Wine Antioxidants and Their Impact on Antioxidant Activity In Vivo

Chapter 12

Wine Antioxidants and Their Impact on Antioxidant Activity In Vivo Simon R. J. Maxwell

Downloaded by CORNELL UNIV on August 23, 2016 | http://pubs.acs.org Publication Date: March 1, 1997 | doi: 10.1021/bk-1997-0661.ch012

Division of Clinical Pharmacology, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom

There has been considerable interest in the role of dietary antioxidants such as flavonoids in retarding the development of cardiovascular disease in human populations. Red wine is a rich source of flavonoid antioxidants. These compounds have been demonstrated to have potent antioxidant properties in vitro but their role as antioxidants in vivo remains to be proven. We have recently developed an enhanced chemiluminescent assay for antioxidant activity in biological fluids. This chapter describes the use of this technique to examine changes in serum antioxidant activity following the ingestion of red wine by healthy human volunteers.

Cardiovascular disease, principally coronary artery disease and stroke, is the major cause of premature death and disability in the developed world. Atherosclerosis in the large arteries supplying blood to the heart or brain is the major pathology underlying most serious cardiovascular events. Atherosclerosis refers to the progressive deposition of cholesterol in the vessel wall which in turn leads to blockage of the vessel, restriction of blood flow to the tissues downstream (ischaemia) and eventually death of those tissues (infarction). In many cases the final event that precipitates blockage is the formation of a blood clot (thrombosis) in the diseased vessel. After cell death has occurred regeneration of cells is impossible and there is irreversible damage to the function of the heart or brain. A reduction in such cardiovascular events will be necessary to produce any significant improvement in life-expectancy in the developed world. The last thirty years have seen an enormous increase in our understanding of the development of atherosclerosis. Four major risk factors - hypercholesterolemia, hypertension, smoking and diabetes - have been identified. Since smoking, the major lifestyle risk factor, is now declining increasing interest is being focused on other aspects of lifestyle such as nutrition and exercise that might influence the rate of

© 1997 American Chemical Society

Watkins; Wine ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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development of vascular disease. Wine is a popular beverage in the Western world. This article will briefly review some of the evidence to suggest that wine flavonoids might have an influence on the development of atherosclerotic vascular disease by acting as antioxidants. In particular, it will examine whether their potent antioxidant effects in vitro may be manifest in vivo and whether wine can be considered as an important source of dietary antioxidants in human nutrition.


Oxidative Stress and Atherosclerosis Oxidative stresses are ubiquitous throughout the body and arise primarily from oxidants produced endogenously (e.g. by mitochondrial electron transport or phagocytic cells) or exogenously by environmental pollution including cigarette smoke or the products of ionising radiation (/). The four electron reduction of molecular oxygen by mitochondria is essential to life in all aerobic organisms. Unfortunately, the process is not completely efficient allowing leakage of the superoxide radical (O2*") which is a precursor for the formation of more potent oxidants such as the peroxide ion (O2 ") and the hydroxyl radical (ΟΗ·). The latter can initiate the peroxidation of lipids and so disrupt biological membranes and render lipoproteins more atherogenic (2). These processes are accelerated in the presence offreetransition metal ions such as Fe ". This sequence of events is of particular relevance to development of atherosclerosis (Figure 1). A characteristic feature of atherosclerosis is the presence of lipid-laden macrophages within the intima of arteries. Macrophages in culture are unable to accumulate significant amounts of native low-density lipoprotein (LDL) because of the low number and affinity of L D L receptors on these cells and their down regulation by accumulated intracellular cholesterol (3). In contrast, the uptake of oxidized L D L is rapid and not subject to down-regulation. Oxidized L D L is also chemotactic for macrophages (4) and cytotoxic to the vascular endothelium (5). Although oxidative mechanisms are clearly not the only pathophysiological events relevant to atherogenesis (6) it is clear that oxidation of lipoproteins represents a significant biological threat and should ideally be prevented. 2

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A number of natural antioxidant mechanisms exist to protect against the everpresent threat of oxidation of L D L . These include proteins such as transferrin, caeruloplasmin and albumin that limit the availability of metal ions. Another very important protective influence is the presence of scavenging antioxidant molecules that are readily sacrificed (oxidized) in preference to more important targets such as L D L . Most of the important sacrificial antioxidants are derived from the diet and include vitamins such as ascorbate (vitamin C) and alpha-tocopherol (vitamin E) and polyphenolic flavonoids derived from sources such as wine, tea and vegetables. The potent antioxidant properties of dietary flavonoids have stimulated interest in whether flavonoid-rich diets might offer any protection against diseases such as atherosclerosis and cancer where oxidation is thought to play an important role. Preliminary evidence from epidemiological studies suggests that high dietary antioxidant intake may indeed be protective against the development of vascular disease (7-9).


WINE: NUTRITIONAL AND THERAPEUTIC BENEFITS


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Figure 1. The oxidative-modification theory of atherosclerosis.


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DoflavonoidsProtect Against Heart Disease? Although most of these studies have concentrated on the better known antioxidants such as vitamin C and vitamin Ε more recent epidemiological studies have focused on the role of flavonoids. The Zutphen Elderly Study (70) assessed the flavonoid intake of 805 men aged 65-84 years in 1985 and followed them up for 5 years. Flavonoid intake analysed in tertiles was significantly inversely associated with death from coronary heart disease (p=0.015) and showed a trend towards an inverse association with myocardial infarction (p=0.08). The relative risk of coronary heart disease in the highest versus the lowest tertile of flavonoid intake was 0.42 (95% CI 0.20-0.88). This relationship persisted after controlling for all other relevant coronary risk factors. Hertog et al (77) also retrospectively reviewed the flavonoid intake of 16 cohorts originally entered into the Seven Countries Study in 1960 calculated from the original dietary questionnaire. Over 25 years of follow-up the flavonoid intake was significantly inversely associated with coronary heart disease mortality and explained 25% of its variance. The only stronger predictor of coronary mortality was saturated fat intake. Knekt et al (12) studied 5133 Finnish men and women aged 30-69 years recruited between 1967 and 1972. The flavonoid intake was calculated from the reported dietary recall of subjects for the year prior to entry into the study and then related to coronary and total mortality over the subsequent 26 years. For women there was a significant inverse gradient of risk for coronary and total mortality with flavonoid intake. The relative risk between the highest and lowest quarters of intake after adjusting for other coronary risk factors was 0.69 (95% CI 0.53-0.90) for total mortality and 0.54 (95% CI 0.33-0.87) for coronary mortality. For men the corresponding values were 0.76 (95% CI 0.63-0.93) and 0.78 (95% CI 0.56-1.08). It was suggested that since the intake of vitamin C in the Finnish diet was low dietary flavonoids may offer an alternative source of antioxidants.

Red Wine Protects against Heart Disease : the French Paradox Flavonoids are derived from many sources in the human diet including fruit, vegetables, red wine and tea. Red wine is a particularly rich source of flavonoids. Previous calculations have suggested that the addition of two glasses of red wine to the Western diet will increase its flavonoid content by 40% (73). Studying the potential impact of red wine flavonoids on coronary heart disease is complicated by the presence of other wine constituents such as alcohol and sugars. Alcohol in particular may have a significant protective effect against vascular disease in its own right (14-18). These effects can probably be attributed to the potential for alcohol to increase protective H D L cholesterol levels and decrease platelet aggregation. However, there have been many claims that there may be benefits associated with the flavonoid content of red wine over and above the effect of alcohol. One of the earliest experiments to suggest this examined the effect of feeding rabbits a high cholesterol diet for three months while also administering alcohol, beer, white wine, red wine or water. These beverages reduced the atherosclerotic lesions over the subsequent 3 months to 75%, 83%, 67% and 40% respectively of those found in the water drinking controls (19).



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The epidemiological evidence for a specific protective effect of flavonoid-rich alcoholic beverages is rather more confused (20). In most countries intakes of saturated fat are directly associated with mortality from coronary heart disease. However, some countries appear to defy this general association. The most notable exception is France where high fat intakes, serum cholesterol and smoking remain associated with a low incedence of coronary heart disease (18). This circumstance has come to be known as the 'French Paradox' and has stimulated interest in local lifestyle factors that may protect the French against heart disease. A likely candidate was the preference of the French for regular consumption of red wine which offers not only the benefits of moderate alcohol consumption but also the potential benefits of a high flavonoid intake. Indeed, it has been reported that wine consumption in several countries shows a remarkable inverse correlation to local rates of coronary heart disease mortality (21).

Mechanism of Flavonoid Protection against Cardiovascular Disease The putative cardioprotective benefits of wine have been largely attributed to its content of flavonoids and their antioxidant activity (22). This, it is suggested, may be important in reducing the tendency of lipoproteins to become oxidised and participate in atherosclerosis. There can be little doubt about the activity of flavonoids as antioxidants in vitro. Many investigators have used a variety of assay systems to study the antioxidant effects of polyphenols derived from wine and other sources. These compounds are able to efficiently scavenge a variety of reactive oxidizing species including superoxide, hydroperoxides and the highly reactive hydroxyl radical (23-30). Flavonoids are not only scavengers of reactive oxygen species but also chelate iron which acts as the template for their formation (31,32). In relation to atherosclerosis it was of particular relevance that flavonoids were not only free radical scavengers but also potent inhibitors of L D L oxidation. Several investigators have been able to demonstrate in vitro that L D L is significantly more resistant to oxidation in the presence of flavonoids derived from red wine and other sources (33-38). Furthermore, flavonoids are also able to spare alpha-tocopherol (vitamin E) the major endogenous radical-scavenging antioxidant of L D L (35). Another important antioxidant effect of flavonoids is related to their ability to inhibit the lipoxygenases, a group of enzymes which might also be involved in initiating L D L oxidation (39-41) and reduction in the production of neutrophil derived oxidants (42-44). Flavonoids may also modify a number of other cardiovascular risk factors including cholesterol, blood pressure and thrombosis. However, it is the potent antioxidant properties of wine flavonoids are thought to be the major mechanism underlying their putative protection against cardiovascular disease (45). Nevertheless, it is yet to be resolved whether these well-documented in vitro effects can be translated into a meaningful alteration in antioxidant protection in vivo.

Impact of Red Wine on Antioxidant Status in vivo Although there is clearly ample evidence of a powerful antioxidant effect of red wine flavonoids from a variety of in vitro assay systems only three studies have addressed



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their potential impact in vivo following acute ingestion (Table I). All of these studies have used the the enhanced chemiluminescent assay for antioxidant activity developed in our laboratory (46). Briefly, a glowing light emission can be produced by mixing the chemiluminescent compound luminol with an oxidant (hydrogen peroxide) and an enhancer phenol (para-iodophenol) in the presence of the enzyme horseradish peroxidase. The light emission can be detected in a conventional luminometer and depends on the constant formation of free radical intermediates of para-iodophenol and luminol (Figure 2). Therefore, serum samples containing radical-scavenging antioxidants interfere with light emission from the glowing chemiluminescent reaction. When all of the added antioxidants are consumed (oxidized) in the reaction light emission resumes (Figure 3). The period of light suppression is then compared with a standard curve created by adding the water-soluble tocopherol analogue trolox (6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) to calculate the antioxidant activity of the test sample (μπιοί trolox Eq./litre). This technique is very reproducible (within- and between-batch coefficient of variation for serum samples are 1.7 and 5.0% respectively), as well as being cheaper and requiring less technical expertise than previously reported antioxidant assays. In our study (47) ten healthy students (5 males, 5 females : mean age - 22.0 years, mean weight - 67.2 kg) attended a clinical investigation unit on two separate afternoons after a four hour fast. Following collection of a basal blood sample each subject consumed a standard meal alone or with red wine (5.7ml French Bordeaux/kg) over 30 minutes. Serial blood samples were taken from an indwelling intravenous cannula for four hours and the serum rapidly separated and cooled. Subjects were given free access to drinking water throughout the study. Following ingestion of red wine there was a rapid increase in serum antioxidant activity to reach a peak after 90 minutes before a gradual decline (Figure 4). The peak antioxidant activity represented a remarkable 14% increase over basal values. Antioxidant activity was still significantly elevated at the end of the study at four hours. No such increase was seen after after the consumption of the meal and water ad libitum. This study was the first (to our knowledge) to suggest that the ingestion of red wine was associated with a significant increase in the antioxidant activity of a human extracellular fluid. Furthermore, the quantitative increases were of a magnitude that had previously been associated with significant inhibition of L D L oxidation in vitro (36). This result is perhaps not surprising since the variety of wine used in our study had an antioxidant activity of 11365μπιο1/1 in the chemiluminescent assay compared to normal serum values of only 350-550μιηο1/1 (46). Indeed, we had found that antioxidant levels of 10-20mmol/l to be common in red wines as previously mentioned. Such values were far superior to the antioxidant activity seen in white wine and a range of other beverages (Figure 5). Assuming all of the antioxidant activity in the red wine given to our volunteers (mean volume 0.38 litres, equivalent to 4353μιηο1 antioxidant activity) had been absorbed the observed mean rise of 66μπιο1/1 suggested a volume of distribution of 66 litres! Clearly, it would require absorption of only a fraction of the available antioxidants in red wine distributed throughout the extracellular fluid compartment to produce the observed increases in serum antioxidant activity. We also concluded from our study that enhanced chemiluminescence offered a simple technique for quantifying antioxidant activity in wines or other beverages and their subsequent impact on body fluids.



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Table I. Studies examining the impact of red wine ingestion on antioxidant activity and urate concentration in human serum. All error values are standard deviations. * The percentage contribution of urate to rises in serum antioxidant activity has been calculated assuming a 1:1 stoichiometric equivalence between urate and the assay standard antioxidant trolox (46). AO A = antioxidant activity, E C L = enhanced chemiluminescence. Study

Maxwell et al [1994]

Whitehead et al [1995]

Day & Stansbie [1995]

AOA Assay

ECL

ECL

ECL

Subjects (M/F)

5/5

4/5

6/0

Wine

Bordeaux Red

Bordeaux Red

Port Wine

Volume (ml)

383 (mean)

300

250

Wine AOA (μπιοΙΛ)

11365

17000

7079

Control

Water

None

Water + 40g ethanol

Follow-Up (min)

240

120

120

Peak Time (min)

90

60

30

Rise in AOA (μιηοΐ/ΐ)

66 (14%)

86(18%)

109 (24%)

Rise in Urate (μπιοΐ/ΐ)

39 (12%)

—

81 (23%)

% rise attributable to Urate *

55%

74%



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Figure 2. The enhanced chemiluminescent reaction. The chemiluminescent reagent luminol (LH") is oxidized to its radical form (L" ) by the hydrogen peroxide (H202)-horseradish peroxidase system. The rate-limiting step of the unenhanced reaction is overcome by the inclusion of enhancer phenols (E-OH) such as paraiodophenol. These are more readily oxidized to their radical form (E-O ) than luminol but can subsequently oxidize luminol and in the process are regenerated to their reduced form. In this way the rate of transfer of electrons from luminol to the enzyme is increased with favourable effects on the characteristics of light emission. Radical-scavenging antioxidants will interfere with the reaction sequence and suppress light emission. #

e


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Sample

Figure 3. The impact of serum, wine and trolox on light emission from the enhanced chemiluminescent reaction. Antioxidant activity in wine and serum are calculated by comparison of their t-value with that of the trolox standard solution to derive values for antioxidant activity in micromoles of trolox equivalents per litre (μηιο1/1).

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Wine Antioxidants and Their Impact on Antioxidant Activity In Vivo

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