Men and women, aged 18 – 50 y, free from any known diseases were included in the study. Smoking, excessive exercise (> 10 h/wk), pregnancy and lactation, present or former alcohol or drug abuse, medications interfering with antioxidants or intake of alcohol, supplementation of vitamins, minerals or probiotics (2 weeks before or during the study) were exclusion criteria. The study was performed according to the guidelines of the Declaration of Helsinki and was approved by the ethics committee of the University of Bonn. All subjects gave their informed written consent.

The study consisted of two parts, a single-dose analysis investigating the effects of a single ingestion of red wine (RW) versus dealcoholized red wine (DRW) and a long-term dietary intervention trial in which RW and DRW were consumed daily for 6 wk. Both trials were prospective, randomized and controlled studies.

After an overnight fast, 27 healthy non-smokers (demographic data are shown in Table 1) were randomized to receive a single dose of 200 mL red wine (group RW; n = 9), 175 mL dealcoholized red wine (group DRW; n = 9), or 200 mL mineral water (controls; n = 9). Total phenolic content, total antioxidant capacity and concentrations of the major antioxidants (ascorbic acid, uric acid, albumin, bilirubin) in plasma, as well as DNA strand breaks in leukocytes (with and without ex vivo H₂O₂ treatment to induce oxidative stress) were measured before, 90 and 360 min after ingestion. Subjects were instructed to abstain from polyphenol rich foodstuff and from any alcoholic drinks 24 h before the first blood sampling until completion of the study. For this purpose they received a list of acceptable foods which are considered to be low in polyphenols. Compliance was controlled by a self-estimated 24-h dietary record. One hour after the first blood sampling, participants could choose from a breakfast buffet composed of foods low in polyphenols (white bread, butter, curd cheese, yogurt, cheese, honey, milk, fruit infusion, mineral water). All subjects were allowed to leave the study center at 90 min, when the initial blood samples had been drawn, and returned at 360 min for the follow-up blood samples. They were allowed to have lunch in between following the dietary restrictions concerning antioxidants.

Seventy-eight healthy subjects were enrolled in the dietary intervention trial. They ingested 200 mL of red wine (group RW, 27 subjects) or 175 mL dealcoholized red wine (group DRW, 26 subjects) daily within one hour after dinner for 6 wk. The control group (25 subjects) did not receive any study drink. Subjects were instructed not to drink more than 2 cups (150 mL each) of coffee, black or green tea, and 2 glasses (200 mL each) of fruit juice per day, and to renounce from grape juice, multivitamin juices, and alcoholic beverages starting one week before the intervention throughout the whole study period.

Blood samples were drawn before and after intervention after an overnight fast (between 07.30 and 09.00 a.m.), and about 12 h after the last ingestion of RW or DRW. In addition to the laboratory parameters measured in the single-dose analysis, α-tocopherol concentration in serum was determined as changes are expected only in the long term 26. To control compliance to dietary restrictions and assess possible changes of dietary patterns due to seasonal variations during the study period, self-estimated 7-day dietary records had to be completed in the week before and in the last week of intervention.

The red wine used in the present studies was Spätburgunder, 1999, Marienthaler Klostergarten, Ahr, Germany. Dealcoholized red wine was produced by vacuum extraction of alcohol from the same batch. The amounts of flavonoids and phenolic acids ingested from a single serving of RW (200 mL) and DRW (175 mL) are listed in Table 2. The application of 175 mL DRW based on the assumption that 12.5% of the volume (25 mL / 200 mL) would be lost due to alcohol extraction, which would increase polyphenol concentration in DRW. However, subsequent analysis revealed a lower polyphenol content in DRW due to the processing. Thus, intake of polyphenols, especially flavonoids, was slightly lower from 175 mL DRW compared to 200 mL RW. The water for the control group in the single-dose analysis was Markus Brunnen "Still" (Vereinte Mineral- und Heilquellen, Rosbach, Germany), a carbonated natural mineral water from which iron is removed.

The subjects received a standardized dietary record which they completed for 24 h (single-dose analysis) or 7 days (dietary intervention trial), respectively. To determine polyphenol intake as exactly as possible, polyphenol rich foods were listed in detail. Calculation of the intake of flavonoids (kaempferol, quercetin, myricetin, catechin, epicatechin, epigallocatechin, gallocatechin, naringenin, cyanidin, peonidin, petunidin, and malvidin) and phenolic acids (salicylic, p-hydroxy benzoic, gallic, syringic, and ellagic acid) was based on data of Linseisen et al. 27 and Radtke et al. 28, which were completed by data for quercetin and kaempferol in tomato products 29 and catechin and epicatechin in apples, red grapes 30, and black tea 31.

Peripheral venous blood was collected in Vacutainer^® tubes (Becton Dickinson, Heidelberg, Germany) containing Li-heparin or no anticoagulant. Samples were protected from light and stored on ice until centrifugation (3000 × g, 20 min, 4°C). Plasma samples for determination of total phenolic content and antioxidant capacity were stored at -70°C, and for determination of albumin, uric acid and bilirubin at -20°C. For measurement of ascorbic acid, plasma was mixed with 5% trichloro acetic acid and centrifuged (3 min, 12000 × g). Supernatants were stored at -70°C. Serum was frozen at -20°C until measurement of α-tocopherol. For determination of DNA damage, heparinized blood was kept in the dark at room temperature until processing 60 – 120 min after sampling.

Total phenolic content in plasma (TPP) was determined by the Folin-Ciocalteu method modified by Serafini et al. 10 to avoid plasma protein interference. Unlike Serafini et al. 10, we centrifuged the thawed plasma samples at 12000 × g for 5 min. To remove plasma proteins completely, 2 mol/L metaphosphoric acid (Merck, Darmstadt, Germany) was used for precipitation and an additional centrifugation step (2700 × g, 3 min) was introduced for the combined supernatants before adding Folin-Ciocalteu reagent (Fluka Chemie, Buchs, Switzerland). Experiments were performed in duplicate.

Plasma antioxidant capacity was determined by the Trolox equivalent antioxidant capacity (TEAC) assay as described previously 32. The antioxidant capacity is given in comparison to a 1 mmol/L standard solution of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma, Deisenhofen, Germany). Experiments were performed in triplicate.

To control, if antioxidants other than polyphenols could have an impact on TEAC, the following major antioxidants in blood were also measured. Vitamin C concentration in plasma was determined colorimetrically according to Speitling et al. 33. Experiments were performed in duplicate. Uric acid, albumin and bilirubin in plasma were determined by routine procedures at the Institute for Clinical Chemistry and Laboratory Medicine, University Duesseldorf, Germany. Concentration of α-tocopherol in serum was measured by High Performance Liquid Chromatography at the Research Centre for Diabetes, Duesseldorf, Germany.

Single Cell Gelelectrophoresis (Comet Assay) was performed to assess DNA strand breaks in peripheral leukocytes. In this assay, cells are embedded in agarose on slides, incubated in PBS (control cells) or H₂O₂ (induction of oxidative stress), and lysed. During subsequent electrophoresis, DNA regions that are relaxed due to strand breaks migrate towards the anode forming a typical comet structure.

This assay was performed according to the standard protocol of Bauch et al. 34 adapted to the use of hydrogen peroxide as a stressor. To simulate the in vivo situation as far as possible and to avoid artifacts by density centrifugation, whole blood was used instead of isolated mononuclear cells. Ten μL heparinized whole blood were diluted with 90 μL PBS (10 mmol/L, pH 7.4, without Ca²⁺/Mg²⁺) (Sigma) to obtain a single cell suspension. After preparation of the slides, they were incubated for 20 min at 4°C with either 300 μmol/L H₂O₂ (Sigma) in PBS to induce DNA strand breaks or in pure PBS (controls). Electrophoresis was performed at 4°C and 1 V/cm for 4 min on a flat-bed electrophoresis apparatus with electrode spacing of 39 cm (Mega Horizontal Gelbox Safety; Molecular Bio-Products, San Diego, USA). Experiments were performed in duplicate.

For analysis, gels were rehydrated in aqua bidest. (10 min, room temperature) and DNA was dyed with 150 μL propidium iodide (Sigma) (25 μmol/L, solved in 125 mmol/L Tris with 123 mmol/L NaCl in aqua bidest., pH 7.5). Measurements were performed with the image analysis software package Comet Assay II (Perceptive Instruments, Suffolk, UK) coupled to a fluorescence microscope (Leitz DM RB, Leica, Bensheim, Germany) with a CCD camera (Cohu, San Diego, USA). Fifty cells were measured on each slide and Tail Intensity (TI) and Tail Moment (TM) were calculated to quantify DNA strand breaks.

DNA strand breaks in untreated cells reflect the steady state between DNA damage and repair in vivo 353637 and are therefore referred to as "endogenous" DNA damage in this paper. "Exogenous" DNA strand breaks are those induced by treatment with H₂O₂ ex vivo and were calculated as the difference between treated and untreated cells.

The sample size calculation was based on plasma antioxidant capacity (TEAC), which was considered as main outcome and based on data from a previous study with healthy subjects who ingested polyphenol rich fruit-juices or a fruit-vegetable concentrate 32. With 6 subjects in each group (RW, ERW, controls) the single-dose analysis has a power of 90% to detect TEAC changes >0.033 mmol/L, which was the mean difference between two measurements in the previous study. To account for possible drop-outs, we included 9 subjects in each group, adding up to a total of 27 participants.

Mean TEAC difference in the previous long-term study was 0.075 mmol/L. To detect changes ≥ 0.075 mmol/L and differences between the groups with a power of 90%, a sample size of 25 subjects in each group (RW, DRW, controls) was calculated. Level of significance was 5% for all tests.

Values given are means ± SD unless indicated otherwise. Differences between the groups were examined using the Mann-Whitney U-test. The effects of the study drinks on the investigated parameters were evaluated by comparing the values obtained at different time intervals with baseline for each group using the Wilcoxon signed rank test. Differences indicated by p < 0.05 were considered statistically significant. Statistical tests were performed with SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA).

The day before the study the subjects ingested 22.9 ± 47.4 mg polyphenols (Flavonoids: 2.1 ± 5.3 mg; Phenolic acids: 20.8 ± 46.2 mg). This corresponds to 8 – 16% of the average intake observed in another German cohort 2728 and in our dietary intervention trial (see below) indicating good compliance to the dietary restrictions.

Results for the antioxidant parameters in plasma are presented in Table 3. Total phenolic content in plasma increased 90 min after ingestion of RW (p = 0.008) or DRW (p = 0.008). After 360 min values were higher than at baseline in groups RW (p = 0.02) and DRW (p = 0.008), but also in control subjects (p = 0.01). Vitamin C concentration in plasma decreased 360 min after RW consumption (p = 0.008), but increased in group DRW (p = 0.02). Significant changes of endogenous antioxidants, i.e. uric acid, albumin or bilirubin, have been observed after consumption of RW and DRW, and also in the control group, but TEAC was not altered significantly in any group.

Endogenous DNA strand breaks increased 360 min after consumption of either RW or DRW compared to baseline values. In group DRW an increase could be observed after 90 min, but only with borderline significance (p = 0.05). Hydrogen peroxide-induced DNA strand breaks decreased in group DRW at 360 min, whereas no alterations could be observed after RW consumption or in controls. Statistical analysis provides the same results for TI (Figure 1) and TM (Table 3).

Four subjects were lost during the study. One woman in group DRW did not tolerate the study drink. The other subjects were allocated to group RW. Of those, one woman fell ill, which was not related to the intervention. One man started smoking during the study and the other one was lost to follow-up. Demographic data of the 74 subjects who completed the study are summarized in Table 1.

Flavonoid intake increased significantly in group RW (28.9 ± 16.6 vs. 76.7 ± 24.5 mg/d; p < 0.001) and DRW (39.1 ± 20.5 vs. 64.2 ± 27.6 mg/d; p < 0.001), but not in controls (37.7 ± 21.3 vs. 41.0 ± 24.8 mg/d). During intervention, the flavonoid intake in group RW exceeded that in group DRW (p = 0.005), and it was higher in group RW (p < 0.001) and DRW (p = 0.005) than in controls. Intake of phenolic acids increased during supplementation of RW (107.8 ± 75.5 vs. 135.1 ± 82.5 mg/d; p = 0.01) and DRW (123.5 ± 83.2 vs. 158.3 ± 102.7 mg/d; p = 0.02), but also in controls (87.2 ± 69.7 vs. 135.6 ± 100.3 mg/d; p = 0.03). The amounts did not differ between the groups before or during intervention.

The difference to the average intake of 54 mg/d flavonoids 27 and 222 mg/d phenolic acids 28 reported by others could be explained by the restriction of polyphenol rich foods, especially coffee, which is a major source of phenolic acids in Germany 28.

Total phenolic content in plasma (Table 4) increased after 6 wk regular consumption of RW (p = 0.002), but did not change in group DRW or controls. A significant increase of plasma uric acid concentration was observed in controls (p = 0.04), and a decrease of bilirubin in group DRW (p = 0.02) (Table 4). In contrast, vitamin C and albumin in plasma, α-tocopherol in serum, and plasma antioxidant capacity did not change significantly in any group (Table 4). There was no significant difference between the groups before and after 6 wk of intervention considering the antioxidant parameters.

Endogenous DNA strand breaks (Figure 2, Table 4) decreased only after regular consumption of RW (TI: p = 0.03; TM: p = 0.04), but not in group DRW and controls. Susceptibility of leukocytes towards H₂O₂-induced DNA damage was not altered in any group. Differences in endogenous or ex vivo induced DNA strand breaks could not be observed between the groups, neither before nor after 6 wk intervention (Figure 2, Table 4).