European Heart Journal (2007) 28, 16831693 doi:10.

1093/eurheartj/ehm149

Review

The red wine hypothesis: from concepts to protective signalling molecules

Lionel H. Opie* and Sandrine Lecour
Department of Medicine, Faculty of Health Sciences, Hatter Cardiovascular Research Institute, University of Cape Town, Private Bag 3, 7935 Observatory, South Africa
Received 30 January 2007; revised 28 March 2007; accepted 5 April 2007; online publish-ahead-of-print 7 June 2007

KEYWORDS
Wine; Alcohol; Resveratrol; Molecular mechanisms; Sirtuin

We review evidence for and against the red wine hypothesis, whereby red wine is more likely to confer cardiovascular benets than white. As background, there is a strong epidemiological and mechanistic evidence for J-shaped relation between alcohol intake and total mortality. However, epidemiological data favouring a specic benet of red over white wine are not strong and the French paradox could at least in part be explained by confounding factors. More convincing evidence is that human studies with de-alcoholized red but not white wine show short-term cardiovascular benets. The specic components of the de-alcoholized wine that are active on cardiovascular endpoints, are the polyphenols found in red wine, especially resveratrol. The effects of resveratrol on isolated tissues or organs are well-described including molecular mechanisms leading to decreased arterial damage, decreased activity of angiotensin-II, increased nitric oxide, and decreased platelet aggregation. Anti-ischaemic effects include stimulation of prosurvival paths, decreased LDL-oxidation, atheroma, and on the ischaemic-benecial metabolic changes. Most recently, the agonist effect of resveratrol on the anti-senescence factor sirtuin has lessened cell death in myocytes from failing hearts. Mechanistic feasibility strengthens the case for prospective therapeutic trials of alcohol vs. red wine vs. resveratrol, for example in those with heart failure.

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Introduction
The harmful effects of risk factors for cardiovascular disease as often linear, as in the case of cigarette smoking and blood lipid abnormalities.1 For alcohol, however, the situation is different, with data for moderate-dose cardiovascular benets2 and high-dose harm. This article will rst review the increasing evidence for this dose-related protection, then evaluate the proposal that wine is more protective than other alcoholic drinks, and thereafter examine the hypothesis that red wine is more protective than white. Important arguments favouring this hypothesis derive from the role of specic protective chemical polyphenols found both in red wine and de-alcoholized red wine. Putative molecular mechanisms involve newly described signals and paths that may help to explain the proposed protective qualities of moderate amounts of red wine.

The J-shaped mortality curve: still controversial?

The major and consistent nding is that alcohol consumption, from whatever source, appears to have a J-shaped curve, whereby a modest intake is benecial and either no intake or an increased intake is harmful. This relation has

* Corresponding author. Tel: 27 21 406 6358; fax: 27 21 447 8789.

E-mail address: lionel.opie@uct.ac.za

been established in a series of studies starting with the Framingham study,3 followed by the British Doctors Study,4 the Cancer Prevention study on about 490 000 persons,5 the Nurses Health Study,6 the Physicians Health Study,7 the British Heart Study,8 the Multiple Risk Factor Intervention Trial,9 and the Copenhagen City Heart Trial,4,5,7,10,11 However, the issue of confounding factors has bedevilled these ndings as shown by two opposing views.12,13 Of particular relevance is a meta-analysis of mortality in 34 outcome studies on more than 1 million subjects.14 Nearly 100 000 deaths were analysed to show a clear J-relationship with decreased total mortality associated with two to four daily drinks for men and one to two for women vs. abstinence or higher drinking rates.14 Of note in relation to the controversy regarding the signicance of confounding factors, adjustment for social factors, and dietary markers made the J-shaped dip shallower and shorter, yet still evident.14 The J-shaped mortality curve might in part be explained by less myocardial infarction6,15 and in part by less incident heart failure,16 but not by decreased hypertension.17 Regarding mechanistic studies, these are many and difcult to explain by the confounding hypothesis. One established mechanism of the cardiovascular benet is through a consistently reported increase in plasma high-density lipoprotein (HDL)-cholesterol levels, thought to be the result of increased hepatic synthesis. The denitive study shows that

& The European Society of Cardiology 2007. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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a genetic variation in hepatic alcohol dehydrogenase, which slows the rate of ethanol metabolism, is associated with higher plasma HDL-levels and lower rates of myocardial infarction.18 This was a prospective study with a high rate of completeness of follow-up data, and part of the carefully monitored Physicians Health and Nurses Health studies. The benets found in moderate drinkers were profoundly altered by the presence or absence of the ADH3 genotype, which is contrary to the confounding hypothesis. About half of the benet of moderate drinking can be related to increased HDL-levels.19 Other possible protective mechanisms include anti-platelet,20 anti-coagulatory,19,21 improved glucose control,19 and anti-inammatory effects as shown in the MONICA study.22 The latter were more evident in moderate drinkers than in abstainers or heavy drinkers. When given acutely for 8 weeks, ethanol reduced triglyceride and insulin levels.23 Of these effects, those on HDL, brinogen, and insulin sensitivity have been established in randomized prospective studies,19 which are especially difcult to explain by the confounding hypothesis. Alcohol also has detrimental high-dose (0.60.9 g/Kg body weight) pro-oxidant effects in humans,24 which together with liver damage may contribute to the right-hand upward slope of the J-curve.

show that in normal persons, alcohol but not de-alcoholized red wine elevates BP by about 23 mmHg.3133 Conversely, alcohol reduction in those men who are fairly heavy drinkers (3 drinks per day) reduces BP by a similar degree.34 Even such small differences may have relatively important epidemiological implications. In hypertensive men, the alcohol-induced rise is even greater.35 These adverse BP data neither exclude alternative benets such as decreased aortic stiffness,36 nor changes in the pattern of arterial wave deections with decreased central systolic BP.37 Overall, for any given level of BP, a lower all-cause mortality is associated with a moderate wine intake (,3 glasses daily).38 This being an observational study, confounding factors are again difcult to exclude.

Patterns of drinking
Its regular intake is protective,19 especially in males.39 Binge-drinking (more than three drinks per hour of any variety of alcohol) is associated with increased mortality after myocardial infarction.19 Binge-drinking is one of several lifestyle factors that may counter the J-shaped curve.40 Regarding the BP, there is little or no pressure rise if alcohol is taken with meals.41 Fitting in with the effect of wine in relation to meals, basic research suggests that red wine could protect against some effects of post-prandial lipaemia.42 While some other indirect evidence also supports the concept of the benets of wine with meals,29 there was no relation to meals in American men who predominantly drink liquor and beer.43
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Is no alcohol really harmful?

In a large multinational study, no alcohol intake was a harmful risk factor for myocardial infarction, especially in women.2 The rst and most obvious explanation would be the lack of the benet of moderate alcohol intake on HDL-cholesterol and, as will be discussed, the absence of the postulated non-ethanolic benets of red wine. Alcohol has benecial effects on insulin and triglyceride levels,23 and abstinence is a risk factor for new type 2 diabetes.25 Alcohol gives experimental cardioprotection against ischaemic-reperfusion injury in isolated hearts.26,27 There is, however, another explanation for the harm of no alcohol intake, which is the recurring problem of confounding factors which are difcult totally to exclude in epidemiological studies. Nonetheless, confounding was discounted in the study on British doctors,12 and also in a very large and up-to-date meta-analysis.14 Yet, Americans who take no alcohol have a different and generally less cardioprotective lifestyle from moderate drinkers.13,28 Regular alcohol intake, generally as wine and with meals, is part of the Mediterranean diet29 and the benecial Mediterranean lifestyle. In France, moderate alcohol and wine drinkers smoke less and have a higher socio-economic status than abstainers.30 However, confounding factors do not mean that the J-shaped protection is lost. Thus men with a healthy lifestyle not only have a lower rate of myocardial infarction but achieve increased protection by moderate alcohol intake.15 Those who take no alcohol might be disadvantaging themselves, not only for lack of alcohol, but also for lack of a benecial lifestyle.

Wine vs. other alcoholic drinks

As already mentioned, many studies have related alcohol to the J-shaped benecial curve.14 An important claim is that wine may have benets that, for example, beer or spirits lack. For example, Gronbaek found all-cause mortality reduced in wine but not in non-wine drinkers in his study on 24 523 Danes over 10 years.10 In a meta-analysis on 209 418 persons, consumption of wine but not that of beer was protective.11 Klatsky followed-up 128 934 adults in Northern California for up to 20 years.44 The J-shaped curve was once again conrmed. Drinkers of any type of wine had a lower mortality risk than did beer or liquor drinkers, in part because wine drinkers were the lighter drinkers. Renauds group studied 36 250 healthy French men, also to show that moderate wine but not other alcohol types reduced all-cause mortality over 1218 years.45 These impressive data from three entirely different countries must be balanced against other studies that differ and the recurrent problem of confounding factors. Thus, the type of alcohol made no difference to its protective effect on myocardial infarction in the 38 077 participants in the US male health professions study over 12 years of follow-up.43 Moderate consumption of both wine and beer equally decreased inammatory markers such as C-reactive protein.22 The real problem is that there are very few long-term prospective studies comparing the possible benecial effects of the types of alcohol on cardiovascular outcomes or total mortality while fully allowing for confounding factors. According to such factors, wine drinkers have a more healthy diet than beer drinkers with increased intake of fruit, vegetables, and bre.28,30,46 In the British

Alcohol and blood pressure

Here there is no J-shaped curve. A common opinion is that wine intake reduces blood pressure (BP) because wine is a relaxant. This only holds for an early effect within hours.31 In long term, the opposite holds. A series of recent articles

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Regional Heart study, 7735 men drawn at random were followed-up for an average of 16.8 years.47 Wine drinkers had a lower age-adjusted risk of coronary heart disease and all-cause mortality than did beer and spirits drinkers, but the wine drinkers also had a better life style (for example, much less smoking). Thus the wine vs. beer data suggest, but do not conclusively give the benet to wine.

known better aroma, palate, and after-taste of French vs. German reds, we still await a large comparative epidemiological study proving the proposed superior cardioprotective effects of French red wines.

Red vs. white wine or vodka: comparative studies

In normal human volunteers, drinking an unspecied amount of red wine but not white or vodka increased coronary owvelocity reserve.57 Drinking red wine raised HDL-cholesterol and plasma antioxidant status more than did an equivalent dose of white wine when given to healthy volunteers.58 Furthermore, in vascular smooth muscle cells, pre-incubation with red wine but not white inhibited the platelet-derived growth factor beta-receptor (PDGF) that is crucial in the development of atherosclerosis.59 Conversely, incubating white wine with grape skins (that contain the polyphenols found in red wine) and alcohol endowed white wine with a similar power of inhibition of LDL-oxidation to that possessed by red wine.60 In human mononuclear cells incubated with VLDL (very low-density lipoproteins), drinking moderate doses of red wine but not vodka inhibited the activation of nuclear factor (NF)-kappa-B61 during post-prandial lipaemia, thus providing an anti-inammatory mechanism.42 This effect was simulated by antioxidants contained in red wine. These comparative studies support the red wine hypothesis.

The red wine hypothesis

It is often thought that epidemiological data from France strongly suggests a protective effect of red wine despite a high-fat diet (the French paradox). This idea goes back to St Leger et al.48 who in 1979 found an inverse relation between coronary heart disease mortality and wine consumption, with France having the lowest mortality. The jump to the specic benet of red wine was made in 1993 when Frankel et al. showed that red wine diluted 1000-fold and contained 10 mmol/L of total phenols that inhibited the oxidation of LDL more than a-tocopherol, an established antioxidant.49 The antioxidant mechanisms involved are scavenging of peroxyl radicals as well as chelation of redox metal ions such a copper.50 Indirect evidence favouring the French paradox and the red wine hypothesis is that the French habitually drink wine with their meals (which are often fatty) and this wine is most often red. Furthermore, Alsace, a white-wine drinking region of France, has a much higher mortality (about 50% higher) than red wine-drinking Mediterranean areas,51 despite having a lower mean serum cholesterol level.20 However, such comparisons need to take into account the many confounding inter-regional differences in diet and life style.30 Furthermore, there are several other explanations for the French paradox, including the time lag hypothesis which states that the French diet had low fat in the past and that it takes about 30 years for any dietary pattern to manifest itself in mortality data.52 A PubMed search could uncover no good epidemiological study rmly to establish any clinical outcome benet of red vs. white wine drinking in Europe. Most large-scale epidemiological data coming from North America suggest that there is no difference between white and red wine,43,44,53 thus concluding that alcohol intake rather than wine colour predicts the eventual cardiovascular outcome. Again there may be confounding factors. The pattern of wine drinking could be different, with less wine with meals and more prior to meals in America than in Europe. Furthermore, American and French diets are typically very different, for example with much more wine used in cooking in France. Marinating meat in red wine substantially decreases formation of potentially toxic heterocyclic amines54 and of lipid peroxides.55

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Red wine: protective effects beyond alcohol

The conclusive studies showing that red wine has qualities beyond alcohol are those on de-alcoholized red wine, which has cardiovascular protective effects in short-term studies on humans.37,62 In patients with coronary artery disease, 250 mL of de-alcoholized Greek red wine decreased arterial stiffness and improved the augmentation index, as derived from arterial wave reection patterns.37 A similar dose of de-alcoholized red wine decreased adverse post-smoking arterial wave reections and lessened the rise in systolic BP.62 Brachial artery ow-mediated vasodilation was improved by 250500 mL of de-alcoholized red wine.63,64 Ingestion of purple grape juice in patients with coronary artery disease (about 640 mL/day for 14 days) similarly improved brachial ow.65 The purple grape juice also reduced LDL-cholesterol susceptibility to oxidation, with the benets being attributed to avonoids. Alcohol-free red but not white wine greatly increased plasma antioxidant capacity 50 min after ingestion.66 Red wine incubated with human plasma strongly inhibited LDL-oxidation and uptake of LDL by macrophages, and the active components were catechins and avonols, whereas ethanol had no effect.50 All these human studies are among the most powerful arguing for the benet of red (but not white) wine beyond alcohol. Supporting data comes from dogs with stenosed coronary arteries, in whom administration of French blended red wine, apparently verging on vintage in age, eliminated cyclic ow reductions caused by periodic acute plateletmediated thrombus formation.67 This markedly benecial effect was also achieved by purple grape juice, but about 2.5 times greater volume was required than in the case of red wine. White wine was not protective, so that the explanation must again lie in the effects of red wine beyond alcohol. Experimentally, the benet of the red wine lies in the polyphenols, found in de-alcoholized red wine extracts

French vs. German reds: effects on nitric oxide

French red wine is better than German reds in the induction of human endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells.56 Presumably, this is a class effect although only six French and three German wines were studied. In view of the similar effects on nitric oxide production by two French reds, the one barrel-fermented and the other not,56 it is very unlikely that better French than German oak can explain these differences. While the human inclination is to add such basic observations to the

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which improve endothelial nitric oxide release,68 and revert the prothrombotic effects of a cholesterol-rich diet.69 De-alcoholized red but not white wine decreased atherosclerosis in apolipoprotein E gene-decient mice.70 However, there is no strict clinical proof that these beyond alcohol effects operate in red wine drinkers to give clinically signicant cardiovascular protection. Rather, there is only indirect observational evidence in a case control study that links high dietary anthocyanidin intake to reduce the risk for acute myocardial infarction independent of alcohol intake.71

A-II-induced production of reactive oxygen species (ROS).84 As the cellular redox state inuences so many reactions, and is sensitive to both free radicals and to resveratrol,80 it is likely but usually assumed rather than directly proven that the antioxidant effect of resveratrol could play a major role in at least some of the other protective mechanisms now to be discussed. Proposed vascular protective mechanisms Resveratrol acts through multiple mechanisms (Figure 1), including the antioxidant effects on platelet formation already discussed. Resveratrol vasodilates isolated human small arteries,61 in response to oestrogen-receptor stimulation with formation of nitric oxide. The vasodilation is absent in arteries from patients with coronary artery disease.61,85 The mechanism involves increased formation and release of nitric oxide associated with increased eNOS activity.68,86 Although there is no direct evidence linking such increased eNOS activity to platelets, there is a wellknown effect of nitric oxide in inhibiting the activation of platelets. Links between resveratrol and reduced platelet aggregation87 could also be mediated by TNF-a.88 Resveratrol at a concentration of only 0.1 mmol/L increases the activity of NF-k-B, which lies on the TNF-a signalling pathway, in human endothelial cells after overnight incubation, thereby providing the mechanism for a possible anti-inammatory and anti-platelet-effect.89 Increased post-infarct neovascularization is a long-term benet.90 Resveratrol interferes with angiotensin-II and epidermal growth factor signalling in vascular smooth muscle cells, which may be a long-term mechanism for inhibition of atherosclerosis.91 Furthermore, inhibition of angiotensin-II and increased synthesis of nitric oxide could, in part, explain renoprotective effects92 and BP reduction in some renal models.93 Cardiac broblasts Resveratrol (525 mmol/L) pretreatment inhibited growth paths stimulated by angiotensin-II, epidermal growth factor, or transforming growth factor-b, which are essential in broblast proliferation and differentiation.94 Thus resveratrol is potentially anti-brotic. Metabolic effects of resveratrol Resveratrol in high doses shifts the physiology of middle-aged obese mice, with features resembling the metabolic syndrome in humans, towards those of non-obese mice on a standard diet.95 The metabolic changes included increased insulin sensitivity, decreased circulating free fatty acids, decreased insulin-like growth factor (IGF-1), increased activity of the energy-sensing enzyme, AMP-activated protein kinase (AMPK), increased activity of peroxisome proliferatoractivated receptor-gamma coactivator-alpha (PGC-1a), and increased mitochondrial number (Figure 2). These diverse effects are mediated by sirtuin (see next paragraph). Whether similar trends can be achieved by the very much lower resveratrol dose associated with red wine drinking is a subject for future research. Pharmacological preconditioning by resveratrol In several studies, resveratrol decreases ischaemic-reperfusion damage (Table 1). A burst of short perfusion by resveratrol or pretreatment before the onset of ischaemic-reperfusion injury stimulates a plethora of preconditioning-like survival paths

Specic protective chemicals in red wine: molecular effects

Polyphenol extracts
Red wine polyphenol extracts have experimental cardioprotective properties,72 and may counter one of the mechanisms underlying atherosclerosis, namely thrombin-induced matrix invasion of vascular smooth muscle cells.73 An extract of Cabernet Sauvignon suppressed endothelin-1 synthesis and release in bovine aortic endothelial cells.74 Another polyphenol extract protected against angiotensinII-induced hypertension in rats by blunting endothelial dysfunction and promoting formation of nitric oxide.75 Yet, another polyphenol extract increased eNOS promoter and nitric oxide release in human umbilical endothelial cells.68 Of the possible polyphenol components tested, the activity only resided in resveratrol which is a stilbene derivative found in grape skins, and hence found much more in red than white wine. Resveratrol also was an active component of a red wine polyphenol extract that transcriptionally inhibited the expression of endothelial vascular cell adhesion molecule, VCAM-1.76 Besides the polyphenols currently attracting most attention are resveratrol and the procyanidins (the latter are avan-3-ols and hence part of the avonoid family).

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Resveratrol
Antioxidant effect Dosing animals with resveratrol gives a variety of cardioprotective results (Table 1) of which the strong antioxidant properties are perhaps the best known,26 and could explain the inhibition of LDL oxidation.49 The antioxidant effect on LDL is also linked to increased expression of the paraoxonase-1 gene.77 Other antioxidant effects are widespread, including cardioprotection where it offsets the adverse pro-oxidant qualities of alcohol.26 Furthermore, resveratrol inhibits the inammatory response by suppressing prostaglandin biosynthesis78 and by downregulating the gene expression of intercellular adhesion molecule-1 (ICAM-1) and NF-k-B, the latter taken as an indirect biomarker of oxidative stress.79 The NF-k-B signalling pathway can be evoked by oxidative stress and is in turn inhibited by resveratrol80 (Figure 1). Not surprisingly, TNF-a-induced activation of NF-k-B is attenuated by resveratrol and this sequence is in part mediated by an antioxidant effect,81 which in turn reduces platelet activation.82 This sequence can also explain the resveratrol-induced inhibition of oxidative stress in platelets induced by TNF-a.83 Resveratrol inhibits angiotensin-II (A-II)-induced cardiomyocyte hypertrophy, which is clearly linked to its antioxidant effects, because it inhibited the

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Table 1 Concentrations of resveratrol that have relevant cardiovascular effects on isolated tissues or organs Tissue Human coronary artery endothelial cells Human umbilical vein endothelial cells Human umbilical vein endothelial cells Human umbilical vein endothelial cells Human umbilical vein endothelial cells Human endothelial progenitor cells Human hepatoma cells Human platelets, ASA-resistant Human small arteries, subcutaneous Rat neonatal ventricular myocytes Rat aortic smooth muscle cells Rat cardiac broblasts Ischaemic-reperfused rat heart Ischaemic-reperfused rat heart Ischaemic-reperfused rat heart, after pretreatment Ischaemic-reperfused rat heart, after pretreatment Ischaemic-reperfused rat heart Ischaemic-reperfused rat heart Myocytes from failing mice hearts Concentration 0.110 mmol/L 0.02282.28 mg/L 0.1 mmol/L 110 mmol/L 10100 mmol/L 5100 mmol/L 1.0 mmol/L 510 mmol/L 10 mmol/L 0.330 mmol/L 0.1100 mmol/L 0.0550 mmol/L 525 mmol/L 10 mmol/L 10 mmol/L Gavage 2.5 mg/kg 18.5 mmol/L in blood after i.p injection 10 mmol/L 10 mmol/L 0.5 mmol/L0.114 mg/L Effect Inhibits TNF-a-induced NF-k-B activation and inammatory markers Increased the inhibitor of TNF-a (NF-k-B) Increased eNOS promoter and nitric oxide release Increased eNOS promoter activity and eNOS protein levels Inhibition of tissue factor activity Increased activity vs. decrease with 50 mM Increased expression of paraoxonase-1 gene Reduced platelet aggregation Dilation but not in those with coronary heart disease Inhibited A-II-induced phosphorylation of ERK and A-II-induced increase of ROS levels Inhibition of A-II and epidermal growth factor-mediated growth signalling path Inhibition of signalling paths mediated by A-II, epidermal and transforming growth factors Expression of anti-apoptotic Bcl-2 Preconditioning of heart via adenosine-3 receptor and survival paths Less infarct size and apoptosis via survival paths Less post-reperfusion depressed function Decreased infarct size Less ischaemic-reperfusion injury Inhibits falling levels of sirtuin (inhibitor of cell death) Reference 81 89 68 86 132 96 77 87 61 84

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91 94 99 99 97 133 26 134 104

Note the wide range of concentrations used of resveratrol, always in trans form (molecular weight 228 g/mol); for relation to circulating concentrations likely to be found after red wine intake in humans, see text. TNF, tumour necrosis factor; NF, nuclear factor; A-II, angiotensin-II; ERK, extracellular signalregulated kinase; ROS, reactive oxygen species.

insulin, may explain the insulin-like effect of resveratrol in diabetic rats.101 Effects on sirtuin (anti-senescent) proteins These effects of resveratrol are the focus of current research (Figure 3). A high resveratrol diet opposes the effects of a high fat diet in mice, increasing insulin sensitivity, decreasing myocardial and aortic damage, and prolonging lifespan.95 Mechanistically, at least some of these effects can be related to the recent discovery that resveratrol is an agonist of the sirtuins (also called SIRT proteins, Silent Information Regulator Two) which belong to the histone deacetylase family.102 By deacetylation they inactivate the histone senescence factor p53. Conversely, overexpression of sirtuins limits premature cellular senescence.103 Activation of sirtuins by resveratrol can experimentally prolong the life of myocytes from failing hearts and decrease myocyte death induced by angiotensin-II.104 Furthermore, resveratrol protects against Alzheimer-like neurodegeneration by increasing sirtuin activity and, thereby, inhibiting adverse NF-k-B signalling,105 thus, experimentally protecting both heart and brain via sirtuins in high doses.104 Resveratrol: male vs. female differences Resveratrol stimulates the oestrogen E-beta receptors in vascular endothelial cells,61 and functions as an agonist for

Figure 1 Proposed multiple vascular protective mechanisms of resveratrol. ROS, reactive oxygen species; NF-k-B, nuclear factor kappa B; eNOS, endothelial nitric oxide synthase; TNFa, tumour necrosis factor alpha; Ang-II, angiotensin II; VSMC, vascular smooth muscle cells.

involving signalling molecules such phosphatidyl-inositol-3phosphate (P-I-3)-kinase-Akt (Figure 2). Other preconditioningassociated proteins include mitogen-activated protein (MAP) kinase kinase,96 and p38-MAP kinase-beta.97 These signals are, at least in part, mediated by the adenosine 3-receptor,98,99 and involve nitric oxide.98,100 An important prosurvival signal is activation of the antiapoptotic enzyme Bcl-2.99 The overlap of some of these paths (P-I-3-kinase-Akt) with those stimulated by

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It would be intriguing to imagine that women could offset this gender disadvantage of alcohol by drinking red rather than white wine, but there are no supporting data. Resveratrol pharmacokinetics Are the protective concentrations used in such studies within the range that could be expected after drinking red wine? (Tables 1 and 2). Resveratrol itself exists in two forms, cis- and trans-forms that are in equilibrium and have similar biological activities, so that the total resveratrol concentration is relevant. However, it varies substantially between wines (Table 2). Resveratrol is only one of several stilbenes in red wine although the best studied. In Brazilian red wine, the total stilbene consumption from drinking half a bottle of some types of red wine per day is about 11 mg,106 which if all were absorbed could give a peak total stilbene concentration of about 10 mmol/L, or about 4 mmol/L of resveratrol. This potential peak value compares well with the lowest in vitro resveratrol concentration having an in vitro biological effect (0.10.3 mmol/L; Table 1), and raises the theoretical possibility that therapeutic resveratrol levels could be reached by drinking highresveratrol red wines. Proof of this hypothesis requires studies in humans of red wine dosage and related resveratrol blood levels. In one study, after 25 mg of resveratrol, as would be found in 2.5 L of some red wines (Table 2), peak total (free and conjugated) resveratrol was about 450 mg/L or about 2 mmol/L,107 20-fold more than the lowest resveratrol concentrations experimentally active (Table 1). Thus half a bottle of such wine should still give enough resveratrol to be active. However, the calculation assumes that resveratrol conjugates are active, which has not yet been studied. The peak free circulating unconjugated resveratrol was just about 5 nmol/L,107 which is 20-fold below the lowest effective concentration reported. In another study, a dose of 1 g as in 10 L of some red wines, gave higher peak free

Figure 2 Proposed anti-ischaemic protective mechanisms of resveratrol. Those myocardial signalling paths involved in resveratrol pharmacological preconditioning are shown on the left, and those giving metabolic protection on the right. Note multiple antioxidant effects. P-I-3 kinase, phosphatidyl-inositol-3 phosphate-kinase; Akt, protein kinase B; BcL-2, mitochondrial anti-apoptotic protein; NF-k-B, nuclear factor kappa-B; LDL, low-dentistry lipoprotein; PGC-1a, peroxisome proliferator-activated receptorgamma coactivator-alpha; IGF-1, insulin-like growth factor; AMPK, AMPactivated protein kinase; mito, mitochondria. Outlines of heart and atheroma modied from Opie LH Heart Physiology, from Cell to Circulation, Lippincott, Williams and Wilkins, Philadelphia, 2004, with permission.

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Table 2 Concentrations of resveratrol in various wine varietals Wine varietal Country of origin Australian Californian Burgundy Spanish Australian California France France Switzerland Brazil Resveratrol concentration 13.4 mg/L 59 mmol/L 5.5 mg/L 4.4 mg/L 5.1 mg/L 1.7 mg/L 0.9 mg/L 3.6 mg/L 3.9 mg/L 5.012.3 mg/L 18.0 mg/L 0.5 mg/L Reference

Pinot noir Pinot noir Pinot noir Pinot noir Cabernet Sauvignon Cabernet Sauvignon Rhone valley reds Bordeaux reds Red, varietal not stated Red, varietal not stated Red grape juice

135 135 135 136 135 135 135 135 135 135 110

Figure 3 Proposed effect of resveratrol in countering heart failure by acting as an agonist to sirtuin, considered as a longevity factor. Hypothetically, heart failure induces overactivity of poly(ADP-ribose) polymerase (PARP), a multifunctional DNA-bound nuclear enzyme, that inhibits NAD-dependent paths thereby decreasing the activity of sirtuin. The proposal is that resveratrol, by increasing the activity of sirtuin, deacetylates and inhibits the pro-apoptotic effector p53 (a transcription factor). The result is that resveratrol protects cells from failing hearts against PARP-mediated cell death. For concepts and data, see Pillai et al.104 and Smith.131 NAD, nicotinamide adenine dinucleotide. Figure of heart from Drugs for the Heart, 6th edn., (Eds) Opie LH and Gersh B, Elsevier Saunders, Philadelphia, 2005, with permission.

oestrogen receptor-mediated transcriptions albeit at a much higher concentration than oestradiol.85 In general, women are more susceptible to the harmful effects of alcohol, with limits of two drinks/day vs. four in men,14 because lower levels of the enzyme alcohol dehydrogenase give higher blood levels than with comparable doses in males.

Concentration of resveratrol in red wines for comparison with concentrations used in various isolated tissue or organ experiments shown in Table 1.

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resveratrol levels of 12 mmol/L.108 Thus, 1 L of such red wine would give levels of 100200 nmol/L, just within the bottom end of the therapeutic range (Table 2). Oral administration of resveratrol to rats in doses similar to a reasonable wine intake gives plasma resveratrol levels of 0.1 mmol/L,109 about one-third of the lowest concentration that can relax isolated human small arteries,61 and one-fth of the lowest concentration that protects mycocytes from failing hearts,104 but about twice the lowest level that interferes with signalling by angiotensin-II.91 There are two further relevant issues. First, the plasma concentrations of the conjugated glucoside form, piceid, can be about six times higher than that of free resveratrol.110 The biological activity of these conjugated compounds is not known. Secondly, with prolonged daily dosing of rats with an amount of resveratrol found in 250 mL of some red wines, there is substantial accumulation in the liver, and further metabolic fates are unknown.109 Overall, resveratrol concentrations reached by drinking within the safe limits of some red wines might be just within or below the therapeutic range, judged by isolated tissue experiments.

avonol and polymeric anthocyanidins.50 However, there are few strict studies relating post-ingestion blood levels found in humans to cardioprotective concentrations in isolated tissues. In this regard, avonoids of the catechin family inhibited the PDGF beta-receptor when used in concentrations similar to those found in the blood after drinking red wine.59

Other active ingredients of red wine

Of the many other polyphenolic components of red wine, the less well-studied avonoids include quercetin and catechin,75 which also have properties of potential cardiovascular importance. In a dietary study in over 10 000 persons for 1 year, those with a higher avonoid intake, particularly quercetin, had less coronary heart disease.113 Both quercetin and catechin given orally can retard progression of atherosclerosis in apolipoprotein E-decient mice, acting by reduction of the susceptibility of LDL to oxidation and aggregation.114 In humans, plasma catechin rises to about 75 nmol/L soon after the equivalent of one to two glasses of red wine.115 These levels contrast with those experimentally protective, such as 50 mmol/L.114 De-alcoholized red wine decreased atherosclerosis in apolipoprotein E genedecient mice, acting by phenolic acids not listed in Table 3.70 The mechanism involved decreased LDL oxidation associated with decreased alpha-tocopheroxyl radicals, the oxidation product of vitamin E. Thus protective effects of vitamin E could be facilitated, overcoming the otherwise adverse excess pro-oxidant effects. In mice, a daily dose of Cabernet Sauvignon similar in dose to half a bottle of wine per day for humans, and with a long list of potentially active polyphenols, was neuroprotective against experimental Alzheimers disease.116 Multiple mechanisms of benet of various ingredients seem probable. What is still missing are human data on the pharmacokinetics and bioavailability of the polyphenols found in red wine.117 Nonetheless of note, at least one laboratory effect of polyphenols, namely inhibition of thrombin-induced matrix invasion of vascular smooth muscle cells, can be found in concentrations similar to those achieved after moderate red wine ingestion.73

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Procyanidins
Resveratrol is not the only protective agent in red wine. Red wine contains a large number of other potentially active avonoids, especially the avan-3-ols which include procynanidins and proanthocyanidins (Table 3). Proanthocyanidin, like resveratrol, is a strong antioxidant and both equally reduce experimental myocardial infarct size and apoptosis in isolated rat hearts.26 The mechanisms include a preconditioning-like effect, shown by induction of heat shock protein (HSP) 70. Taking inhibition of endothelin-1 production by aortic endothelial cells as another endpoint, oligomeric procyanidins inhibit ET-1 synthesis at concentrations similar to those in some red wines, with some correlation between the procyanidin activity of the wine varietal and the longevity.111 Oligomeric procyanidins are also inhibitors of ACE activity, in concentrations similar to those found in red wines.112 The antioxidant effect of red wine on LDL oxidation is mediated chiey by catechins but also by the

Table 3 Potentially active polyphenols in wine125 Polyphenols have multiple aromatic rings possessing hydroxyl groups A. Flavonoids Anthocyanins: cyanidin, delphindin (both as glycosides) Flavonols: quercetin, as glycoside Flavan-3-ols (not found as glycosides): Monomers: catechins Oligomers: procyanidins,111 Polymers: proanthocyanidins (oligomers and polymers contain catechins and epicatechin) B. Non-avonoids Resveratrol Peceid, conjugated resveratrol glycoside Gallic acid Others
The italicized polyphenols are those most clearly linked to benecial changes in cardiovascular experiments.

Proposed protective components in white wine and beer

White wine may have different protective qualities Red and white wine have equal effects on brinolytic factors in a short-term cross-over study.118 Equal effects on collagen-induced platelet aggregation are also reported.119 In addition, white wines may contain anti-inammatory protective substances that lessen cytokine release, as also found in extra virgin olive oil.120 Thereby, the inammatory reaction that promotes atherosclerosis could be lessened. De-alcoholized beer may also give some protection Some epidemiological studies have not shown less protection by beer than wine drinking.22 De-alcoholized beer inhibits platelet and thrombogenic activity in young adults.121 However, the dose was high (3 L) so that if translated into the amount of alcohol in standard beer, such intake would be harmful.

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Overall perspective
There are several sequential proposals leading from observational population studies. Thus (i) only modest alcohol intake gives a J-shaped pattern of protection; (ii) wine is more protective than beer; and (iii) red wine is more protective than white. Of these, the J-shaped curve is best established, but in every case confounding factors are difcult to exclude.107,122 However, in the case of the J-shaped curve, the genetic links between alcohol dehydrogenase activity, increased HDL-cholesterol, and decreased myocardial infarction, and the prospective trial data on HDL, brinogen, and insulin, are powerful arguments against confounding. Moderate red wine drinking could be, at least in part, a surrogate for a healthy life style. In this case the cardiovascular data with de-alcoholized red but not white wine and studies on isolated tissues or organs give decisive mechanistic insights that strengthen the case already made for randomized prospective controlled trials with cardiovascular endpoints.122 The rst trial should simply compare alcohol with red wine with abstinence, for example in postinfarct patients.107 Similar prospective studies would be needed conclusively to prove the red-better-than-white wine hypothesis. As these studies may never be done, attention has shifted to the benecial components of red wine. Resveratrol remains as the most powerful of the polyphenols and one of the most likely to give biological protection.123 The recent discovery that resveratrol is an agonist of the sirtuin (SIRT) proteins may lead to a dose-response trial of resveratrol and red wine in heart failure patients. However, pharmacokinetic data remain incomplete. Experimentally, other polyphenolic components of red wine also play a protective role. One hypothesis would be that no single component of red wine confers all the benet, but rather several components contribute by different mechanisms. For example, resveratrol and procyanidins are both antioxidants, but resveratrol interacts with SIRT, whereas the procyanidins inhibit endothelin-1 synthesis. Given that there will always be wine drinkers, do current data help in choice of wine? First, all drinkers of any alcohol should avoid overdosing and binge-drinking. Then they should consider the incomplete but emerging evidence that red wine is more cardioprotective (and possibly, neuroprotective) than white. Furthermore, as red wine-drinkers can choose a specic varietal, pinot noir is high both in resveratrol124 and in procyanidins.125 The price of pinot is high enough to ensure drinking only in moderate amounts and preferably with meals. Some may be inuenced by the neuroprotective qualities of Cabernet Sauvignon given in low doses to rodents. And others, especially those inuenced by American epidemiological data, will settle for ethanol as the major cardioprotective agent in wine. Teetotallers will be consoled by the substantial data linking de-alcoholized red wine to experimental cardiovascular benets and turn to purple grape juice. Hopefully, in the future, low alcohol yet still protective wines will make their debut.126 Special patient groups who need abstinence or specic limitations Those with gout are traditionally warned against red wine, which does increase blood uric acid levels.127 Those with

alcoholic cardiomyopathy may not take any alcohol, despite the ambiguous effects of ethanol on myocardial function.128 Those with atrial brillation should be safer with less than 35 drinks/week.19 Hypertensive males may need to limit drinks to not more than two/day34,129 with, presumably, even fewer for hypertensive females. However, as alcohol only elevates BP by a few millimetres of mercury, a reasonable alternative would be to monitor by BP measurements the contrasting policies of (i) allowing the normal moderate amounts of alcohol while watching the BP and, if needed, adjusting doses of anti-hypertensive drugs; and (ii) keeping the medication unchanged but cutting the alcohol. Post-myocardial infarction, moderate alcohol does not impose a greater risk of overt heart failure on those already having systolic dysfunction.130 Also, it is important to remember that alcohol interacts with many drugs such as nitroglycerin, anxiolytics, neuroleptics, and oral contraceptives.
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Summary
There are strong epidemiological data favouring the view that modest alcohol but neither zero nor more than modest intake benecially reduces total mortality and cardiovascular risk. As there is only a narrow window of opportunity for optimal intake, it becomes important to consider which is the most benecial type of alcohol. The red-better-than white hypothesis rests on1 human studies showing short-term cardiovascular benets achieved by de-alcoholized red but not white wine; and2 many studies on isolated tissues or organs assessing the effects of polyphenols in red wine, especially resveratrol. Thus, red wine potentially has benecial effects beyond alcohol. The molecular mechanisms involved are many and complex as revealed by studies on isolated tissues or organs. Besides, the well known antioxidant effects of polyphenols, resveratrol effects include activation of preconditioning-like prosurvival signalling paths similar to those responding to insulin, anti-apoptotic protection mediated by the antisenescence factor sirtuin, and inhibition of adverse signalling by TNF-a, NF-k-B, angiotensin-II, and growth factors (epidermal and transforming growth factor-beta). Mechanistic feasibility strengthens the case for prospective therapeutic trials.

Acknowledgements
We would like to thank D. Blackhurst for very helpful comments. Conict of interest: none declared.

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The red wine hypothesis

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