- Email: [email protected]
The effect of chronic consumption of red wine on cardiovascular disease risk factors in postmenopausal women
Atherosclerosis 185 (2006) 438–445
The effect of chronic consumption of red wine on cardiovascular disease risk factors in postmenopausal women Mary Naissides, John C.L. Mamo, Anthony P. James, Sebely Pal ∗ Department of Nutrition, Dietetics and Food Science, School of Public Health, Curtin University of Technology, Kent Street, Bentley, WA 6102, Australia Received 12 April 2005; received in revised form 31 May 2005; accepted 21 June 2005 Available online 10 August 2005
Abstract Background: Moderate red wine has been shown to reduce cardiovascular disease (CVD) risk, however the effects on certain CVD risk factors are unclear. In this study we have investigated the effects of dealcoholised red wine (DRW) and full-complement red wine (RW) on several cardiovascular risk factors in mildly hypercholesterolaemic postmenopausal women. Objectives: To elucidate whether the chronic consumption of red wine polyphenols improves risk factors associated with CVD in hypercholesterolaemic postmenopausal women. Design: Forty-five hypercholesterolaemic postmenopausal women were randomly assigned to consume 400 mL/day of either water, DRW or RW for 6 weeks following a 4-week washout. Fasting measures of lipids, lipoproteins, insulin and glucose were taken at 0 and 6 weeks. Results: DRW consumption had no effect of fasting concentrations of lipids, lipoproteins, insulin and glucose. However, chronic consumption of RW significantly reduced fasting LDL cholesterol concentrations by 8% and increased HDL cholesterol concentrations by 17% in hypercholesterolaemic postmenopausal women. Conclusions: Collectively, regular consumption of full-complement red wine reduces CVD risk by improving fasting lipid levels in hypercholesterolaemic postmenopausal women. This study uniquely demonstrated the LDL cholesterol-lowering effects of red wine in individuals at high CVD risk, which has not previously been shown. © 2005 Published by Elsevier Ireland Ltd. Keywords: Cardiovascular disease; Cholesterol; LDL; HDL; Apolipoprotein B48; Insulin; Red wine; Polyphenols; Postmenopausal women
1. Introduction Moderate red wine consumption has been shown to reduce the risk of cardiovascular disease (CVD) to a greater extent than other alcoholic beverages, such as beer and spirits [1,2]. The polyphenolic compounds present in red wine are thought to explain the putative beneficial effects on CVD [3]. However presently, the effect of red wine and its polyphenolic constituents on certain CVD risk factors is unclear and requires elucidation through well-controlled clinical trials. ∗ Corresponding author at: Curtin University, School of Public Health, GPO Box U1987, Perth, WA 6845, Australia. Tel.: +61 8 92664755; fax: +61 8 92662958. E-mail address: [email protected] (S. Pal).
0021-9150/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2005.06.027
In vitro and animal studies have provided strong scientific evidence of the potent lipid- and lipoprotein-lowering effects of red wine and its polyphenolic constituents [4,5]. Studies from our laboratory have demonstrated a significant reduction in apolipoprotein B100 secretion, as well as an increase in LDL receptor expression and HMG-CoA reductase activity, following the incubation of HepG2 cells with full-complement red wine and dealcoholised red wine (polyphenol component), compared to controls [4]. In addition, significant reductions in chylomicron secretion were observed using the intestinal CaCO2 cell line following incubation with red wine [6]. Consistent with these findings, Vinson et al. recently showed a significant reduction in the concentration of total cholesterol and LDL cholesterol in dyslipidaemic hamsters consuming red wine and dealco-
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
holised red wine over a 10-week period, compared to controls [5]. However, human studies investigating the effect of red wine and its polyphenol components on lipid and lipoprotein metabolism have reported only modest or insignificant changes. The majority of these clinical studies have been conducted in healthy, normolipidaemic subjects rather than dyslipidaemic subjects where an effect may more readily be identified [7,8]. We suspect that a reduction in baseline cholesterol levels in already healthy individuals may be difficult to achieve. The lipid and lipoprotein lowering effects of red wine have not been examined in a high-risk group. In this study we have investigated hypercholesterolaemic postmenopausal women who are at an increased risk of developing CVD. The potential lipid-lowering effects of red wine may be influenced by the duration (acute versus chronic) of consumption. We have previously described the effect of acute consumption of red wine (RW) and dealcoholised red wine (DRW) on postprandial lipid and lipoprotein metabolism, as well as insulin homeostasis, in dyslipidaemic postmenopausal women [9]. Neither RW nor DRW were efficacious in acutely modulating postprandial lipaemia and insulin homeostasis. It was speculated that a single-dose of red wine polyphenols may be insufficient to achieve a concentration in tissue and plasma which is biologically active. Studies using animal models have demonstrated significant improvements in lipid and lipoprotein concentrations following the regular consumption of red wine and red wine polyphenols over an extended period of time [5]. Based on these findings we propose that chronic consumption of red wine and its polyphenolic constituents may be required to observe cardiovascular benefits in humans. The aim of this study was to investigate the effect of chronic consumption of red wine (with and without alcohol) on the metabolism of lipids, lipoproteins, insulin and glucose in moderately hypercholesterolaemic, postmenopausal women.
2. Subjects and methods 2.1. Subjects Forty-five moderately hypercholesterolaemic postmenopausal women, between the ages of 50 and 70 years, were recruited from the community. Subjects were screened for plasma cholesterol concentrations of ≥5.5 mmol/L and plasma triglyceride concentrations of <2 mmol/L. All subjects who met these inclusion criteria underwent a medical. Exclusion criteria included hormone replacement therapy, lipid-lowering medication, use of steroids and other agents that may influence lipid metabolism, use of warfarin, smoking, hyper- or hypothyroidism, diabetes mellitus, cardiovascular events within last 6 months, psychological unsuitability, major systemic diseases, gastrointestinal problems, proteinuria, liver and renal failure, apolipoprotein genotype (E2/E2 exclusion). All procedures were approved by the Ethics Com-
439
mittee of Curtin University and conformed to the Helsinki Declaration. Subjects provided written informed consent prior to participation in the study. 2.2. Experimental protocol The study was a randomised parallel-arm design examining the effects of chronic consumption of red wine and red wine polyphenols on lipid, lipoprotein, insulin and glucose metabolism in hypercholesterolaemic postmenopausal women. Forty-five subjects were randomised into either a water, dealcoholised wine (DRW) or red wine (RW) drinking group using the method of randomly permuted blocks. The number of subjects in each intervention group at the end of the study was: 16 in the water group, 15 in the DRW group and 14 in the RW group. Initially, subjects underwent a 4-week washout period where they were required to abstain from alcohol and red wine consumption, monitor intake of polyphenolic-rich food as well as maintain their usual dietary intake and physical activity level. The maintenance of dietary intake over the course of the trial was monitored through the completion of 3-day food diaries at the start and finish of the study. Following the 4-week washout, subjects arrived at Curtin University after a 10–12 h fast for baseline measurements. Subsequent to a mandatory rest period of approximately 30 min, fasting blood samples drawn by venepucture for analysis of various indicators of atherosclerotic risk. The subjects were then randomised to a 6-week intervention period consisting of 400 mL of either water, DRW or RW (approximately 13% (v/v) alcohol ∼40 g) taken with their evening meal. Total polyphenol content between the DRW and RW was identical. The collection of fasting blood samples was repeated after 6 weeks of beverage consumption. Compliance to beverage consumption was monitored regularly and serum ␥-glutamyl transpeptidase (␥GT) levels were measured at baseline and 6 weeks subsequent to beverage consumption. Serum and plasma were collected from blood samples after centrifugation at 3000 rpm at 4 ◦ C for 10 min for determination of apolipoprotein B48 (apo B48), total apolipoprotein B (apo B), total cholesterol, LDL cholesterol, HDL cholesterol, triacylglycerol, insulin and blood glucose. Samples were either analysed immediately or stored at −80 ◦ C for subsequent analysis. 2.3. Standardised diets Standardised meals, including main meals and snacks, were provided for 2 days prior to each intervention day. Standardised diets were based on the subjects usual energy intake, as well as reports from the National Nutrition Survey of 1995 [10] on the average ‘typical’ dietary intake of Australians aged 55–64 years, the Australian dietary goals [11] and National Heart Foundation (NHF) guidelines. Instructions on the correct consumption of the standard meals were
440
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
given to the subjects by a dietitian and compliance was monitored.
colorimetry (TRACE Scientific Ltd., Melbourne, Australia). Serum LDL cholesterol was determined by using a modified version of the Friedewald equation [14].
2.4. Measurement of total polyphenols in wine 2.8. Measurement of insulin and HOMA The total polyphenols in a 2001 Cabernet Sauvignon (13.4% (v/v) alcohol, Ferngrove Vineyard Ltd., Frankland, WA) was quantified using a colorimetric assay with FolinCiocalteau reagent according to the method published by the Association of Official Analytical Chemists [12]. Polyphenols are estimated at a wavelength of 760 nm in relation to a standard curve for gallic acid. The total polyphenol content in both DRW and RW was approximately 2.5 g/L. Therefore, a total of 1 g of red wine polyphenols was consumed by subjects on daily basis. 2.5. Dealcoholised wine preparation Ethanol was removed from red wine by rotary evaporation at <40 ◦ C to approximately 50% of the original volume. The dealcoholised wine was then made to 400 mL with water, ensuring that the concentration of polyphenols was identical to that in the full-complement red wine. 2.6. Apolipoprotein B48 and total apolipoprotein B determination Apolipoprotein B48 (apo B48) was quantitated using a Western blotting/chemiluminescence procedure as previously described [13]. Briefly, apolipoproteins were separated by SDS-PAGE using a Novex Mini-cell Electrophoresis system (Invitrogen, Carlsbad, USA) with precast NUPAGE 3–8% Tris–acetate gels according to the manufacturer’s instructions. After electrophoresis, separated lipoproteins where transferred to a PVDF membrane (0.45 m; Immobilon PTM ; Millipore Corporation, Bedford, MA, USA). apo B48 bands were identified and visualized using an antibody to apo B (DAKO A/S, Glostrup, Denmark) and subsequently antirabbit IgG antibody (HRP conjugated; Amersham, Little Chalfont, UK) using enhanced chemiluminescence (ECLTM ; Amersham, Little Chalfont, UK). Densitometric scanning of apo B48 bands and standardisation to known apo B48 protein mass allowed quantification of the protein. Total apolipoprotein B (apo B) was measured in sera samples by an immunonephelometric assays using Behring reagents on a Behring Nephelometer II (BN-II) system (Dade Behring Inc., Marburg, Germany). 2.7. Measurement of plasma lipids Serum triglyceride (TG) and total cholesterol was measured by enzymatic colorimetric kits (TRACE Scientific Ltd., Melbourne, Australia). Serum HDL cholesterol was determined after precipitation of apo B-containing lipoproteins with phosphotungstic acid and MgCl2 , the supernatant containing the HDL cholesterol was determined by enzymatic
Plasma insulin was measured by an ELISA kit (Dako Diagnostic, Japan). The HOMA score was used as a surrogate estimate of the state of insulin sensitivity by multiplying fasting insulin concentration (mIU/L) with fasting glucose concentration (mM) and divide by 22.5 [15]. 2.9. Statistical analysis Statistical analysis was done using SPSS 11 for Windows (SPSS Inc., Chicago, IL) and SAS 8 (SAS Institute Inc., Cary, NC, USA). Data are expressed as mean (S.E.M.) and assessed for normality. Comparison of baseline characteristics between each group was done by analysis of variance with Tukey’s post hoc analysis. Between and within group comparisons of the water, DRW and RW groups were made using general linear models with adjustment for baseline values. Statistical differences were analysed further by post hoc analysis using the least square differences (LSD) method. Statistical significance was considered at p < 0.05.
3. Results 3.1. Subject characteristics The clinical characteristics of subjects in the water, DRW and RW groups measured at screening are shown in Table 1. There was no significant difference between groups in the anthropometric or lipid parameters. According to NCEP ATP III guidelines, the postmenopausal women in this study displayed moderate to high total cholesterol and LDL cholesterol concentrations, with mean concentrations of 6.2 and 4.1 mmol/L, respectively. Table 1 Clinical characteristics of subjects at screening Characteristics
Water
DRW
RW
Age (years) Weight (kg) BMI (kg/m2 ) Waist (cm) W:H ratio Oestradiol (pmol/L) TG (mmol/L) TC (mmol/L) Glucose (mmol/L) Systolic BP (mmHg) Diastolic BP (mmHg)
59.3 (1.4) 70.0 (2.7) 26.7 (1.2) 84.3 (3.1) 0.8 (0.02) <150 1.2 (1.2) 6.5 (0.12) 5.3 (0.11) 127.1 (3.76) 75.3 (2.1)
57.6 (1.3) 72.8 (3.3) 26.3 (0.9) 86.1 (2.9) 0.8 (0.02) <150 1.4 (0.1) 7.1 (0.3) 5.4 (0.1) 127.3 (3.48) 78 (2.06)
58.4 (1.3) 69.7 (4.6) 25.9 (1.6) 83.4 (3.9) 0.8 (0.02) <150 1.3 (0.2) 6.56 (0.2) 5.2 (0.1) 131.9 (4.82) 79.3 (3.55)
Data represents mean (S.E.M.) of clinical characteristics of postmenopausal participants at screening. There was no significant difference between subject characteristics at screening. W:H ratio, waist to hip ratio; TG, triglyceride; TC, total cholesterol; BP, blood pressure.
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
441
Table 2 Dietary intake of subjects at the start and finish of the intervention Water
Energy (kJ) Carbohydrate (% of total kJ) Protein (% of total kJ) Total fat (% of total kJ) SFA (% of total kJ) MUFA (% of total kJ) PUFA (% of total kJ) Cholesterol (mg) Alcohol (% of total kJ)
DRW
RW
Start
Finish
Start
Finish
Start
Finish
8452.9 (463.47) 43.9 (0.02) 18.5 (0.01) 37.5 (0.02) 14.0 (1.03) 14.4 (1.37) 5.1 (0.36) 276.2 (23.53) –
9687.5 (692.01) 42.1 (0.02) 18.7 (0.01) 39.1 (0.02) 15.1 (0.77) 15 (1.09) 4.9 (0.46) 331.0 (30.56) –
9031.7 (508.10) 46.3 (0.03) 19.2 (0.01) 34.3 (0.03) 13.9 (1.41) 12.2 (1.25) 4.5 (0.45) 266.0 (31.63) –
8575.9 (937.93) 42.2 (0.02) 19.0 (0.01) 38.6 (0.02) 15.7 (1.09) 13.0 (0.93) 5.6 (0.77) 407.6 (106.52) –
10033 (609.11) 46.3 (0.03) 19.4 (0.01) 34.2 (0.03) 12.43 (1.22) 12.0 (1.05) 5.1 (0.66) 362.0 (80.31) 11.24
9281.5 (472.59) 48.9 (0.04) 18.2 (0.01) 32.9 (0.04) 12.87 (2.19) 11.8 (1.15) 4.6 (0.54) 253.68 (25.64) 12.0
Data represents are mean (S.E.M.) of the nutritional data reported in the 3-day food diary at the start and finish of the intervention. There was no significant difference in dietary intake at the start and finish of the intervention. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
3.2. Nutritional assessment There were no significant differences in macronutrient intake by subjects at the start and finish of the intervention (Table 2). There was no change in serum ␥GT levels in subjects consuming water and DRW, confirming that subjects in these groups abstained from alcohol consumption over the 6-week intervention period. In subjects consuming RW, as expected, a progressive rise in serum ␥GT (p < 0.05) was observed over the 6-week period. 3.3. Changes in fasting lipid concentrations There was no significant change in fasting LDL cholesterol, HDL cholesterol, total cholesterol and triglyceride concentrations following the consumption of DRW, compared to water (Table 3). However, RW consumption significantly decreased fasting LDL cholesterol concentration by 8% (p < 0.05) after 6 weeks, compared to water (Fig. 1). A significant 17% (p < 0.05) increase in HDL cholesterol concentration was observed following 6 weeks of RW consumption, compared with water (Fig. 2). Despite the improvement in LDL cholesterol concentration there was no change in total cholesterol concentration following the consumption of RW compared to water (Table 3). In addition, a significant
negative correlation (r = −0.533; p = 0.05) between baseline total cholesterol concentration and the percent change in LDL cholesterol concentration (baseline to 6 weeks) was observed in the RW group. RW consumption had no effect of triglyceride concentration, compared to water (Table 3). The results did not change following adjustments for body weight, BMI and waist circumference. 3.4. Changes in fasting lipid ratios The total cholesterol (TC):HDL ratio and the LDL:HDL ratio did not change with DRW consumption, compared to water. However, RW consumption significantly decreased the TC:HDL ratio by 14% (p < 0.05) after 6 weeks, compared to water (Fig. 3). In addition, we detected a significant decrease of 20% (p < 0.01) in the LDL:HDL ratio following 6 weeks of RW consumption, compared to water (Fig. 4). The results were not affected by adjustments for body weight, BMI and waist circumference. 3.5. Changes in fasting apolipoprotein B concentrations Fasting plasma chylomicron concentration, as measured by apo B48, was not altered by the consumption of DRW or RW over the 6-week intervention period (Table 3). Similarly,
Table 3 The change in fasting concentrations of cardiovascular disease risk factors Water
TC (mmol/L) TG (mmol/L) Apo B48 (g/mL) Apo B (g/L) Glucose (mmol/L) Insulin (IU/mL) HOMA
DRW
RW
Baseline
6 weeks
Baseline
6 weeks
Baseline
6 weeks
6.17 (0.15) 1.24 (0.12) 12.01 (0.59) 1.10 (0.04) 5.11 (0.11) 5.48 (0.87) 1.28 (0.22)
6.29 (0.15) 1.21 (0.10) 12.25 (0.68) 1.13 (0.05) 5.11 (0.10) 5.32 (0.58) 1.23 (0.14)
6.27 (0.26) 1.33 (0.17) 14.09 (0.85) 1.14 (0.06) 5.16 (0.11) 6.07 (0.81) 1.39 (0.18)
6.19 (0.32) 1.31 (0.14) 14.09 (0.84) 1.15 (0.07) 5.07 (0.06) 5.04 (0.39) 1.14 (0.09)
6.26 (0.14) 1.19 (0.15) 12.40 (0.58) 1.11 (0.04) 5.06 (0.08) 5.37 (1.07) 1.23 (0.26)
6.25 (0.17) 1.29 (0.18) 12.56 (0.57) 1.08 (0.06) 5.24 (0.09) 5.77 (1.57) 1.36 (0.39)
Values are mean (S.E.M.). There was no significant difference in certain cardiovascular disease risk factors following DRW and RW consumption. TC, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; HDL:LDL; HDL to LDL ratio; TG, triglycerides; Apo B48, apolipoprotein B48; Apo B, total apolipoprotein B; HOMA, homeostasis model assessment.
442
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
Fig. 1. Changes in fasting serum LDL cholesterol concentrations. The percentage change in fasting LDL cholesterol concentrations from baseline to 6 weeks following consumption of water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
Fig. 4. Changes in fasting LDL:HDL ratio. The percentage change in the LDL:HDL ratio from baseline to 6 weeks following consumption of either water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
3.6. Changes in fasting concentrations of blood glucose, plasma insulin and HOMA Fasting blood glucose, plasma insulin and HOMA score were not altered following the DRW or RW interventions, compared to water (Table 3). The results remained unchanged following adjustments for body weight, BMI and waist circumference.
4. Discussion Fig. 2. Changes in fasting serum HDL cholesterol concentrations. The percentage change in fasting HDL cholesterol concentrations from baseline to 6 weeks following consumption of either water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
serum concentrations of total apo B were unaffected by the consumption of DRW and RW compared to water, although a trend towards a decrease in apo B concentrations was noted following RW consumption (p = 0.4; Table 3). The results were not affected by adjustments for body weight, BMI and waist circumference.
Fig. 3. Changes in fasting TC:HDL ratio. The percentage change in the TC:LDL ratio from baseline to 6 weeks following consumption of either water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
We have previously demonstrated that acute consumption of DRW (polyphenol component) does not affect postprandial lipaemia or insulin sensitivity in dyslipidaemic postmenopausal women [9]. Furthermore, it was shown that an acute dose of full-complement RW exacerbates postprandial lipid and insulin levels, suggesting that neither red wine nor its polyphenolic components confer cardiovascular benefits acutely through improvements in postprandial lipid and lipoprotein metabolism. Based on these findings, as well as extensive reports in animal studies supporting improvements in CVD risk factors following long-term consumption of red wine and red wine polyphenols, it was proposed that chronic ingestion of red wine and its polyphenolic constituents may be essential for favourable physiological effects to occur, including improvements in lipid and lipoprotein metabolism. In this study, we have extended our previous findings from cell-culture and clinical studies by examining the effect of chronic consumption of DRW and RW on lipid, lipoprotein, insulin and glucose metabolism in moderately hypercholesterolaemic postmenopausal women. Our findings show that DRW consumption had no effect on fasting lipid and lipoprotein levels, and insulin sensitivity, over a 6-week period. Rather, chronic consumption of full-complement RW was required to observe improvements in fasting lipid and lipoprotein concentrations. Although the reduction in CVD mortality associated with moderate red wine consumption is well established, the certain mechanisms responsible for this risk reduction require further elucidation.
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
We have provided novel and unequivocal evidence that chronic red wine consumption significantly reduces serum LDL cholesterol, increases serum HDL cholesterol and improves fasting lipid ratios in postmenopausal women, which may partly explain the inverse association between red wine consumption and CVD risk. In addition, the findings of this study suggest that the improvements observed in cardiovascular disease risk factors with red wine consumption may not be independent of its alcohol component. The alcohol and polyphenol components in red wine may be working synergistically to produce biological effects. Studies have postulated that ethanol may facilitate the absorption of polyphenols found in red wine, resulting in greater plasma concentrations of these compounds than when consumed in the absence of ethanol [16]. However, in our study we cannot exclude the exclusive cardiovascular effects of the alcohol component in red wine. Several epidemiological studies show that the CVD benefits observed with alcohol consumption are independent of beverage type [17], although clinical studies are inconclusive [18]. Further studies comparing the effects of chronic consumption of ethanol compared to red wine in postmenopausal women are required and are currently underway in our laboratory. Interestingly, red wine polyphenols per se have been shown to improve lipid, lipoprotein and insulin levels in cell-culture and animal studies [4,5]. However, our results did not show any changes in fasting levels of lipid, lipoproteins or insulin following 6 weeks of DRW consumption by hypercholesterolaemic postmenopausal women. Evidence is emerging that cell-culture studies investigating the biological effects of polyphenols are inherently flawed, leading to inaccurate interpretation of the data [19]. The majority of in vitro studies have assessed the biological activity of polyphenols by administering them in their natural phytochemical form, for example as aglycones and various glycosides [20]. However, it is now clear that polyphenols are extensively modified in the digestive tract, as well as in circulation, to form conjugates and metabolites of the parent compound [21]. As a consequence, the polyphenol metabolites are likely to possess different biological properties and distribution patterns within cells and tissue to the parent compounds. In addition, in vitro studies have administered polyphenol concentrations which are substantially greater than the plasma concentration attained in humans following consumption of a polyphenol-rich meal [19]. Therefore, data from many in vitro trials reporting the biological effects of polyphenols cannot be extrapolated to humans. In addition, the absorption and metabolism of polyphenols has primarily been elucidated in animal models, however, data on human metabolism of these compounds is scarce. It is possible that following the consumption of a polyphenol-rich meal, the metabolites occurring in circulation are different between human and animal models in terms of chemistry, concentration and potency. Potential differences in circulating metabolites between the two models may explain the discrepancies in cardiovascular effects observed in our study following DRW consumption.
443
Elevated concentrations of LDL cholesterol are strongly associated with the development of atherosclerosis; therefore its reduction in circulation is critical in the attenuation of CVD mortality. Contrary to the findings of previous studies, we have demonstrated unequivocally that chronic RW consumption significantly reduces LDL cholesterol in hypercholesterolaemic postmenopausal women. The positive correlation between baseline total cholesterol levels and the percent change in LDL cholesterol suggest that the extent of improvement in atherogenic lipid levels may be related to the severity of hypercholesterolaemia. These findings are in support of our initial hypothesis as they imply that reductions in LDL cholesterol following RW consumption may be more readily observed in hypercholesterolaemic populations. Furthermore, the duration and beverage consumption could play a significant role in potential reductions of circulating lipid levels. In this study, changes in LDL cholesterol were observed only after 6 weeks of RW consumption. However, the majority of studies in humans have involved a modest intervention period of approximately 2–4 weeks, which may also account for the lack of significant change in LDL cholesterol reported [7,18]. The mitigation of LDL cholesterol observed following RW consumption was accompanied by a non-significant decrease in total apo B. Since apo B48 levels remained unchanged, the decrease in total apo B levels may reflect an alcohol-induced attenuation of apo B100 concentrations. Apo B100 is exclusively associated with hepatically derived lipoproteins in circulation, providing a useful estimate of LDL particle number. The simultaneous reduction in LDL cholesterol and apo B100 may be indicative of a decrease in LDL-particle number, which may occur either through a decrease in production and/or enhanced hepatic clearance of LDL particles. Cell-culture studies from our laboratory have reported a 30% decrease in apo B100 synthesis and secretion in HepG2 cells following incubation with RW [4]. The reduction in apo B100 was shown to result from a decrease in intracellular cholesterol synthesis and an up-regulation in LDL receptor expression, enhancing the clearance of these particles [4]. Human studies investigating the specific mechanisms responsible for the reduction in LDL cholesterol are required. Improvements in HDL cholesterol following moderate alcohol consumption, including RW, are well documented [22]. In fact, approximately 50% of the cardiovascular benefits observed with moderate alcohol consumption are thought to be mediated through an increase in HDL [23]. Several studies have documented an increase in HDL cholesterol in healthy individuals consuming regular doses of alcohol similar to those administered in this study [7,18]. To our knowledge, this is the first study to report improvements in HDL cholesterol in a hypercholesterolaemic subject group. Moreover, the magnitude of change in HDL cholesterol observed in our study appears to be greater than the changes observed in several other studies administering similar quantities of alcohol to healthy volunteers [7,18,24]. The
444
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
hypercholesterolaemic nature of our subject group may contribute to the enhanced effects of RW on HDL cholesterol. Plasma lipid ratios, such as the TC:HDL ratio and the LDL:HDL ratio, have been shown to be strong predictors of CHD [25]. A TC:HDL ratio of <3.5 is considered optimal, an increase beyond this cut-off value is associated with increased risk of CHD [25]. The consumption of DRW did not improve plasma lipid ratios, suggesting the RW polyphenols may not confer cardiovascular benefits through improvements in circulating lipid levels. However, a significant decrease in TC:HDL ratio from 4.3 to 3.7 was observed in the RW group, compared to controls. Furthermore, lipidlowering drug trials assessing the association between lipid ratios and CHD have shown that for every 1% decrease in the TC:HDL ratio, CHD risk decreases by 1.3% [26]. We observed an overall decrease 14% in the TC:HDL ratio in hypercholesterolaemic postmenopausal women, which indicates an 18% reduction in CHD risk. Similarly, reductions in the LDL:HDL ratio following RW consumption are comparable to improvements observed following treatment with lipid-lowering drugs which has been associated with significant reductions in CHD risk [26]. Chylomicron remnants have been implicated in the development of atherosclerosis [27]. The effect of chronic consumption of RW polyphenols (±alcohol) on chylomicron metabolism in humans has not been investigated previously. At baseline, the concentration of apolipoprotein B48 (surrogate marker for chylomicrons) was shown to be elevated in hypercholesterolaemic postmenopausal women. However, fasting apo B48 levels remained unchanged following chronic consumption of DRW and RW, suggesting that neither polyphenols, nor full-complement RW, modulate intestinal lipoprotein metabolism. Our results confirm recent findings from Daher et al. showing no changes in apo B48 concentrations in rats consuming moderate doses of alcohol over a 10-week period [28]. It is also worthy to note that in contrast to acute RW consumption, chronic RW consumption did not induce hypertriglyceridaemia in moderately hypercholesterolaemic postmenopausal women. Similar results have been observed in other clinical studies following chronic consumption of similar doses of RW and alcohol, although findings are inconsistent [7,18,29]. It is also possible that RW consumption improves CVD risk via improvements in insulin sensitivity. Several observational studies have associated moderate RW and alcohol consumption with enhanced insulin sensitivity, low plasma insulin and glucose levels, and reduced risk of type II diabetes mellitus [30], although some have not observed this association [31]. Controlled clinical trials elucidating the effects of alcohol consumption, specifically red wine, on insulin and glucose metabolism are scarce and their findings inconsistent [29,32]. Moreover, we are unaware of any human experimental trials which have examined the effect of chronic consumption of red wine polyphenols (DRW) on glucose tolerance and insulin sensitivity. In this group of hypercholesterolaemic postmenopausal women, chronic consumption of DRW did
not influence insulin or glucose metabolism. Interestingly, chronic RW consumption neither improved nor exacerbated plasma insulin levels, blood glucose levels and insulin sensitivity. In summary, we have found that chronic consumption of DRW does not affect fasting lipid and lipoprotein levels, or insulin sensitivity in moderately hypercholesterolaemic postmenopausal women. In contrast, full-complement RW consumption improved circulating lipid and lipoprotein concentrations, as well as the atherogenic risk profile in this group of women. Collectively, our findings suggest that chronic consumption of full-complement red wine may reduce cardiovascular disease risk through improvements in basal levels of lipid and lipoproteins in hypercholesterolaemic postmenopausal women. Furthermore, it appears that the beneficial effects on lipid and lipoprotein metabolism observed with red wine consumption are not independent of its alcohol component. Caution should be exercised when advocating regular alcohol consumption as excessive alcohol consumption may result in adverse health effects.
Acknowledgements The project was supported by grants from the National Heart Foundation of Australia and the ATN Centre for Metabolic Fitness. The statistical advice received from S. Dhaliwal is appreciated. We are grateful to the Chemistry Centre, Perth, Australia for their assistance in the dealcoholising of red wine. References [1] Renaud S, De Lorgeril M, Salen MD, et al. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 1992;339:1523–6. [2] St Leger AS, Cochrane AL, Moore F. Factors associated with cardiac mortality in developed countries with particular reference to the consumption of wine. Lancet 1979;1:1017–20. [3] Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 1993;342:1007–11. [4] Pal S, Ho N, Santos C, et al. Red wine polyphenolics increase LDL receptor expression and activity and suppress the secretion of ApoB100 from human HepG2 cells. J Nutr 2003;133:700–6. [5] Vinson JA, Teufel K, Wu N. Red wine, dealcoholised red wine, and especially grape juice, inhibit atherosclerosis in a hamster model. Atherosclerosis 2001;156:67–72. [6] Pal S, Ho SS, Takechi R. Red wine polyphenolics suppress the secretion of ApoB48 from human intestinal CaCo-2 Cells. J Agric Food Chem 2005;53:2767–72. [7] Goldberg DM, Garovic-Kocic V, Diamandis EP, Pace-Asciak CR. Wine: does the colour count? Clin Chim Acta 1996;246:183–93. [8] Lavy AFB, Markel A, Danker G, et al. Effect of dietary supplementation of red or white wine on human blood chemistry, hematology and coagulation: favourable effect of red wine on plasma high-density lipoproteins. Ann Nutr Metabol 1994;38:287–94. [9] Naissides M, Mamo JC, James AP, Pal S. The effect of acute red wine polyphenol consumption on postprandial lipaemia in postmenopausal women. Atherosclerosis 2004;177:401–8.
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445 [10] Australian Food and Nutrition Monitoring Unit. Comparable data on food and nutrient intake and physical measurements from the 1983, 1985 and 1995 national nutrition surveys. Commonwealth of Australia; 2001. [11] Nutbeam D, Wise M, Bauman A, Harris E, Leeder S. Goals and targets for Australia’s health in the year 2000 and beyond. Sydney, Australia: Department of Public Health, University of Sydney; 1993. [12] Helrick K. Official methods of analysis of the Association of Official Analytical Chemists. 15th ed. Arlington, VA: AOAC; 1990. [13] Smith D, Proctor SD, Mamo JC. A highly sensitive assay for quantitation of apolipoprotein B48 using an antibody to human apolipoprotein B and enhanced chemiluminescence. Ann Clin Biochem 1997;34:185–9. [14] Bairaktari E, Hatzidimou K, Tzallas C, et al. Estimation of LDL cholesterol based on the Friedewald formula and on apo B levels. Clin Biochem 2000;33:549–55. [15] Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [16] Miyagi Y, Miwa K, Inoue H. Inhibition of human low-density lipoprotein oxidation by flavonoids in red wine and grape juice. Am J Cardiol 1997;80:1627–31. [17] Rimm EB, Klatsky A, Grobbee D, Stampfer MJ. Review of moderate alcohol consumption and reduced risk of coronary heart disease: is the effect due to beer, wine, or spirits. Br Med J 1996;312:731–6. [18] Senault C, Betoulle D, Luc G, et al. Beneficial effects of a moderate consumption of red wine on cellular cholesterol efflux in young men. Nutr Metab Cardiovasc Dis 2000;10:63–9. [19] Kroon PA, Clifford MN, Crozier A, et al. How should we assess the effects of exposure to dietary polyphenols in vitro? Am J Clin Nutr 2004;80:15–21. [20] Gebhardt R. Variable influence of kaempferol and myricetin on in vitro hepatocellular cholesterol biosynthesis. Planta Med 2003; 69:1071–4. [21] Donovan JL, Bell JR, Kasim-Karakas S, et al. Catechin is present as metabolites in human plasma after consumption of red wine. J Nutr 1999;129:1662–8.
445
[22] Goldberg DM, Hahn SE, Parkes JG. Beyond alcohol: beverage consumption and cardiovascular mortality. Clin Chim Acta 1995;237:155–87. [23] Linn S, Carroll M, Johnson C, et al. High-density lipoprotein cholesterol and alcohol consumption in US white and black adults: data from NHANES II. Am J Public Health 1993;83:811–6. [24] Baer DJ, Judd JT, Clevidence BA, et al. Moderate alcohol consumption lowers risk factors for cardiovascular disease in postmenopausal women fed a controlled diet. Am J Clin Nutr 2002;75: 593–9. [25] Castelli WP. Cholesterol and lipids in the risk of coronary artery disease—the Framingham Heart Study. Can J Cardiol 1988;4(Suppl A):5A–10A. [26] Rader DJ, Davidson MH, Caplan RJ, Pears JS. Lipid and apolipoprotein ratios: association with coronary artery disease and effects of rosuvastatin compared with atorvastatin, pravastatin, and simvastatin. Am J Cardiol 2003;91:20C–3C [discussion 23C–4C]. [27] Mamo J, Yu K, Elsgood C, et al. Is atherosclerosis exclusively a postprandial phenomenon? Clin Exp Pharmacol Physiol 1997;24: 288–93. [28] Daher CF, Berberi RN, Baroody GM. Effect of acute and chronic moderate alcohol consumption on fasted and postprandial lipemia in the rat. Food Chem Toxicol 2003;41:1551–9. [29] Cordain L, Melby CL, Hamamoto AE, et al. Influence of moderate chronic wine consumption on insulin sensitivity and other correlates of syndrome X in moderately obese women. Metabolism 2000;49:1473–8. [30] Facchini F, Chen YD, Reaven GM. Light-to-moderate alcohol intake is associated with enhanced insulin sensitivity. Diab Care 1994;17:115–9. [31] Meyer KA, Conigrave KM, Chu NF, et al. Alcohol consumption patterns and HbA1c C-peptide and insulin concentrations in men. J Am Coll Nutr 2003;22:185–94. [32] Davies MJ, Baer DJ, Judd JT, et al. Effects of moderate alcohol intake on fasting insulin and glucose concentrations and insulin sensitivity in postmenopausal women: a randomized controlled trial. JAMA 2002;287:2559–62.
The effect of chronic consumption of red wine on cardiovascular disease risk factors in postmenopausal women Mary Naissides, John C.L. Mamo, Anthony P. James, Sebely Pal ∗ Department of Nutrition, Dietetics and Food Science, School of Public Health, Curtin University of Technology, Kent Street, Bentley, WA 6102, Australia Received 12 April 2005; received in revised form 31 May 2005; accepted 21 June 2005 Available online 10 August 2005
Abstract Background: Moderate red wine has been shown to reduce cardiovascular disease (CVD) risk, however the effects on certain CVD risk factors are unclear. In this study we have investigated the effects of dealcoholised red wine (DRW) and full-complement red wine (RW) on several cardiovascular risk factors in mildly hypercholesterolaemic postmenopausal women. Objectives: To elucidate whether the chronic consumption of red wine polyphenols improves risk factors associated with CVD in hypercholesterolaemic postmenopausal women. Design: Forty-five hypercholesterolaemic postmenopausal women were randomly assigned to consume 400 mL/day of either water, DRW or RW for 6 weeks following a 4-week washout. Fasting measures of lipids, lipoproteins, insulin and glucose were taken at 0 and 6 weeks. Results: DRW consumption had no effect of fasting concentrations of lipids, lipoproteins, insulin and glucose. However, chronic consumption of RW significantly reduced fasting LDL cholesterol concentrations by 8% and increased HDL cholesterol concentrations by 17% in hypercholesterolaemic postmenopausal women. Conclusions: Collectively, regular consumption of full-complement red wine reduces CVD risk by improving fasting lipid levels in hypercholesterolaemic postmenopausal women. This study uniquely demonstrated the LDL cholesterol-lowering effects of red wine in individuals at high CVD risk, which has not previously been shown. © 2005 Published by Elsevier Ireland Ltd. Keywords: Cardiovascular disease; Cholesterol; LDL; HDL; Apolipoprotein B48; Insulin; Red wine; Polyphenols; Postmenopausal women
1. Introduction Moderate red wine consumption has been shown to reduce the risk of cardiovascular disease (CVD) to a greater extent than other alcoholic beverages, such as beer and spirits [1,2]. The polyphenolic compounds present in red wine are thought to explain the putative beneficial effects on CVD [3]. However presently, the effect of red wine and its polyphenolic constituents on certain CVD risk factors is unclear and requires elucidation through well-controlled clinical trials. ∗ Corresponding author at: Curtin University, School of Public Health, GPO Box U1987, Perth, WA 6845, Australia. Tel.: +61 8 92664755; fax: +61 8 92662958. E-mail address: [email protected] (S. Pal).
0021-9150/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2005.06.027
In vitro and animal studies have provided strong scientific evidence of the potent lipid- and lipoprotein-lowering effects of red wine and its polyphenolic constituents [4,5]. Studies from our laboratory have demonstrated a significant reduction in apolipoprotein B100 secretion, as well as an increase in LDL receptor expression and HMG-CoA reductase activity, following the incubation of HepG2 cells with full-complement red wine and dealcoholised red wine (polyphenol component), compared to controls [4]. In addition, significant reductions in chylomicron secretion were observed using the intestinal CaCO2 cell line following incubation with red wine [6]. Consistent with these findings, Vinson et al. recently showed a significant reduction in the concentration of total cholesterol and LDL cholesterol in dyslipidaemic hamsters consuming red wine and dealco-
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
holised red wine over a 10-week period, compared to controls [5]. However, human studies investigating the effect of red wine and its polyphenol components on lipid and lipoprotein metabolism have reported only modest or insignificant changes. The majority of these clinical studies have been conducted in healthy, normolipidaemic subjects rather than dyslipidaemic subjects where an effect may more readily be identified [7,8]. We suspect that a reduction in baseline cholesterol levels in already healthy individuals may be difficult to achieve. The lipid and lipoprotein lowering effects of red wine have not been examined in a high-risk group. In this study we have investigated hypercholesterolaemic postmenopausal women who are at an increased risk of developing CVD. The potential lipid-lowering effects of red wine may be influenced by the duration (acute versus chronic) of consumption. We have previously described the effect of acute consumption of red wine (RW) and dealcoholised red wine (DRW) on postprandial lipid and lipoprotein metabolism, as well as insulin homeostasis, in dyslipidaemic postmenopausal women [9]. Neither RW nor DRW were efficacious in acutely modulating postprandial lipaemia and insulin homeostasis. It was speculated that a single-dose of red wine polyphenols may be insufficient to achieve a concentration in tissue and plasma which is biologically active. Studies using animal models have demonstrated significant improvements in lipid and lipoprotein concentrations following the regular consumption of red wine and red wine polyphenols over an extended period of time [5]. Based on these findings we propose that chronic consumption of red wine and its polyphenolic constituents may be required to observe cardiovascular benefits in humans. The aim of this study was to investigate the effect of chronic consumption of red wine (with and without alcohol) on the metabolism of lipids, lipoproteins, insulin and glucose in moderately hypercholesterolaemic, postmenopausal women.
2. Subjects and methods 2.1. Subjects Forty-five moderately hypercholesterolaemic postmenopausal women, between the ages of 50 and 70 years, were recruited from the community. Subjects were screened for plasma cholesterol concentrations of ≥5.5 mmol/L and plasma triglyceride concentrations of <2 mmol/L. All subjects who met these inclusion criteria underwent a medical. Exclusion criteria included hormone replacement therapy, lipid-lowering medication, use of steroids and other agents that may influence lipid metabolism, use of warfarin, smoking, hyper- or hypothyroidism, diabetes mellitus, cardiovascular events within last 6 months, psychological unsuitability, major systemic diseases, gastrointestinal problems, proteinuria, liver and renal failure, apolipoprotein genotype (E2/E2 exclusion). All procedures were approved by the Ethics Com-
439
mittee of Curtin University and conformed to the Helsinki Declaration. Subjects provided written informed consent prior to participation in the study. 2.2. Experimental protocol The study was a randomised parallel-arm design examining the effects of chronic consumption of red wine and red wine polyphenols on lipid, lipoprotein, insulin and glucose metabolism in hypercholesterolaemic postmenopausal women. Forty-five subjects were randomised into either a water, dealcoholised wine (DRW) or red wine (RW) drinking group using the method of randomly permuted blocks. The number of subjects in each intervention group at the end of the study was: 16 in the water group, 15 in the DRW group and 14 in the RW group. Initially, subjects underwent a 4-week washout period where they were required to abstain from alcohol and red wine consumption, monitor intake of polyphenolic-rich food as well as maintain their usual dietary intake and physical activity level. The maintenance of dietary intake over the course of the trial was monitored through the completion of 3-day food diaries at the start and finish of the study. Following the 4-week washout, subjects arrived at Curtin University after a 10–12 h fast for baseline measurements. Subsequent to a mandatory rest period of approximately 30 min, fasting blood samples drawn by venepucture for analysis of various indicators of atherosclerotic risk. The subjects were then randomised to a 6-week intervention period consisting of 400 mL of either water, DRW or RW (approximately 13% (v/v) alcohol ∼40 g) taken with their evening meal. Total polyphenol content between the DRW and RW was identical. The collection of fasting blood samples was repeated after 6 weeks of beverage consumption. Compliance to beverage consumption was monitored regularly and serum ␥-glutamyl transpeptidase (␥GT) levels were measured at baseline and 6 weeks subsequent to beverage consumption. Serum and plasma were collected from blood samples after centrifugation at 3000 rpm at 4 ◦ C for 10 min for determination of apolipoprotein B48 (apo B48), total apolipoprotein B (apo B), total cholesterol, LDL cholesterol, HDL cholesterol, triacylglycerol, insulin and blood glucose. Samples were either analysed immediately or stored at −80 ◦ C for subsequent analysis. 2.3. Standardised diets Standardised meals, including main meals and snacks, were provided for 2 days prior to each intervention day. Standardised diets were based on the subjects usual energy intake, as well as reports from the National Nutrition Survey of 1995 [10] on the average ‘typical’ dietary intake of Australians aged 55–64 years, the Australian dietary goals [11] and National Heart Foundation (NHF) guidelines. Instructions on the correct consumption of the standard meals were
440
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
given to the subjects by a dietitian and compliance was monitored.
colorimetry (TRACE Scientific Ltd., Melbourne, Australia). Serum LDL cholesterol was determined by using a modified version of the Friedewald equation [14].
2.4. Measurement of total polyphenols in wine 2.8. Measurement of insulin and HOMA The total polyphenols in a 2001 Cabernet Sauvignon (13.4% (v/v) alcohol, Ferngrove Vineyard Ltd., Frankland, WA) was quantified using a colorimetric assay with FolinCiocalteau reagent according to the method published by the Association of Official Analytical Chemists [12]. Polyphenols are estimated at a wavelength of 760 nm in relation to a standard curve for gallic acid. The total polyphenol content in both DRW and RW was approximately 2.5 g/L. Therefore, a total of 1 g of red wine polyphenols was consumed by subjects on daily basis. 2.5. Dealcoholised wine preparation Ethanol was removed from red wine by rotary evaporation at <40 ◦ C to approximately 50% of the original volume. The dealcoholised wine was then made to 400 mL with water, ensuring that the concentration of polyphenols was identical to that in the full-complement red wine. 2.6. Apolipoprotein B48 and total apolipoprotein B determination Apolipoprotein B48 (apo B48) was quantitated using a Western blotting/chemiluminescence procedure as previously described [13]. Briefly, apolipoproteins were separated by SDS-PAGE using a Novex Mini-cell Electrophoresis system (Invitrogen, Carlsbad, USA) with precast NUPAGE 3–8% Tris–acetate gels according to the manufacturer’s instructions. After electrophoresis, separated lipoproteins where transferred to a PVDF membrane (0.45 m; Immobilon PTM ; Millipore Corporation, Bedford, MA, USA). apo B48 bands were identified and visualized using an antibody to apo B (DAKO A/S, Glostrup, Denmark) and subsequently antirabbit IgG antibody (HRP conjugated; Amersham, Little Chalfont, UK) using enhanced chemiluminescence (ECLTM ; Amersham, Little Chalfont, UK). Densitometric scanning of apo B48 bands and standardisation to known apo B48 protein mass allowed quantification of the protein. Total apolipoprotein B (apo B) was measured in sera samples by an immunonephelometric assays using Behring reagents on a Behring Nephelometer II (BN-II) system (Dade Behring Inc., Marburg, Germany). 2.7. Measurement of plasma lipids Serum triglyceride (TG) and total cholesterol was measured by enzymatic colorimetric kits (TRACE Scientific Ltd., Melbourne, Australia). Serum HDL cholesterol was determined after precipitation of apo B-containing lipoproteins with phosphotungstic acid and MgCl2 , the supernatant containing the HDL cholesterol was determined by enzymatic
Plasma insulin was measured by an ELISA kit (Dako Diagnostic, Japan). The HOMA score was used as a surrogate estimate of the state of insulin sensitivity by multiplying fasting insulin concentration (mIU/L) with fasting glucose concentration (mM) and divide by 22.5 [15]. 2.9. Statistical analysis Statistical analysis was done using SPSS 11 for Windows (SPSS Inc., Chicago, IL) and SAS 8 (SAS Institute Inc., Cary, NC, USA). Data are expressed as mean (S.E.M.) and assessed for normality. Comparison of baseline characteristics between each group was done by analysis of variance with Tukey’s post hoc analysis. Between and within group comparisons of the water, DRW and RW groups were made using general linear models with adjustment for baseline values. Statistical differences were analysed further by post hoc analysis using the least square differences (LSD) method. Statistical significance was considered at p < 0.05.
3. Results 3.1. Subject characteristics The clinical characteristics of subjects in the water, DRW and RW groups measured at screening are shown in Table 1. There was no significant difference between groups in the anthropometric or lipid parameters. According to NCEP ATP III guidelines, the postmenopausal women in this study displayed moderate to high total cholesterol and LDL cholesterol concentrations, with mean concentrations of 6.2 and 4.1 mmol/L, respectively. Table 1 Clinical characteristics of subjects at screening Characteristics
Water
DRW
RW
Age (years) Weight (kg) BMI (kg/m2 ) Waist (cm) W:H ratio Oestradiol (pmol/L) TG (mmol/L) TC (mmol/L) Glucose (mmol/L) Systolic BP (mmHg) Diastolic BP (mmHg)
59.3 (1.4) 70.0 (2.7) 26.7 (1.2) 84.3 (3.1) 0.8 (0.02) <150 1.2 (1.2) 6.5 (0.12) 5.3 (0.11) 127.1 (3.76) 75.3 (2.1)
57.6 (1.3) 72.8 (3.3) 26.3 (0.9) 86.1 (2.9) 0.8 (0.02) <150 1.4 (0.1) 7.1 (0.3) 5.4 (0.1) 127.3 (3.48) 78 (2.06)
58.4 (1.3) 69.7 (4.6) 25.9 (1.6) 83.4 (3.9) 0.8 (0.02) <150 1.3 (0.2) 6.56 (0.2) 5.2 (0.1) 131.9 (4.82) 79.3 (3.55)
Data represents mean (S.E.M.) of clinical characteristics of postmenopausal participants at screening. There was no significant difference between subject characteristics at screening. W:H ratio, waist to hip ratio; TG, triglyceride; TC, total cholesterol; BP, blood pressure.
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
441
Table 2 Dietary intake of subjects at the start and finish of the intervention Water
Energy (kJ) Carbohydrate (% of total kJ) Protein (% of total kJ) Total fat (% of total kJ) SFA (% of total kJ) MUFA (% of total kJ) PUFA (% of total kJ) Cholesterol (mg) Alcohol (% of total kJ)
DRW
RW
Start
Finish
Start
Finish
Start
Finish
8452.9 (463.47) 43.9 (0.02) 18.5 (0.01) 37.5 (0.02) 14.0 (1.03) 14.4 (1.37) 5.1 (0.36) 276.2 (23.53) –
9687.5 (692.01) 42.1 (0.02) 18.7 (0.01) 39.1 (0.02) 15.1 (0.77) 15 (1.09) 4.9 (0.46) 331.0 (30.56) –
9031.7 (508.10) 46.3 (0.03) 19.2 (0.01) 34.3 (0.03) 13.9 (1.41) 12.2 (1.25) 4.5 (0.45) 266.0 (31.63) –
8575.9 (937.93) 42.2 (0.02) 19.0 (0.01) 38.6 (0.02) 15.7 (1.09) 13.0 (0.93) 5.6 (0.77) 407.6 (106.52) –
10033 (609.11) 46.3 (0.03) 19.4 (0.01) 34.2 (0.03) 12.43 (1.22) 12.0 (1.05) 5.1 (0.66) 362.0 (80.31) 11.24
9281.5 (472.59) 48.9 (0.04) 18.2 (0.01) 32.9 (0.04) 12.87 (2.19) 11.8 (1.15) 4.6 (0.54) 253.68 (25.64) 12.0
Data represents are mean (S.E.M.) of the nutritional data reported in the 3-day food diary at the start and finish of the intervention. There was no significant difference in dietary intake at the start and finish of the intervention. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
3.2. Nutritional assessment There were no significant differences in macronutrient intake by subjects at the start and finish of the intervention (Table 2). There was no change in serum ␥GT levels in subjects consuming water and DRW, confirming that subjects in these groups abstained from alcohol consumption over the 6-week intervention period. In subjects consuming RW, as expected, a progressive rise in serum ␥GT (p < 0.05) was observed over the 6-week period. 3.3. Changes in fasting lipid concentrations There was no significant change in fasting LDL cholesterol, HDL cholesterol, total cholesterol and triglyceride concentrations following the consumption of DRW, compared to water (Table 3). However, RW consumption significantly decreased fasting LDL cholesterol concentration by 8% (p < 0.05) after 6 weeks, compared to water (Fig. 1). A significant 17% (p < 0.05) increase in HDL cholesterol concentration was observed following 6 weeks of RW consumption, compared with water (Fig. 2). Despite the improvement in LDL cholesterol concentration there was no change in total cholesterol concentration following the consumption of RW compared to water (Table 3). In addition, a significant
negative correlation (r = −0.533; p = 0.05) between baseline total cholesterol concentration and the percent change in LDL cholesterol concentration (baseline to 6 weeks) was observed in the RW group. RW consumption had no effect of triglyceride concentration, compared to water (Table 3). The results did not change following adjustments for body weight, BMI and waist circumference. 3.4. Changes in fasting lipid ratios The total cholesterol (TC):HDL ratio and the LDL:HDL ratio did not change with DRW consumption, compared to water. However, RW consumption significantly decreased the TC:HDL ratio by 14% (p < 0.05) after 6 weeks, compared to water (Fig. 3). In addition, we detected a significant decrease of 20% (p < 0.01) in the LDL:HDL ratio following 6 weeks of RW consumption, compared to water (Fig. 4). The results were not affected by adjustments for body weight, BMI and waist circumference. 3.5. Changes in fasting apolipoprotein B concentrations Fasting plasma chylomicron concentration, as measured by apo B48, was not altered by the consumption of DRW or RW over the 6-week intervention period (Table 3). Similarly,
Table 3 The change in fasting concentrations of cardiovascular disease risk factors Water
TC (mmol/L) TG (mmol/L) Apo B48 (g/mL) Apo B (g/L) Glucose (mmol/L) Insulin (IU/mL) HOMA
DRW
RW
Baseline
6 weeks
Baseline
6 weeks
Baseline
6 weeks
6.17 (0.15) 1.24 (0.12) 12.01 (0.59) 1.10 (0.04) 5.11 (0.11) 5.48 (0.87) 1.28 (0.22)
6.29 (0.15) 1.21 (0.10) 12.25 (0.68) 1.13 (0.05) 5.11 (0.10) 5.32 (0.58) 1.23 (0.14)
6.27 (0.26) 1.33 (0.17) 14.09 (0.85) 1.14 (0.06) 5.16 (0.11) 6.07 (0.81) 1.39 (0.18)
6.19 (0.32) 1.31 (0.14) 14.09 (0.84) 1.15 (0.07) 5.07 (0.06) 5.04 (0.39) 1.14 (0.09)
6.26 (0.14) 1.19 (0.15) 12.40 (0.58) 1.11 (0.04) 5.06 (0.08) 5.37 (1.07) 1.23 (0.26)
6.25 (0.17) 1.29 (0.18) 12.56 (0.57) 1.08 (0.06) 5.24 (0.09) 5.77 (1.57) 1.36 (0.39)
Values are mean (S.E.M.). There was no significant difference in certain cardiovascular disease risk factors following DRW and RW consumption. TC, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; HDL:LDL; HDL to LDL ratio; TG, triglycerides; Apo B48, apolipoprotein B48; Apo B, total apolipoprotein B; HOMA, homeostasis model assessment.
442
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
Fig. 1. Changes in fasting serum LDL cholesterol concentrations. The percentage change in fasting LDL cholesterol concentrations from baseline to 6 weeks following consumption of water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
Fig. 4. Changes in fasting LDL:HDL ratio. The percentage change in the LDL:HDL ratio from baseline to 6 weeks following consumption of either water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
3.6. Changes in fasting concentrations of blood glucose, plasma insulin and HOMA Fasting blood glucose, plasma insulin and HOMA score were not altered following the DRW or RW interventions, compared to water (Table 3). The results remained unchanged following adjustments for body weight, BMI and waist circumference.
4. Discussion Fig. 2. Changes in fasting serum HDL cholesterol concentrations. The percentage change in fasting HDL cholesterol concentrations from baseline to 6 weeks following consumption of either water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
serum concentrations of total apo B were unaffected by the consumption of DRW and RW compared to water, although a trend towards a decrease in apo B concentrations was noted following RW consumption (p = 0.4; Table 3). The results were not affected by adjustments for body weight, BMI and waist circumference.
Fig. 3. Changes in fasting TC:HDL ratio. The percentage change in the TC:LDL ratio from baseline to 6 weeks following consumption of either water, dealcoholised red wine (DRW) or red wine (RW) in hypercholesterolaemic, postmenopausal women. Data are mean ± S.E.M. Statistically significant differences from control are indicated by * p < 0.05.
We have previously demonstrated that acute consumption of DRW (polyphenol component) does not affect postprandial lipaemia or insulin sensitivity in dyslipidaemic postmenopausal women [9]. Furthermore, it was shown that an acute dose of full-complement RW exacerbates postprandial lipid and insulin levels, suggesting that neither red wine nor its polyphenolic components confer cardiovascular benefits acutely through improvements in postprandial lipid and lipoprotein metabolism. Based on these findings, as well as extensive reports in animal studies supporting improvements in CVD risk factors following long-term consumption of red wine and red wine polyphenols, it was proposed that chronic ingestion of red wine and its polyphenolic constituents may be essential for favourable physiological effects to occur, including improvements in lipid and lipoprotein metabolism. In this study, we have extended our previous findings from cell-culture and clinical studies by examining the effect of chronic consumption of DRW and RW on lipid, lipoprotein, insulin and glucose metabolism in moderately hypercholesterolaemic postmenopausal women. Our findings show that DRW consumption had no effect on fasting lipid and lipoprotein levels, and insulin sensitivity, over a 6-week period. Rather, chronic consumption of full-complement RW was required to observe improvements in fasting lipid and lipoprotein concentrations. Although the reduction in CVD mortality associated with moderate red wine consumption is well established, the certain mechanisms responsible for this risk reduction require further elucidation.
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
We have provided novel and unequivocal evidence that chronic red wine consumption significantly reduces serum LDL cholesterol, increases serum HDL cholesterol and improves fasting lipid ratios in postmenopausal women, which may partly explain the inverse association between red wine consumption and CVD risk. In addition, the findings of this study suggest that the improvements observed in cardiovascular disease risk factors with red wine consumption may not be independent of its alcohol component. The alcohol and polyphenol components in red wine may be working synergistically to produce biological effects. Studies have postulated that ethanol may facilitate the absorption of polyphenols found in red wine, resulting in greater plasma concentrations of these compounds than when consumed in the absence of ethanol [16]. However, in our study we cannot exclude the exclusive cardiovascular effects of the alcohol component in red wine. Several epidemiological studies show that the CVD benefits observed with alcohol consumption are independent of beverage type [17], although clinical studies are inconclusive [18]. Further studies comparing the effects of chronic consumption of ethanol compared to red wine in postmenopausal women are required and are currently underway in our laboratory. Interestingly, red wine polyphenols per se have been shown to improve lipid, lipoprotein and insulin levels in cell-culture and animal studies [4,5]. However, our results did not show any changes in fasting levels of lipid, lipoproteins or insulin following 6 weeks of DRW consumption by hypercholesterolaemic postmenopausal women. Evidence is emerging that cell-culture studies investigating the biological effects of polyphenols are inherently flawed, leading to inaccurate interpretation of the data [19]. The majority of in vitro studies have assessed the biological activity of polyphenols by administering them in their natural phytochemical form, for example as aglycones and various glycosides [20]. However, it is now clear that polyphenols are extensively modified in the digestive tract, as well as in circulation, to form conjugates and metabolites of the parent compound [21]. As a consequence, the polyphenol metabolites are likely to possess different biological properties and distribution patterns within cells and tissue to the parent compounds. In addition, in vitro studies have administered polyphenol concentrations which are substantially greater than the plasma concentration attained in humans following consumption of a polyphenol-rich meal [19]. Therefore, data from many in vitro trials reporting the biological effects of polyphenols cannot be extrapolated to humans. In addition, the absorption and metabolism of polyphenols has primarily been elucidated in animal models, however, data on human metabolism of these compounds is scarce. It is possible that following the consumption of a polyphenol-rich meal, the metabolites occurring in circulation are different between human and animal models in terms of chemistry, concentration and potency. Potential differences in circulating metabolites between the two models may explain the discrepancies in cardiovascular effects observed in our study following DRW consumption.
443
Elevated concentrations of LDL cholesterol are strongly associated with the development of atherosclerosis; therefore its reduction in circulation is critical in the attenuation of CVD mortality. Contrary to the findings of previous studies, we have demonstrated unequivocally that chronic RW consumption significantly reduces LDL cholesterol in hypercholesterolaemic postmenopausal women. The positive correlation between baseline total cholesterol levels and the percent change in LDL cholesterol suggest that the extent of improvement in atherogenic lipid levels may be related to the severity of hypercholesterolaemia. These findings are in support of our initial hypothesis as they imply that reductions in LDL cholesterol following RW consumption may be more readily observed in hypercholesterolaemic populations. Furthermore, the duration and beverage consumption could play a significant role in potential reductions of circulating lipid levels. In this study, changes in LDL cholesterol were observed only after 6 weeks of RW consumption. However, the majority of studies in humans have involved a modest intervention period of approximately 2–4 weeks, which may also account for the lack of significant change in LDL cholesterol reported [7,18]. The mitigation of LDL cholesterol observed following RW consumption was accompanied by a non-significant decrease in total apo B. Since apo B48 levels remained unchanged, the decrease in total apo B levels may reflect an alcohol-induced attenuation of apo B100 concentrations. Apo B100 is exclusively associated with hepatically derived lipoproteins in circulation, providing a useful estimate of LDL particle number. The simultaneous reduction in LDL cholesterol and apo B100 may be indicative of a decrease in LDL-particle number, which may occur either through a decrease in production and/or enhanced hepatic clearance of LDL particles. Cell-culture studies from our laboratory have reported a 30% decrease in apo B100 synthesis and secretion in HepG2 cells following incubation with RW [4]. The reduction in apo B100 was shown to result from a decrease in intracellular cholesterol synthesis and an up-regulation in LDL receptor expression, enhancing the clearance of these particles [4]. Human studies investigating the specific mechanisms responsible for the reduction in LDL cholesterol are required. Improvements in HDL cholesterol following moderate alcohol consumption, including RW, are well documented [22]. In fact, approximately 50% of the cardiovascular benefits observed with moderate alcohol consumption are thought to be mediated through an increase in HDL [23]. Several studies have documented an increase in HDL cholesterol in healthy individuals consuming regular doses of alcohol similar to those administered in this study [7,18]. To our knowledge, this is the first study to report improvements in HDL cholesterol in a hypercholesterolaemic subject group. Moreover, the magnitude of change in HDL cholesterol observed in our study appears to be greater than the changes observed in several other studies administering similar quantities of alcohol to healthy volunteers [7,18,24]. The
444
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445
hypercholesterolaemic nature of our subject group may contribute to the enhanced effects of RW on HDL cholesterol. Plasma lipid ratios, such as the TC:HDL ratio and the LDL:HDL ratio, have been shown to be strong predictors of CHD [25]. A TC:HDL ratio of <3.5 is considered optimal, an increase beyond this cut-off value is associated with increased risk of CHD [25]. The consumption of DRW did not improve plasma lipid ratios, suggesting the RW polyphenols may not confer cardiovascular benefits through improvements in circulating lipid levels. However, a significant decrease in TC:HDL ratio from 4.3 to 3.7 was observed in the RW group, compared to controls. Furthermore, lipidlowering drug trials assessing the association between lipid ratios and CHD have shown that for every 1% decrease in the TC:HDL ratio, CHD risk decreases by 1.3% [26]. We observed an overall decrease 14% in the TC:HDL ratio in hypercholesterolaemic postmenopausal women, which indicates an 18% reduction in CHD risk. Similarly, reductions in the LDL:HDL ratio following RW consumption are comparable to improvements observed following treatment with lipid-lowering drugs which has been associated with significant reductions in CHD risk [26]. Chylomicron remnants have been implicated in the development of atherosclerosis [27]. The effect of chronic consumption of RW polyphenols (±alcohol) on chylomicron metabolism in humans has not been investigated previously. At baseline, the concentration of apolipoprotein B48 (surrogate marker for chylomicrons) was shown to be elevated in hypercholesterolaemic postmenopausal women. However, fasting apo B48 levels remained unchanged following chronic consumption of DRW and RW, suggesting that neither polyphenols, nor full-complement RW, modulate intestinal lipoprotein metabolism. Our results confirm recent findings from Daher et al. showing no changes in apo B48 concentrations in rats consuming moderate doses of alcohol over a 10-week period [28]. It is also worthy to note that in contrast to acute RW consumption, chronic RW consumption did not induce hypertriglyceridaemia in moderately hypercholesterolaemic postmenopausal women. Similar results have been observed in other clinical studies following chronic consumption of similar doses of RW and alcohol, although findings are inconsistent [7,18,29]. It is also possible that RW consumption improves CVD risk via improvements in insulin sensitivity. Several observational studies have associated moderate RW and alcohol consumption with enhanced insulin sensitivity, low plasma insulin and glucose levels, and reduced risk of type II diabetes mellitus [30], although some have not observed this association [31]. Controlled clinical trials elucidating the effects of alcohol consumption, specifically red wine, on insulin and glucose metabolism are scarce and their findings inconsistent [29,32]. Moreover, we are unaware of any human experimental trials which have examined the effect of chronic consumption of red wine polyphenols (DRW) on glucose tolerance and insulin sensitivity. In this group of hypercholesterolaemic postmenopausal women, chronic consumption of DRW did
not influence insulin or glucose metabolism. Interestingly, chronic RW consumption neither improved nor exacerbated plasma insulin levels, blood glucose levels and insulin sensitivity. In summary, we have found that chronic consumption of DRW does not affect fasting lipid and lipoprotein levels, or insulin sensitivity in moderately hypercholesterolaemic postmenopausal women. In contrast, full-complement RW consumption improved circulating lipid and lipoprotein concentrations, as well as the atherogenic risk profile in this group of women. Collectively, our findings suggest that chronic consumption of full-complement red wine may reduce cardiovascular disease risk through improvements in basal levels of lipid and lipoproteins in hypercholesterolaemic postmenopausal women. Furthermore, it appears that the beneficial effects on lipid and lipoprotein metabolism observed with red wine consumption are not independent of its alcohol component. Caution should be exercised when advocating regular alcohol consumption as excessive alcohol consumption may result in adverse health effects.
Acknowledgements The project was supported by grants from the National Heart Foundation of Australia and the ATN Centre for Metabolic Fitness. The statistical advice received from S. Dhaliwal is appreciated. We are grateful to the Chemistry Centre, Perth, Australia for their assistance in the dealcoholising of red wine. References [1] Renaud S, De Lorgeril M, Salen MD, et al. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 1992;339:1523–6. [2] St Leger AS, Cochrane AL, Moore F. Factors associated with cardiac mortality in developed countries with particular reference to the consumption of wine. Lancet 1979;1:1017–20. [3] Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 1993;342:1007–11. [4] Pal S, Ho N, Santos C, et al. Red wine polyphenolics increase LDL receptor expression and activity and suppress the secretion of ApoB100 from human HepG2 cells. J Nutr 2003;133:700–6. [5] Vinson JA, Teufel K, Wu N. Red wine, dealcoholised red wine, and especially grape juice, inhibit atherosclerosis in a hamster model. Atherosclerosis 2001;156:67–72. [6] Pal S, Ho SS, Takechi R. Red wine polyphenolics suppress the secretion of ApoB48 from human intestinal CaCo-2 Cells. J Agric Food Chem 2005;53:2767–72. [7] Goldberg DM, Garovic-Kocic V, Diamandis EP, Pace-Asciak CR. Wine: does the colour count? Clin Chim Acta 1996;246:183–93. [8] Lavy AFB, Markel A, Danker G, et al. Effect of dietary supplementation of red or white wine on human blood chemistry, hematology and coagulation: favourable effect of red wine on plasma high-density lipoproteins. Ann Nutr Metabol 1994;38:287–94. [9] Naissides M, Mamo JC, James AP, Pal S. The effect of acute red wine polyphenol consumption on postprandial lipaemia in postmenopausal women. Atherosclerosis 2004;177:401–8.
M. Naissides et al. / Atherosclerosis 185 (2006) 438–445 [10] Australian Food and Nutrition Monitoring Unit. Comparable data on food and nutrient intake and physical measurements from the 1983, 1985 and 1995 national nutrition surveys. Commonwealth of Australia; 2001. [11] Nutbeam D, Wise M, Bauman A, Harris E, Leeder S. Goals and targets for Australia’s health in the year 2000 and beyond. Sydney, Australia: Department of Public Health, University of Sydney; 1993. [12] Helrick K. Official methods of analysis of the Association of Official Analytical Chemists. 15th ed. Arlington, VA: AOAC; 1990. [13] Smith D, Proctor SD, Mamo JC. A highly sensitive assay for quantitation of apolipoprotein B48 using an antibody to human apolipoprotein B and enhanced chemiluminescence. Ann Clin Biochem 1997;34:185–9. [14] Bairaktari E, Hatzidimou K, Tzallas C, et al. Estimation of LDL cholesterol based on the Friedewald formula and on apo B levels. Clin Biochem 2000;33:549–55. [15] Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [16] Miyagi Y, Miwa K, Inoue H. Inhibition of human low-density lipoprotein oxidation by flavonoids in red wine and grape juice. Am J Cardiol 1997;80:1627–31. [17] Rimm EB, Klatsky A, Grobbee D, Stampfer MJ. Review of moderate alcohol consumption and reduced risk of coronary heart disease: is the effect due to beer, wine, or spirits. Br Med J 1996;312:731–6. [18] Senault C, Betoulle D, Luc G, et al. Beneficial effects of a moderate consumption of red wine on cellular cholesterol efflux in young men. Nutr Metab Cardiovasc Dis 2000;10:63–9. [19] Kroon PA, Clifford MN, Crozier A, et al. How should we assess the effects of exposure to dietary polyphenols in vitro? Am J Clin Nutr 2004;80:15–21. [20] Gebhardt R. Variable influence of kaempferol and myricetin on in vitro hepatocellular cholesterol biosynthesis. Planta Med 2003; 69:1071–4. [21] Donovan JL, Bell JR, Kasim-Karakas S, et al. Catechin is present as metabolites in human plasma after consumption of red wine. J Nutr 1999;129:1662–8.
445
[22] Goldberg DM, Hahn SE, Parkes JG. Beyond alcohol: beverage consumption and cardiovascular mortality. Clin Chim Acta 1995;237:155–87. [23] Linn S, Carroll M, Johnson C, et al. High-density lipoprotein cholesterol and alcohol consumption in US white and black adults: data from NHANES II. Am J Public Health 1993;83:811–6. [24] Baer DJ, Judd JT, Clevidence BA, et al. Moderate alcohol consumption lowers risk factors for cardiovascular disease in postmenopausal women fed a controlled diet. Am J Clin Nutr 2002;75: 593–9. [25] Castelli WP. Cholesterol and lipids in the risk of coronary artery disease—the Framingham Heart Study. Can J Cardiol 1988;4(Suppl A):5A–10A. [26] Rader DJ, Davidson MH, Caplan RJ, Pears JS. Lipid and apolipoprotein ratios: association with coronary artery disease and effects of rosuvastatin compared with atorvastatin, pravastatin, and simvastatin. Am J Cardiol 2003;91:20C–3C [discussion 23C–4C]. [27] Mamo J, Yu K, Elsgood C, et al. Is atherosclerosis exclusively a postprandial phenomenon? Clin Exp Pharmacol Physiol 1997;24: 288–93. [28] Daher CF, Berberi RN, Baroody GM. Effect of acute and chronic moderate alcohol consumption on fasted and postprandial lipemia in the rat. Food Chem Toxicol 2003;41:1551–9. [29] Cordain L, Melby CL, Hamamoto AE, et al. Influence of moderate chronic wine consumption on insulin sensitivity and other correlates of syndrome X in moderately obese women. Metabolism 2000;49:1473–8. [30] Facchini F, Chen YD, Reaven GM. Light-to-moderate alcohol intake is associated with enhanced insulin sensitivity. Diab Care 1994;17:115–9. [31] Meyer KA, Conigrave KM, Chu NF, et al. Alcohol consumption patterns and HbA1c C-peptide and insulin concentrations in men. J Am Coll Nutr 2003;22:185–94. [32] Davies MJ, Baer DJ, Judd JT, et al. Effects of moderate alcohol intake on fasting insulin and glucose concentrations and insulin sensitivity in postmenopausal women: a randomized controlled trial. JAMA 2002;287:2559–62.