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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:722-728, April 2005

Biochemical and Molecular Actions of Nutrients

Grape Powder Polyphenols Attenuate Atherosclerosis Development in Apolipoprotein E Deficient (E0) Mice and Reduce Macrophage Atherogenicity1

Bianca Fuhrman2, Nina Volkova, Raymond Coleman and Michael Aviram

Lipid Research Laboratory and Department of Anatomy and Cell Biology, Technion Faculty of Medicine, Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel

2To whom correspondence should be addressed. E-mail: Fuhrman{at}tx.technion.ac.il.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The beneficial health effects of red wine have been attributed to the antioxidant activity of its polyphenols. The present study investigated the effects of a standardized freeze-dried powder made from fresh grapes, rich in grape-specific polyphenols and free of alcohol, on oxidative stress, atherogenicity of macrophages, and the development of atherosclerotic lesions in apolipoprotein E deficient (E0) mice. Thirty E0 mice were assigned to 3 groups. Mice consumed water alone (control), 150 µg total polyphenols/d in the form of grape powder (grape powder), or the equivalent amount of glucose and fructose (placebo) in drinking water for 10 wk. Consumption of grape powder reduced the atherosclerotic lesion area by 41% (P < 0.0002) compared to the control or placebo mice. The antiatherosclerotic effect was at least partly due to a significant 8% reduction in serum oxidative stress, an up to 22% increase in serum antioxidant capacity, a significant 33% reduction in macrophage uptake of oxidized LDL, and a 25% decrease in macrophage-mediated oxidation of LDL relative to controls. Grape powder directly protected both plasma LDL and macrophages from oxidative stress in vitro. We conclude that polyphenols from fresh grape powder directly affect macrophage atherogenicity by reducing macrophage-mediated oxidation of LDL and cellular uptake of oxidized LDL. Both of these processes can eventually reduce macrophage cholesterol accumulation and foam cell formation and hence attenuate atherosclerosis development.

KEY WORDS: • grape • polyphenols • wine • lipid peroxides • macrophage

The oxidative hypothesis of atherosclerosis development has stimulated extensive investigation of a possible preventive role of antioxidants. The formation of macrophage foam cells during early atherogenesis depends on the balance between pro-oxidants and antioxidants in arterial wall cells, as well as in plasma lipoproteins (1). Antioxidants that prevent oxidative stress, such as vitamin E or polyphenolic flavonoids, as well as polyphenol-rich foods, protect LDL from oxidation and, in parallel, reduce the development of atherosclerotic lesions (2–5). However, oxidative stress can attack lipids not only in plasma lipoproteins, but also in arterial macrophages (6,7). Such "oxidized lipid-rich macrophages" exhibit atherogenic characteristics, including increased ability to oxidize LDL and enhanced uptake of oxidized LDL (Ox-LDL)3 (8). Enrichment of macrophages with antioxidants increases cell resistance to oxidation and in parallel reduces cellular atherogenicity (8,9).

The "French Paradox," i.e., a low incidence of cardiovascular events in southern France despite a diet rich in saturated fat, was attributed to the regular drinking of red wine (10). The beneficial effect of red wine consumption against the development of atherosclerosis was attributed to both the antioxidant activity of its polyphenols (11–13) and its alcohol component (14,15). We have previously shown that red wine consumption by humans (16) or by apolipoprotein E deficient (E0) mice (17) inhibits oxidation of LDL and attenuates the development of atherosclerosis (18). However, grape juice (19) or dealcoholized wine (20–22) also inhibits atherosclerosis in animal models by mechanisms independent of serum lipid peroxidation inhibition.

The aim of the present study was to investigate the antiatherosclerotic activity in vivo of a standardized freeze-dried powder preparation made from fresh grapes and rich in grape-specific polyphenols and to determine whether this activity is related to the antioxidant capacity of the polyphenols. The study was performed with atherosclerotic E0 mice. These mice were created by gene-targeting techniques, and they are characterized by accelerated development of atherosclerosis along with increased oxidative state.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Experimental protocol

E0 mice were kindly provided by Dr. Jan Breslow, Rockefeller University. At 6 wk of age, 30 E° mice were assigned randomly to 3 groups, 10 mice in each group. The mice received their standard, nonpurified diet,4 supplemented for 10 wk (via their drinking water) with the following: the control group received tap water; the placebo group received a 1:1 mixture of glucose and fructose, 10 g/L; and the grape powder group received 30 mg grape powder/d, equivalent to 150 µg of total polyphenols/d.

Water intake did not differ among mice. Each mouse consumed ~5 mL of water/d and 4–5 g of food/d. Grape powder intake did not cause the mice to lose their appetite.

At the end of the experiment, mouse peritoneal macrophages (MPM), heart, aorta, and blood samples were collected from all mice.

The experimental protocol was approved by the Animal Care and Use Committee of the Technion, No. IL-066–10-2001.

Methods

    Grape freeze-dried extract preparation. Freeze-dried grape powder was prepared and supplied by the California Table Grape Commission. The freeze-dried grape powder is a composite of fresh red, green, and blue-black California grapes (seeded and seedless varieties) that have been frozen, ground with food-quality dry ice, freeze-dried, and reground. The powder was processed and stored to preserve the integrity of biologically active compounds found in fresh grapes. The dry powder contains 90% sugar, which is half glucose and half fructose, and ~1% moisture (fresh grapes contain ~82% moisture). Thus, 100 g of fresh grapes corresponds to ~18.2 g of powder. Selected phytochemical profiles of the freeze-dried grape powder are shown in Table 1. Because sugar makes up the majority of the powder, we prepared an appropriate control consisting of a mixture of glucose and fructose (1:1, w:w), which was administered to the mice in a similar manner as the grape powder.


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  		TABLE 1 Selected phytochemicals in freeze-dried grape powder

 
Grape powder was dissolved in water at a concentration of 10 g/L. The suspension was stirred using a magnetic stirrer for 5 h at room temperature. Then the suspension was left overnight at 4°C so that the insoluble materials would settle. The upper liquid phase was separated and used for supplementation and for the in vitro studies.

    Polyphenol measurement. Total polyphenols were determined spectrophotometrically and analyzed with Folin and Ciocalteu’s phenol reagent by the method of Singleton and Rossi, modified for small volumes (23). Quercetin (3, 6, 13, and 19 µg) served as a standard. Flavanols were analyzed by reaction with vanillin; anthocyanins were analyzed spectrophotometrically; flavonols and resveratrol were analyzed by HPLC after acid hydrolysis.

    LDL preparation. LDL was separated from plasma of normal healthy volunteers by discontinuous density-gradient ultracentrifugation (24) and dialyzed against saline with EDTA (1 mmol/L). Before the oxidation study, LDL was diluted in phosphate-buffered saline (PBS) to 1 g protein/L and dialyzed overnight against PBS at 4°C to remove the EDTA. LDL protein concentration was determined with the Folin phenol reagent (25). LDL was radioiodinated by the iodine monochloride method as modified for lipoproteins (26).

    LDL oxidation. LDL (100 mg of protein/L) was incubated for 10 min at room temperature with or without (for Ox-LDL preparation) water extract from grape powder (25, 37.5, 50, 75, and 125 mg/L). Oxidation of LDL was carried out at 37°C under air in a shaking water bath or in the spectrophotometer cuvettes. LDL was incubated for 2 h at 37°C with freshly prepared CuSO4 (5 µmol/L) or with 5 mmol/L of 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH). Oxidation was terminated by refrigeration at 4°C. Radioiodinated Ox-LDL was prepared by oxidation of 125I-LDL.

LDL oxidation was determined by continuous monitoring of the formation of conjugated dienes by measuring the increase in absorbance at 234 nm (27) or by measuring the amounts of TBARS and lipid peroxides (28). Determination of LDL-associated lipid peroxide is based on the oxidative activity of lipid peroxides that convert iodide to iodine and is measured spectrophotometrically.

    Macrophage oxidation. A J-774A.1 macrophage-like cell line was obtained from the American Tissue Culture Collection. Cells were preincubated with increasing concentrations of grape powder water extract. Intracellular oxidative stress was assayed through the oxidation of 2',7'-dichlorofluorescein diacetate (DCFH-DA) (29) and monitored by flow cytometry (30,31). For determination of macrophage resistance to oxidation, the cells were incubated for 5 h at 37°C with FeSO4 (50 µmol/L) or AAPH (5 mmol/L). Then the cells were washed; cellular lipids were extracted with hexane isopropanol (3:2, v:v); and the hexane phase was separated, evaporated, and used to measure lipid peroxides, as described for LDL.

    Macrophage-mediated LDL oxidation. J-774A.1 macrophages were incubated with LDL (100 mg of protein/L) under conditions of oxidative stress in the presence of CuSO4 (2 µmol/L) for 5 h at 37°C. After the incubation the extent of LDL oxidation in the medium was measured by the TBARS assay (28). Values obtained for LDL incubated under similar conditions in the absence of cells were subtracted from values obtained in the presence of cells.

    Serum lipids. Within each group, blood samples were analyzed individually (n = 10).

Serum total cholesterol and triglycerides were measured using commercially available kits (Raichem, Clinical Chemistry Reagents, Cat. 85464 and Cat. 84098, respectively).

    Serum lipid peroxidation. Serum was diluted 1:4 with PBS and incubated with 100 mmol/L of AAPH. Serum susceptibility to oxidation was determined by measuring lipid peroxides and TBARS formation (28).

    MPM preparation. Mice were killed by an overdose of anesthesia. MPM were harvested before removing the heart and aorta from the peritoneal fluid of the E0 mice (15–25 g) 4 d after intraperitoneal injection of 3 mL of thioglycolate (24 g/L) in saline into each mouse. The cells (10–20 x 106/mouse) were washed and centrifuged 3 times with PBS at 1000 x g for 10 min and then resuspended to 109/L in DMEM containing 15% horse serum (heat-inactivated at 56°C for 30 min), 0.1 U/L penicillin, 100 mg/L streptomycin, and 2 mmol/L glutamine. The cell suspension was dispensed into 35-mm plastic petri dishes and incubated in a humidified incubator (5% CO2, 95% air) for 2 h. The dishes were washed once with 5 mL DMEM to remove nonadherent cells, and the monolayer was then incubated under similar conditions for 18 h before beginning the experiment.

    Uptake of lipoproteins by MPM. Radioiodinated oxidized LDL (125I-Ox-LDL) or native LDL (125I-LDL), at a concentration of 10 mg of protein/L, was incubated with the cells at 37°C for 5 h. Lipoprotein cellular degradation was measured in the collected medium as the trichloroacetic acid (TCA) soluble, nonlipid radioactivity, which was not due to free iodide (32). Lipoprotein degradation in a cell-free system measured under identical conditions was minimal (<10%) and was subtracted from the total degradation. The remaining cells were washed 3 times with cold PBS and dissolved in 0.1 mol/L of NaOH for the determination of protein.

    mRNA expression of CD36 in MPM. mRNA expression of CD36 in MPM was analyzed by RT-PCR. Total RNA was extracted from the cells with Tri-reagent (Molecular Research Center). cDNA was generated from 1 µg of total RNA using RT (Boehringer-Mannheim). GAPDH served as a housekeeping gene. Products of the RT reaction were diluted 1:10 and subjected to PCR amplification into 40 µL using specific primers (Genset SA): CD36: sense, 5'-TGC GAA CTG TGG GCT CAT TG-3', antisense, 5'-CCT CGG GGT CCT GAG TTA TAT TTT C-3'; GAPDH: sense, 5'-CTG CCA TTT GCA GTG GCA AAG TGG-3', antisense, 5'-TTG TCA TGG ATG ACC TTG GCC AGG-3'.

    Histopathology of aortic atherosclerotic lesions. After 10 wk of treatment, mice were anesthetized with ethyl ether in a local nasal container. Each heart and entire aorta was rapidly dissected out and immersion fixed in 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer with 0.01% calcium chloride, pH 7.4, at room temperature. The histopathological development of the lesions was analyzed as previously described (17).

    Statistical analysis. Student’s t test was used to compare grape powder and control solutions in vitro. The groups of mice were compared by ANOVA and post hoc t tests. Values are means ± SD or means ± SEM for the in vitro and in vivo experiments, respectively. Differences were considered significant at P < 0.05.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
After 10 wk of treatment, body weights, serum cholesterol, and serum triglycerides did not differ among the 3 groups (data not shown). Aortic arches of all mice in the control and placebo groups showed extensive atherosclerotic lesions. On the contrary, in the mice that consumed grape powder, 1 did not develop atherosclerotic lesions at all, and in the other 9 the lesion area was 41% smaller (P < 0.0002) than in the control and placebo groups (Fig. 1). In the control and placebo-treated mice, the lesions were large and consisted of small groups of lipid-laden macrophage foam cells in the tunica intima. In mice that consumed grape powder, the lesions were much smaller in size, with only a few foam cells present.



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  		FIGURE 1 The effect of grape powder consumption by atherosclerotic E0 mice on foam cell formation and the size of atherosclerotic lesions. (A) Values are means ± SEM, n = 10. Means without a common letter differ, *P < 0.0002. Photomicrographs of typical atherosclerotic lesions of the aortic arch of E0 mice in the control (B), placebo (C), and grape powder–treated (D) groups.

 
Grape powder consumption reduced MPM capacity to oxidize LDL by 25% (P < 0.01) (Fig. 2A) and reduced MPM uptake of Ox-LDL by 33% (P < 0.003) (Fig. 2B), compared with control or placebo consumption. CD36 mRNA expression in MPM did not differ among the 3 groups (Fig. 2C), suggesting that the reduction in Ox-LDL uptake induced by grape powder was not mediated via downregulation of the CD36 receptor at the gene transcription level. MPM uptake of native LDL did not differ among the 3 groups (52 ± 6, 62 ± 15, and 58 ± 9 µg of LDL protein/mg cell protein in control, placebo, and grape groups, respectively).



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  		FIGURE 2 Effect of grape powder consumption by E0 mice on macrophage-mediated LDL oxidation (A) and macrophage uptake of oxidized LDL (B) by MPM from control, placebo, and grape groups. (A) Macrophage-mediated oxidation of LDL (100 mg protein/L) by MPM determined in the medium by the TBARS assay. Means without a common letter differ, P < 0.01. (B) Degradation of 125I-Ox-LDL (10 mg lipoprotein protein/L). Values are means ± SEM, n = 10, Means without a common letter differ, *P < 0.003. (C) Expression of CD36 and LDL receptor mRNA in MPM from control, placebo, and grape groups. GAPDH cDNA product was used as internal standard.

 
Consumption of grape powder reduced the basal oxidative stress in plasma, as demonstrated by an 8% (P < 0.01) reduction in plasma lipid peroxide concentration (from 585 ± 37 µmol/L and 596 ± 24 µmol/L in the control and placebo groups, respectively, to 536 ± 21 µmol/L in the grape group). The antioxidant capacity of serum did not differ between placebo and control groups, but it was reduced by 16 and 22% (P < 0.02) when measured as lipid peroxide formation (Fig. 3A) or as TBARS formation (Fig. 3B), respectively, in the grape group compared with the control and placebo groups.



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  		FIGURE 3 Effect of grape powder consumption by E0 mice on serum oxidation. AAPH-induced oxidation of serum from control, placebo, or grape groups was determined as lipid peroxide formation (A) or as TBARS formation (B). Values are individual data and means ± SEM, n = 10. Means without a common letter differ, *P < 0.02.

 
The addition of increasing concentrations of grape powder (25–125 mg/L) to LDL dose-dependently inhibited copper ion–induced LDL oxidation (Fig. 4A, B). The IC50 (inhibition of LDL oxidation by 50%) of grape powder was 48 mg/L for TBARS (Fig. 4A) and 45 mg/L for lipid peroxide formation (Fig. 4B). The maximum inhibitory effect (>99%) occurred at 125 mg/L for both TBARS and lipid peroxide formation. Incubation of LDL (100 mg of protein/L) with 5 µmol/L CuSO4 resulted in the formation of oxidized LDL after a lag time of 45 min, as evidenced by conjugated diene formation. However, incubation of LDL with CuSO4 in the presence of increasing concentrations of grape powder retarded the onset of LDL lipid peroxidation in a dose-dependent manner (Fig. 4C). Grape powder at 150 mg/L prolonged the lag phase by >100% (up to 100 min). Similarly to the effect of grape powder in the copper ion–induced oxidation system, the addition of increasing concentrations of grape powder (25–125 mg/L) also inhibited AAPH-induced LDL oxidation in a dose-dependent manner. The IC50 for grape powder was 52 µg/L for TBARS formation (Fig. 4D) and 49 mg/L for lipid peroxide formation (Fig. 4E). The maximum inhibitory effect occurred at 0.25 mg/L (by 92% for TBARS and by 74% for lipid peroxides). These results suggested that grape powder directly inhibits lipid peroxidation in LDL.



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  		FIGURE 4 The effect of grape powder on LDL oxidation induced by copper ion (A–C) or by AAPH (D,E). LDL (100 mg protein/L) were incubated with 25–125 mg/L grape powder. LDL oxidation was measured as TBARS (A, D), as kinetic formation of conjugated dienes (C), or as lipid peroxide formation (B, E). The IC50 values represent the inhibition of LDL oxidation by 50%. Values are means ± SD, n = 3.

 
Grape powder was tested next for a direct antioxidative effect on macrophages. Preincubation of macrophages in vitro with increasing concentrations of grape powder reduced cellular oxidative stress, determined by DCF fluorescence, by up to 50% compared to control cells (Fig. 5A). Incubation of J-774 A.1 macrophages with AAPH or with iron ions increased cellular lipid peroxide content by 27 and 78% (P < 0.01), respectively (Fig. 5B). However, preincubation of the cells for 30 min with grape powder (250 mg/L) inhibited formation of lipid peroxides in macrophages by 17 and 18% (P < 0.01), respectively (Fig. 5B). Furthermore, preincubation of the cells for 30 min with grape powder (125 or 250 mg/L) reduced the capacity of the cells to oxidize LDL by 46 and 90% (P < 0.01), respectively (Fig. 5C).



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  		FIGURE 5 Effect of grape powder on cellular oxidative stress in J-774 A.1 macrophages. (A) Representative histogram of DCFH-DA fluorescence intensity generated by 10,000 cells. (B) Lipid peroxides in control and grape powder–treated macrophages exposed to AAPH (5 mmol/L) or to FeSO4 (50 µmol/L) for 5 h at 37°C. Values are means ± SD, n = 3. For (–) grape powder, means marked b and c differ from that marked a, P < 0.01. #, Different from respective untreated macrophages marked *, P < 0.01. (C) LDL oxidation by control macrophages or by grape powder–treated macrophages (125 and 250 mg/L) determined by the TBARS assay. Values are means ± SD, n = 3. Means marked b and c differ from that marked a, P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The present study demonstrates that consumption of fresh grape freeze-dried powder has a remarkable inhibitory effect on the development of atherosclerotic lesions in atherosclerotic apolipoprotein E deficient mice, which could be related to the antioxidant capacity of the grape powder polyphenols. Grape powder polyphenols reduced macrophage uptake of Ox-LDL, in parallel to the reduction in oxidative stress in serum and in macrophages.

In a previous study, we demonstrated that the uptake of Ox-LDL by macrophages positively correlates with the oxidative status of the cells (8). It is possible then that the reduced uptake of Ox-LDL due to grape powder was secondary to the grape powder–mediated reduction in oxidative stress in the cells. Our results demonstrated that grape powder can directly reduce the basal oxidative stress in macrophages and the oxidative capacity of macrophages toward LDL, ex vivo (when harvested from mice after the consumption of grape powder) and in vitro (after macrophage incubation with grape powder). Furthermore, in the in vitro study, we demonstrated that grape powder also increased the resistance of the cells to oxidation induced by free radicals or by iron ions. Taken together these results evidence that grape powder has a direct effect on macrophages by reducing their oxidative capacity. However, macrophage oxidative stress is determined not only by the balance between oxidants and antioxidants within the cells, but also by the oxidative stress in the environment, i.e., the serum lipoproteins (1). Therefore, the increase in serum antioxidant capacity ex vivo due to grape powder consumption may have also contributed to the reduction in macrophage oxidative stress. Consequently, reduced macrophage oxidative stress can lead to reduced cellular uptake of Ox-LDL, in agreement with previous results (8). Therefore, the inhibition in atherosclerosis development observed in E0 mice that consumed grape powder could be related, at least in part, to the reduction in oxidative stress.

In a previous study (17), we demonstrated that treatment of E0 mice with whole red wine (equivalent to 50 µg polyphenols/d) reduced atherosclerotic lesion development by 48%, compared with a 41% reduction in the present study after administration of 150 mg grape powder polyphenols/d. Thus, it is possible that the alcohol in red wine also contributed to the inhibition in the development of atherosclerotic lesions. Indeed, previous studies showed that alcohol itself has antiatherosclerotic effects (14,15), thus increasing the antiatherosclerotic effect of red wine beyond that of pure polyphenols.

In both cases, the reduction in atherosclerotic lesion development could be related to a reduction in serum lipid oxidative stress. However, the present study extends the previous findings by showing that grape polyphenols exert an antioxidative protective effect not only on serum lipids, but also on macrophages. This demonstrates that polyphenols are the main constituents in grape powder, as well as in red wine, that confer their antiatherosclerotic effects, probably due to their antioxidant capacity.

Recent studies demonstrated, however, that dealcoholized red wine inhibited atherosclerosis in E0 mice independently of its effect on lipid peroxidation (20,21). This conclusion was based on measurement of F2-isoprostanes and hydroxyeicosatetraenoic acid as biomarkers for lipid peroxidation. Although both of these fatty acid peroxidation products are regarded as good markers for in vivo lipid peroxidation, they represent only a minor component of fatty acids in biological systems and may not represent the bulk of in vivo serum lipid peroxidation (33,34). Thus, although isoprostanes are specific end products of PUFA peroxidation, they do not qualify as ideal biomarkers because they represent a minor fraction of serum lipid peroxidation; also, the amount formed is influenced by variables such as molecular oxygen concentration and saturation (35). In the present study, we evaluated lipid peroxidative damage in serum and in LDL by measuring lipid peroxides and TBARS formation, as well as by kinetic monitoring of the formation of conjugated dienes. Lipid peroxidation in cells was evaluated by the DCFH assay and by cellular oxidative capacity toward LDL. Furthermore, we evaluated the resistance of the cells to oxidation induced by iron ions or by AAPH (both are inversely related to the initial oxidative state of the cells). In all of these assays, we demonstrated that grape powder reduced the oxidative stress in plasma in vivo, as well as in macrophages ex vivo and in vitro, thus leading to the conclusion that, at least in part, grape powder reduces foam cell formation via a direct inhibition of oxidative stress in both macrophages and serum. Stocker and O’Halloran (20) have also found a decrement in lipid peroxidation, measured as cholesterylester hydroperoxides, in plasma obtained from E0 mice treated with dealcoholized red wine (but not in their aortas). However, in this study the mice consumed the dealcoholized red wine for a period of 24 wk, whereas in the present study, grape powder was administered to mice for a period of 10 wk only. This may explain the lack of inhibition of lipid peroxidation in the aorta in their study, because lipid peroxidation in E0 mice increases with age (8), in parallel with the stage of atherosclerosis (36), and the oxidative damage probably overcomes the protection offered by the polyphenols.

In summary, we conclude that grape powder consumption reduces macrophage uptake of Ox-LDL secondary to its antioxidative effect against lipid peroxidation in plasma and in macrophages. These conclusions are further strengthened by our previous results (8), which showed that administration of antioxidants (vitamin E or the licorice-derived polyphenol glabridin) to E0 mice reduced both the oxidative stress in macrophages and, in parallel, macrophage degradation of Ox-LDL. Thus, consumption of fresh grape-derived powder rich in polyphenols may confer a health benefit against macrophage cholesterol accumulation and may inhibit enhanced development of atherosclerosis.


   FOOTNOTES
 
1 Supported by a grant from the California Table Grape Commission, Fresno, CA. Back

3 Abbreviations used: AAPH, 2,2-azobis(2-amidinopropane dihydrochloride); DCFH-DA, 2',7'-dichlorofluorescein diacetate; E0, apolipoprotein E deficient; MPM, mouse peritoneal macrophages; Ox-LDL, oxidized LDL; PBS, phosphate buffered saline; TCA, tricholoroacetic acid. Back

4 Food was supplied by Koffolk Ltd, Tel-Aviv, Israel. Its ingredients are as follows: 210 g/kg total protein, 40 g/kg total fat, 45 g/kg cellulose, 70 g/kg ash, 8–12 g/kg calcium, 7–9 g/kg phosphorous, 3 g/kg chlorides, and 2.5 g/kg sodium. Back

Manuscript received 29 September 2004. Initial review completed 20 October 2004. Revision accepted 21 January 2005.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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