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英文文献Red wine consumption increases antioxidant status and decreases oxidative stress in the circulation of both young and old humansMichelle Micallef, Louise Lexisand Paul LewandowskiAbstractBackground: Red wine contains a naturally rich source of antioxidants, which may protect the body from oxidative stress, a determinant of age-related disease. The current study set out to determine the in vivo effects of moderate red wine consumption on antioxidant status and oxidative stress in the circulation.Methods: 20 young (1830 yrs) and 20 older ( 50 yrs) volunteers were recruited. Each age group was randomly divided into treatment subjects who consumed 400 mL/day of red wine for two weeks, or control subjects who abstained from alcohol for two weeks, after which they crossed over into the other group. Blood samples were collected before and after red wine consumption and were used for analysis of whole blood glutathione (GSH), plasma malondialdehyde (MDA) and serum total antioxidant status.Results: Results from this study show consumption of red wine induced significant increases in plasma total antioxidant status (P 0.03), and significant decreases in plasma MDA (P 0.001) and GSH (P 70%) reduction in H2O2 induced genetic damage after 1hour post consumption of 300 mL of red wine. These findings are also supported by a similar study by Szeto and Benzie , showing that DNA damage was significantly reduced in a H2O2 challenge, with treatment of caffeic acid, a polyphenol found in red wine.Oxidative damage to a range of biomolecules is of particular interest to researchers. The tripeptide glutathione(GSH) functions as an antioxidant, which scavenges free radical species in circulation. GSH is oxidized as the enzyme glutathione peroxidase catalyzes the degradation of H2O2 . Increasing evidence demonstrates GSH plays an integral role in the protection against oxidative stress in the circulation due to its ability to facilitate the recycling of oxidized -tocopherol and ascorbic acid, two important antioxidants in the circulation and is widely used as a biomarker of circulating antioxidant levels . Within plasma fatty acid residues of phospholipids and LDL, are extremely susceptible to oxidative damage by free radical intermediates resulting in oxidized fatty acids and peroxidation byproducts, such as conjugated diennes (CD) and malondialdehyde (MDA) derivatives . MDA appears to be one of the most toxic and mutagenic aldehydes generated by lipid peroxidation of polyunsaturated fatty acids of cell membranes . It is also a popular measurement used to quantify the effects of radical damage to cellular lipids.A large body of evidence which indicates that free radical production can directly or indirectly play a major role in cellular processes implicated in atherosclerosis and CVD,.Therefore the aim of this study were firstly to under stand how moderate red wine consumption (400 ml/day) for two weeks effected circulating lipids, antioxidant level and total antioxidant capacity in the circulation and secondly assess the differences in bioefficacy of red wine in young and older populations.MethodsRecruitment of volunteersThis study protocol was approved by the Human Research Ethics Committee of Victoria University (HRETH.SET 15/05). Forty volunteers were selected based upon their responses to a general health questionnaire and after giving written informed consent. Those who were taking any anti-coagulant or anti-inflammatory medications or had a history of cardiovascular or liver disease were excluded. Two age groups were selected, these were 20 volunteers aged between 1830 years old (young group) and 20 volunteers aged older then 50 years old (older group). Volunteers were randomly assigned to begin in the red wine or control group within their respective age group (Figure 1).Intervention designPrior to drinking the red wine or control period volunteers were asked to abstain from consuming any alcohol, grapes or grape products for one week. After this one week lead in subjects had three 10 mL tubes of fasting blood collected via venipuncture to determine baseline measures of MDA, GSH, and total antioxidant capacity and BMI (kg/m2) calculated, after which they began the red wine or control period. During the red wine period participants consumed 400 mL of red wine each day (Cabernet Sauvignon) over a period of two consecutive weeks and abstained from other alcohol, grapes or grape products. A placebo such as alcohol free wine was not used due to difficulties in matching the flavour and mouth feel of the redwine used. Instead a crossover design was used whereby after completing either the red wine or control period volunteers were given a two week washout period before crossing over into the other group. During the control period volunteers abstained from consuming any source of alcohol, grapes or grape products for two weeks. Three 10 mL tubes of fasting blood were again collected after the treatment or control phase (see Figure 1). Participants were also encouraged to maintain their usual diet and exercise habits throughout the entire study phase which was monitored by participants keeping a food and activity diary before and during the study. There were no specific instructions given to avoid foods containing large amounts of phenolic compounds, other than abstain from consuming any alcohol, grapes or grape products as previously described.Wine supplementationThe red wine used throughout this study was a Cabernet Sauvignon, supplied as a cask wine to prevent the oxidation of the wine. This style was chosen since it is known to be palatable to most people and to the volunteers in the study. Participants consumed the wine at any time during the day, however, it was suggested that they do so at a time when they would normally consume alcohol (e.g. with an evening meal). Importantly, during the period of supplementation participants were asked to refrain from consuming any other sources of alcohol, grapes or grape products.Wine compositionThe concentrations of total anthocyanins, degree of anthocyanin ionisation, total phenolic compounds, red wine colour (density and hue) and two indices providing a measure of polymerisation of monomeric forms (Chemical age index #1 and #2) were determined by spectropho tometric methods . Determination of the concentration of free and bound sulphur dioxide in the wine was made using the method of Rankine and Pocock. Alcohol content was provided by the wine producer. The composition of the wine used in this study was analysed can be seen in Table 1. All components of the wine used in this study, except for red wine colour hue and free sulfur dioxide, were slightly higher than the red wine used in a study by Greenrod et al.Analyses of glutathione Glutathione was measured as it is an important antioxidant in the circulation using a commercially available colorimetric kit (Northwest Life Sciences) based on the method of Teitze following the manufactures instructions. Blood was collected via venipuncture using EDTAcoated tubes and stored at 4C. Whole blood samples were then deproteinated mixing aliquots with 100 ul of cold 5% metaphosphoric acid followed by centrifugation at 1500 g for 5 min, the supernatant was then removed and stored at -20C awaiting further analysis. All sampleswere then assayed for reduced GSH as a batch. This involved mixing 50L of calibrators or samples with 50L DTNB reagent and 50 L glutathione reductase reagent in the wells of microplate. This reaction mix was then incubated at ambient temperature for 3 min after which 50 ul NADPH reagent was added to all wells and absorbance values read at 405 nm with data collected at 15 sec intervals for 3 min. Absorbance values were then plotted as a function of time for each calibrator and sample. A calibration curve was then constructed by plotting the A405/min for each calibrator as a function of the GSHconcentration and the equation for the calibration curve was then used to calculate the concentration of GSH in all samples. Analyses of malondialdehydePlasma malondialdehyde was as a marker of lipid peroxidation using a commercially available colorimetric kit (Northwest Life Sciences) following the manufactures instructions. Blood was collected via venipuncture using EDTA coated tubes, stored at 4C and plasma separated within 2 hrs by centrifugation at 3000 g for 10 minutes at room temperature. Plasma samples were then stored at -20C awaiting further analysis. All samples were then assayed for MDA as a batch. This involved mixing 250 ul calibrator or sample with 10 uL of Butylated hydroxytoluene reagent, 250 ul Phosphoric acid reagent and 250 ul 2-Thiobarbituric acid reagent. This reaction mix was then incubated at 60C for 60 min followed by centrifugation at 10000 g for 3 min. Absorbance of calibrators and samples was then read at 532 nm in a spectrophotometer (Biorad). Absorbance values for calibrators were thenused to construct a calibration curve and the equation for calibration curve was then used to calculate the concentration of MDA in all samples.Analyses of total antioxidant statusSerum total antioxidant status (TAS) was determined for a quantitative assessment of in vivo antioxidant status using a commercially available kit (Randox) based on the troloxequivalent antioxidant capacity method of Miller following the manufactures instructions. Blood was collected via venipuncture using serum separator tubes, stored at 4C and serum separated within 2 hrs. Serum samples were then stored at -20C awaiting further analysis. All samples were then assayed for TAS as a batch. This involved mixing 20 L calibrator (6-hydroxy-2,5,7,8-etramethylchroman-2-carboxylic acid 1.79 mmol/L) or sample with 1 ml of chromogen (metmyoglobin 6.1mol/L, ABTS 610 mol/L) and incubating at 37C for 3 min. Initial absorbance was then read at 600 nm in a spectrophotometer (Biorad). After which 200 L of substrate(hydrogen peroxide 250 mol/L) was added to calibrator and sample and incubated at 37C for 3 min. Final absorbance was then read at 600 nm. The change in absorbance value for samples relative to the change in absorbance of the calibrator was then to calculate the TAS in all samples. The total antioxidant status of the red wine (Cabernet Sauvignon) used in this study was also meas ured using the same assay.Analyses of serum glucose and plasma lipidsSerum glucose was determined using a commercial glucose oxidase reagent and standard (Thermo Electron Corporation). This involved mixing 3 L of calibrator or sample with 450 L of glucose oxidase reagent and incubating at 37C for 5 min. Absorbance of calibrators and samples was then read at 500 nm in a spectrophotometer (Biorad). The absorbance value of samples relative to the absorbance of the calibrator was then to calculate the glucose level in all samples. Plasma triglycerides were determined using commercially available colorimetric kit (Thermo Electron Corporation). This involved mixing 6 L of calibrator or sample with 600 L of triglyceride reagent and incubating at 37C for 3 min. Absorbance of calibrators and samples was then read at 500 nm in a spectrophotometer (Biorad). The absorbance value of samples relative to the absorbance of the calibrator was then to calculate the triglyceride level in all samples. Total cholesterol was determined using commercially available colorimetric kit (Thermo Electron Corporation). This involved mixing 6 L of calibrator or sample with 600L of cholesterol reagent and incubating at 37C for 3 min. Absorbance of calibrators and samples was then read at 500 nm in a spectrophotometer (Biorad). The absorbance value of samples relative to the absorbance of the calibrator was then to calculate the cholesterol level in all samples. HDL cholesterol was determined using commercially available colorimetric kit (Thermo Electron Corporation). This involved mixing 4 L of calibrator or sample with 300 L of HDL reagent 1 and incubating at 37C for 5 min. After which 100L of HDL reagent 2 was added to calibrator and sample and incubated at 37C for 3 min. Absorbance of calibrators and samples was then read at 600 nm in a spectrophotometer (Biorad). The absorbance value of samples relative to the absorbance of the calibrator was then to calculate the triglyceride level in all samples. LDL cholesterol, a risk factor for cardiovascular disease, was calculated by subtracting HDL cholesterol values, a negative risk factor for cardiovascular disease, from total cholesterol.Statistical analysisStatistical analysis was performed using the SPSS statistical package (version 12.0, SPSS Inc.). All data were distributed normally and expressed as mean standard error of the mean (SEM). Data from young and older individuals were analyzed using a three way ANOVA to determine the effect of wine consumption within the young or old group, any difference between young and old groups and any difference between pre samples with the young or old group. Due to the cross over design of the study any difference between are not included in the analysis s the primary focus of the research was to determine the effect of red wine consumption. In all cases a P value of 0.05 was considered statistically significant.Results Whole blood glutathione was measured as it is an important circulating antioxidant. Before and after red wine consumption GSH levels were elevated in older volunteers compared to young volunteers (P 0.001, Figure 2). Despite this difference between young and old volunteers consumption of red wine had the same effect with both the young and old groups causing significant reductions in GSH levels after red wine consumption, young with wine (P = 0.004) and older with wine periods (P 0.001, Figure 2). No significant changes in GSH level were observed in young and older groups without red wine.Plasma malondialdehyde was measured as a biomarker of lipid peroxidation. Before and after red wine consumption MDA levels were reduced in older volunteers compared to young volunteers (P 0.05, Figure 3). Despite this difference between young and old volunteers consumption of red wine had the same effect within both the young and old group causing significant reductions in MDA levels after red wine consumption, young with wine (P 0.001) and older with wine periods (P 0.001, Figure3). No significant changes in MDA level were observed in young and older groups without red wine.Serum total antioxidant status was calculated for samples from each study group. Before red wine consumption TAS levels were decreased in older volunteers compared to young volunteers (P 0.001, Figure 4). Despite this difference between young and old volunteers consumption of red wine had the same effect within both the young and old group demonstrating a significant increase in total antioxidant status after red wine consumption, young with wine (P = 0.026) and older with wine periods (P=0.01, Figure 4). These changes correspond to the changes in GSH and MDA with red wine consumption for both young and older groups. The total antioxidant status of the red wine consumed by all treatment subjects in this study contained 1.53 0.027 mmol/L of antioxidant capacity (Figure 4).There was no significant difference in both age (yrs) and BMI (kg/m2) between red wine and abstinence periods for both young and older population groups (Table 2). Similarly there were no differences in serum glucose concentrations between pre and post samples for both young and older control and treatment groups (Table 2). Plasma lipid profiles for each study group were examined through the determination of plasma cholesterol, plasma triglycerides, plasma HDL-cholesterol and plasma LDL-cholesterol values. No statistical significance was found for any of the blood lipid profiles within each study group (Table2).References1. Morton LW, Abu-Amsha Caccetta R, Puddey IB, Croft KD: Chemistry and biological effects of dietary phenolic compounds：relevance to cardiovascular disease. Clin Exp Pharmacol Physiol 2000, 27(3):152-9.2. Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB: Therelative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic Res 1995, 22(4):375-83.3. German JB, Walzem RL: The health benefits of wine. Annu Rev Nutr 2000, 20:561-93.4. Renaud S, de Lorgeril M: Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 1992339(8808):1523-6.5. Gronbaek M: Factors influencing the relation between alcohol and mortality with focus on wine. 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