Nutrient Interactions and Toxicity

Thermally Oxidized Dietary Fats Increase the Susceptibility of Rat LDL to

Lipid Peroxidation but Not Their Uptake by Macrophages1
Klaus Eder,2 Uta Keller, Frank Hirche and Corinna Brandsch
Institute of Nutritional Sciences, Martin-Luther-University of Halle-Wittenberg, Emil-Abderhalden-Stra␤e 26,
D-06108 Halle/Saale, Germany

ABSTRACT The aim of this study was to investigate the effect of dietary oxidized fats on the lipoprotein profile
and the atherogenicity of LDL. Two experiments with male Sprague-Dawley rats were conducted. In Experiment 1,
diets with either fresh fat or oxidized fat, prepared by heating at temperatures of 50, 105 or 190°C, containing either
25 or 250 mg ␣-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 macro-
phages 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.
J. Nutr. 133: 2830 –2837, 2003.

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 con-
centrations 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 possi-
ble proatherogenic effects of thermally oxidized fat, the cho-
lesterol metabolism, particularly the concentrations of choles-
terol in LDL and HDL, is of special interest. Elevated levels of
LDL cholesterol constitute a major risk factor for atheroscle-
rotic 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 ex-
isting 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 be-
tween heated fat and oxidatively modified LDL seems reason-
able. Oxidized LDL appear to affect several steps of the athero-
genic 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)3 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 ox-
idative 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

1
Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG).
2 3
To whom correspondence should be addressed. Abbreviations used: apo, apolipoprotein; DiI, 1,1⬘dioctadecyl-3,3,3⬘3⬘-tetra-
E-mail: eder@landw.uni-halle.de. methylindocarbocyanine perchlorate; POV, peroxide value.

0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences.

Manuscript received 13 May 2003. Initial review completed 1 June 2003. Revision accepted 19 June 2003.

2830
 THERMOXIDIZED FATS AND LDL IN RATS
the susceptibility of LDL against oxidation is strongly influ-
enced by their vitamin E concentration (13), we used diets
with either nutritionally adequate or excess vitamin E con-
centrations.
Because the in vitro oxidation assay does not give informa-
tion about possible oxidative modification of LDL in vivo, in
the second experiment of this study, we examined the apoli-
poprotein pattern of LDL and the uptake of LDL by macro-
phages via their LDL and scavenger receptors. Oxidatively
modified LDL differ in their apolipoprotein pattern from un-
modified LDL; as a consequence, they are taken up via scav-
enger 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 macro-
phages.
It has been shown that primary and secondary lipid peroxi-
dation products exert different physiologic effects. Primary
lipid peroxidation products are highly toxic when adminis-
tered 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 treat-
ment of oils depends on the conditions during heating. Treat-
ing 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 per-
oxidation products in the first experiment, we used fats that
were heated at different temperatures.
MATERIALS AND METHODS
Animals. Two experiments were carried out with male Sprague-
Dawley rats supplied by Charles River (Sulzfeld, Germany). In Ex-
periment 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 guide-
lines 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 composi-
tion of the diets was identical in both experiments. The concentra-
tion 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.
In Experiment 1, fats treated under various conditions were used
(see below). The vitamin E concentrations of the diets were 25 vs.
250 mg ␣-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 consider-
ation of the native tocopherol concentrations of the dietary fats, the
diets were supplemented individually with all-rac-␣-tocopheryl ace-
tate 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
2831
TABLE 1
Composition of the basal diet used in Experiments 1 and 2
Component g/kg
Casein 200
Cornstarch 300
Sugar 298
Fat1 100
Cellulose 40
Mineral mixture2 40
Vitamin mixture3 20
DL methionine 2
1 See Materials and Methods.
2 Mineral mixture supplied the following (per kg diet): 10.0 g of
potassium sulfate, 9.8 g of calcium carbonate, 9.4 g of dicalcium
phosphate, 3.0 g of sodium chloride, 0.9 g of magnesium oxide, 160 mg
of ferrous sulfate hydrate, 51 mg of zinc oxide, 24 mg of manganese
oxide, 24 mg of copper sulfate pentahydrate, 0.32 mg of calcium iodate
and 0.33 mg of sodium selenite pentahydrate.
3 Vitamin mixture supplied the following (per kg diet): 1.34 mg of
all-trans-retinol, 25 ␮g of cholecalciferol, 7.5 mg of menadion sodium
bisulfite, 5 mg of thiamine hydrochloride, 6 mg of riboflavine, 6 mg of
pyridoxine hydrochloride, 15 mg of calcium pantothenate, 30 mg of
nicotinic acid, 2 mg of folic acid, 0.2 mg of biotin and 1000 mg of
choline chloride.
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 sun-
flower 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 con-
jugated 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-poly-
ethylene tubes. Plasma were obtained by centrifugation of the blood
(1800 ⫻ 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 ⫻ g at 4°C for 3 h. In Experiment 1, an aliquot of the LDL
2832 EDER ET AL.
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 macro-
phages (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 Interna-
tional, Darmstadt, Germany, Cat.-No. 1.14830). The fatty acid com-
position 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). Concen-
trations of ␣-, ␤-, ␥- 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 ␮mol/L).
The reaction took place in 96-well UV-plates (Greiner bio-one,
Frickenhausen, Germany) in a total volume of 200 ␮L. The kinetics
of LDL oxidation were determined by monitoring the change in
absorbance at 234 nm at room temperature with a microplate absor-
bance reader (SpectraFluor, Tecan, Crailsheim, Germany). From the
kinetic profile of each probe, the lag time and rate of diene produc-
tion during the propagation phase were determined.
LDL uptake. LDL were labeled with the fluorochrome 1,1⬘di-
octadecyl-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 ␮L
of a 100 mmol/L solution of ascorbic acid to prevent oxidation and 4
␮L 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 ⫻ g, 1 h, 4°C) and dialyzed at 4°C for 12 h against PBS.
For uptake studies, the adherent growing murine monocyte-mac-
rophage cell line, J774 A.1 (ACC 170; DSMZ, Braunschweig, Ger-
many), 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 ␮L serum-free DMEM containing 1 ␮g
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 ␮L isopropanol. The lysates were centrifuged at 10,000
⫻ 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 rela-
tion 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 ␮g LDL/mg
cell protein. The mean protein concentration of the cell monolayers
was 115 ␮g/well. To determine the specificity of the uptake, incuba-
tion 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 sig-
nificantly different at P ⬍ 0.05.
RESULTS
Characterization of the experimental fats. Within each
experiment, the fatty acid composition was similar in the fresh
fat and the oxidized fats (Table 2).
Experiment 1. The fresh fat contained 202 mg ␣-tocoph-
erol/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, respec-
tively, 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 ␣-tocoph-
erol/kg; concentrations of other tocopherols were below the
detection limit of 1 mg/kg. In the oxidized fat, the concentra-
tions 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 concen-
tration 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
 THERMOXIDIZED FATS AND LDL IN RATS 2833

TABLE 2
Characteristics of the dietary fats used in Experiments 1 and 2

Experiment 1 Experiment 2

Fresh fat Oxidized fat 1 Oxidized fat 2 Oxidized fat 3 Fresh fat Oxidized fat

Composition
Sunflower oil, g/100 g fat 31 50 50 50 19 50
Lard, g/100 g fat 69 50 50 50 81 50
Treatment, temperature, time None 50°C, 38 d 105°C, 81 h 190°C, 24 h None 55°C, 49 d
Major fatty acids, g/100 g fatty acids
16:0 19.2 17.4 17.8 17.5 22.5 20.9
18:0 11.5 9.8 10.4 10.2 13.8 12.8
18:1 35.6 36.9 35.6 34.7 35.5 36.4
18:2 (n-6) 26.1 26.9 26.6 27.2 20.6 20.2
18:3 (n-3) 0.6 0.3 0.3 0.3 0.8 0.2
Peroxidation products
Before inclusion in the diet
Peroxide value, mEq O2/kg 1.6 804 150 3.5 3.1 666
TBARS1, mmol/kg ⬍0.01 10.0 2.2 0.3 ND1 ND
Total polar compounds, % 2.2 43.7 46.8 37.6 3.2 53.0
Acid value, g KOH/kg 0.8 2.9 2.2 2.0 0.6 3.2
Total carbonyls, mmol/kg ⬍3 53.7 53.2 45.6 ⬍3 59.9
After inclusion in the diet
Peroxide value, mEq O2/kg 4.5 918 231 39 21 1159
TBARS, mmol/kg ⬍0.01 21.9 2.2 0.2 2.3 7.4
Conjugated dienes, mmol/kg 17 349 310 178 12 206

1 ND, not determined.

TABLE 3
Plasma total, LDL and HDL cholesterol concentrations in rats fed either fresh fat or various types of oxidized fats with either 25 or
250 mg ␣-tocopherol equivalents/kg (Experiment 1) and rats fed fresh fat or oxidized fat with 25 mg ␣-tocopherol
equivalents/kg (Experiment 2)1

Treatment
(Temperature,
Fat time) Vitamin E Total LDL HDL Plasma/HDL

mg/kg diet mmol cholesterol/L plasma

Experiment 1
Fresh2 — 25 1.72b 0.63b 0.74b 2.33a
Fresh2 — 250 1.86ab 0.70b 0.73b 2.28a
Oxidized3 50°C, 38 d 25 1.56b 0.64b 0.85a 1.86b
Oxidized3 50°C, 38 d 250 1.66b 0.72b 0.90a 1.91b
Oxidized3 105°C, 81 h 25 1.74ab 0.76ab 0.89a 1.97b
Oxidized3 105°C, 81 h 250 1.98a 0.93a 0.91a 2.24a
Oxidized3 190°C, 24 h 25 1.71b 0.71b 0.90a 1.92b
Oxidized3 190°C, 24 h 250 1.84ab 0.86a 0.89a 2.05ab
Pooled SD 0.27 0.16 0.01 0.26
ANOVA (P-value)
Fat ⬍0.05 ⬍0.05 ⬍0.001 ⬍0.001
Vitamin E ⬍0.05 ⬍0.05 NS NS
Fat ⫻ vitamin E NS NS NS NS
Experiment 2
Fresh4 — 25 2.40 0.49 1.37 1.76
Oxidized3 55°C, 49 d 25 2.03 0.77 1.15 1.53
Pooled SD 0.30 0.41 0.37 0.20
t Test ⬍0.05 NS NS NS

1 Values are means, n ⫽ 10 for each treatment group in both experiments. Values in a column in Experiment 1 not sharing a letter differ, P ⬍ 0.05.
NS, not significant (P ⬎ 0.05).
2 Sunflower oil and lard (31:69, wt/wt).
3 Sunflower oil and lard (1:1, wt/wt).
4 Sunflower oil and lard (19:81, wt/wt).
2834 EDER ET AL.

TABLE 4
Vitamin E concentrations, fatty acid composition, lag time and rate of diene production during copper-induced lipid peroxidation
in LDL of rats fed either fresh fat or various types of oxidized fats with either 25 or 250 mg ␣-tocopherol
equivalents/kg (Experiment 1)1

Oxidized fat 13 Oxidized fat 23 Oxidized fat 33 Results of ANOVA

Fat Treatment Fresh fat2 50°C, 38 d 105°C, 81 h 190°C, 24 h P-value
(temperature, time) Pooled Fat
Vitamin E 25 250 25 250 25 250 25 250 SD Fat Vitamin E ⫻ vitamin E

Vitamin E
LDL, ␮mol/mmol4 7.1c 26.4a 7.2c 24.8a 6.3c 21.3b 6.2c 20.6b 2.7 ⬍0.001 ⬍0.001 ⬍0.05
Fatty acids of LDL total
lipids, mol/100 mol
16:0 19.4b 20.4ab 21.1a 21.6a 20.7ab 20.4ab 19.5b 20.0b 1.2 ⬍0.005 NS NS
18:0 14.9 15.8 15.1 14.6 17.3 16.9 15.9 16.9 1.9 ⬍0.05 NS NS
18:1 16.1 13.0 17.7 16.5 15.4 12.8 15.1 13.9 3.7 NS ⬍0.05 NS
18:2 (n-6) 18.1a 18.0a 14.2b 14.5b 15.9b 15.6b 17.1a 16.6ab 1.6 ⬍0.001 NS NS
20:4 (n-6) 15.7b 20.2a 16.8b 20.6a 19.0ab 22.2a 21.1a 21.5a 2.9 ⬍0.005 ⬍0.001 NS
SFA 34.9b 36.5ab 36.6ab 36.5ab 38.3a 37.6a 35.7b 37.2ab 1.7 ⬍0.01 NS NS
PUFA 35.2b 39.3a 32.4c 36.6b 36.4b 39.2a 39.6a 39.5a 2.3 ⬍0.001 ⬍0.001 ⬍0.05
Lag time, min 21ab 29a 18ab 26a 15b 16b 16b 19ab 8.7 ⬍0.05 ⬍0.05 NS
Rate of oxidation, ␮mol
dienes/(min 䡠 g protein) 4.0a 3.2b 3.5ab 2.4bc 3.2b 2.1c 4.5a 2.7bc 0.8 ⬍0.05 ⬍0.05 NS

1 Values are means, n ⫽ 10 for each treatment group. Values in a column not sharing a letter differ, P ⬍ 0.05. NS, non significant (P ⬎ 0.05).
2 Sunflower oil and lard (31:69, wt/wt).
3 Sunflower oil and lard (1:1, wt/wt).
4 (Triglycerides ⫹ cholesterol).

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 con-
centrations 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 concen-
trations, 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 signif-
icantly 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.
In Experiment 1, the LDL cholesterol concentrations were
significantly influenced by the dietary fat and by the dietary
vitamin E concentration. Rats fed oxidized fat heated at 50 or
190°C had concentrations of LDL cholesterol similar to those
fed fresh fat; rats fed oxidized fat heated at 105°C had higher
concentrations than those of all other groups. Rats fed diets
containing high dietary vitamin E concentrations had higher
concentrations of LDL cholesterol than those fed diets con-
taining low vitamin E concentrations. In Experiment 2, the
LDL cholesterol concentration did not differ significantly be-
tween the groups.
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 choles-
terol did not differ between the two groups of rats.
Vitamin E concentration, fatty acid composition and cop-
per-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 ␮mol/(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 dis-
tinctly higher concentrations of vitamin E in LDL than those
fed diets with low vitamin E concentrations.
The effects of the dietary fat and the dietary vitamin E
concentration on the fatty acid composition of LDL total
lipids were small but significant. The largest effect was on the
amount of linoleic acid, which was lower in rats fed diets with
the oxidized fat heated at 50 or 105°C than in those fed diets
with the fresh fat or diets with oxidized fat heated at 190°C.
 THERMOXIDIZED FATS AND LDL IN RATS
Rats fed the diets with the oxidized fat heated at 50°C also had
significantly lower amounts of total PUFA than those fed the
diets with fresh fat or fats heated at 105 or 190°C. The
amounts of palmitic, stearic, oleic and arachidonic acid varied
less between rats fed the fresh fat diet and those fed the diets
containing the three types of oxidized fat.
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 in-
fluenced 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 ␮g 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 incor-
porated (2.58 ⫾ 2.10 ␮g 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 ␮g 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.
DISCUSSION
In this study, we investigated the effect of thermally oxi-
dized fats on selected variables associated with the develop-
ment of atherosclerosis, well aware of the fact that there are
many other events involved in this complex process. When
investigating oxidized fats, special consideration must be given
to the lipid peroxidation products formed during the heating
process. The characterization of thermally treated fats is very
difficult because the treatment generates a large number of
different products, which are partly unstable and decompose
during heating and storage. To provide a rough estimate, we
determined the POV, the concentrations of conjugated dienes,
TBARS and carbonyls and the percentage of total polar com-
pounds. For the POV and the concentrations of dienes and
TBARS, we found that diets with fats heated at low temper-
atures over a long period contained high concentrations of
primary lipid peroxidation products. The fat heated at medium
temperatures had medium concentrations and the fat heated at
high temperature had the lowest concentrations of primary
lipid peroxidation products. This might be due to the fact that
the unstable peroxidation products decompose during expo-
sure to high temperatures in volatile components, which are
stripped from the fat by air. The percentages of polar com-
pounds were similar in all heated fats and much higher than in
fresh fat. Because these compounds are formed during later
stages of lipid peroxidation, we assume that the concentrations
2835
of secondary lipid peroxidation products were high in all
oxidized fats used in this study. The observation that feeding
the oxidized fats had no marked effect on body weights of the
rats indicates that the fats used in these studies were moder-
ately oxidized. However, heating of fats leads to decomposition
of PUFA and therefore to changes in the fatty acid composi-
tion. The dietary fatty acids influence the fatty acid composi-
tion of the LDL and therefore the susceptibility of LDL to
oxidation (33). To avoid this kind of interference, the fatty
acid composition of fresh and heated fats was standardized
within each experiment by combining different fats.
In the first experiment, we investigated the effect of oxi-
dized fat on the cholesterol concentrations in plasma and
lipoproteins. Only a few, rather conflicting data exist concern-
ing cholesterol concentrations in plasma of animals fed oxi-
dized fats. Recently, we found reduced concentrations of cho-
lesterol 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) com-
pared 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 concen-
trations 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 cho-
lesterol 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 to-
gether 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). Be-
cause 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 oxi-
dized 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 concen-
trations prolonged the lag time of LDL, which was expected
and described earlier (13,38). The protective effect of vitamin
2836 EDER ET AL.
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 proathero-
genic 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 prepara-
tions 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 apoli-
poproteins. 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 reduc-
tion 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 peroxida-
tion 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 distribu-
tion.
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 peroxida-
tion products. As was observed in Experiment 1, fats heated at
different temperatures that contained various amounts of pri-
mary and secondary lipid peroxidation products could have
different effects on the antioxidant status and lipid peroxida-
tion in LDL. The effects of dietary fats heated at high tem-
peratures 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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