© 1997 American Heart Association, Inc.
Articles |
Reduced Progression of Atherosclerosis in Apolipoprotein E–Deficient Mice Following Consumption of Red Wine, or Its Polyphenols Quercetin or Catechin, Is Associated With Reduced Susceptibility of LDL to Oxidation and Aggregation
From the Lipid Research Laboratory (T.H., B.F., M.R., P.B., M.A.), and the Division of Morphological Sciences (R.C.), Technion Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences; and Rambam Medical Center, Haifa, and Migal, Galilee Technological Center, Kiryat Shmona (J.V., P.B.) and Meir Hospital, Kfau Saba (A.E.), Israel.
Correspondence to Dr Michael Aviram, The Lipid Research Laboratory, Rambam Medical Center, Haifa, 31096, Israel. E-mail aviram{at}tx.technion.ac.il
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Abstract The effect of consuming red wine, or its major polyphenol constituents catechin or quercetin, on the development of atherosclerotic lesions, in relation to the susceptibility of plasma LDL to oxidation and to aggregation, was studied in atherosclerotic apolipoprotein E deficient (E°) mice. Forty E° mice at the age of 4 weeks were divided into four groups, 10 mice in each group, and were supplemented for up to 6 weeks in their drinking water with placebo (1.1% alcohol); catechin or quercetin (50 µg/d per mouse), or red wine (0.5 mL/d per mouse). Consumption of catechin, quercetin, or red wine had no effect on plasma LDL or HDL cholesterol levels. The atherosclerotic lesion area was smaller in the treated mice by 39%, 46%, and 48%, respectively, in comparison with E° mice that were treated with placebo. In accordance with these findings, cellular uptake of LDL derived after catechin, quercetin, or red wine consumption was found to be reduced by 31%, 40%, and 52%, respectively. These results were associated with reduced susceptibility to oxidation (induced by different modes such as copper ions, free radical generator, or macrophages) of LDL isolated after red wine or quercetin and, to a lesser extent after catechin consumption, in comparison with LDL isolated from the placebo group. Similar results were obtained when LDL was preincubated in vitro with red wine or with the polyphenols prior to its oxidation. Even in the basal oxidative state (not induced oxidation), LDL isolated from E° mice that consumed catechin, quercetin, or red wine for 2 weeks was found to be less oxidized in comparison with LDL isolated from E° mice that received placebo, as evidenced by 39%, 48%, and 49% reduced content of LDL-associated lipid peroxides, respectively. This effect could be related to enhanced serum paraoxonase activity in the polyphenol-treated mice. LDL oxidation was previously shown to lead to its aggregation. The present study demonstrated that the susceptibility of LDL to aggregation was reduced in comparison with placebo-treated mice, by 63%, 48%, or 50% by catechin, quercetin, and red wine consumption, respectively, and this effect could be shown also in vitro. The inhibition of LDL oxidation by polyphenols could be related, at least in part, to a direct effect of the polyphenols on the LDL, since both quercetin and catechin were found to bind to the LDL particle via the formation of an ether bond. We thus conclude that dietary consumption by E° mice of red wine or its polyphenolic flavonoids quercetin and, to a lesser extent, catechin leads to attenuation in the development of the atherosclerotic lesion, and this effect is associated with reduced susceptibility of their LDL to oxidation and aggregation.
Key Words: lipid peroxidation • apolipoprotein E • atherosclerosis • red wine • polyphenols • paraoxonase
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Introduction |
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Consumption of phenolic flavonoids in the diet was shown to be inversely associated with morbidity and mortality from coronary heart disease.1 2 3 Different classes of flavonoids are present in different fruits and vegetables and in beverages such as tea or wine. Phenolic flavonoids possess antioxidative properties toward LDL lipid peroxidation,4 5 6 and they have been reported to exert free radical–scavenging capabilities.7 8 The antioxidant activity of the flavonoids is related to their chemical structure.9 10 Dietary supplementation in humans of nutrients rich in polyphenols, such as black or green tea,11 olive oil,12 13 licorice root extract,14 15 or red wine16 was shown to be associated with increased resistance of their plasma LDL to oxidation and an increase in plasma antioxidant capacity. The "French Paradox," ie, low incidence of cardiovascular events in spite of diets high in saturated fat, was attributed to the regular drinking of red wine.17 Red wine, a dietary source of polyphenols, contains the flavonols quercetin and myricetin and also the three-flavanol catechin and epi(gallo)catechin.10 Since oxidative modification of LDL is thought to play a key role in the pathogenesis of early atherosclerosis,18 19 20 the beneficial effect of red wine consumption against the development of this disease was attributed to the antioxidant activity of the polyphenols in the red wine. Ingestion of red wine was shown to be associated with increased serum antioxidant activity,21 22 and flavonoids extracted from red wine were shown to protect the LDL against in vitro oxidation.23 We have recently demonstrated16 24 25 that red wine consumption by healthy volunteers reduced the susceptibility of their LDL to lipid peroxidation and also increased plasma HDL concentration.26 However, other studies27 28 29 using different types of red wine did not confirm the above results.
Aggregation of LDL represents another lipoprotein modification of atherogenic properties, since aggregated LDL is taken up by macrophages at increased rate, leading to foam cell formation.30 31 Recently, it was shown that extensive oxidation of LDL leads to its aggregation32 33 34 and that both of these modified forms of LDL are present in the atherosclerotic lesion.35 Today, there is no information on the possible beneficial effects of polyphenolic flavonoids against development of atherosclerosis or against LDL oxidation and aggregation under in vivo pathological conditions of oxidative stress. Thus, in the present study, we investigated the effect of red wine consumption, and its polyphenols quercetin and catechin, on development of atherosclerotic lesions, in association with the susceptibility of LDL to aggregation and oxidation under conditions of oxidative stress. For this purpose, we have used apolipoprotein E–deficient (E°) mice, since their LDL is highly susceptible to oxidation and aggregation.36
The results of the present study clearly showed that red wine, catechin, or quercetin consumption exhibited an inhibitory effect on development of aortic atherosclerotic lesions and on atherogenic modifications of LDL, in atherosclerotic E° mice.
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Methods |
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Materials
Na2 EDTA and 1,1 diphenyl-2-picryl-hydrazyl (DPPH) were purchased from Sigma. 2,2'-Azobis 2-amidinopropane hydrochloride (AAPH) was from Wako Chemical Industries, Ltd.
Animals
E° mice were kindly provided by Dr Jan Breslow, Rockefeller
University, New York, NY. At 4 weeks of age, 40 E° mice were assigned
randomly to four groups, 10 mice in each group. For the studies of the
atherosclerotic lesion areas, a second group of 40 mice was introduced
into the study 2 weeks later. The mice received their regular chow diet
and were supplemented for 6 weeks via their drinking water with (1)
placebo (alcoholized water, 1.1% alcohol); (2) red wine (cabernet
sauvignon) containing 1.1% alcohol, 0.5 mL/d per mouse (50 µg
equivalents of catechin); (3) catechin, 50 µg/d per mouse in
1.1% alcoholized solution; (4) quercetin 50 µg/d per mouse in
1.1% alcoholized solution.
Blood was collected from the retroorbital plexus under
anesthesia with ether into Eppendorf tubes with 1
mmol/L Na2 EDTA after 2 weeks and 6 weeks of
treatment. From each group of animals, three separate pools of plasma
were prepared, each one consisting of 
3 mL plasma. LDL
(d=1.006 to 1063 g/mL) was isolated from each 3 mL of
pooled plasma by sequential density ultracentrifugation
as previously described,37 resulting in three different
LDL samples from each group. The LDL protein content was determined by
the method of Lowry et al.38 LDL vitamin E content was
measured by HPLC with 
-tocopherol as a standard.
LDL Oxidation
LDL was dialyzed for 24 hours against PBS before oxidation, to
remove the EDTA. Oxidation of LDL was carried out in a shaking water
bath at 37°C under air. For metal ion–dependent oxidation, LDL (100
µg of protein per milliliter) was incubated for 2 hours at 37°C
with freshly prepared CuSO4 (10 µmol/L). For
metal ion–independent oxidation, LDL was incubated for 2 hours at
37°C with 5 mmol/L AAPH, which is an aqueous soluble azo
compound that thermally decomposes to produce peroxyl radicals at a
constant rate. EDTA (0.1 mmol/L) was added to the
incubation medium to chelate adventitious metal ions that could
otherwise contribute to the radical initiator-induced oxidation. LDL
oxidation was terminated by refrigeration at 4°C and addition of
0.1 mmol/L EDTA to the CuSO4 system. LDL
oxidation was determined by measuring the amount of
TBARS39 and lipid peroxide formation40 or by
continuous monitoring the formation of conjugated dienes by measuring
the increase in absorbance at 234 nm.41
For macrophage-mediated oxidation, J-774 A.1 macrophages (2x106 cells per 35-mm dish) obtained from the American Type Culture Collection, Rockville, Md, were incubated for 18 hours at 37°C in medium Ham's F-10 in presence of 2 µmol/L CuSO4, with LDL (100 µg of protein per milliliter) derived from mice that consumed placebo, catechin, quercetin, or red wine. The oxidation of LDL was measured directly in the medium by the TBARS assay. LDL incubated under similar conditions in absence of cells (cell-free) served as control. Cell-mediated oxidation was calculated by subtracting the cell-free value from the value obtained in presence of cells.
Cellular Uptake of LDL by Macrophages
LDL cholesterol uptake by J-774 A.1
macrophages was estimated by measurement of the stimulation of
[3H]oleate incorporation into cholesteryl
ester.42 Cells were incubated in the presence of 25 µg
cholesterol per milliliter of the lipoproteins for 18 hours
at 37°C. During the last 2 hours of incubation,
[3H]oleate in complex with albumin (2.7
mmol/L, 83 nmol oleate per milligram albumin, 10
µCi/mL) was added to the medium. At the end of the incubation,
cellular lipids were extracted with hexane/isopropanol (3:2,
vol/vol), and the cholesteryl ester was separated by thin-layer
chromatography using hexane/ether/acetic acid
(130:30:1.5, vol/vol/vol), visualized by iodine vapor,
scraped into vials containing 3 mL of scintillation fluid, and counted
in a beta scintillation counter.
Free Radical–Scavenging Capacity
The free radical–scavenging capacity of catechin, quercetin,
and red wine was analyzed by the DPPH assay. Aliquots of the
polyphenols (100 µg/mL) and of red wine (1%) were mixed with
100 µmol/L DPPH (in ethanol) in a cuvette. The time
course of the change in the OD at 517 nm was then kinetically
monitored.43
LDL Aggregation
LDL (100 µg of protein per milliliter) was vigorously mixed by
vortex, and the OD at 680 nm was monitored every 10 seconds against a
PBS blank solution.
Serum PON Activity
PON activity was measured with 1.0 mmol/L paraoxon
(Sigma) in a total volume of 800µL. Enzyme activity was measured in
50 mmol/L glycine/NaOH at pH 10.5. Ten microliters of serum
was added to start the reaction, and the increase in absorbance at 412
nm was recorded.44 The amount of
p-nitrophenol was calculated from the molar extinction
coefficient at pH 10.5, which was 18 290
mol/L-1 cm-1.
The blank contained substrate without the enzyme. One unit of PON
activity is defined as 1 nmol of p-nitrophenol formed per
minute, and the activity was expressed as units per liter of serum.
Analyses of Catechin and Quercetin in LDL
Catechin and quercetin were extracted from LDL with ethyl
acetate using 1 vol LDL to 3 vol ethyl acetate (x3). Recovery was
tested by incubating standards of catechin and quercetin with LDL (40
µg/mL each standard with 1 mg LDL protein per milliliter) and
was found to be 67% to 75%.
The separation and detection of catechin and quercetin were performed on Hewlett Packard HPLC model 1100, with HP UV-visible detector, coupled to an HP Chem Station. Samples were injected into a C18 column (Merck; 25-cm length, 0.4-cm diameter, 5-µm particle size).
Catechin and quercetin were analyzed by detecting their absorbance at 208 nm and 370 nm, respectively, using 0.025 mol/L KH2PO4, pH 2.4, and acetonitrile. The eluent flow was 0.9 mL/min at 80:20 ratio (KH2PO4 buffer/acetonitrile, vol/vol), for up to 5 minutes, followed by a gradient to 60:40 (vol/vol) from 5 to 10 minutes, and then a third gradient of 10:90 for 10 minutes was performed. The retention times for catechin and quercetin were 4.0 and 13.5 minutes, respectively. As polyphenols can possibly form ether or ester bonds with LDL constituents, we also measured the polyphenol content in LDL after hydrolysis. Hydrolysis of ether bonds (eg, glycosides) was conducted according to Hertog et al.45 In brief, a sample of dried extract was dissolved in 200 µL of methanol and 200 µL of 2.0 mol/L HCl, heated at 90°C for 2 hours, and then injected directly to the HPLC column (loop of 20 mL).
Hydrolysis of ester bond was conducted by saponification of the LDL samples according to Hodis et al46 with a slight modification. In brief, a sample of dried extract was dissolved in 0.5 mL diethyl ether, and 0.5 mL of 20% KOH in MeOH was added. The remaining head space of the vial was filled with nitrogen, and the reaction mixture was left with stirring in the dark at room temperature during 3 hours. The mixture was neutralized by addition of 0.5 mL of 25% citric acid in water and the upper organic phase removed. The remaining aqueous layer was washed twice with 1 mL of diethyl ether and the collected organic layers were combined, dried (sodium sulfate), filtered, and evaporated under nitrogen.
Analysis of Aortic Atherosclerotic Lesions
At the end of the experimental period, the mice were killed. The
heart and entire aorta were rapidly dissected out and immersion fixed
in 3% glutaraldehyde in 0.1 mol/L sodium
cacodylate buffer (pH 7.4) with 0.01% calcium chloride at room
temperature. Since it was shown that the aortic origin region with the
valves and bifurcation is the most susceptible for
atherosclerosis,47 48 49 50 we restricted our
study to the aortic arch for comparative histomorphometric studies of
atherosclerotic lesion development. Thus, the aortic arch was dissected
free from the surrounding fatty tissue and the first 4 mm of the
ascending aorta (beginning with the aortic valves) removed and cut
transversely with razor blades into four blocks of 1 mm each.
The samples were kept in the fixative overnight prior to rinsing and
storage in 0.1 mol/L sodium cacodylate buffer containing 7.5%
wt/vol sucrose. This step was followed by treatment with 1% aqueous
solution of osmium tetroxide for 4 hours, cacodylate rinse, dehydration
in ascending ethanols and propylene oxide, and embedding in epoxy resin
(Eponate 12, Pelco). Transverse sections (1 µm) were cut for
light microscopy. The prolonged osmium treatment stains the
intraluminal, intramural, and intracellular lipid a dense black color.
Osmium staining en bloc is an excellent method for lipids and shows up
very well the atherosclerotic lesions without further staining in thin
epoxy-embedded sections at much higher resolution than oil red O
staining in frozen sections. Moreover, the sections can be cut on the
ultramicrotome and stained with alkaline toluidine blue to provide even
better resolution.
Atherosclerotic lesion was defined as the area of (abnormal) pathological structural change. Lesional areas were determined by using a computerized quantitative image-analysis system (Cue-2, Olympus Corp) with appropriate morphometric software. The imaging system consists of a Zeiss Universal R photomicroscope (x10 objective) fitted with a Panasonic WV-CD50 video camera and 14-inch Sony color monitor and IBM-compatible PC. Image analysis was performed on aortic arches from placebo mice (n=20) and mice treated with red wine (n=20), catechin (n=19), and quercetin (n=20). Approximately 80 transverse sections (0.05 mm separation) were taken in total from each animal, and of these, sections with atherosclerotic lesions were selected and marked for image analysis. In the present study, standardized "windows" (fields of measurement) with an area of 176 758 µm2 were used as follows: placebo mice (n=31), red wine (n=25), catechin (n=49), and quercetin (n=31). The results were pooled for each group and presented as average cross-sectional lesion area per experimental group.
Statistical Analyses
ANOVA was used to analyze the significance of the
results. Results are given as mean±SD.
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Results |
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Macrophage Foam Cells and Lesion Formation
E° mice were supplemented for up to 6 weeks with 50 µg/d pure catechin, quercetin, or red wine containing 1.1% alcohol and compared with mice supplemented with placebo (1.1% alcohol). Consumption of catechin, quercetin, or red wine for either 2 or 6 weeks had no significant effect on plasma concentrations of LDL cholesterol (580±42, 620±53, and 540±50 mg/dL, respectively, in comparison with 590±51 mg/dL in the placebo group) or on plasma HDL cholesterol concentrations (41±4, 32±3, 46±4 mg/dL, respectively, versus 39±3 mg/dL in the placebo group).
Analyses of the aortic arch lesions after 6 weeks of polyphenol
consumption revealed that the atherosclerotic lesion area in mice that
consumed red wine, quercetin, or catechin was significantly
(P<.05) reduced by 48%, 46%, and 39%, respectively, in
comparison with the atherosclerotic lesions area in mice that received
placebo (Fig 1
). Only 3 of 20 mice
examined in the group that received the red wine or the polyphenols
developed significant aortic lesions, as characterized by the formation
of well-defined fatty streaks, whereas 16 of the 20 mice treated with
placebo developed such lesions.
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Fig 2 demonstrates photomicrographs of
typical atherosclerotic lesions of aortic arch of E° mice at the age
of 3 months that were treated with placebo (A). The lesions are large
and consist of small groups of lipid-laden macrophages in the
tunica intima. After treatment with red wine (B), quercetin (C), or
catechin (D), these lesions are much smaller, with fewer foam cells
present.
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Since foam cells in early atherosclerotic lesions are due to increased uptake of LDL by macrophages, we further investigated the macrophage uptake of LDL derived from E° mice that consumed polyphenols.
We measured the cellular cholesterol esterification rate
induced by LDL derived after consumption of catechin, quercetin, or red
wine, as a measure for cellular lipoprotein uptake. Incubation of J-774
A.1 macrophages for 5 hours with 10 µg of protein per
milliliter of plasma LDL derived from E° mice after consumption of
catechin, quercetin, or red wine resulted in 31%, 40%, and 52%
reduced LDL-induced cellular cholesterol esterification,
respectively, in comparison with the effect of LDL from the placebo
group (Fig 3). To determine whether the
decreased LDL-induced cellular cholesterol esterification
is due to a direct effect of the polyphenols on the acetyl coenzyme A
acyltransferase enzyme, we incubated J-774 A.1 macrophages with
increasing concentrations of catechin, quercetin, or red wine for 5
hours, after which we determined the acetyl coenzyme A acyltransferase
activity by measuring incorporation of [3H]oleic acid
into cellular cholesteryl esters. No significant effect could be found
on the cellular cholesterol esterification rate at any
polyphenol concentration studied (data not shown).
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These results suggest that the reduced atherosclerotic lesion formation in E° mice that consumed polyphenols may be attributed to reduced uptake of their LDL by macrophages. Since enhanced cellular uptake of LDL is associated with lipoprotein modifications, we further investigated the effect of polyphenol consumption on LDL atherosclerotic modifications, ie, oxidation and aggregation.
Propensity of LDL From E° Mice to Oxidation
We have previously shown that consumption of red wine with meals
by healthy volunteers reduces the susceptibility of their plasma LDL to
lipid peroxidation. The susceptibility to oxidation of LDL derived from
E° mice that were dietary supplemented for 2 weeks with placebo,
catechin, quercetin, or red wine was studied by incubation of the LDLs
(100 µg protein per milliliter) with copper ions (10
µmol/L) or with the free radical initiator AAPH (5
mmol/L), for 2 hours at 37°C, or with J-774A.1-cultured
macrophages under oxidative stress (2 µmol/L
CuSO4). Continuous monitoring of conjugated diene formation
by measuring the absorbance at 234 nm revealed that copper ion-induced
oxidation of LDL derived from E° mice that consumed quercetin or red
wine was delayed by 120 minutes, whereas the onset of lipid
peroxidation in LDL derived from E° mice that consumed catechin was
retarded by only 40 minutes, in comparison with LDL from the placebo
group.
Determination of the extent of LDL oxidation by measuring the formation
of TBARS after 2 hours of LDL incubation with CuSO4
(10 µmol/L) or AAPH (5 mmol/L) or after 18
hours of LDL incubation with J-774A.1 macrophages under
oxidative stress (in the presence of 2 µmol/L
CuSO4) revealed that quercetin or red wine consumption
resulted in a 54% and 43% reduction in copper ion-induced oxidation,
respectively (Fig 4A), an 83% and 81%
reduction in AAPH-induced oxidation, respectively (Fig 4B), and a 33%
and 30% inhibition in macrophage-mediated oxidation,
respectively (Fig 4C). No significant inhibition in TBARS formation
measured under these experimental conditions could be demonstrated with
LDL derived after catechin consumption.
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Measurement of lipid peroxides formation gave similar results (data not
shown). In vitro enrichment of LDL, derived from nontreated E° mice
at the age of 2 months, with polyphenols (18-hour incubation of the LDL
at 37°C with 50 µmol/L catechin or quercetin or 10%
red wine, followed by extensive dialysis to remove nonbound materials)
resulted in a remarkable delay in the onset of LDL oxidation (up to 360
minute after LDL enrichment with quercetin or red wine and 90 minute
after LDL enrichment with catechin (Fig 5A). When LDL oxidation was measured by
the TBARS assay, a 95% inhibition was obtained after enrichment of the
LDL with red wine or quercetin, whereas LDL enrichment with catechin
showed no significant inhibition in LDL oxidation under these
experimental conditions (Fig 5B). These results suggest that quercetin,
catechin, and red wine possess the capacity to directly inhibit LDL
oxidation. However, catechin possesses lower capacity than quercetin
against LDL oxidation.
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Plasma LDL Oxidative State in E° Mice
We have also analyzed the effect of dietary
supplementation of polyphenols on the oxidative state of LDL from E°
mice under basal conditions (not induced by copper ions or by AAPH).
LDL derived from E° mice that consumed catechin, quercetin, or red
wine for 2 weeks was found to be less oxidized in comparison with LDL
isolated from mice that consumed placebo. This was evidenced by 39%,
48%, and 49% reduced levels of LDL-associated lipid peroxides,
respectively (Fig 6).
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The extent of LDL oxidative modification in plasma is determined by intrinsic factors within the LDL particle, as well as by extrinsic plasmatic factors.
Among the intrinsic factors, the vitamin E content in LDL derived from mice that consumed catechin or quercetin was lower in comparison with its content in LDL derived from the placebo group (0.07 and 0.13 nmol/mg protein, respectively, versus 0.46 nmol/mg LDL protein in LDL from the placebo group).
Among the extrinsic factors that affect LDL oxidation, plasma HDL was reported to inhibit LDL oxidation,51 and HDL-associated PON was suggested to be involved in this effect.52 53 PON measurements in serum derived from E° mice after 2 weeks of polyphenol consumption, in comparison with serum derived from the placebo group, revealed 14%, 113%, and 75% higher activity after the consumption of catechin, quercetin, or red wine, respectively, in comparison with the placebo group (serum PON activities were 23±3, 26±4, 49±9, and 40±8 U/mL in mice that received placebo, catechin, quercetin, or red wine, respectively). The increased levels of serum PON in E° mice that consumed polyphenols can contribute to the reduction in LDL oxidative state by PON action on LDL-associated lipid peroxides. High serum PON activity in these mice may have also resulted from the reduced oxidative stress in the presence of the polyphenolic antioxidants. Serum PON activity in E° mice (whose plasma is oxidized and highly susceptible to oxidation) was indeed found to be lower by 27% in comparison with that found in control mice (23±3 U/mL in E° mice versus 33±2 U/mL in control mice). Furthermore, incubation of HDL (200 µg of protein per milliliter), the major serum PON carrier, with 10 µmol/L CuSO4 or 5 mmol/L AAPH for 2 hours at 37°C, resulted in a 21% and 30% reduction in HDL-associated PON activity, respectively (from 42±2 U/mL in control HDL to 33±3 U/mL and to 30±5 U/mL in HDL that was incubated with copper ions or with AAPH, respectively). These results show that serum PON activity is reduced under oxidative stress and may suggest that polyphenol consumption prevents the reduction in PON activity by reducing the oxidative stress in E° mice and thus may in turn contribute to PON peroxidase activity against LDL oxidation.
Susceptibility of LDL to Aggregation
Recently, it was shown that extensive oxidation of LDL leads to
lipoprotein aggregation.32 Fig 7A demonstrates that consumption of
quercetin, red wine, or catechin by the E ° mice resulted in a
reduced susceptibility of their LDL to aggregation induced by vortex by
48%, 50%, and 63%, respectively, in comparison with LDL from
placebo-treated mice. This inhibitory effect can be
associated with a direct effect of the polyphenols on LDL, as the
aggregation of LDL induced by vortexing in vitro was also reduced by
70%, 50%, and 30% after 2 hours of incubation with catechin,
quercetin, or red wine, respectively (Fig 7B). The antioxidative, as
well as the antiaggregative effects of polyphenol consumption,
persisted for longer than 2 weeks, since LDL isolated after 6 weeks of
polyphenol supplementation demonstrated similar results to those
obtained with LDL isolated after 2 weeks of dietary polyphenol
supplementation (data not shown).
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Mechanisms for the Antioxidative Effect of Polyphenols Against
LDL Oxidation
To compare the antioxidant capability of catechin with that of
quercetin, we performed the DPPH assay. Addition of 100
µmol/L quercetin to the DPPH solution decreased the optical
absorbance at 517 nm from 1.047 to 0.133 OD within 8 minutes, whereas
during a similar period, catechin reduced the absorbance of the
solution to only 0.676 OD. These results suggest that quercetin
possesses a better radical-scavenging capacity than catechin, and this
characteristic may contribute to its higher potency to inhibit LDL
oxidation.
To find out whether catechin and quercetin offer their antioxidative protection to LDL due to their binding to the lipoprotein, we preincubated LDL (1 mg protein per milliliter) for 18 hours at 37°C with 50 µmol/L pure polyphenols or with 10% red wine, followed by removal of the unbound materials by dialysis. Then, we measured the content of LDL-associated catechin or quercetin by reverse-phase HPLC with UV detection. No significant levels of either catechin or quercetin could be detected in the LDL samples using this procedure.
As polyphenols may interact with surface components of LDL such as fatty acids (ester bond) or sugar residues (ether bond), we performed an alkaline hydrolysis (saponification) prior to the HPLC analysis of the polyphenols.
The alkaline hydrolysis of the LDL samples did not result in the identification of the polyphenols. However, when acidic hydrolysis was performed on the LDL samples before the HPLC analysis, both catechin (0.35 nmol/mg LDL protein) and quercetin (1.00 nmol/mg LDL protein) were clearly identified and quantified within the LDL particle. LDL derived after red wine consumption contained 3.65 nmol of catechin and 3.00 nmol of quercetin per milligram LDL protein. No polyphenols could be measured in LDL derived from the placebo group. These results suggest that catechin and quercetin probably bind to the LDL particle by forming an ether (glycosidic) bond with the LDL particle.
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Discussion |
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The present study demonstrates that dietary consumption by the atherosclerotic E° mice of red wine or its major polyphenols catechin and quercetin inhibited the progression of aortic atherosclerosis in these mice. This effect was associated with a reduced LDL oxidative state and also a reduced propensity of their LDL to aggregation and oxidation under different modes of oxidative stress. The inhibitory effect of polyphenols on the ex vivo oxidation of LDL was probably related to the binding of polyphenols to the LDL particle via the formation of ether bonds.
The LDL oxidation hypothesis of atherosclerosis suggests that inhibition of LDL oxidation should result in the attenuation of the rapid clearance of oxidized LDL from plasma via the macrophage scavenger receptors. This effect in turn leads to a reduction in macrophage cholesterol accumulation and foam cell formation. Indeed, we have shown that LDL derived from the E° mice after dietary consumption of polyphenols was taken up by cultured macrophages at reduced rate in comparison with LDL derived from mice consuming placebo. The mechanism responsible for this effect may lie in the reduced oxidation state, as well as the reduced aggregation state of the LDL that was supplemented with polyphenols.
However, LDL derived after catechin consumption exhibited contradictory antioxidative effects when measured under different conditions. Determination of LDL oxidation by continuous monitoring of conjugated diene formation revealed that catechin in comparison with placebo retarded the onset of LDL oxidation by 40 minutes, in comparison with 360 minutes' prolongation of the lag phase by quercetin. Determination of LDL oxidation by measuring LDL-associated TBARS or lipid peroxides formation at one time point (2 hours), however, showed no inhibitory effect of catechin, as at this time point this polyphenol no longer affects the lag phase. It is important to note, however, that both quercetin and catechin significantly reduced the basal oxidative state of the LDL, and this may be related to the fact that both polyphenols are continuously present in the blood and can act as free radical scavengers as long as they are not consumed.
LDL oxidation is a dynamic process characterized by a lag time during which the polyunsaturated fatty acids in the LDL are protected from oxidation by the LDL-associated antioxidants. After consumption of the intrinsic LDL antioxidants, the polyunsaturated fatty acids in the LDL are rapidly oxidized (the propagation phase), followed by a decomposition phase to end products such as aldehydes.41 Therefore, determination of LDL oxidation by methods measuring formation of end products, such as the TBARS assay, at only one time point, does not provide enough information on the whole LDL oxidation process. For this reason, in the present study, we performed several different determinations of LDL oxidation to assess the whole oxidation process.
Thus, catechin proved to act as an antioxidant, but to a lesser extent than quercetin. Although catechin and quercetin share a similar hydroxyl group arrangement, the electron-donating ability of the flavonol catechin is lower than that of the flavonol quercetin, which is in agreement with its lower capacity to scavenge free radicals, as demonstrated in the DPPH assay. Quercetin was indeed demonstrated, in different oxidation systems, to act more efficiently as an antioxidant than catechin.54 55 56 Quercetin demonstrated a twofold higher antioxidative potential against free radicals, and this was attributed to an altered bonding in its C ring, which allows delocalization between the A and B rings, thus stabilizing the aryloxyl radical after hydrogen donation.54
Differences in the capacity of catechin and quercetin to bind to the LDL particle may also have contributed to their different inhibitory capacities toward LDL oxidation. Incubation of LDL with similar concentrations of pure catechin or quercetin resulted in a threefold increased binding of quercetin to the lipoprotein in comparison with catechin. Furthermore, LDL that was incubated with red wine, which contains about 20-fold more catechin than quercetin, binds similar amounts of catechin and quercetin, which may explain the fact that red wine consumption affected LDL oxidation similarly to pure quercetin, although catechin is quantitatively the major polyphenol in red wine.
The present results obtained with the E° mice are in accordance with our previous study with healthy humans,16 suggesting that the inhibitory effect of the polyphenols on LDL oxidation is expressed in both normocholesterolemic and hypercholesterolemic subjects.
The mechanism by which polyphenol consumption inhibits LDL oxidation in vivo may involve their effects on intrinsic factors within the LDL particle, as well as on extrinsic factors, in its milieu. The protective effect of polyphenols against LDL oxidation in the E° mice was not mediated via conservation of the vitamin E in the LDL, as vitamin E consumption was reduced, not increased, in the presence of polyphenols. Extrinsic factors in the plasma could also be affected by polyphenol consumption, and indeed, serum PON activity was higher in mice that consumed polyphenols than in mice that consumed placebo. Paraoxonase is a calcium- dependent, HDL-associated, organophosphate hydrolase, which inhibits copper ion-induced LDL oxidation.52 We have shown in the present study that serum PON activity is reduced under oxidative stress in vitro, and in the E° atherosclerotic mice, serum PON activity was lower than that found in control mice. Serum PON activity was also shown to be lower in subjects who had suffered myocardial infarction57 and in populations with heterozygous familial hypercholesterolemia,58 in comparison with healthy subjects. These patients are characterized by high oxidative stress,59 similar to the hypercholesterolemic E° mice.35 Thus, it may be suggested that polyphenol consumption, which reduces the oxidative stress (and LDL oxidation), is the cause for the increased serum PON activity, which can in turn protect LDL from oxidation by possible peroxidase-like activity.52
Lipoprotein atherogenicity was attributed not only to its oxidation but also to its aggregation.19 LDL aggregation induced by vortexing is believed to result from interaction between the lipoprotein hydrophobic domains, which are exposed during vortexing of LDL.60 As polyphenols are multidentate ligands able to bind simultaneously to more than one molecule on the lipoprotein surface,61 62 their binding to the LDL particle can reduce the susceptibility of the lipoprotein to aggregation forces. Reduced LDL aggregation was shown after dietary consumption of polyphenols, as well as following in vitro LDL incubation with these polyphenols. However, in the in vivo study, the oxidizability of the lipoprotein could also contribute to its enhanced aggregability, since it was previously shown that LDL oxidation leads to its subsequent aggregation.32
Thus, we conclude that consumption of red wine or its polyphenols, such as quercetin and catechin, is an antiatherogenic intervention means, as it is associated with reduced LDL oxidation, reduced LDL aggregation, reduced foam cell formation, and most importantly, attenuation of atherosclerotic lesion progression.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a Fund for the Promotion of Research at the Technion (R. Coleman) and a grant from the Rappaport Family Institute for Research in the Medical Sciences (M. Aviram). E° mice were kindly provided by Dr Jan Breslow, Rockefeller University, New York, NY. We thank Ludmilla Mazor for her technical help in the histomorphometric studies.
Received January 30, 1997; accepted April 18, 1997.
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C. Auger, B. Caporiccio, N. Landrault, P. L. Teissedre, C. Laurent, G. Cros, P. Besancon, and J.-M. Rouanet Red Wine Phenolic Compounds Reduce Plasma Lipids and Apolipoprotein B and Prevent Early Aortic Atherosclerosis in Hypercholesterolemic Golden Syrian Hamsters (Mesocricetus auratus) J. Nutr., June 1, 2002; 132(6): 1207 - 1213. [Abstract] [Full Text] [PDF]
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H. B. Loest, S. K. Noh, and S. I. Koo Green Tea Extract Inhibits the Lymphatic Absorption of Cholesterol and {alpha}-Tocopherol in Ovariectomized Rats J. Nutr., June 1, 2002; 132(6): 1282 - 1288. [Abstract] [Full Text] [PDF]
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M. Kaplan, T. Hayek, A. Raz, R. Coleman, L. Dornfeld, J. Vaya, and M. Aviram Pomegranate Juice Supplementation to Atherosclerotic Mice Reduces Macrophage Lipid Peroxidation, Cellular Cholesterol Accumulation and Development of Atherosclerosis J. Nutr., August 1, 2001; 131(8): 2082 - 2089. [Abstract] [Full Text] [PDF]
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T. O. Cheng, L. M. Blanco-Colio, C. Bustos, M. Ortego, M. A. Hernandez-Presa, P. Cancelas, J. Gomez-Gerique, J. Egido, M. Valderrama, L. A. Alvarez-Sala, et al. Conundrum of the "French Paradox" Response Circulation, June 26, 2001; 103 (25): e132 - e133. [Full Text] [PDF]
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J. L. Donovan, V. Crespy, C. Manach, C. Morand, C. Besson, A. Scalbert, and C. Rémésy Catechin Is Metabolized by Both the Small Intestine and Liver of Rats J. Nutr., June 1, 2001; 131(6): 1753 - 1757. [Abstract] [Full Text]
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P. N. Durrington, B. Mackness, and M. I. Mackness Paraoxonase and Atherosclerosis Arterioscler. Thromb. Vasc. Biol., April 1, 2001; 21(4): 473 - 480. [Abstract] [Full Text] [PDF]
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J. F. Bentzon, E. Skovenborg, C. Hansen, J. Moller, N. S.-C. de Gaulejac, J. Proch, and E. Falk Red Wine Does Not Reduce Mature Atherosclerosis in Apolipoprotein E-Deficient Mice Circulation, March 27, 2001; 103(12): 1681 - 1687. [Abstract] [Full Text] [PDF]
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 I. J. Goldberg, L. Mosca, M. R. Piano, and E. A. Fisher Wine and Your Heart : A Science Advisory for Healthcare Professionals From the Nutrition Committee, Council on Epidemiology and Prevention, and Council on Cardiovascular Nursing of the American Heart Association Stroke, February 1, 2001; 32(2): 591 - 594. [Full Text] [PDF]
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I. J. Goldberg, L. Mosca, M. R. Piano, and E. A. Fisher Wine and Your Heart : A Science Advisory for Healthcare Professionals From the Nutrition Committee, Council on Epidemiology and Prevention, and Council on Cardiovascular Nursing of the American Heart Association Circulation, January 23, 2001; 103(3): 472 - 475. [Full Text] [PDF]
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Y. Miura, T. Chiba, I. Tomita, H. Koizumi, S. Miura, K. Umegaki, Y. Hara, and M. Ikeda Tea Catechins Prevent the Development of Atherosclerosis in Apoprotein E-Deficient Mice J. Nutr., January 1, 2001; 131(1): 27 - 32. [Abstract] [Full Text]
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A. Theriault, Q. Wang, S. C. Van Iderstine, B. Chen, A. A. Franke, and K. Adeli Modulation of hepatic lipoprotein synthesis and secretion by taxifolin, a plant flavonoid J. Lipid Res., December 1, 2000; 41(12): 1969 - 1979. [Abstract] [Full Text]
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Bart De Geest, D. Stengel, M. Landeloos, M. Lox, L. Le Gat, D. Collen, P. Holvoet, and E. Ninio Effect of Overexpression of Human Apo A-I in C57BL/6 and C57BL/6 Apo E-Deficient Mice on 2 Lipoprotein-Associated Enzymes, Platelet-Activating Factor Acetylhydrolase and Paraoxonase : Comparison of Adenovirus-Mediated Human Apo A-I Gene Transfer and Human Apo A-I Transgenesis Arterioscler. Thromb. Vasc. Biol., October 1, 2000; 20 (10): e68 - e75. [Abstract] [Full Text] [PDF]
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M. Tomas, M. Senti, F. Garcia-Faria, J. Vila, A. Torrents, M. Covas, and J. Marrugat Effect of Simvastatin Therapy on Paraoxonase Activity and Related Lipoproteins in Familial Hypercholesterolemic Patients Arterioscler. Thromb. Vasc. Biol., September 1, 2000; 20(9): 2113 - 2119. [Abstract] [Full Text] [PDF]
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L. M. Blanco-Colio, M. Valderrama, L. A. Alvarez-Sala, C. Bustos, M. Ortego, M. A. Hernandez-Presa, P. Cancelas, J. Gomez-Gerique, J. Millan, and J. Egido Red Wine Intake Prevents Nuclear Factor-{kappa}B Activation in Peripheral Blood Mononuclear Cells of Healthy Volunteers During Postprandial Lipemia Circulation, August 29, 2000; 102(9): 1020 - 1026. [Abstract] [Full Text] [PDF]
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 J.-H. Moon, R. Nakata, S. Oshima, T. Inakuma, and J. Terao Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2000; 279(2): R461 - R467. [Abstract] [Full Text] [PDF]
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M. Aviram, L. Dornfeld, M. Rosenblat, N. Volkova, M. Kaplan, R. Coleman, T. Hayek, D. Presser, and B. Fuhrman Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice Am. J. Clinical Nutrition, May 1, 2000; 71(5): 1062 - 1076. [Abstract] [Full Text] [PDF]
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B. Fuhrman, M. Rosenblat, T. Hayek, R. Coleman, and M. Aviram Ginger Extract Consumption Reduces Plasma Cholesterol, Inhibits LDL Oxidation and Attenuates Development of Atherosclerosis in Atherosclerotic, Apolipoprotein E-Deficient Mice J. Nutr., May 1, 2000; 130(5): 1124 - 1131. [Abstract] [Full Text]
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K. Iijima, M. Yoshizumi, M. Hashimoto, S. Kim, M. Eto, J. Ako, Y.-Q. Liang, N. Sudoh, K. Hosoda, K. Nakahara, et al. Red Wine Polyphenols Inhibit Proliferation of Vascular Smooth Muscle Cells and Downregulate Expression of Cyclin A Gene Circulation, February 22, 2000; 101(7): 805 - 811. [Abstract] [Full Text] [PDF]
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S. Parthasarathy, N. Santanam, S. Ramachandran, and O. Meilhac Oxidants and antioxidants in atherogenesis: an appraisal J. Lipid Res., December 1, 1999; 40(12): 2143 - 2157. [Abstract] [Full Text] [PDF]
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J. L. Donovan, J. R. Bell, S. Kasim-Karakas, J. B. German, R. L. Walzem, R. J. Hansen, and A. L. Waterhouse Catechin Is Present as Metabolites in Human Plasma after Consumption of Red Wine J. Nutr., September 1, 1999; 129(9): 1662 - 1668. [Abstract] [Full Text]
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T. Murase, N. Kume, T. Hase, Y. Shibuya, Y. Nishizawa, I. Tokimitsu, and T. Kita Gallates Inhibit Cytokine-Induced Nuclear Translocation of NF-{kappa}B and Expression of Leukocyte Adhesion Molecules in Vascular Endothelial Cells Arterioscler. Thromb. Vasc. Biol., June 1, 1999; 19(6): 1412 - 1420. [Abstract] [Full Text] [PDF]
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 M. Rosenblat, P. Belinky, J. Vaya, R. Levy, T. Hayek, R. Coleman, S. Merchav, and M. Aviram Macrophage Enrichment with the Isoflavan Glabridin Inhibits NADPH Oxidase-induced Cell-mediated Oxidation of Low Density Lipoprotein. A POSSIBLE ROLE FOR PROTEIN KINASE C J. Biol. Chem., May 14, 1999; 274(20): 13790 - 13799. [Abstract] [Full Text] [PDF]
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M. Aviram, S. Billecke, R. Sorenson, C. Bisgaier, R. Newton, M. Rosenblat, J. Erogul, C. Hsu, C. Dunlop, and B. La Du Paraoxonase Active Site Required for Protection Against LDL Oxidation Involves Its Free Sulfhydryl Group and Is Different From That Required for Its Arylesterase/Paraoxonase Activities : Selective Action of Human Paraoxonase Allozymes Q and R Arterioscler. Thromb. Vasc. Biol., October 1, 1998; 18(10): 1617 - 1624. [Abstract] [Full Text] [PDF]
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