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Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269
2To whom correspondence should be addressed. E-mail: tosca.zern{at}uconn.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • grape polyphenols • atherosclerosis • plasma triglycerides • guinea pigs
In the United States, 
13 million people have coronary heart disease (CHD) (1); of this number of individuals, 450,000 people die of CHD each year (1). Risk factors include elevated plasma total and LDL cholesterol as well as elevated triglyceride (TG) levels (2,3). Gender is yet another risk factor for CHD. Compared with premenopausal women, men have a much higher risk of developing CHD. However, due to the loss of estrogen, this relationship changes when menopause occurs (4). Some of the metabolic changes in plasma lipid levels observed in postmenopausal women are increased total cholesterol, LDL cholesterol and TG concentrations (5,6). These higher levels of LDL cholesterol may increase the risk for LDL oxidation, arterial deposition and ultimately CHD (1).
Researchers have suggested that there is an inverse relationship between the intake of flavonoids and CHD mortality (7,8). Grapes are composed of a wide variety of polyphenols, including flavonoids, flavans, flavanols and anthocyanins. In numerous studies, flavonoids and their derivatives have been reported to reduce LDL oxidation in both humans and animal models. Short-term ingestion of grape juice by patients with coronary artery disease (CAD) increased the mean lag time of LDL oxidation (7). Studies using flavonoids have also shown reductions in plasma lipids and multiple effects on lipoprotein metabolism. For example, the grapefruit flavonoid, naringenin, has been shown to reduce the secretion of apolipoprotein (apo)-B–containing lipoproteins (9). In addition, a naringenin-supplemented diet decreased plasma total and hepatic cholesterol concentrations in rats (10).
The goals of the present study were to evaluate the effects of a grape preparation on plasma lipids, lipoprotein cholesterol distribution, LDL size and composition, and atherosclerosis in ovariectomized guinea pigs, a model for menopausal women. Previous studies in our laboratory have shown that ovariectomized guinea pigs are a good model for menopausal women because they have higher concentrations of plasma total and LDL cholesterol and TG levels similar to those found after menopause (11). Overall, guinea pigs have been reported to be a good model for human cholesterol metabolism (12).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cholesterol oxidase, cholesterol esterase, peroxidase, and kits to measure plasma triglycerides and cholesterol were purchased from Boehringer Mannheim (Indianapolis, IN). Kits for the measurement of free cholesterol and phospholipid were obtained from Wako (Osaka, Japan). Quick-seal ultracentrifuge tubes were from Beckman (Palo Alto, CA) and halothane from Halocarbon (Hackensack, NJ). Oleoyl-[1-14C] CoA and cholesteryl-[1
,2 (n)-3H] oleate were purchased from Amersham Biosciences (Clearbrook, IL). Glass silica gel plates were purchased from EM Science (Gibbstown, NJ). Lyophilized grape powder was provided by the California Table Grape Commission (Fresno, CA).
Diets.
Diets were prepared and pelleted by Research Diets (New Brunswick, NJ). Diets were equal in composition except for the amount of grape powder. The grape powder was prepared from fresh red, green and blue-black California grapes. Approximately 18.2 g of the grape powder corresponded to 100 g of fresh grapes. The chemical composition of the grape powder is as follows: 4 g/100 g protein, 90 g/100g sugar and 6 g/100 g fiber. Selected phytochemical components were determined, and values are as follows: total phenols 0.58 g/100 g, flavans 0.41 g/100 g, anthocyanins 0.077 g/100 g, quercetin 10.2 µmol/100 g, myricetin 0.8 µmol/100 g, kaempferol 1.1 µmol/100 g and resveratrol 0.7 µmol/100 g. Total phenols of the grape powder were analyzed with Folin and Ciocalteu’s phenol reagent. Flavans were analyzed by reaction with vanillin, and anthocyanins were analyzed spectrophotometrically. Flavonols and reservatrol were analyzed by HPLC after acid hydrolysis (13). The concentration of grape powder used for the diet formulation was 10 g/100 g (Table 1). Dietary cholesterol was maintained at 0.33 g/100 g to raise plasma cholesterol concentrations and ensure the development of atherosclerosis. This amount of dietary cholesterol corresponds to 2475 mg/d in the human diet (14). The fat mixture was rich in lauric and myristic acids, which induce endogenous hypercholesterolemia in guinea pigs (11).
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Female ovariectomized guinea pigs weighing 250–300g were purchased from Charles River (Boston, MA). Guinea pigs were randomly allocated to the control (n = 12) or to the grape (n = 11) diets. Treatment was for 12 wk, an amount of time that results in a steady-state plasma cholesterol level and development of atherosclerosis (15). Two guinea pigs were housed per cage in a light cycle room (light from 700 to 1900h) at 22°C. Diet and water were consumed ad libitum. All guinea pig experiments were conducted in accordance with U.S. Public Health Service/U.S. Department of Agriculture guidelines. Experimental protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee.
Plasma lipids.
Plasma total cholesterol and HDL cholesterol were determined by enzymatic methods (16). Plasma TG were determined by an enzymatic method (17). HDL cholesterol was analyzed after precipitation of apo-B–containing lipoproteins with dextran sulfate (18) using a modified method (19).
Lipoprotein isolation.
Fed guinea pigs were anesthetized under halothane vapors and blood was obtained via heart puncture. Plasma samples were collected and preservation cocktail was added to the samples (5 mL/L aprotinin, 1 mL/L phenylmethylsulfonyl fluoride and 1 mL/L sodium azide). Plasma from each guinea pig (1 mL) was stored at 4°C for analysis of plasma lipids; the rest was used for lipoprotein isolation and LDL oxidation.
Lipoprotein isolation was done by sequential ultracentrifugation in an L8-M ultracentrifuge (Beckman Instruments, Fullerton, CA). VLDL were isolated in a density range of 1.006–1.019 kg/L at 125,000 x g at 15°C for 19 h in a Ti50 rotor. LDL were isolated in a density range of 1.019–1.09 kg/L at 150,000 x g for 3 h using a Vti 62.5 rotor (19). LDL samples were dialyzed in 0.09 g/100 g NaCl, 0.01 g/100 g EDTA, pH 7.2 for 24 h and stored at 4°C for composition analysis.
LDL size determination.
The Lipoprint LDL system (Quantimetrix Redondo Beach, CA) was used to identify the size of LDL using a nongradient high resolution polyacrylamide gel electrophoresis system. Briefly, 25 µL of plasma was added to precast polyacrylamide gel tubes and overlaid with 200 µL of loading gel. Tubes were then photopolymerized for 30 min and placed into the electrophoresis chamber. Electrophoresis buffer (Tris-hydroxymethyl aminomethane 66.1 g/100 g, boric acid 33.9 g/100 g, pH 8.2–8.6) was added to the top and bottom portions of the chamber. The gel was run for 60 min at 36 mV or until the HDL fraction was 1 cm from the end of the gel. Gels were allowed to sit for 30 min and scanned with a densitometer.
VLDL and LDL characterization.
VLDL and LDL composition was calculated by determining free and esterified cholesterol (16), protein by a modified Lowry method (20) and TG and PL by enzymatic kits. VLDL apo B was selectively precipitated with isopropanol (21). The number of constituent molecules of LDL was calculated on the basis of 1 apo B/particle with a molecular mass of 412 kDa (22). The molecular weights were 885.4, 386.6, 645 and 734 for TG, free and esterified cholesterol, and PL, respectively (23). LDL diameters were calculated according to Van Heek and Zilversmit (24).
Plasma lecithin cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) determinations.
LCAT and CETP activities were determined according to Ogawa and Fielding (25). Physiologic CETP activity was determined without inhibiting LCAT by measuring the mass transfer of cholesterol ester between HDL and apo B–containing lipoproteins. Samples were incubated at 37°C for 6 h in a shaking water bath, and total and free plasma cholesterol and HDL cholesterol were measured. LCAT activity was determined by mass analysis of the decrease in plasma free cholesterol between 0 and 6 h at 37°C. Assays were carried out concurrently with measurements of CETP. Both of these methods have been standardized for guinea pig plasma (26).
Hepatic lipids.
Livers were excised from guinea pigs after exsanguination and were stored at -20°C for lipid analysis. Liver lipids were extracted from 0.4 g of liver sliced into small pieces and combined with 10 mL of chloroform/methanol (2:1) overnight (27). The mixture was then filtered by gravity filtration; the filtrate was mixed with acidified water and separated into two phases with a separatory funnel. An aliquot of 0.2 mL, taken from the lower phase, was evaporated completely and reconstituted with 0.2 mL ethanol for enzymatic determination of total and free cholesterol (16). Cholesteryl esters were determined by subtracting free cholesterol from total cholesterol. Hepatic triglycerides were determined according to Carr et al. (17).
Hepatic acyl-CoA cholesterol acyltransferase (ACAT) activity.
Hepatic microsomes were isolated as previously described (23). Hepatic ACAT (E.C.2.3.1.26) activity was assayed by the method of Smith et al. (28). Microsomes (0.8–1 mg protein/assay) were preincubated with albumin (84 g/L) and buffer (50 mmol/L KH2PO4, 1 mol/L sucrose, 50 mmol/L KCl, 30 mmol/L EDTA and 50 mmol/LNaF) to a final volume of 0.18 mL. After 5 min at 37°C, 20 µL (500 µmol/L) of oleoyl-[1-14C] coenzyme (0.15 GBq/pmol) was added, and the reaction proceeded for 15 min at the same temperature. The reaction was stopped by adding 2.5 mL of chloroform/methanol, 2:1; [3H] cholesterol oleate (0.045 GBq/assay) was added as an internal recovery standard, and the samples were mixed and allowed to stand overnight at room temperature. The aqueous phase was removed and the organic phase was dried under nitrogen. The samples were resuspended in 0.150 mL of chloroform containing 30 µg of unlabeled cholesteryl oleate. Samples were applied to glass silica gel TLC plates and developed in hexane/diethyl ether, 9:1. Cholesteryl esters were visualized with iodine vapors, scraped from the TLC plates, and counted in a scintillation counter.
Concentration of cholesterol in aortas.
The heart and aorta were removed from guinea pigs and stored in formalin at 2–8°C. Aortas were thoroughly cleaned of excess tissue and fat. From the cleaned tissue, aortic lipids were extracted from 0.1 g aortic arch using 10 mL of chloroform/methanol (2:1) overnight (27). The extraction and determination of aortic lipids were similar to the hepatic lipids procedures previously described.
Statistical analysis.
Student’s t test was used to evaluate significant differences in plasma and hepatic lipids, LCAT, and CETP activities, lipoprotein composition, ACAT activity and concentration of cholesterol in aorta. Data are presented as means ± SD. P-values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The concentration of cholesterol in the aorta in the grape-fed group was 33% lower (P < 0.05) than in the control group (Fig. 1). Guinea pigs fed the grape diet had 4.7 ± 1.9 mmol cholesterol/g tissue, whereas values for controls were 7.1 ± 1.7 mmol/g (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma lipids and lipoprotein metabolism.
Previous studies have reported an increase in plasma triglyceride levels due to grape juice consumption. Investigators have attributed this increase to the large amount of carbohydrates found in this juice (7). In contrast to these findings, the grape preparation used in the current study decreased plasma triglycerides and VLDL cholesterol levels. Similar to our findings, studies using Hep G2 cells have shown that naringenin, a grapefruit flavonoid, decreases apo B secretion, thereby reducing the concentration of TG secreted into the medium (9,29). Results from these studies (9,29) suggest that although intake of grape polyphenols did not affect VLDL composition in the present study, the number of secreted particles may have been decreased, resulting in a reduction in the concentration of plasma triglycerides.
In guinea pigs, the major carrier of cholesterol is LDL (12); therefore, the lowering in VLDL cholesterol does not have a major effect on plasma total cholesterol concentrations. The lower concentrations of VLDL cholesterol in guinea pigs fed the grape diet suggest that grape polyphenols could have affected either VLDL secretion or VLDL removal from the plasma. Because dietary treatment did not modify VLDL composition or plasma CETP activity, we speculate that grape polyphenols reduced the number of VLDL particles secreted by the liver, which resulted in lower concentrations of VLDL cholesterol. Studies using triton WR-1339, an inhibitor of lipoprotein lipase activity, have shown that other dietary treatments, such as high consumption of complex carbohydrates (30), reduce the number of secreted particles in guinea pigs, resulting in lower levels of VLDL cholesterol.
Grape polyphenols also modified the LDL particles, resulting in less TG and PL molecules in LDL derived from guinea pigs fed the grape diet. Although modifications in LDL composition were observed, there was no difference in size between LDL from the control or grape-fed groups when measured by two different methods. It is possible that these compositional modifications might have affected the susceptibility of the LDL particles to oxidation and decreased LDL uptake by the aorta.
Hepatic cholesterol metabolism.
Grape intake decreased ACAT activity, which may play a role in lipoprotein metabolism and composition, thereby altering the deposition of cholesterol in the aorta. As previously noted, naringenin, a grapefruit polyphenol, has been shown to decrease plasma lipids by decreasing the secretion of apo B–containing lipoproteins. Borradaile et al. (29) showed that naringenin selectively inhibits ACAT activity and therefore decreases CE concentrations in the microsomal lumen.
Naringenin has also been shown to inhibit microsomal triglyceride transfer protein (MTP), thereby reducing overall TG accumulation within the endoplasmic reticulum (9,29). MTP and ACAT play vital roles in the production of lipoprotein key components. Because of the inhibitory effects of naringenins on MTP and ACAT and consequent reduction in the accumulation of TG and CE, investigators have speculated that these modifications reduced the overall secretion of apo B–containing lipoproteins (29). Although MTP activity was not measured in the current study, similar to the studies using naringenin (9,29), the intake of grape polyphenols reduced ACAT activity, plasma VLDL cholesterol and TG concentrations.
Accumulation of cholesterol in the aorta.
In this study, ovariectomized guinea pigs, a model for menopausal women, were used to assess the protective effects of grape polyphenols against cholesterol deposition in the periphery and development of the atherosclerotic process in the absence of estrogen. Although no changes in the total pool of hepatic cholesterol or plasma cholesterol concentrations were observed, the modifications induced in the LDL particle were sufficient to decrease the deposition of cholesterol in the aorta.
The observed decrease in aortic cholesterol accumulation is similar to published studies using dietary polyphenolic compounds. Yamakoshi et al. (31) reported reduced aortic cholesterol in rabbits fed either 0.1 or 1.0 g/100 g proanthocyanidin extract from grape seeds (31). Similarly, red wine phenolics have been shown to decrease the percentage of aortic fatty streak area in hamsters (32). The findings of the current study demonstrate that grape polyphenols reduced the deposition of cholesterol in the aorta, the most important metabolic alteration associated with decreased risk for coronary heart disease. Although no reductions in plasma cholesterol were observed, the potential atherogenicity of the LDL particle was reduced by grape intake, leading to less accumulation of cholesterol in the aorta. Similar to these findings, studies with coronary heart disease patients have demonstrated that although their concentrations of plasma cholesterol are not elevated, they have a higher potential for LDL modification and oxidation, resulting in increased LDL uptake by macrophages and increased lesion formation (33).
Results from these studies contribute to our understanding of the role of polyphenols in protecting against heart disease. The data suggest that even in the absence of estrogen, polyphenols exert their protective effects. The current study also emphasizes the importance of hepatic ACAT activity and modifications in LDL composition on the development of atherosclerosis and CHD despite the fact that polyphenols did not alter plasma cholesterol concentrations.
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FOOTNOTES |
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3 Abbreviations used: ACAT: acyl CoA, cholesteryl acyltransferase; apo, apolipoprotein; CE, cholesteryl ester; CETP, cholesterol ester transfer protein; CHD, coronary heart disease; FC, free cholesterol; LCAT, lecithin:cholesterol acyltransferase; MTP, microsomal triglyceride transfer protein; PL, phospholipids; TG, triglycerides.
Manuscript received 28 January 2003. Initial review completed 19 February 2003. Revision accepted 17 April 2003.
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