Volume 87, Issue 1, January (2002), pp. 30237
? The Corresponding Authors 2002
Jennifer L. Donovan*, Sidika Kasim-Karakas J. Bruce German and Andrew L. Waterhouse
(Received 1 May 2001 ? Revised 25 July 2001 ? Accepted 31 August 2001)
Little is known about flavonoid metabolism and excretion in man. In the present study, the urinary excretion of a major flavonoid in wine, catechin, and its metabolites, were measured after nine human subjects each consumed 120 ml red wine (RW) on one day and de-alcoholized red wine (DRW) on a separate day. Both the RW and DRW contained 120 (SEM 3) mmol catechin (35 mg). GCMS analyses of the trimethylsilylated derivatives of catechin and 39 and 49 methylcatechin were performed before and after hydrolysis of conjugates by b-glucuronidase and sulfatase. Baseline urine samples collected prior to wine consumption contained 0·013 (SEM 0·005) mmol catechin and metabolites. During the 8 h period following consumption of RW and DRW, 6·6 (SEM 0·9) and 5·3 (SEM 0·6) mmol catechin and metabolites were excreted in 893 (SEM 94) and 740 (SEM 101) ml urine respectively. This corresponded to 3·010·3 % of the dose after RW and 2·18·2 % of the dose after DRW. The amount of catechin and metabolites excreted in urine was 20 % higher after RW compared with DRW (P=0·06). Catechin in all urine samples was present as metabolites and there were no differences in the proportions of individual metabolites after RW and DRW. As with other flavonoids, the fate of most ingested catechin is not yet known.
Wine: Catechin: Catechin metabolites: Urinary excretion: Man
Flavonoids are abundant components of fruits, vegetables, red wine, tea and chocolate (Kühnau, 1976; Scalbert & Williamson, 2000; Arts et al. 2001). They display a multitude of biological effects in vitro and in vivo after consumption of flavonoid-containing foods (Walle et al. 1995; Clifford et al. 1996; So et al. 1996; Teissedre et al. 1996; Da Silva et al. 1998; Nidgikar et al. 1998; Day et al. 2000; Rein et al. 2000a, b; Young et al. 2000; Schramm et al. 2001). Epidemiologic studies show strong associations between consumption of flavonoid-containing foods, especially wine, and reduced mortality from cardiovascular disease (St Leger et al. 1979; Hertog et al. 1993; Klatsky & Armstrong, 1993; Criqui & Ringel, 1994; Sasaki & Kesteloot, 1994; Grønbæk et al. 1995; Hertog et al. 1995; Renaud & de Lorgeril, 1992).
Little is known about flavonoid metabolism, distribution and excretion after consumption of common foods. There have been some clinical studies reporting the absorption and excretion of flavonols (Hollman et al. 1995, 1997; de Vries et al. 1998, 2001; Manach et al. 1998; flavones (Nielsen et al. 1999), anthocyanins (Lapidot et al. 1998), flavanones (Fuhr & Kummert, 1995), flavanols (Lee et al. 1995; Richelle et al. 1999; Rein et al. 2000a) and isoflavonoids (Xu et al. 1994, 1995; Ameer et al. 1996) after consumption from food sources. However, the vast majority of studies were performed using gram-size doses, which gives little insight into the fate of flavonoids when consumed in smaller quantities as food constituents (Das, 1971; Das & Sothy, 1971; Gugler et al. 1975; Shaw & Griffiths, 1980; Shaw et al. 1982; Hackett et al. 1983; Wermeille et al. 1983).
The present study focused on the urinary excretion of the flavonoid catechin and its metabolites following consumption of a single serving of red wine (RW) or de-alcoholized red wine (DRW). Catechin was chosen as the flavonoid of interest because it is a major wine flavonoid and it is found in many different foods (Hollman & Arts, 2000; Arts et al. 2000a,b; de Pascual-Teresa et al. 2000). Studies have shown its potent antioxidant activity in vitro (Teissedre et al. 1996; Plumb et al. 1998) and its efficacy in reducing atherogenesis in an animal model (Xu et al. 1998).
There are no previous reports of the urinary excretion of catechin after doses that are normally present in wine or foods. Earlier studies on the urinary excretion of catechin by human subjects used large doses of purified catechin. Das (1971) recovered 8 % of a 4 g dose as catechin and its glucuronide and sulfate conjugates over 24 h, most of which were excreted during the first 6 h. Hackett et al. (1983) also measured the excretion of methylated catechin metabolites and reported that 48 % of a 2 g dose was recovered in urine as conjugates of catechin or methylated catechin over a 24 h period.
The pharmacokinetics of catechin and its metabolites in human plasma after consumption of RW and DRW have been reported (Donovan et al. 1999a). In that study, catechin metabolites reached a concentration of 100 nmol/l plasma 1 h after a single serving of RW. When the results were compared with those of earlier studies that used larger doses, it was apparent that the dose influenced both the amount of catechin in plasma as well as the pattern of catechin metabolites. Few differences were observed between the amounts of metabolites in plasma after consumption of RW and DRW. Ethanol in RW, however, reduced the elimination half-life of metabolites from plasma. Thus, it is important to determine if ethanol also has an effect on urinary elimination. The present study reports the urinary excretion of catechin and its metabolites in the same human subjects over an 8 h period after RW and DRW consumption.
Chemicals and reagents
(+)-Catechin was obtained from Fluka Chemicals (Ronkonkoma, NY, USA) and (+)-taxifolin from Apin Chemicals Ltd (Abingdon, Oxford, UK). 39-O-methylcatechin (39MC) and 49-O-methylcatechin (49MC) were synthesized, purified and identified as previously described (Donovan et al. 1999b). The derivatizing reagent, N,O-bis(trimethylsilyl)-trifluoroacetamide, was purchased from Pierce (Rockford, IL, USA). b-Glucuronidase (G-0376), aryl sulfatase (S-9754) and D-saccharic acid 14 lactone were purchased from Sigma Chemical Co. (St Louis, MO, USA). All solvents and reagents were Fisher HPLC or Optima grade, and other reagents were either purchased from Fisher (Pittsburgh, PA, USA) or Aldrich (Milwaukee, WI, USA).
Subjects and study design
The experimental design, preparation and analysis of the RW and DRW, and the human subject selection criteria have been described previously (Donovan et al. 1999a; Bell et al. 2000). Briefly, nine volunteers (Five men, Four women, age 29 (SEM 3) years, weight 78 (SEM 4) kg consumed a flavonoid-free diet for 2 d prior to ingestion of wine. After a 14 h fast, a baseline urine sample was collected from each subject who then consumed, in random order, 120 ml either RW or DRW (100 % Cabernet Sauvignon, 1996), providing 120 (SEM 3) mmol of (+)-catechin (35 mg) as analysed by HPLC (Ritchey & Waterhouse, 1999). The RW contained 103 g/l ethanol and provided 12·4 g ethanol in 120 ml. The crossover design allowed each subject to serve as his or her own control. Each subject consumed the second wine sample after resuming the flavonoid-free diet for at least 2 d. The subjects urinated as needed over the 8 h time period but were asked to empty their bladders near 8 h. The individual specimens were collected in plastic cups and the time and volume of each urine collection was recorded. A 1 ml aliquot of urine was mixed with 25 ml phosphate-buffered ascorbic acid (0·4 mol NaH2PO4/l, 200 g ascorbic acid/l, 1 g EDTA/l, pH 3·6) and was flushed with N2 and immediately frozen at -70°C. The clinical protocol was approved by the Human Subjects Committee at the University of California at Davis.
Analysis of urine
To prevent oxidation of the analytes during sample preparation, all solvents and reagents were deoxygenated by purging with N2 and were kept cold during extraction. The samples were thawed and 20 ml urine were mixed with 450 ml sodium acetate buffer (50 mmol/l, pH 5·5) and 250 ml CaCl2 (0·6 mol/l). Taxifolin was used as an internal standard and a final concentration of 20 mmol/l was added to all urine samples. Samples for measurement of total catechin, total 3>MC and total 4>MC were incubated at 37°C in a shaker-water bath for 1 h in N2-flushed tubes containing 100 units sulfatase and 2500 units b-glucuronidase dissolved in 120 ml water. Individual conjugate forms were determined after incubation without enzymes (free), or with b-glucuronidase (glucuronide conjugates), or with sulfatase and 0·2 mol saccharic acid 1,4 lactone/l (sulfate conjugates). Conjugates containing both the glucuronide and sulfate residues were then estimated by subtracting the free, sulfate and glucuronide forms from the total values.
After incubation, the urine was extracted with 2 ml ethyl acetate and centrifuged at 4500 g for 3 min at 4°C. The supernatant fraction was removed and the remaining fraction was extracted a second time with 1·5 ml ethyl acetate. The combined ethyl acetate extracts were passed through anhydrous sodium sulfate packed in a Pasteur pipette, dried under N2, redissolved in 20 ml pyridine and derivatized with 30 ml N,O-bis (trimethylsilyl)-trifluoroacetamide at 6575°C for 2 h.
GCMS analyses were performed on a Hewlett-Packard 6890 GC equipped with a 5973 quadropole MS and a DB-23 capillary GC column (
For quantitation, standard curves were prepared in blank urine (i.e. urine containing undetectable levels of the analytes). Catechin, 39MC and 49MC were added to the blank urine at final concentrations ranging from 0·2 to 34·4 mmol/l. Taxifolin was added at a final concentration of 20 mmol/l. The urine was then incubated without addition of enzymes, extracted and analysed as described for the samples. Standard curves were linear and had average r2 values of 0·998. The slopes of five standard curves prepared over 10 d varied <5 % (CV). The limit of detection (signal:
Concentrations of catechin metabolites in urine were multiplied by the respective urinary volume to obtain the amount excreted during a given sampling time. All values are expressed as mean values with the standard errors for the nine subjects. Each subject served as his or her own control in this study and levels of catechin metabolites after RW and DRW were compared for each subject using the paired t test. To determine the changes in metabolite composition over time, the differences in the percentages of the individual conjugate forms in the first urine sample (near 1 h) were compared with the percentages in the final urine sample (near 8 h) using the paired t test.
Volumes of urine collected prior to RW and DRW consumption were 55 (SEM 13) and 70 (SEM 22) ml respectively. The average number of urine samples during the 8 h collection period after RW and DRW consumption was 4 (SEM 1). The total volumes of urine collected during the 8 h period after RW and DRW consumption were 893 (SEM 94) and 740 (SEM 101) ml respectively (P=0·1).
Total excretion of catechin
Urine samples collected before RW and DRW consumption respectively contained a total of 0·008 (SEM 0·003) and 0·020 (SEM 0·007) mmol total catechin (catechin and all measured metabolites). The cumulative excretion of total catechin in the urine of each subject over the 8 h period after RW and DRW consumption is shown in Fig. 1
Metabolites of catechin
The total amounts of the individual forms of catechin metabolites excreted over the 8 h period after RW and DRW consumption are shown in Table 1
Sulfated conjugates of catechin accounted for 40 (SEM 4) % total metabolites in urine after RW and 42 (SEM 6) % after DRW. Catechin was also excreted in a form with both glucuronide and sulfate and these accounted for 37 (SEM 4) % after RW and 35 (SEM 6) % after DRW. Only a small amount of catechin was excreted as a glucuronide conjugate.
Catechin was also methylated in the 39 position. Over the 8 h urine collection period, 19 (SEM 2) and 18 (SEM 1) % of the total metabolites were methylated in the 39 position after RW and DRW. The majority of these 39 methylated metabolites were also conjugated with glucuronide. However, smaller levels 39 methylated metabolites were also present as sulfate conjugates, glucuronidesulfate conjugates or in unconjugated form.
Extremely small amounts of 4> methylated metabolites were also excreted in urine. An average value of 0·027 mmol were excreted after RW and an average value of 0·018 mmol were excreted after DRW over the 8 h period. These metabolites accounted for <0·5 % catechin metabolites. Because the amounts of 49 methylated metabolites in urine were so low, it was not possible to determine accurately all the forms of the conjugates in all urine samples. However, in the most concentrated urine samples (obtained 24 h after consumption), an average of 48 % catechin metabolites were present as glucuronide conjugates, 10 % as sulfate conjugates and 16 % as both glucuronide and sulfate conjugates.
The percentages of the conjugate forms of catechin as well as 39MC in the first urine samples (about 1 h) and the final urine samples (about 8 h) after RW consumption are shown in Fig. 2
There is an inverse relationship between wine consumption and cardiovascular disease mortality (Renaud & de Lorgeril 1992). Ethanol in wine appears to be responsible for only a fraction of the health benefits enjoyed by wine-drinking populations (St Leger et al. 1979; Klatsky & Armstrong, 1993; Sasaki & Kesteloot, 1994; Gronbæk et al. 1995). Flavonoids in wine may provide additional protection against cardiovascular disease by acting as antioxidants (de Whalley et al. 1990; Fuhrman et al. 1995; Whitehead et al. 1995; Cao et al. 1996; Serafini et al. 1996, 1998; Teissedre et al. 1996; Hayek et al. 1997; Nidgikar et al. 1998), or reducing platelet activity and counteracting the increased activity of platelets after ethanol withdrawal (Seigneur et al. 1990; Demrow & Folts, 1994; Demrow et al. 1995; Ruf et al. 1995; Pace-Asciak et al. 1996; Renaud & Ruf, 1996; Hayek et al. 1997; Osman et al. 1998, Rein et al. 2000b). Flavonoids also have anti-inflammatory and vasorelaxing activities (Fitzpatrick et al. 1993; Schramm et al. 1997). Understanding the contribution of each of these mechanisms to vascular health requires comprehensive knowledge of the metabolism, distribution and excretion of flavonoids in wine. It is not known if there are additional biological effects when flavonoids are consumed in the presence of ethanol. Flavonoids and ethanol are co-ingested in wine, but similar considerations can arise from the simultaneous consumption of other sources of ethanol with foods that contain flavonoids.
We previously reported that ethanol reduced the elimination half-life of total catechin after RW consumption without affecting the pattern of catechin metabolites (Donovan et al. 1999a; Bell et al. 2000). In the present study, 20 % more catechin metabolites were excreted in urine over an 8 h period after RW compared with DRW. Although the significance level was not very high (P=0·06), this result, coupled with our previous finding in plasma does indicate a role of ethanol in enhancing the rate of catechin elimination. The urine volume was increased by 17 % after wine with ethanol suggesting that the increased elimination is due to a diuretic effect (Rozman & Klaassen, 1996). Further studies, however, are necessary to fully understand the effect of ethanol on the elimination of flavonoids as well as its mechanism of action.
Several investigators have measured the urinary excretion of other flavonoids and their conjugates after consumption of foods. In all of these studies, only a small fraction of the flavonoid dose was recovered in urine (Xu et al. 1994; Fuhr & Kummert, 1995; Lee et al. 1995; Ameer et al. 1996; Nielsen et al. 1997, 1999; de Vries et al. 1998, 2001; Lapidot et al. 1998; Watanabe et al. 1998; Young et al. 1999). Another study showed that even when absorption of flavonol glycosides is >50 %, elimination in urine is <1 % (Hollman et al. 1995).
We also found that only a small fraction of the dose of catechin is recovered in urine after RW consumption. In the present study we used a fairly short collection period, compared with other studies of flavonoid excretion. However, catechin has a much shorter half-life in plasma than other flavonoids (only 23 h) and most metabolites were excreted during the first 4 h (see Fig. 1). Thus, 5 % of the ingested dose represents the vast majority of catechin excreted by this mechanism after wine consumption.
Differences in the proportions of specific catechin metabolites in plasma and urine indicate that certain metabolites may not be exclusively eliminated by urine. Large, extensively conjugated metabolites are more likely to be eliminated by bile (Rozman & Klaassen, 1996). Metabolites conjugated with both the glucuronide and sulfate residues made up the majority of catechin metabolites in plasma after wine consumption (Donovan et al. 1999a). However, these metabolites accounted for only 39 % of the total catechin in urine, suggesting that biliary excretion may also be occurring. Conversely, the metabolites that were conjugated with just sulfate were rapidly eliminated from plasma, and their increased proportion in urine indicates that these metabolites are preferentially excreted in urine.
Some catechin may also have been eliminated in forms that do not contain the intact flavonoid ring as reported in the early studies by Das (1971) and Hackett et al. 1983. Recent experiments also indicate that microflora present in the large intestine can metabolise catechin as well as other flavanols to smaller phenolic acids and lactones (Meselhy et al. 1997; Déprez et al. 2000; Li et al. 2000). These metabolites were not measured in the present study as they can originate from many phenolic components in wine. However, this may represent an important elimination mechanism that should be investigated further in the future.
The results of the present study show that after RW consumption 310 % ingested catechin is eliminated in urine over an 8 h period. They also suggest that ethanol reduced the elimination half-life of catechin in plasma by increasing elimination in urine. Differences in the proportions of individual metabolites in plasma and urine indicate that some metabolites are not eliminated exclusively in urine. Other mechanisms of elimination, such as excretion by bile and metabolism within the large intestine must be studied further for a complete understanding of the fate of flavonids in man.
We gratefully acknowledge the advice of Alan Buckpitt, Rosemary Walzem and Robert Hansen. Funding was provided by E&J Gallo Winery, the Wine Spectator and the Wine Institute.
Abbreviations: DRW; de-alcoholized red wine; 39MC; 39-O-methylcatechin; 49MC; 49-O-methylcatechin; RW; red wine containing alcohol
CACorresponding author: Dr Jennifer L. Donovan, present address Laboratory of Drug Disposition and Pharmacogenetics,Department of Psychiatry, Room 246N, Medical University of South Carolina, 67 President Street, Charleston, SC 29425, USA, fax +1 843 792 6318, email email@example.com
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