Human Nutrition and Metabolism Research Communication Red Wine Is a Poor Source of Bioavailable Flavonols in Men1
al. 1990, Frankel et al. 1993, Kanner et al. 1994, Kinsella et al. 1993, Kondo et al. 1994, Lanningham-Foster et al. 1995, Rice-Evans et al. 1996). The wine phenolics quercetin, epicatechin and resveratrol protected LDL from oxidation in vitro (Frankel et al. 1993, Kerry and Abbey 1997, Margetts and Nelson 1991). The strongest effects were seen for quercetin and epicatechin. However, data from studies investigating ex vivo effects on protection of LDL oxidation are contradictory. Some studies (Fuhrman et al. 1995, Nigdikar et al. 1998) found that LDL oxidation was inhibited after the drinking of red wine, whereas others (Caccetta et al. 2000, de Rijke et al. 1996) did not find this effect. Thus, it is still not proved that phenolics in red wine truly have an effect. To have any direct effect on LDL oxidation, phenolics have to be absorbed into the blood. However, data on the absorption of phenolics from wine, especially flavonols, are sparse, and it is not yet known to what extent these components are bioavailable. Hollman et al. (1996) developed a sensitive and accurate method to determine flavonols in plasma and urine. Using this method, we found that quercetin and kaempferol from onions, apples and tea were absorbed (de Vries et al. 1998, Hollman et al. 1995), but the extent of absorption differed (Hollman et al. 1997 and 1999). To determine whether the bioavailability of quercetin from wine is similar to that of other sources in the diet, we compared quercetin levels in plasma and urine after the consumption of equal amounts of quercetin from red wine, tea or onions. In addition, we determined the levels of kaempferol and isorhamnetin, two other major flavonols.
(Manuscript received 9 August 2000. Initial review completed 13 September 2000. Revision accepted 11 December 2000.) Jeanne H. M. de Vries,*2 Peter C. H. Hollman,† Ingrid van Amersfoort,* Margreet R. Olthof* and Martijn B. Katan* *Division of Human Nutrition and Epidemiology, Wageningen University, 6703 HD Wageningen, the Netherlands and †State Institute for Quality Control of Agricultural Products (RIKILT), 6708 PD Wageningen, the Netherlands
ABSTRACT Red wine is a source of polyphenolic antioxidants, of which flavonols such as quercetin are representatives. Red wine might therefore prevent LDL oxidation and atherosclerosis. However, data on the bioavailability of flavonols from wine are lacking. Therefore, we compared the bioavailability of flavonols, especially quercetin, from red wine with that from the major dietary sources, yellow onions and black tea. Twelve healthy men consumed 750 mL red wine, 50 g fried onions or 375 mL of black tea, each for 4 d in random order. These supplements provided similar amounts of quercetin (14 –16 mg). There was a washout period of 3 d between each period of supplementation. The plasma quercetin concentration after the consumption of wine was lower than that after onions (P < 0.05) and not different from that after tea. Urinary excretion of quercetin after wine did not differ from that after onions and was higher than that after tea (P < 0.05). We conclude that flavonols from red wine are absorbed. However, because one glass of red wine provides fewer available flavonols than one portion of onions or one glass of tea, red wine appears to be a poorer source of flavonols than these other two sources. J. Nutr. 131: 745–748, 2001. KEY WORDS: ● flavonoids
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humans
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red wine
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quercetin
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MATERIALS AND METHODS Subjects and study design. The Medical Ethics Committee of the Division of Human Nutrition and Epidemiology approved the study. We recruited subjects via posters and local newspapers. To ensure that subjects could tolerate the alcohol in the wine, we only allowed men with a consumption of at least seven drinks per week to participate. The subjects were thoroughly informed about the study, and all gave their written informed consent. They were medically evaluated and considered healthy by a physician. The mean ⫾ SD age of the 12 subjects was 24.8 ⫾ 10.4 y, and their body mass index was 22.1 ⫾ 2.1 kg/m2 . The subjects were randomly assigned to one of three groups. They followed three treatment periods of 1 wk each. On d 4 –7 of each treatment period, the subjects consumed red wine, onions or tea. In this way there was a washout period of 3 d between the periods. Each of the three groups received the treatments in a different order. Throughout the study, the subjects consumed a diet low in flavonols. They were asked not to consume anything from a list with vegetables and fruits containing ⬎15 mg quercetin/kg, beverages containing ⬎4 mg quercetin/L (Hertog et al. 1992 and 1993) and vegetables containing ⬎5 mg kaempferol/kg. Supplements. During treatment periods, the subjects consumed either six 125-mL glasses of red wine, 50 g fried yellow onions or three 125-mL cups of black tea per d. The wine originated from the Me´doc region in France (Chateau Latour St. Bonnet 1993) and was selected
flavonols
Red wine contains phenolics such as flavonols, catechins, resveratrol (Constant 1997, Goldberg et al. 1996, Hertog et al. 1993, Ku¨hnau 1976, Siemann and Creasy 1992), anthocyanins and proanthocyanidins (Mazza 1995). These compounds are thought to have antioxidative properties that could protect LDL from oxidation (Abu-Amsha et al. 1996, De Whalley et
1 This work was supported by grants of the Netherlands Heart Foundation (93.084) and the Foundation for Nutrition and Health Research. 2 To whom correspondence should be addressed at Division of Human Nutrition and Epidemiology, Wageningen University, Bomenweg 2, 6703 HD Wageningen, the Netherlands. E-mail:
[email protected]
0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences.
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for its high content of quercetin. All bottles of wine were taken from one lot, and the concentration of quercetin was 19 ⫾ 0.3 mg/L (n ⫽ 5). The yellow onions were provided in aluminum trays, and the black instant tea was made by the subjects from 0.5 g extract/cup. The daily amount of quercetin provided by the three foods was almost the same. Thus, the wine provided 14.2 ⫾ 0.3 mg (⫾SD) quercetin, 1.5 ⫾ 0.1 mg kaempferol, 1.6 ⫾ 0.0 mg isorhamnetin and 6.7 ⫾ 0.1 mg myricetin per d. The fried onions provided 15.9 ⫾ 0.5 mg quercetin, 0.1 ⫾ 0.0 mg kaempferol, 0.6 ⫾ 0.0 mg isorhamnetin and no detectable amounts of myricetin per d. The tea provided 13.7 ⫾ 1.6 mg quercetin, 8.7 ⫾ 1.6 mg kaempferol, no isorhamnetin and 2.7 ⫾ 0.3 mg myricetin per d. We told the subjects to consume one third of the wine or tea at lunch, one third at dinner and one third between 2200 and 2400 h; the consumption of onions was divided between lunch and dinner. All empty wine bottles, onion trays and tea bags were returned to the division. We checked dietary compliance by 24-h recalls once a week. Energy and nutrient intakes were calculated using the Dutch nutrient database (Stichting Nederlands Voedingsstoffenbestand 1993), and intakes of quercetin and kaempferol were calculated using our published values for contents in vegetables, fruit and beverages (Hertog et al. 1992 and 1993). Medications were not allowed except for contraceptives and acetaminophen (paracetamol). We provided all subjects with a diary and asked them to record all deviations from the guidelines. Blood sampling. Venous blood samples were taken in each period as previously described (Hollman et al. 1996). All subjects provided a baseline blood sample on the morning of d 4 of the 2nd wk, after they had followed the dietary guidelines for 3 d and before they had started to consume the second series of supplements. On d 7 of each period, the subjects provided two blood samples: one between 1400 and 1500 h and one between 1700 and 1800 h. Urine sampling. Five of 12 subjects collected urine for a baseline value on d 3 of wk 3 after they had consumed the low flavonol diet for 2 d and before they consumed the third series of supplements. All 12 subjects collected their urine during 24 h on d 7, the last day of each treatment period. We provided each subject with ten 500-mL bottles with 2.5 mL thymol dissolved in isopropanol as a preservative and one 1000-mL bottle with 5 mL thymol. Subjects collected all urine samples until the next morning, including the first urine after getting up. The urine samples were immediately put into polystyrene boxes containing dry ice. Within 1–5 d, the urine samples were thawed at 40°C. Urine samples were pooled for each subject per day and stored at ⫺40°C until analyses. To check the completeness of urine sampling, we used lithium as a marker. Each morning for 21 d the subjects consumed 235 mol lithium (2 mg) as lithium chloride dissolved in 10 mL water as a marker. The dose is 1% of that used for patients with bipolar disorder (anonymous 1980). The recovery of orally ingested lithium over a period of 6 d is ⬃95% (Sanchez-Castillo et al. 1987a and 1987b). Analytical methods. Quercetin, kaempferol and their conjugates were simultaneously extracted from plasma or urine and hydrolyzed to the aglycone with 2 mol HCl/L in aqueous methanol (Hollman et al. 1997) and determined by HPLC with fluorescence detection (Hollman et al. 1996). The limit of detection was 0.003 mol/L for quercetin and 0.002 mol/L for kaempferol and isorhamnetin in plasma and urine. A separate undiluted urine sample was acidified and analyzed for lithium through atomic absorption spectrophotometry (anonymous 1976). Lithium recovery. Recoveries of lithium in urine were 96 ⫾ 12% after wine, 100 ⫾ 6% after onions and 98 ⫾ 9% after tea. The recovery of 12 of the 36 urine samples was ⬍85% or ⬎105%. This could mean that subjects did not take their lithium or sampled too much or too little urine, and urinary excretion could be considered mistakenly too high or too low. However, reanalysis of our data with correction for lithium recovery did not change the differences we found among treatments. The diets. According to diaries, the number of empty packages and 24-h recalls, the subjects complied well with the dietary guidelines. They consumed negligible amounts of flavonol-rich foods dur-
FIGURE 1 Estimated plasma concentrations of quercetin that will be reached after the consumption of one portion of wine (100 mL), fried yellow onions (15 g) or black tea (125 mL), all with average quercetin concentration as found in commercially available foods (Hertog et al. 1992 and 1993). The differences in bioavailability among the three foods are based on plasma concentrations of 12 men who consumed 750 mL red wine, 50 g fried onions or 375 mL strong black tea.
ing the study. The only differences in intake from the diet without the supplements between the treatments were the intake of energy, which was lower in the tea period than in the wine period, and the intake of alcohol, which was significantly higher in the tea period. Statistical analysis. To analyze the effects of red wine, onions and tea in plasma, the mean flavonol concentrations of the two afternoon blood samples were used. To achieve normality, the amounts of quercetin, kaempferol and isorhamnetin analyzed in plasma and urine were converted to log10 values. The effects of red wine, onions and tea were analyzed by using the General Linear Model procedure (analysis of variance, fixed effect) of the Statistical Analysis System (SAS Institute 1989) with subject and treatment as class variables. We found no treatment order effect. The significance of differences was determined by paired t tests (least significant difference procedure). A P-value of ⬍0.05 was considered to be significant. We used Pearson correlation coefficients to determine the relationship between plasma concentrations and urinary excretions of flavonols and that between the first and second times that blood was sampled, and 95% confidence intervals (CI)3 were calculated using Fisher’s Z transformation. We calculated within- and between-person variations for quercetin levels in plasma and urine using the SAS procedure VARCOMP. To compare the bioavailability of quercetin from one common portion of red wine with that of onions and tea (Fig. 1), we first calculated the relative bioavailability from each food as the rise in plasma quercetin divided by the amount of quercetin consumed in this study. We then multiplied this figure by the amount of quercetin provided by one average portion (Donders-Engelen et al. 1997, Hertog et al. 1992 and 1993). Thus, for red wine, one glass contains 100 mL, which provides 0.8 mg (average concentration 8 mg/L). This was then multiplied by the concentration of 26 nmol/L found in plasma and divided by the14.2 mg quercetin/d given in this study.
RESULTS After the three treatments, flavonol concentrations in plasma were up to five times and excretions in urine were up to nine times higher than values during baseline (Tables 1 and 2). Plasma concentrations of quercetin and of the sum of the flavonols quercetin, kaempferol and isorhamnetin were lower after treatment with wine than after onions (P ⬍ 0.05) and not different from those after black tea (Table 1). Urinary excretions suggested that the absorption of quercetin from wine was between absorptions from onions and tea. The absolute urinary excretion of quercetin after red wine 3
Abbreviation used: CI, confidence interval.
POOR BIOAVAILABILITY OF FLAVONOLS FROM RED WINE
TABLE 1 Baseline concentrations and effects of daily consumption of 750 mL red wine (14 mg quercetin), 50 g fried onions (16 mg quercetin) and 375 mL black tea (14 mg quercetin) on concentrations of flavonols in plasma of healthy men1 Treatment Flavonol
Baseline
Wine
Onions
Tea
nmol/L Quercetin* Kaempferol* Isorhamnetin Sum
10 ⫾ 3 3⫾3 3⫾3 16
26 ⫾ 10a 3 ⫾ 3a 9 ⫾ 6b 38
53 ⫾ 17b 3 ⫾ 0a 6 ⫾ 3a 62
26 ⫾ 13a 14 ⫾ 7b 3 ⫾ 3a 43
1 Values are means ⫾ SD, n ⫽ 12; average of two blood samples per subject. Values in a row with different superscript letters differ significantly, P ⬍ 0.05.
tended to be lower than that after onions (P ⫽ 0.09), but it was significantly higher (P ⬍ 0.05) than that after black tea (Table 2). The amount of quercetin excreted in urine relative to intake was on average 0.8% after red wine, 1.0% after onions and 0.6% after tea. The excretion of the sum of all determined flavonols, i.e., quercetin plus kaempferol plus isorhamnetin, was lowest after wine. Plasma concentrations of flavonols agreed well between the blood samples taken between 1400 and 1500 h and between 1700 and 1800 h This was shown by high Pearson correlation coefficients between the quercetin concentrations of flavonols of all treatment periods (n ⫽ 36): 0.93 (95% CI 0.76 – 0.98) for quercetin, 0.70 (95% CI 0.48 – 0.84) for kaempferol and 0.85 (95% CI 0.72– 0.92) for isorhamnetin. The variation in plasma quercetin within persons between the first and second blood samples on the same day was 9% after all supplements, and the variation between subjects was 14% after wine, 11% after onions and 20% after tea. DISCUSSION We investigated whether the bioavailability of quercetin from red wine is higher than that from other sources from the diet. We found that plasma concentrations of quercetin after the intake of similar amounts of quercetin from red wine were lower than those after the consumption of onions and not different from those after the consumption of tea. Urinary excretions of quercetin were between those of tea and onions. These results suggest that the bioavailability of quercetin from red wine is less than that from onions and only slightly better than that from tea. In addition, the sum of the three flavonols, quercetin, kaempferol and isorhamnetin, was lowest after red wine consumption in both plasma and urine. To increase the precision of the measurements, we selected wine for its high quercetin concentration and used tea that was about twice as strong as tea usually consumed in our country (Hertog et al. 1993). We estimate that the consumption of one glass (100 mL) of average red wine with a concentration of quercetin of 8 mg/L (Hertog 1994) would produce plasma quercetin levels much lower than those after the consumption of 1 cup (125 mL) of average tea or 1 spoonful (15 g) of fried onions (Fig. 1). We did not directly measure the absorption of flavonols but rather measured it indirectly through an assessment of their
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concentrations in plasma and urine. It is possible that flavonols from red wine are metabolized differently than those from onions or tea. For example, the alcohol in the wine could have influenced the rate of metabolism. However, the urinary excretion of quercetin after wine and onion consumption was similar, which argues against such an effect of alcohol. In addition, the extent to which plasma total (⫹)-catechin, another polyphenol, increased after the consumption of red wine was not affected by the coingestion of alcohol (Bell et al. 2000). Plasma quercetin after wine consumption indicates that the bioavailability of quercetin from red wine is only half of that from onions. However, urinary excretion suggests that these differences in bioavailability are smaller. In our previous studies (de Vries et al. 1998, Hollman et al. 1997), the bioavailability of flavonols as determined by the area under the plasma concentration time curve correlated well with urinary excretion. The higher plasma concentration of quercetin due to onion consumption might be caused by the fact that the daily quercetin intake was provided by only two portions of onions, whereas it was provided by three portions of wine or tea. Also, excretions of quercetin relative to intake after the consumption of onions or tea in this study were similar to those we found previously (de Vries et al. 1998, Hollman et al. 1995). Thus, urinary excretions appear to be suitable to compare the bioavailability of quercetin in different foods. The absorption of flavonols from foods depends on the form in which quercetin is present (Hollman et al. 1997 and 1999). Quercetin is linked with rutinoside or glucoside. The bioavailability of quercetin from the rutinoside is only 20% of that from glucoside (Hollman et al. 1999). The two components are likely absorbed differently; quercetin rutinoside is absorbed from the colon (Hollman et al. 1999), whereas quercetin glucoside is actively absorbed from the small intestine (Hollman et al. 1999). The rutinosides are major flavonoids in tea and wine, whereas onions contain only glucosides (Goldberg et al. 1996). Thus, the results from our study, especially with tea, are consistent with former findings that quercetin conjugated with rutinoside as present in red wine and tea is not as well absorbed as is quercetin glucoside in onions. We found variations of plasma quercetin within persons of 10% and between persons of 10 –20%. Variations within persons were rather high, but there was a good agreement between the quercetin concentrations of the first and second blood samples. This confirms that quercetin concentrations
TABLE 2 Baseline levels and effects of daily consumption of 750 mL red wine (14 mg quercetin), 50 g fried onions (16 mg quercetin) and 375 mL black tea (14 mg quercetin) on concentrations of flavonols excreted in 24-h urine samples of healthy men1 Treatment Flavonol
Baseline
Wine
Onions
Tea
nmol/24 h Quercetin* Kaempferol* Isorhamnetin* Sum
53 ⫾ 33 80 ⫾ 35 89 ⫾ 47 222
371 ⫾ 109b 252 ⫾ 87a 335 ⫾ 184b 958
507 ⫾ 219b 332 ⫾ 486a 174 ⫾ 82a 1013
252 ⫾ 149a 706 ⫾ 405b 92 ⫾ 85a 1050
1 Values are means ⫾ SD, n ⫽ 12; those in a row with different superscript letters differ, P ⬍ 0.05.
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stabilize in 4 d, as was shown previously (Hollman et al. 1996). Variations between persons of quercetin concentrations can be explained by differences in absorption and metabolism. The different forms in which quercetin is present in foods might contribute to the small differences in variance between subjects found for wine, onions and tea. We conclude that flavonols from red wine are absorbed. However, the bioavailability of flavonols from red wine is not better than that from onions or tea. Because one glass (125 mL) of red wine provides lower amounts of available flavonols than one portion of onions (15 g) or one glass of tea (125 mL), red wine appears to be a poorer source of flavonols in the diet than these other two sources. ACKNOWLEDGMENT We thank Michel Buijsman for technical assistance.
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