ratios between plasma and HDL cholesterol than rats fed the diet containing the fresh fat. As determined from the lag time, the susceptibility of LDL to copper-induced lipid peroxidation was higher in rats fed oxidized fats heated at 105 or 190°C than in rats fed the diets containing the fresh fat or the oxidized fat treated at 50°C, irrespective of the dietary vitamin E concentration. However, in Experiment 2, the composition of LDL apolipoproteins and uptake of LDL by macrophages were not different between rats fed the fresh fat diet and those fed the oxidized fat diet. We conclude that ingestion of oxidized fats does not adversely affect the lipoprotein profile in the rat model used, and does not cause modifications of apolipoproteins that would lead to enhanced uptake of LDL via macrophage scavenger receptors.
KEY WORDS: • antioxidant status • LDL • oxidized fat • rats
Thermally treated dietary fats, which are an important source of fat in typical Western diets, contain increased concentrations of a large number of lipid peroxidation products. It has been shown that feeding thermally oxidized fats increases the concentrations of lipid peroxidation products in tissues (1,2). Accumulating evidence suggests that oxidized fats and lipid oxidation products in the diet can also contribute to the pathogenesis of atherosclerosis (3). When investigating possible proatherogenic effects of thermally oxidized fat, the cholesterol metabolism, particularly the concentrations of cholesterol in LDL and HDL, is of special interest. Elevated levels of LDL cholesterol constitute a major risk factor for atherosclerotic diseases, whereas elevated levels of HDL have beneficial effects. To date, little information exists concerning the effects of thermoxidized fats on the lipoprotein profile, and the existing data are contradictory (4,5). One aim of this study, therefore, was to investigate the effect of oxidized fats on the lipoprotein profile in rats. Dietary oxidized fats could also enhance oxidative modification of the LDL through their lipid peroxidation products. Studies by Penumetcha et al. (6) with intestinal cell lines suggested that oxidized fatty acids can be readily absorbed by the intestine, esterified into complex lipids and incorporated into lipoproteins. Hence, a relationship between heated fat and oxidatively modified LDL seems reasonable. Oxidized LDL appear to affect several steps of the atherogenic process. There is evidence that they play a role in the initiation of atherosclerotic lesions (7). A number of animal studies even suggested that atherosclerotic lesion development is increased through intake of heated oil (8,9). Oxidatively modified LDL also promote atherosclerosis by chemoattraction of monocytes and by stimulation of their adhesion to the endothelium and their retention in the intima (10,11). The differentiation of the monocytes to mature macrophages takes place in the intima and is accompanied by the induction of scavenger receptor expression. Because oxidatively modified LDL have different apolipoprotein (apo)2 patterns, binding and uptake by the strongly regulated LDL receptors in liver and peripheral organs, which is mediated by the apo B protein, are no longer possible; instead LDL may be taken up in unrestricted ways by macrophage scavenger receptors in the intima, leading to foam cell formation, a main constituent of atherosclerotic plaques (12). Another objective of this study, therefore, was to determine whether oxidized fats induce oxidative modification of LDL. To assess the susceptibility of LDL against lipid peroxidation, in the first experiment of this study, we conducted an in vitro oxidation assay by incubating LDL of rats fed fresh or oxidized fats with copper ions. Because the susceptibility of LDL against oxidation is strongly influenced by their vitamin E concentration (13), we used diets with either nutritionally adequate or excess vitamin E concentrations.
Because the in vitro oxidation assay does not give information about possible oxidative modification of LDL in vivo, in the second experiment of this study, we examined the apolipoprotein pattern of LDL and the uptake of LDL by macrophages via their LDL and scavenger receptors. Oxidatively modified LDL differ in their apolipoprotein pattern from unmodified LDL; as a consequence, they are taken up via scavenger receptor mechanisms (10,12). If dietary oxidized fats provoked oxidative modification of LDL, we would expect an increased uptake of LDL by scavenger receptors of macrophages.
It has been shown that primary and secondary lipid peroxidation products exert different physiologic effects. Primary lipid peroxidation products are highly toxic when administered parenterally but less toxic when given orally, probably due to low digestibility (14,15). The formation of primary and secondary lipid peroxidation products during thermal treatment of oils depends on the conditions during heating. Treating fats for a long period at low temperatures without catalysts produces mainly primary lipid peroxidation products such as peroxides and hydroperoxides. Because the primary lipoxy radical is unstable, fats treated at high temperatures or in the presence of catalysts contain predominantly secondary lipid peroxidation products such as carbonyls or dimeric, trimeric, polymeric and cyclic fatty acids (16). To compare fats with different concentrations of primary and secondary lipid peroxidation products in the first experiment, we used fats that were heated at different temperatures.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two experiments were carried out with male Sprague-Dawley rats supplied by Charles River (Sulzfeld, Germany). In Experiment 1, rats (n = 80) with an initial body weight (±SD) of 103 ± 7 g were assigned to eight groups of 10 rats each; in Experiment 2, rats (n = 20) with an initial body weight of 133 ± 7 g were assigned to two groups of 10 rats each. The animals were housed individually in Macrolon cages in a room maintained at a temperature of 23°C and 50–60% relative humidity with lighting from 0600 to 1800 h. All experimental procedures described followed established guidelines for the care and handling of laboratory animals and were approved by the council of Saxony-Anhalt.
Diets.
Semisynthetic diets were used (Table 1). The composition of the diets was identical in both experiments. The concentration of fat was 100 g/kg. The diets contained sufficient amounts of minerals and vitamins based on recommendations by the AIN (17) for rodent diets. The concentrations of vitamin E were selected in accordance with the experimental design.
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-tocopherol equivalents/kg diet. In Experiment 2, only the type of fat (fresh fat vs. oxidized fat, see below) was varied. The vitamin E concentration in both diets was 25 mg -tocopherol equivalents/kg diet. To adjust dietary vitamin E concentrations, we analyzed the native concentrations of tocopherols in the fresh fats and in the oxidized fats after the thermal treatment. With consideration of the native tocopherol concentrations of the dietary fats, the diets were supplemented individually with all-rac--tocopheryl acetate whose biopotency is 
67% that of -tocopherol. Diets were prepared by mixing the dry components with fat and water and subsequent freeze-drying. The residual water content of the diet was <5 g/100 g diet. In both experiments, diets were administered in restricted amounts to standardize the diet intake. The rats were fed once daily at 0800 h. The amount of diet administered was 20% less than the amounts of identical diets with fresh fats consumed ad libitum by rats in preliminary studies. The daily amount of diet was increased continuously during the experiment from 8.3 to 17.4 g (Experiment 1) and from 10.0 to 18.8 g (Experiment 2). Thus, all of the rats within one experiment consumed identical amounts of diet. Water was freely available from nipple drinkers. The experimental diets were fed for 9 wk in Experiment 1 and for 8 wk in Experiment 2.
Preparation of the oxidized fats.
In Experiment 1, a fresh fat consisting of a mixture of sunflower oil and lard (31:69, wt/wt) and three different types of oxidized fats consisting of mixtures of sunflower oil and lard (1:1, wt/wt) were used. The first type of oxidized fat was prepared by heating for 38 d at a temperature of 50°C; the second type of oxidized fat was prepared by heating for 81 h at a temperature of 105°C; the third type of oxidized fat was prepared by heating for 24 h at a temperature of 190°C. In Experiment 2, the fresh fat consisted of a mixture of sunflower oil and lard (19:81, wt/wt). The oxidized fat used in Experiment 2 was prepared by heating a mixture of sunflower oil and lard (1:1, wt/wt) at a temperature of 55°C for 49 d. For heat treatment, the fats were put into quartz glass beakers which were placed in a drying oven set at the intended temperature. Throughout the heating process, air was continuously bubbled through the fats. This treatment caused a loss of PUFA, a complete loss of tocopherols and raised the concentrations of lipid peroxidation products in the fats. We equalized the fatty acid composition of fresh and oxidized fats within one experiment by varying the ratio of lard to sunflower oil in the mixtures of the fresh fats. The extent of lipid peroxidation in the fat was estimated by assaying the peroxide value (POV) (18), concentration of TBARS (19), concentration of conjugated dienes (20), acid values (18), the percentage of total polar compounds (21) and the concentration of total carbonyls (22). To determine the concentrations of those lipid peroxidation products of the dietary fats after they have been incorporated into the diets, the fats were extracted from the diets with a mixture of hexane and isopropanol [3:2, v/v, according to (23)].
Sample collection.
After completion of the feeding periods, the rats were starved for 12 h and killed by decapitation under light anesthesia with diethyl ether. Blood was collected into EDTA-polyethylene tubes. Plasma were obtained by centrifugation of the blood (1800 x g, 10 min, 4°C). LDL, 1.006 < 
< 1.063 kg/L) and HDL, > 1.063 kg/L) were isolated by ultracentrifugation of plasma (Mikro-Ultrazentrifuge, Sorvall Products, Bad Homburg, Germany) at 900,000 x g at 4°C for 3 h. In Experiment 1, an aliquot of the LDL fraction was used immediately for studying the susceptibility to in vitro oxidation (see below). In Experiment 2, an aliquot of the LDL fraction was processed immediately for uptake studies using macrophages (see below). The remaining LDL, HDL and plasma were stored at a temperature of -20°C until analysis.
Analysis.
Concentrations of cholesterol in plasma, LDL and HDL were determined using enzymatic reagent kits (VWR International, Darmstadt, Germany, Cat.-No. 1.14830). The fatty acid composition of dietary fats and LDL total lipids was determined by GC as described recently (24). Total lipids of fats and LDL samples were extracted with a mixture of hexane/isopropanol (3:2, v/v). Concentrations of -, ß-, 
- and 
- tocopherol in dietary fats and LDL were determined by HPLC using a Hewlett-Packard system (HP 1100; Waldbronn, Germany) according to Coors (25) with modifications described recently (24).
LDL oxidation.
LDL were dialyzed for 12 h at 4°C against PBS, pH 7.4, which was purged with nitrogen before use. The protein content was determined using the method of Bradford (26). The in vitro oxidation was performed according to Esterbauer et al. (27) with modifications. The protein concentration was adjusted to 0.05 g/L with dialysis buffer. Oxidation was initiated by adding a freshly prepared copper sulfate solution (final concentration 350 µmol/L). The reaction took place in 96-well UV-plates (Greiner bio-one, Frickenhausen, Germany) in a total volume of 200 µL. The kinetics of LDL oxidation were determined by monitoring the change in absorbance at 234 nm at room temperature with a microplate absorbance reader (SpectraFluor, Tecan, Crailsheim, Germany). From the kinetic profile of each probe, the lag time and rate of diene production during the propagation phase were determined.
LDL uptake.
LDL were labeled with the fluorochrome 1,1'dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) according to Zouhair et al. (28) with modifications. Briefly, 1.2 mL of LDL (mean protein concentration, 437 mg/L) was mixed with 2 µL of a 100 mmol/L solution of ascorbic acid to prevent oxidation and 4 µL of DiI in dimethyl sulfoxide (36 mmol/L). The mixture was incubated for 6 h at 37°C in the dark, under nitrogen and gentle agitation. The DiI-labeled LDL were reisolated by ultracentrifugation (415,000 x g, 1 h, 4°C) and dialyzed at 4°C for 12 h against PBS.
For uptake studies, the adherent growing murine monocyte-macrophage cell line, J774 A.1 (ACC 170; DSMZ, Braunschweig, Germany), was used. The cells were cultured in DMEM containing 10% fetal bovine serum and 0.5% gentamicin in a CO2-incubator (5% CO2 in air) at 37°C. J774 A.1 were seeded in 24-well plates and cultured until confluence was reached. On the day of the experiment, the medium was removed and cells were washed with serum-free DMEM. Thereafter, 200 µL serum-free DMEM containing 1 µg DiI-LDL was added to the cells and incubation was carried out for 2 h at 37°C. After incubation, the cells were washed twice with PBS and lysed in 300 µL isopropanol. The lysates were centrifuged at 10,000 x g for 5 min, and fluorescence was measured in the supernatant with the fluorescence detector of the HP 1100 (excitation wavelength: 520 nm, emission wavelength: 580 nm). All incubations were replicated 8 times per animal. To determine the concentration of the bound LDL, the fluorescence intensity of the probes was expressed in relation to the fluorescence intensity of the DiI-LDL preparation used. Cellular protein content was measured in the same samples of lysed cells according to Bradford (26). Uptake was expressed as µg LDL/mg cell protein. The mean protein concentration of the cell monolayers was 115 µg/well. To determine the specificity of the uptake, incubation of the cells was carried out in the presence and absence of polyinosinic acid (5 mg/L DMEM), which is a potent inhibitor of scavenger receptors (29,30), and in presence and absence of heparin (80,000 U/L DMEM), which inhibits the LDL receptor–mediated uptake of LDL (31). Specific uptake was calculated by subtracting uptake in the presence of the inhibitors from uptake in the absence of the inhibitors, for both inhibitors separately. As a positive control we used LDL oxidized in vitro by a 1-h incubation with 25 mmol/L copper sulfate at 37°C. Incubation of cells with these oxidized LDL in the presence of polyinosinic acid revealed 47% specific uptake via the scavenger receptor.
Apolipoprotein analysis.
The pattern of LDL apolipoproteins was assayed qualitatively by denaturing electrophoresis. The proteins were separated on a 6–19% polyacrylamide gradient gel with 0.01 g/L SDS. Gels were stained with silver nitrate according to Nesterenko (32). The bands of apolipoproteins were identified by comparison with molecular weight standards and analyzed as digitized video pictures (apparatus and software from Syngene, Cambridge, UK).
Statistical analysis.
Results of Experiment 1 were analyzed by two-way ANOVA, using fat type, vitamin E concentration as well as their interaction as factors. For significant F-values, individual means were compared by Fisher’s least significant difference test. Results of Experiment 2 were compared by t test. Means were considered significantly different at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Within each experiment, the fatty acid composition was similar in the fresh fat and the oxidized fats (Table 2).
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The fresh fat contained 202 mg -tocopherol/kg; concentrations of other tocopherols were below the detection limit of 1 mg/kg. In the dietary fats heated at 50 or 105°C, the concentrations of all tocopherols were <1 mg/kg. The fat heated at 190°C contained 28 mg -tocopherol/kg; concentrations of other tocopherols were <1 mg/kg. Before inclusion in the diet, the POV of the oxidized fat treated at 50°C was 500 times higher, that of oxidized fat treated at 105°C was nearly 100 times higher and that of oxidized fat treated at 190°C was 100% higher than that of the fresh fat. The concentrations of TBARS of oxidized fats treated at 50, 105 and 190°C were 1000, 200 and 30 times higher, respectively, than that of the fresh fat. The percentages of total polar compounds were similar in all three types of oxidized fats. In the fats treated at 50, 105 and 190°C, they were 19, 20 and 16 times higher, respectively, than in the fresh fat. The acid values of the three oxidized fats were similar, and were 1.5–2.5 times higher than that of the fresh fat. The concentrations of total carbonyls were also similar in the three types of oxidized fat, and were at least 14 times higher than in the fresh fat. After inclusion into the diet, the POV of oxidized fats treated at 50, 105 and 190°C were 200, 50 and 8 times higher, respectively, than that of the fresh fat. The TBARS levels rose even more sharply, with concentrations 2000, 200 and 20 times higher in oxidized fats treated at 50, 105 and 190°C, respectively, than in the fresh fat. The concentrations of conjugated dienes did not differ in oxidized fats treated at 50 and 105°C, and were 100% higher than in the oxidized fat treated at 190°C and 18 times higher than in the fresh fat (Table 2).
Experiment 2.
The fresh fat contained 90 mg -tocopherol/kg; concentrations of other tocopherols were below the detection limit of 1 mg/kg. In the oxidized fat, the concentrations of all tocopherols were <1 mg/kg. The POV of the oxidized fat was 200 times higher than that of the fresh fat. In the oxidized fat, the percentage of total polar compounds was 18 times higher, the acid value was 4 times higher and the concentration of total carbonyls was 19 times higher than in the fresh fat. After the oxidized fat was included in the diet, its POV was 54 times higher than in the fresh fat and nearly 100% higher than before inclusion into the diet. The concentration of TBARS was 2.2 times higher and the concentration of dienes was 16 times higher compared with the level in the fresh fat (Table 2).
Diet intake and body weights of rats.
The diet intake of the rats in each of the two experiments did not differ. In the first experiment, food intake averaged 14.3 g/d over the entire period; in the second experiment it was 15 g/d. In Experiment 1, final body weights differed significantly among the eight groups. Rats fed diets containing low vitamin E concentrations and oxidized fats heated at 50 or 190°C had lower final body weights than those fed diets containing low vitamin E concentrations and fresh fat or oxidized fat heated at 105°C (fresh fat: 388 ± 12 g; oxidized fat heated at 50°C: 364 ± 12 g; oxidized fat heated at 105°C: 376 ± 13 g; oxidized fat heated at 190°C: 362 ± 16 g; means ± SD, n = 10; P < 0.05). Within the groups of rats fed diets containing high vitamin E concentrations, there was no effect of the dietary fat on the final body weights (fresh fat: 378 ± 18 g; oxidized fat heated at 50°C: 374 ± 11 g, oxidized fat heated at 105°C: 385 ± 8 g, oxidized fat heated at 190°C: 382 ± 12 g; means ± SD, n = 10 for each group). In Experiment 2, final body weights did not differ between groups (fresh fat: 365 ± 16 g; oxidized fat: 358 ± 19 g; means ± SD, n = 10).
Cholesterol concentrations in plasma, LDL and HDL.
The concentrations of total cholesterol in plasma were significantly influenced by the dietary fat, in Experiments 1 and 2, and by the dietary vitamin E concentration in Experiment 1 (Table 3). In Experiment 1, rats fed the oxidized fat heated at 50°C had lower concentrations of cholesterol in plasma than those fed fresh fat or oxidized fat heated at 105 or 190°C. Rats fed diets with high dietary vitamin E concentrations had higher concentrations of cholesterol in plasma than those fed diets with low vitamin E concentrations. In Experiment 2, rats fed the oxidized fat had lower cholesterol concentrations in plasma than those fed the fresh fat.
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In Experiment 1, HDL cholesterol concentrations were influenced by the type of fat. Rats fed all types of oxidized fats had higher HDL cholesterol concentrations than those fed the fresh fat, independent of the dietary vitamin E concentration. In Experiment 2, the HDL cholesterol concentrations did not differ significantly between the groups. In Experiment 1, the ratios between plasma cholesterol and HDL cholesterol were lower in all groups fed oxidized fats than in those fed fresh fat. In Experiment 2, the ratio between plasma and HDL cholesterol did not differ between the two groups of rats.
Vitamin E concentration, fatty acid composition and copper-induced lipid peroxidation of LDL (Experiment 1).
In LDL, -tocopherol was the only tocopherol that could be detected; the concentrations of other tocopherol isomers were below the detection limit of 0.1 µmol/(mmol triglycerides + cholesterol). There was a significant interaction between the type of fat and the dietary vitamin E concentration on the vitamin E concentrations in LDL (Table 4). Rats fed the high vitamin E diet with oxidized fat heated at 105 or 190°C had lower vitamin E concentrations in LDL than those fed the diet with fresh fat or the diet with oxidized fat heated at 50°C. Within the groups fed the low vitamin E diet, the vitamin E concentration of LDL was not influenced by the dietary fat. Rats fed diets with high vitamin E concentrations had distinctly higher concentrations of vitamin E in LDL than those fed diets with low vitamin E concentrations.
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Lag times and rates of diene production during incubation of LDL with copper ions were significantly influenced by the dietary fat and vitamin E concentration (Table 4). Rats fed oxidized fat heated at 105 or 190°C had shorter lag times than those fed diets with fresh fat or oxidized fat heated at 50°C. Rats fed diets with oxidized fats heated at 50 or 105°C had lower rates of diene production than those fed diets with fresh fat or oxidized fat heated at 190°C. Rats fed diets with high vitamin E concentrations had longer lag times and lower rates of diene production than rats fed diets with low vitamin E concentrations.
Macrophage uptake of DiI-labeled LDL (Experiment 2).
The uptake of DiI-labeled LDL by macrophages was not influenced by the dietary fat. The amounts of LDL taken up by the cells without inhibition did not differ in the two groups (2.64 ± 2.16 µg DiI-LDL/mg cell protein; mean ± SD, n = 20). The LDL taken up corresponded to 17% of the added LDL. The inhibition of the scavenger receptor mechanism by polyinosinic acid had no effect on the amount of LDL incorporated (2.58 ± 2.10 µg DiI-LDL/mg cell protein; mean ± SD, n = 20). Inhibition of the LDL receptor–mediated endocytosis of LDL by heparin resulted in 57% less incorporated LDL, independent of the dietary fat type (1.50 ± 1.28 µg DiI-LDL/mg cell protein; mean ± SD, n = 20).
Apolipoprotein composition of LDL (Experiment 2).
The proportions of LDL apolipoproteins were not influenced by the dietary fat. The mean relative amounts of apolipoproteins were (g/100 g): apo B 100: 12.1 ± 1.6; apo B 48: 45.7 ± 10.5; apo E: 42.2 ± 9.6 (means ± SD, n = 6). The mobility of the apolipoproteins in the electrophoresis did not differ in the two groups.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the first experiment, we investigated the effect of oxidized fat on the cholesterol concentrations in plasma and lipoproteins. Only a few, rather conflicting data exist concerning cholesterol concentrations in plasma of animals fed oxidized fats. Recently, we found reduced concentrations of cholesterol in plasma and LDL in miniature pigs fed an oxidized fat (34). In rats fed oxidized fats, cholesterol concentrations in plasma were not altered (4,35) or even increased (36) compared with controls fed fresh fat. In this study, the only effect on the cholesterol concentrations in plasma was caused by the oxidized fat, which had the highest concentrations of primary peroxidation products. This fat lowered plasma cholesterol, but had no effect on LDL cholesterol. We cannot yet explain why the two oxidized fats containing medium or low concentrations of primary lipid peroxidation products increased the concentration of LDL. However, the finding that feeding all three oxidized fats used in Experiment 1 enhanced HDL cholesterol and decreased the plasma to HDL cholesterol ratio shows that oxidized fats are not necessarily proatherogenic, at least when considering these variables. In studies with LDL receptor knock-out mice, dietary intake of oxidized fatty acids showed proatherogenic effects only when administered together with cholesterol (37). Hence, an additional supply of cholesterol seems to be crucial for a proatherogenic role of oxidized fat. Intake of thermally oxidized fat in a low fat diet without cholesterol supplementation as used in our study likely has no unfavorable effect on the cholesterol concentration or distribution in plasma.
The determination of the lag time before onset of lipid peroxidation during incubation of LDL with copper ions shows that the dietary fats heated at 105 or 190°C increased the susceptibility of LDL to lipid peroxidation. The susceptibility of LDL to lipid peroxidation depends mainly on their PUFA contents and their concentrations of antioxidants (27). Because the percentages of total PUFA in LDL total lipids were not different between rats fed the fats heated at 105°C or 190°C and the rats fed the fresh fat, we assume that the increased susceptibility of LDL to lipid peroxidation of rats fed the heated fats was due to their lower concentrations of vitamin E. Other studies also demonstrated that dietary oxidized fats enhance the consumption of vitamin E and decrease vitamin E concentrations in tissues and lipoproteins (2,35). The findings that the oxidized fat heated at 50°C, which had the highest POV and TBARS concentrations, does not reduce vitamin E concentrations in LDL and the lag time during oxidation compared with the fresh fat suggest that primary lipid peroxidation products do not increase the susceptibility of LDL to oxidation. The lower rate of diene production in the LDL of rats fed oxidized fats heated at 50 or 105°C compared with rats fed fresh fat or oxidized fat heated at 190°C might be due in part to lesser amounts of linoleic acid in the LDL.
Supplementation of the diet with high vitamin E concentrations prolonged the lag time of LDL, which was expected and described earlier (13,38). The protective effect of vitamin E, however, was weaker in rats fed oxidized fats heated at 105 or 190°C than in rats of the other groups. This suggests that there are unidentified decomposition products in these two oxidized fats, which consumed more -tocopherol. However, all of these in vitro effects cannot be equated with a proatherogenic action.
To our knowledge this is the first study describing the uptake of LDL by macrophages after feeding oxidized fats to rats. The total amount of LDL taken up by the cells in this study is comparable to that determined by others (39). The cell line, J-774A.1, used for the uptake studies, expresses LDL as well as scavenger receptors and hence has the ability to take up native and oxidized LDL (39,40). In our study, the uptake of LDL of rats fed fresh fat and LDL of rats fed an oxidized fat by LDL and scavenger receptors did not differ, suggesting that the dietary fat used in Experiment 2 did not increase oxidative modification of LDL in vivo. The addition of polyinosinic acid as an inhibitor of scavenger receptor–mediated uptake did not reduce the uptake of LDL, suggesting that no measurable oxidatively modified LDL were present in the LDL preparations of the two groups of rats. The uptake of LDL in the presence of heparin was strongly inhibited in both groups of rats, indicating that LDL was taken up specifically by LDL receptor–mediated endocytosis. The apolipoprotein pattern of LDL also did not differ between rats fed the fresh fat and rats fed the oxidized fat, also suggesting that the dietary oxidized fat did not cause strong oxidative modification of LDL in vivo. Strong oxidation of LDL leads to modifications of the apolipoproteins. In studies in which rats have been exposed to enhanced oxidative stress in vivo, an abnormal lipoprotein composition was detected. Brunet et al. (41) described reduction of apo B protein amounts in LDL of iron-loaded rats, whereas Kamiyam et al. (42) observed increased amounts of apo B protein in rats of a special strain exposed to vitamin C and/or E deficiency. In both studies, increased lipid peroxidation was measured in LDL. It should be noted, however, that small oxidative modifications of lipoproteins might not alter the distribution of apolipoproteins in LDL. Therefore, we cannot exclude minor oxidative modification of LDL, which would not be reflected by an altered apolipoprotein distribution.
Considering the results of this study, we assert that the dietary oxidized fat used in Experiment 2 did not enhance oxidative modification of LDL. The fat used in this experiment was heated at a low temperature over a long period and contained very high concentrations of primary lipid peroxidation products. As was observed in Experiment 1, fats heated at different temperatures that contained various amounts of primary and secondary lipid peroxidation products could have different effects on the antioxidant status and lipid peroxidation in LDL. The effects of dietary fats heated at high temperatures containing high concentrations of secondary lipid peroxidation products such as those present in fried foods on in vivo oxidation of LDL require further investigation.
In conclusion, on the basis of the variables measured in this study, we cannot conclude that ingestion of thermally oxidized fats exerts proatherogenic effects. Dietary intake of oxidized fats increases the susceptibility of LDL to oxidation in vitro by lowering their vitamin E concentrations. Notable oxidative modification of LDL in vivo resulting in an increased uptake by scavenger receptor mechanisms, however, did not occur in rats fed an oxidized fat, rich in primary lipid peroxidation products.
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FOOTNOTES |
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3 Abbreviations used: apo, apolipoprotein; DiI, 1,1'dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate; POV, peroxide value.
Manuscript received 13 May 2003. Initial review completed 1 June 2003. Revision accepted 19 June 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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