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Biochemical and Molecular Actions of Nutrients

Lipoprotein Oxidation Mediated by J774 Murine Macrophages Is Inhibited by Individual Red Wine Polyphenols but Not by Ethanol

Department of Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903

¹To whom correspondence should be addressed. E-mail: khachaav{at}umdnj.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The in vitro capacities of individual polyphenols in red wine to inhibit the cell-mediated oxidation of lipoproteins and their effects on cell viability were determined. LDL and HDL were incubated with J774.A1 macrophages and 2 and 4 µmol/L copper, respectively, in the absence and presence of polyphenols in ethanol at concentrations found in red wine. A mixture of polyphenols in amounts found in red wine equivalent to 0.2 g/L ethanol and 0.05 g/L ethanol inhibited thiobarbituric acid-reactive substance production from LDL by 91.7 and 45.9%, respectively, compared with ethanol controls (P < 0.01). HDL oxidation was inhibited 85 and 82.4% by the polyphenols at 0.2 and 0.05 g/L ethanol (P < 0.01). The effects of the polyphenol mixture on LDL oxidation were confirmed by measuring production of conjugated dienes and lipid peroxides, and trinitrobenzene sulfonic acid reactivity. Catechin at the concentration found in red wine (1.32 µmol/L) at an ethanol concentration equivalent to 0.2 g/L inhibited LDL oxidation by 83.2%, while epicatechin (0.56 µmol/L) and gallic acid (1.02 µmol/L) inhibited by 60.6 and 26.9%, respectively (P < 0.05). At 1 µmol/L, LDL oxidation was inhibited by epicatechin, catechin and quercetin by 86.2, 79.9 and 69.4%, respectively (P < 0.05). Incubation of macrophages with ethanol alone and with polyphenols in ethanol did not affect cell viability. Our results indicate that catechin and epicatechin are the major contributors to the antioxidant activity of red wine.

KEY WORDS: • wine • polyphenol • lipoprotein oxidation • macrophage • cell viability

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Several lines of evidence indicate that the oxidative modification of LDL and their accelerated uptake by artery wall macrophages contribute to the formation of atherosclerotic plaques (1 ,2 ). The results of in vitro studies suggest that oxidized HDL have a diminished role in reverse cholesterol transport (3 ), an effect that is also potentially atherogenic. We have previously determined the in vitro effects of red and white wines and ethanol on LDL and HDL oxidation mediated by J774.A1 macrophages. At a concentration equivalent to 0.2 g/L ethanol, red wine had three- to fourfold higher antioxidant activity than white wine as assessed by conjugated diene formation, thiobarbituric acid-reactive substance (TBARS)² production and trinitrobenzene sulfonic acid (TNBS) reactivity (4 ). Ethanol at 1 g/L did not affect LDL or HDL oxidation, indicating that the inhibitory effect of red wine was not due to its ethanol content. Because the antioxidant activity of red wine correlates with its polyphenol content (5 ,6 ) we studied the effects of several polyphenols found in red wine, individually and in combination, on the cell-mediated oxidation of lipoproteins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Chemicals

Cell culture reagents, media and serum, reagents for lipoprotein oxidation and cell viability assays, polyphenols, butylated hydroxytoluene (BHT) and vitamin E (DL--tocopherol) were obtained from Sigma-Aldrich (St. Louis, MO). Copper nitrate solution (1 g/L) was from Fisher Scientific (Springfield, NJ). Bicinchoninic acid protein assay reagents were from Pierce (Rockford, IL). The Cabernet Sauvignon (12.5% alcohol by volume) was from Robert Mondavi (Woodbridge, CA). An aqueous solution of polyphenols and ethanol at concentrations found in California red wine (7 ) was prepared; it contained 660 µmol/L catechin, 510 µmol/L gallic acid, 280 µmol/L epicatechin, 20 µmol/L quercetin, 6 µmol/L trans-resveratrol and 100 g ethanol/L. The polyphenol mixture and individual polyphenols were added to incubations based on a final ethanol concentration of 0.2 g/L or as indicated; the concentrations of the polyphenols in the incubations are listed in Results.

Lipoprotein preparation

Initial experiments (see Fig. 1 ) were performed using lipoproteins isolated from four fasting healthy adults (two males and two females) whose serum lipid concentrations were within normal ranges. The subjects maintained a constant diet and level of physical activity and did not consume any antioxidant vitamin supplements for 4 wk before or any wine or other alcoholic beverages for 2 wk before and during the course of the study. Because similar results were obtained using samples from each subject, subsequent experiments were performed using serum obtained on several occasions from one of the men whose serum antioxidant vitamin concentrations and total antioxidant status were within normal ranges (4 ). Use of human subjects was approved by the Robert Wood Johnson Medical School institutional review board. LDL were isolated at 1.019 kg/L < d < 1.063 kg/L and HDL were isolated at 1.063 kg/L < d < 1.21 kg/L from serum by sequential ultracentrifugation (8 ) as previously described (9 ). After isolation, the lipoproteins were dialyzed against 150 mmol/L sodium chloride, 0.2 g/L ethylenediamine tetraacetic acid (EDTA) and 2 mmol/L sodium phosphate (pH 7.4) and passed through a 0.45-µm filter. Protein content of the fractions were estimated by the bicinchoninic acid method (10 ) using bovine serum albumin as the standard.

View larger version (24K): [in this window] [in a new window]   FIGURE 1 Effects of the polyphenol mixture and red wine (equivalent to 0.2 g/L ethanol) on LDL oxidation (A) and HDL oxidation (B) in the absence and presence of J774 macrophages. Control incubations contained 0.2 g/L ethanol. Values are mean ± SEM; n = 7. Means with different letters differ (P < 0.01; two-way ANOVA and Bonferroni’s post-hoc test).

Murine monocyte-derived J774.AI macrophages were obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture in Dulbecco’s minimum essential medium (DMEM) with phenol red and 100 mL/L bovine calf serum, 100,000 U/L penicillin and 100 mg/L streptomycin at 37°C under 5% CO₂ in air. Cells in DMEM with serum and antibiotics were plated in 24-well plates (100,000 cells/well in 1 mL) for lipoprotein oxidation measurements or 96-well plates (20,000 cells/well in 200 µL) for cell viability measurements. In some experiments, medium was added to wells without cells. After 24 h the wells were washed with serum-free Hank’s balanced salt solution (HBSS) without phenol red before the addition of experimental media. Subsequent cell incubations were done at 37°C.

Cell-mediated lipoprotein oxidation

LDL and HDL were dialyzed at 4°C against phosphate-buffered saline (PBS) with calcium, magnesium and 10 mmol/L sodium bicarbonate (pH 7.4) to remove EDTA. Incubations were conducted at 37°C in serum-free DMEM without phenol red. Copper is required to catalyze cell-mediated lipoprotein oxidation in this medium (11 ). Macrophages are found in atherosclerotic lesions, and there is evidence indicating that arterial wall cells modify LDL in vivo (12 ). We have previously demonstrated (4 ) that J774 macrophages stimulate lipoprotein oxidation in vitro. LDL (200 mg protein/L) were incubated without and with macrophages for 20 h in DMEM containing 2 µmol/L copper. HDL (400 mg protein/L) was incubated without and with macrophages as above for 24 h in DMEM containing 4 µmol/L copper. HDL were added at a higher protein concentration so that incubations with LDL and HDL were closer in amounts of lipoprotein lipid. The copper to protein ratio was kept constant (13 ). Results of previous studies (9 ,11 ) indicated that copper at these concentrations does not negatively affect the capacity of cells to oxidize lipoproteins in vitro. Samples of media were taken for assays of LDL and HDL oxidation. Incubation of macrophages under these conditions with ethanol, polyphenols in ethanol, or wine did not affect their appearance or their adherence to the incubation wells as determined by light microscopy.

Assays of lipoprotein oxidation

Lipoprotein oxidation was quantified in all experiments by TBARS production, and additionally in the experiment shown in Table 1 by conjugated diene formation measured at 234 nm, lipid peroxide production, and loss of TNBS-reactive lysine amino groups on the apolipoproteins. TBARS are expressed as nmol malondialdehyde (MDA) equivalents/mg lipoprotein protein using tetramethoxypropane as the standard (14 ). Change in absorbance at 234 nm (A234) was determined after a 1/10 dilution with 0.1 g/L EDTA, 150 mmol/L sodium chloride and 10 mmol/L sodium bicarbonate (pH 7.6) (15 ) and is expressed relative to unmodified LDL. Lipid peroxides were measured by reaction with ammonium ferrous sulfate and xylenol orange and expressed as nmol/mg protein using hydrogen peroxide as the standard (16 ). TNBS reactivity is expressed as the percentage of lysine that remains reactive with TNBS after oxidation compared with reactive lysine in unincubated, unmodified LDL (17 ). A decrease in TNBS reactivity is an indication of increased derivitization of apoproteins resulting from the formation of apoprotein adducts of lipid oxidation products. Samples of experimental incubation media were dialyzed against 0.01% EDTA, 150 mmol/L sodium chloride and 10 mmol/L sodium bicarbonate (pH 7.6) before being assayed for A234 and TNBS reactivity. We have previously shown that TBARS were not detected in cell incubations that did not contain lipoproteins (13 ). Experiments were performed using two to four replicates per condition.

Cell viability was measured by the conversion of dimethylthiazol-diphenyltetrazolium bromide (MTT) to its formazan by mitochondrial dehydrogenases (18 ). Macrophages were incubated for 20 h in the absence and presence of LDL (200 mg protein/L) in DMEM containing 2 µmol/L copper without and with the various polyphenols or BHT, all at 1 µmol/L. After this incubation, 50 µg of MTT in 20 µL of HBSS were added directly to each well without removing the media. After 3 h at 37°C, 100 µL of 15 g/L sodium dodecyl sulfate in 500 mL/L dimethyl formamide were added to each well. After 20 h at room temperature the absorbances were measured at 540 nm in a microtiter plate spectrophotometer. Experiments were performed using four replicates.

Data analysis

Data were entered into the GraphPad Prism statistical analysis program (GraphPad, San Diego, CA). Comparisons of results from various incubation conditions were done by one-way, two-way or repeated measures analysis of variance (ANOVA). Where significant effects were found, post-hoc analysis using Dunnett’s, Bonferroni’s, or Tukey’s tests were performed as described in the table footnotes and figure legends. Results are expressed as mean ± SEM; n = number of independent observations.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
LDL oxidation measured by TBARS production was threefold higher in the presence of J774 macrophages compared with incubations in the absence of cells (Fig. 1 A). When LDL were incubated without and with red wine and the polyphenol mixture at 0.2 g/L ethanol, there were 80.9 and 77.6% reductions in TBARS production in the absence of cells and 91.7 and 93.8% reductions in the presence of cells with the addition of the polyphenols and red wine, respectively (Fig. 1 A). The effects of the polyphenols on LDL oxidation were confirmed by additional measurements of lipoprotein oxidation based on changes in A234, lipid peroxide formation and loss of TNBS reactivity (Table 1) .

In the presence of J774 macrophages, HDL oxidation measured by TBARS production was twofold higher compared with incubations in the absence of cells (Fig. 1 B). In the presence and absence of red wine and the polyphenol mixture at 0.2 g/L ethanol, there were 77 and 80.9% reductions in TBARS production in the absence of cells and 85 and 88.3% reductions in the presence of cells with the addition of the polyphenol mixture and red wine, respectively.

The effects of incubating LDL and HDL with various concentrations of ethanol, red wine and the polyphenol mixture on cell-mediated oxidation are shown in Figure 2 . For both LDL (Fig. 2 A) and HDL (Fig. 2 B) oxidation, TBARS production was inhibited to a similar extent by the polyphenol mixture and the red wine. Ethanol alone did not inhibit the cell-mediated oxidation of either lipoprotein. Polyphenols and wine at 0.05 g/L ethanol inhibited cell-mediated LDL oxidation by 45.9 and 61.2% (Fig. 2 A). The concentrations for 50% inhibition of LDL oxidation by the polyphenol mixture and red wine approximated from the data shown in Figure 2 A were 0.05 and 0.04 g/L ethanol, respectively. In additional experiments, the polyphenol mixture without catechin or epicatechin at 0.2 g/L ethanol inhibited TBARS production by 72.6 and 89.1%, respectively, compared with 90.2% by the complete mixture. Polyphenols and wine at 0.05 g/L ethanol inhibited cell-mediated TBARS production from HDL by 82.4 and 80.9% (Fig. 2 B).

View larger version (23K): [in this window] [in a new window]   FIGURE 2 Concentration-dependent effect of ethanol, the polyphenol mixture and red wine on cell-mediated oxidation of LDL (A) and HDL (B). Values are mean ± SEM; n = 3. An "a" above a data point indicates that means for ethanol differ from polyphenols or red wine (P < 0.002; repeated measures analysis of variance (ANOVA) and Bonferroni’s post-hoc test).

View larger version (59K): [in this window] [in a new window]   FIGURE 3 Cell-mediated oxidation of LDL in the absence and presence of various polyphenols at concentrations found in red wine, compared with the polyphenol mixture and red wine. The concentrations were as follows: catechin, 1.32 µmol/L; gallic acid, 1.02 µmol/L; epicatechin, 0.56 µmol/L; quercetin, 0.04 µmol/L; resveratrol, 0.012 µmol/L. All incubations contained ethanol at 0.2 g/L. The mean thiobarbituric acid-reactive substance (TBARS) production in the control incubation (represented as 100%) was 18.5 ± 0.2 nmol malondialdehyde (MDA)/mg LDL protein. Values are mean ± SEM; n = 4. Means with different letters differ (P < 0.05; one-way analysis of variance (ANOVA) and the post-hoc Tukey’s test).

View larger version (12K): [in this window] [in a new window]   FIGURE 4 Cell-mediated oxidation of LDL in the absence and presence of epicatechin, catechin and resveratrol at various concentrations. All incubations contained ethanol at 0.2 g/L. Results are the mean ± SEM. Data were not analyzed statistically because of the small numbers of observations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We compared the ability of several polyphenols found in red wine, individually and in combination, to inhibit cell-mediated oxidation of LDL and HDL. A mixture of polyphenols at concentrations found in red wine was as effective as red wine in inhibiting the oxidation of LDL and HDL, suggesting that these polyphenols are associated with the antioxidant activity of red wine. Miller and Rice-Evans (7 ) calculated the average concentrations of 10 polyphenols from the data of Frankel et al. (5 ), who determined the levels of these components in 14 California red wines, seven of which were produced from Cabernet Sauvignon grapes. Based on these reported values we used concentrations of polyphenols proportional to an ethanol concentration that could be attained in the blood after consumption of a moderate amount of red wine. The polyphenol mixture almost completely inhibited LDL and HDL oxidation mediated by macrophages in vitro. We studied catechin, epicatechin and gallic acid because they are present at the highest concentrations among the known polyphenols in red wine, quercetin because of its high antioxidant activity and resveratrol because red wine, in addition to peanuts, is the only source of this compound in the diet (19 ). The concentration of resveratrol varies among red wines and is lower in California red wine than catechin, epicatechin or quercetin (5 ,7 ).

Our results with cell-mediated lipoprotein oxidation are in agreement with the results of other earlier studies which suggest that polyphenols are major contributors to the inhibition of cell-free, copper-mediated lipoprotein oxidation by red wine. Frankel et al. (5 ) demonstrated that the ability of several red and white wines to inhibit copper-catalyzed LDL oxidation correlated with the polyphenol concentrations of the wines. Kerry and Abbey (6 ) reported that a red wine fraction containing catechins inhibited copper-catalyzed LDL oxidation to the greatest extent, and that red wine stripped of phenolic compounds by treatment with polyvinyl polypyrrolidine did not have an antioxidant activity. The results of the present experiments shown in Figure 2 demonstrate that, even at low concentrations, red wine and the polyphenol mixture have similar antioxidant activities. Data reported by Miller and Rice-Evans (7 ) indicate that the polyphenols used in our experiments account for 93.8% of the antioxidant activity of the phenolic compounds identified in California red wine but 21.4% of the total antioxidant activity of these wines, suggesting the presence of other antioxidants. These investigators determined antioxidant activity using the ferrylmyoglobin/azino-di-3-ethylbenzthiazoline sulphonate assay, which may explain the differences between these findings and the present results.

Resveratrol probably is not an important contributor to the antioxidant activity of California red wines because of its low concentration in most red wines (20 ) and its low antioxidant activity in cell-mediated lipoprotein oxidation. The results of Frankel et al. (21 ) indicated that resveratrol at 10 µmol/L inhibited cell-free, copper-catalyzed LDL oxidation measured by hexanal formation to nearly the same extent as 10 µmol/L epicatechin or quercetin. Sanchez-Moreno et al. (22 ) found that resveratrol at 1.1 µmol/L was comparable with catechin at 1.38 µmol/L in inhibiting cell-free LDL oxidation determined by measuring lag time and extent of conjugated diene formation. Resveratrol is thought to inhibit in vitro lipoprotein oxidation by chelating copper and is not as effective as catechin, epicatechin or quercetin in inhibiting lipoprotein oxidation induced by a free radical generator (20 ). This may be an explanation for the low activity of resveratrol in inhibiting cell-mediated LDL oxidation at the concentrations used in the present study. Although transition metals appear to be required for the initiation of lipoprotein oxidation in vitro, there is evidence that reactive substances such as thiyl (11 ), tyrosyl (23 ,24 ), oxygen (25 ) and lipid hydroperoxyl radicals (26 ) are the important cellular mediators of lipoprotein oxidation.

The extent of the inhibition of LDL oxidation by consumption of red wine measured ex vivo (27 –29 ) is not as great as the in vitro inhibition by wine or the polyphenol mixture observed in the present study. The effectiveness of polyphenols in vivo would depend upon the amount absorbed and the antioxidant activity of their metabolites. Thus, some of the concentrations of polyphenols used in this study might have been higher than are physiologically relevant. The absorption of polyphenols in humans is not well studied. There are reports that polyphenol absorptions range from 24 to 52% for quercetin, from 9 to 21% for soy isoflavones such as genistein and from 0.2 to 0.9% for catechins. The remainder of the ingested compounds are either metabolized in the intestine or excreted, but the extent of the absorption of polyphenol metabolites and their contribution to the antioxidant activity in plasma are not known (30 ). The differences between the in vitro and ex vivo studies may also be due to the loss of lipoprotein-associated polyphenols during the isolation procedure used in ex vivo studies (31 ); however, the degree of loss has not been studied in detail. Studies in animal models indicate that effective levels of polyphenols are absorbed and remain associated with isolated LDL during assays of ex vivo susceptibility to oxidation. Hayek et al. (32 ) found that catechin and quercetin were bound by an ether linkage to isolated LDL from mice after being fed red wine for 6 wk. This LDL had a reduced susceptibility to oxidation. Xia et al. (33 ) demonstrated that administration of grape polyphenols in the diet diminished the increase in LDL oxidation induced by 2 mo of ethanol administration to rats. Our results confirm these authors’ finding that ethanol added in vitro has no effect on lipoprotein oxidation. Polyphenols could also exert their antioxidant action in solution in the blood or arterial wall even if they are not bound to the lipoprotein. Better assays of lipoprotein oxidation in vivo and ultimately clinical trials (34 ) may be needed to evaluate the effects of consumption of red wine or its polyphenol components.

	ACKNOWLEDGMENTS

 
We thank Shelley Greenhaus for clinical assistance and Frank Caputo and Prasuna Gourkanthi for technical assistance.

	FOOTNOTES

 
² Abbreviations used: ANOVA, analysis of variance; BHT, butylated hydroxytoluene; DMEM, Dulbecco’s minimum essential medium; EDTA, ethylenediamine tetraacetic acid; HBSS, Hank’s balanced salt solution; MDA, malondialdehyde; MTT, dimethylthiazol-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; TBARS, thiobarbituric acid-reactive substance; TNBS, trinitrobenzene sulfonic acid.

Manuscript received 26 December 2001. Initial review completed 26 February 2002. Revision accepted 1 July 2002.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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