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Articles

Catechins Are Bioavailable in Men and Women Drinking Black Tea throughout the Day^{1 ,2}

Beverly A. Warden³, Lametta S. Smith^*, Gary R. Beecher, Douglas A. Balentine and Beverly A. Clevidence^**

Analytical Sciences, Incorporated, Statistics and Public Health Research, Durham, NC 27713; ^* Florida International University, Department of Medical Laboratory Sciences, Miami, FL 33199; U.S. Department of Agriculture/ARS, Beltsville Human Nutrition Research Center, Food Composition Laboratory and ^** Phytonutrients Laboratory, Beltsville, MD 20705; and Unilever Health Institute, Unilever Research, 3133 AT Vlaardingen, The Netherlands

³To whom correspondence should be addressed. E-mail: bwarden{at}asciences.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS Study design RESULTS DISCUSSION REFERENCES

 
Tea consumption has been associated with reduced risk of both cancer and cardiovascular disease in population studies, but clinical data demonstrating bioavailability of the individual catechins and other polyphenolic components of tea are limited. This study assessed the apparent bioavailability of the prominent catechins from black tea in humans drinking tea throughout the day. After 5 d of consuming a low flavonoid diet, subjects drank a black tea preparation containing 15.48, 36.54, 16.74, and 31.14 mg of (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG), respectively, at four time points (0, 2, 4 and 6 h). Blood, urine and fecal specimens were collected over a 24- to 72-h period and catechins were quantified by HPLC with coularray detection. Plasma concentrations of EGC, EC and EGCG increased significantly relative to baseline (P < 0.05). Plasma EGC, EC and EGCG peaked after 5 h, whereas ECG peaked at 24 h. Urinary excretion of EGC and EC, which peaked at 5 h, was increased relative to baseline amounts (P < 0.05) and fecal excretion of all four catechins was increased relative to baseline (P < 0.05). Approximately 1.68% of ingested catechins were present in the plasma, urine and feces, and the apparent bioavailability of the gallated catechins was lower than the nongallated forms. Thus, catechins were bioavailable. However, unless they are rapidly metabolized or sequestered, the catechins appeared to be absorbed in amounts that were small relative to intake.

KEY WORDS: • bioavailability • catechins • human study • HPLC-coularray • black tea

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS Study design RESULTS DISCUSSION REFERENCES

 
Oxidative stress ;Fn1-3>is thought to play a role in the etiology of certain degenerative diseases including cancer, cardiovascular disease and neurological disorders (1 2 3) . Epidemiologic studies have shown that consumption of a diet high in antioxidant nutrients is associated with reduced risk of these disorders (4 ,5) .

Recently, much attention has been focused on the antioxidant properties of flavonoids, a large class of polyphenolic compounds derived from plants. Evidence suggests that these compounds may protect tissues against damage caused by oxygen free radicals and lipid peroxidation (6) . Observational studies have suggested an inverse relationship between flavonoid intake and risk of cardiovascular disease in humans (7 8 9 10) . In vitro and animal studies have demonstrated a protective effect of flavonoids in the prevention of cancer (11) ; however, epidemiologic studies in humans have been inconclusive and more research in this area is required (8 ,12 13 14) . A number of other studies, which have been reviewed, illustrated that tea consumption might provide protection against stroke, osteoporosis, liver disease, and bacterial and viral infections (10) .

When tea drinking was evaluated separately as a source of flavonoids for the Zutphen Elderly Study (7) , the risk of coronary heart disease (CHD)⁴ was found to be significantly lower in men with a high intake of tea (15) . Using the same cohort, tea drinking also showed an inverse correlation with incidence of stroke (9) . However, other epidemiologic studies, including the Nurse’s Health Study (16) , the Male Health Professional Study (17) and the Caerphilly Study (18) showed little or no inverse association between flavonoid consumption and incidence of heart disease. In fact, for the Caerphilly cohort, CHD increased directly with intake of tea, the major source of flavonoids for these subjects.

Currently, information about the bioavailability of tea polyphenols in humans is limited. It has been suggested that the planning of future human epidemiologic and cancer prevention studies would be facilitated by additional data about the bioavailability and metabolism of tea polyphenols (19 ,20) . Therefore, further study of the absorption, distribution and metabolism of tea polyphenols is warranted (10) . A few studies have demonstrated that consumption of green tea, black tea and decaffeinated black and green teas leads to increased concentrations of tea polyphenols in plasma, whole blood, urine and feces. The results of these studies suggest that tea polyphenols can be absorbed by humans, that a greater intake of polyphenols produces higher blood concentrations, and that the concentration of polyphenols excreted in the urine and feces is directly proportional to intake (21 22 23 24 25) .

The objective of the current study was to follow the absorption and excretion profiles of (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) for 24 h (72 h for feces) in subjects administered a black tea preparation at four times points over 6 h. The study was designed to mimic the tea intake of a regular tea drinker during a day. To our knowledge, this is the first report to describe the extraction and analysis of individual catechins in biological fluids of subjects drinking black tea under controlled conditions.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS Study design RESULTS DISCUSSION REFERENCES

 
Chemicals and reagents.

EGC, EC, EGCG and ECG were purchased from Indofine Chemical (Sommerville, NJ). The black tea powder was prepared from the leaves of Camellia sinensis and was provided by Lipton (Englewood Cliffs, NJ). ß-Glucuronidase (G-7896) and sulfatase (S-9754) were obtained from Sigma Chemical (St. Louis, MO). All other reagents and HPLC-grade solvents were from Fisher Scientific (Pittsburgh, PA). Stock solutions of EGC, EC, EGCG and ECG were prepared by dissolving 1 mg of each compound in 1 mL of methanol. These stock solutions were stable for at least 1 mo. The vitamin C-EDTA (Vc-EDTA) solution was composed of 0.4 mmol/L sodium phosphate (monobasic), 200 g/L ascorbic acid and 1.0 g/L EDTA, pH 3.6.

Instrumentation.

The samples were assayed by HPLC with coularray detection. The system consisted of a Beckman AS507 autosampler, two Beckman Model 126 pumps with a Beckman Gradient Controller attached to a Beckman System Gold computer (Beckman Coulter, Fullerton, CA), an ESA Model 5500 coulochem array system with a Dec pc 450 D2LP computer and a Hewlett-Packard cse 660 printer (ESA, Bedford, MA). The column was a C-18 reversed-phase column (150 x 4.6 mm) with a particle size of 5 µm (ESA)

	Study design

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS Study design RESULTS DISCUSSION REFERENCES

 
Diet and tea administration.

For 5 d before tea administration, free-living subjects ate a self-selected diet with the exception that they avoided flavonoid-rich foods. Subjects were given a list of foods to exclude from their diet for the entire study period, including alcoholic beverages, apples, apricots, celery, chocolate, citrus, coffee, fruit juices, grapes, green leafy vegetables, herbal tea, nuts, onions, peaches, potatoes, tea, tomatoes and tomato products. On the day of tea administration, subjects remained at the USDA/BHNRC human study facility for the entire day eating breakfast (0800 h), lunch (1200 h) and dinner (1630 h) on site. Subjects selected meals from a variety of low flavonoid foods provided by the diet kitchen. Upon leaving the study facility, subjects returned to a free-living, self-selected low flavonoid diet.

During the day at the study facility, subjects drank a tea preparation with sugar cookies over a 15-min period. One serving (250 mL) of tea preparation contained 15.48 mg EGC, 36.54 mg EGCG, 16.74 mg EC and 31.14 mg ECG. This tea preparation was administered at four time points (0, 2, 4 and 6 h) for a total catechin intake of 400 mg. Each 250-mL serving had a catechin content equivalent to 3 cups (750 mL) of brewed tea. Subjects ingested four 250-mL servings, which had a catechin content equivalent to 12 cups (3000 mL) of brewed tea

Sample collection and storage.

Blood samples were collected in heparinzed tubes from an inlying catheter immediately before each administration of the tea preparation and at 1, 3, 5, 7, 8 and 24 h after ingestion of the first tea preparation. After collection, blood samples were placed immediately on ice, transported to the processing room and centrifuged at 20°C for 20 min at 15000 x g. After centrifugation, plasma samples were mixed with 20 µL Vc-EDTA/mL plasma and stored at -80°C until analysis.

Urine samples were collected immediately before tea consumption (baseline) and for 24 h after the consumption of the first tea preparation. Urine samples were collected in individual containers at 0, 1, 2, 3, 4, 5, 6, 7 and 8 h (i.e., 2 h after the last dose of tea) and identified by time of collection. In addition, a 9- to 24-h urine specimen was collected and stored on ice in a single container provided to each subject. Aliquots of urine (20 mL) were mixed with 20 mg of ascorbic acid and 0.5 mg EDTA and stored at -80°C until assay.

Feces were collected in the 24-h period preceding the ingestion of tea (baseline), during the day of tea ingestion and for 72 h after tea consumption. Each fecal specimen was collected in a separate container, identified by date and time of collection and stored on dry ice in a cooler provided to each subject. Fecal dye markers were used to identify the specimen of interest. Subjects ingested one 220-mg capsule of the fecal dye marker carmine red at 1830 h the night before the study began and one 220-mg capsule of the fecal dye marker indigo carmine after the blood donation at the 8-h time point. Fecal samples were stored at -80°C until analysis.

Subject selection.

Healthy nonsmoking men and women aged 25–65 y were recruited from the Beltsville, MD area. Potential subjects completed a health questionnaire and their height and weight were measured. Fifteen of the screened applicants were selected for the study. Subjects were selected without regard to race or gender. Participants were tea drinkers, nonsmokers and in good health; they had no history of cancer, hypertension, hyperlipidemia, diabetes, gout, endocrine disorders or peripheral vascular, heart, liver or kidney disease. In addition, subjects passed a health screening and were within 85–120% of desired body mass index according to the 1983 Metropolitan Life Insurance Tables (26) . Exclusion criteria included the use of lipid-lowering drugs and thyroid medications as well as the use of nutritional supplements.

Study participants were informed of all procedures and requirements for the study. Before entering the study, subjects read and signed consent forms, which detailed the objectives of the study as well as the risks and benefits associated with participation. Subjects received monetary compensation for the time and inconvenience associated with study participation. All procedures were in strict compliance with the study protocol, which was approved by an Institutional Review Board, the Johns Hopkins University Committee on Human Research.

Analysis of plasma and urine samples.

Plasma and urine samples were analyzed by an adaptation of the method of Lee et al. (23) . Briefly, 200 µL thawed plasma or diluted urine (50 µL urine in 150 µL of 0.1 mol/L sodium phosphate buffer, pH 6.5) were mixed with 20 µL Vc-EDTA, 10 µL ß-D-glucuronidase in 0.1 mol/L sodium phosphate, pH 6.5 (500 U) and 10 µL sulfatase in 0.1 mol/L sodium acetate, pH 5.0 (40 U). This mixture was incubated at 37°C for 45 min. After incubation of plasma samples, 500 µL of methylene chloride and 300 µL of water were added to stop the enzyme reaction. All plasma samples were vortex-mixed for 4 min and 400 µL of the aqueous phase was transferred to a microcentrifuge tube. After a second extraction with 300 µL of water, the combined aqueous phase was vortex-mixed for 4 min with 1000 µL ethyl acetate and centrifuged; then 700 µL of the organic phase were transferred to a second microcentrifuge tube. After a second extraction with 700 µL of ethyl acetate, the combined extracts were mixed with 10 µL of 100 g/L ascorbic acid and dried under N₂. The dried sample was reconstituted with 200 µL of 10% (v/v) acetonitrile in 0.04 mol/L EDTA, centrifuged for 15 min at 16000 x g and 50 µL were assayed by HPLC. Urine samples were processed as described above for plasma with the omission of the methylene chloride/water extraction step. Samples were eluted at 35°C and 1 mL/min in a linear gradient from 96% Buffer A [30 mmol/L NaH₂PO₄, 2.37% (v/v) acetonitrile, 0.12% (v/v) tetrahydrofuran (THF), pH 3.35] and 4% Buffer B [30 mmol/L NaH₂PO₄, 40% (v/v) acetonitrile, 6.65% (v/v) THF, pH 3.45] to 76% Buffer A and 24% Buffer B over 24 min after which the gradient was changed linearly to 5% Buffer A and 95% Buffer B over 11 min. The gradient was maintained at 5% Buffer A and 95% Buffer B for 7 min before returning to starting conditions (96% Buffer A and 4% Buffer B). The eluent was monitored at -90, -10, 70, and 150 mV and four chromatograms were obtained simultaneously.

Analysis of fecal samples.

Each frozen fecal specimen was pulverized to produce a uniform sample, then homogenized in 5 mL of 10% (v/v) Vc-EDTA/g of sample. The homogenate was lyophilized overnight and the dry weight of feces was determined. Dry feces (1 g) were emulsified in 5 mL of 10% (v/v) Vc-EDTA in a Teflon tube; 10 mL of methylene chloride was added and the sample was vortex mixed for 4 min, then centrifuged for 15 min at 10000 x g. The aqueous (upper) fraction (1 mL) was transferred to a second Teflon tube followed by the addition of 2 mL of ethyl acetate. Subsequently, the sample was vortex-mixed for 4 min and centrifuged at 10000 x g for 15 min after which 1.5 mL of the ethyl acetate fraction was transferred to a microcentrifuge tube. This fraction was mixed with 10 µL of 100 g/L ascorbic acid and evaporated to dryness under nitrogen. Before analysis by HPLC, the dried extract was reconstituted in 750 µL of 10% (v/v) acetonitrile in 0.04 mol/L EDTA. The reconstituted samples were assayed by HPLC using the gradient elution conditions described above for plasma and urine. All samples were analyzed in duplicate unless otherwise indicated.

Assay standardization.

The catechins in the plasma, urine and fecal specimens were quantified using external standardization. Standard curves were prepared by spiking plasma, urine and feces specimens with EGC, EC, EGCG and ECG. Standard curves for plasma and urine were prepared by spiking zero-time plasma and urine specimens with known concentrations of EGC, EC, EGCG and ECG (0–13059, 0–13780, 0–8726 and 0–9042 nmol/L, respectively, for plasma and 0–52236, 0–55120, 0–34904 and 0–36168 nmol/L, respectively, for urine). To prepare the feces standard curve, EGC, EC, EGCG and ECG (0–3265, 0–3445, 0–2181 and 0–2269 nmol/L, respectively) were added to a baseline feces sample and extracted according to the procedure described above.

Plasma standard curves displayed linearity from 0 to 13059, 0 to 13780, 0 to 8726 and 0 to 9042 nmol/L for EGC, EC, EGCG and ECG, respectively, with r = 0.994. 0.990, 0.978, and 0.980 for EGC, EC, EGCG and ECG, respectively. Urine standard curves were linear from 0 to 52236, 0 to 55120, 0 to 34904 and 0 to 36168 nmol/L for EGC, EC, EGCG and ECG, respectively, with r = 0.999, 0.998, 0.995, and 0.993 for EGC, EC, EGCG and ECG, respectively. Feces standard curves displayed linearity from 0 to 3265, 0 to 3445, 0 to 2181 and 0 to 2269 nmol/L for EGC, EC, EGCG and ECG, respectively, with r = 0.9835, 0.9734., 0.9943 and 0.9949 for EGC, EC, EGCG and ECG, respectively. The recovery for the sample extraction for plasma was 94, 94, 93 and 95% for EGC, EC, EGCG and ECG, respectively. For urine, the recovery was 99, 97, 98 and 98% for EGC, EC, EGCG and ECG, respectively and for feces was 101, 97, 82 and 83% for EGC, EC, EGCG and ECG, respectively. The detection limits, defined as three times the standard deviation of the blank, were 3.3, 3.4, 2.2 and 6.8 nmol/L for EGC, EC, EGCG and ECG, respectively.

Calculations.

The compound concentrations (nmol/L) were used to determine whether a significant increase in plasma EGC, EC, EGCG and ECG occurred during tea consumption. To estimate the bioavailability of the catechins, the milligrams of EGC, EC, EGCG, and ECG present in peak plasma samples were calculated, normalized for blood volume (69 mL/kg body for men and 65 mL/kg body for women), and the percentage of ingested catechins found in the plasma was calculated (27) . The level (nmol/L) of EGC, EC, EGCG, and ECG in urine was corrected for the total volume of urine and the total excretion (nmol/h) of EGC, EC, EGCG and ECG by each subject over the 24-h period was calculated. The levels (nmol/L) of EGC, EC, EGCG and ECG in feces were corrected for sample dilution and grams of fecal sample (wet weight). Samples demarcated by fecal dye markers and baseline samples were used to calculate the amount (µmol) of EGC, EC, EGCG and ECG excreted in the feces after ingestion of four doses of tea. The mean mass of EGC, EC, EGCG and ECG was computed for plasma, urine and feces. Finally, the percentage of ingested EGC, EC, EGCG and ECG found in the plasma, urine and feces was determined.

Balance calculations.

The total mass of EGC, EC, EGCG and ECG excreted in the urine was calculated by totaling the mass excreted for each time point (0, 1, 2, 3, 4, 5, 6, 7, 8 and 9–24 h). The net mass of EGC, EC, EGCG and ECG excreted in the feces was calculated by subtracting the baseline sample from the sum of the samples containing dye marker. It was sometimes difficult to detect the dye marker, and one subject did not appear to excrete either of the dye markers during the 72-h period of sample collection. Two subjects had no baseline sample. These subjects were excluded from the balance calculations. Apparent catechin retention (ACR) was calculated according to the following formula:

Statistical analysis.

All statistical analyses were conducted using Microsoft EXCEL. Regression analysis was used to evaluate the linearity of the HPLC catechin analyses. Student’s t test ( = 0.05) was used to determine whether a significant difference could be demonstrated between men and women in concentrations of EGC, EC, EGCG and ECG in plasma, urine and feces. Because no differences by gender were found, data for men and women were pooled. The paired t test ( = 0.05) was used to assess whether there was a significant difference in the plasma concentrations of EGC, EC, EGCG and ECG at peak relative to the baseline. In addition, the paired t test ( = 0.05) was used to determine whether the peak urinary excretion of EGC, EC, EGCG, and ECG was significantly greater than baseline excretion. Finally, the paired t test ( = 0.05) was used to evaluate the difference in the amount of EGC, EC, EGCG and ECG in baseline fecal samples relative to those demarcated by fecal dye marker.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS Study design RESULTS DISCUSSION REFERENCES

 
Study population.

Eight women and 7 men aged 25 to 56 y (mean = 37 y) completed the study. Medications continued during the study period were PremPro (1 subject), orthotrycyden (1 subject), Claritin (1 subject) and Accutane (1 subject). All other medications were discontinued at the beginning of the study. No relationship between intake of any of these medications and catechin levels in plasma, urine or feces was noted.

Catechins in plasma and urine samples.

Plasma levels of EGC, EC and EGCG increased in response to each successive dose of tea, reaching a plateau at between 5 and 8 h with peak levels of 145, 174 and 20.1 nmol/L, respectively, which returned to baseline levels by 24 h. Plasma ECG levels increased in a linear fashion over the 24-h period, peaking at 50.6 nmol/L (Fig. 1 ). Peak plasma levels of EGC, EC and EGCG were significantly different from baseline values (Table 1 ). Urinary excretion of EGC and EC paralleled the rise in plasma levels after the initial dose of tea. Urinary excretion of EGC, EC and ECG peaked at 5 h at 836, 743 and 41.1 nmol/h, respectively, and urinary excretion of EGCG peaked at 1 h at 47.8 nmol/h (Table 2 ). Figure 2 depicts the cumulative excretion of EGC, EC, EGCG and ECG in the urine during the 24-h period of urine collection. Peak urinary excretions of EGC and EC were significantly different from baseline excretions (Table 2) .

View larger version (18K): [in this window] [in a new window]   Figure 1. Plasma concentrations of (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) in men and women consuming a black tea preparation at 0, 2, 4 and 6 h. Each data point represents the mean ± SEM, n = 15. Means with a letter are significantly different from baseline (P < 0.05) for EGC (a), EC (b) and EGCG (c), respectively.

View larger version (31K): [in this window] [in a new window]   Figure 2. Cumulative urinary excretion of (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) over 24 h in men and women (n = 15) consuming a black tea preparation at 0, 2, 4 and 6 h.

Fecal catechin excretion varied widely from subject to subject. Excluding subjects with no baseline sample, the range of values was from 0 to 15.0, 0 to 11.7, 0 to 3.71 and 0 to 0.520 µmol for EGC, EC, EGCG and ECG, respectively, after correcting for baseline excretion. The amount of catechins in fecal samples demarcated by dye markers was significantly different from the amount in baseline fecal samples for EGC, EC, EGCG and ECG (Table 3 ).

The total mass (mg) of catechins in the body plasma pool at the peak was used to calculate the percentage of ingested catechins. The peak levels of EGC, EC, EGCG and ECG in the body plasma pool were 0.224, 0.244, 0.00440 and 0.118 mg, which represented 0.36, 0.36, 0.030 and 0.095% of the ingested dose, respectively. In addition, the percentage of total catechins in plasma was estimated from the urinary excretion. The average mass (mg) of EGC, EC, EGCG and ECG excreted in the urine was 2.27, 1.76, 0.219, and 0.151 mg, respectively. This represents 3.7, 2.6, 0.14 and 0.12% of the ingested dose, for EGC, EC, EGCG and ECG, respectively. The average mass of EGC, EC, EGCG and ECG excreted in the demarcated feces samples was 0.687, 0.609, 0.294 and 0.0780 mg, which represented 1.1, 0.9, 0.2 and 0.06% of the ingested dose for each, respectively. Using peak plasma levels in milligrams as an estimate of plasma catechins after tea ingestion over 6 h, 0.16% of the total catechins consumed (400 mg) were accounted for in plasma; 1.1 and 0.42% were accounted for in the urine and feces, respectively. The ACR (± SD) for total catechins was 399 ± 1.21 mg, suggesting that 99.9% of the ingested dose of catechins was absorbed. However, only 0.16% of the ingested catechins was detected in the plasma, suggesting very low levels of absorption. These two contradictory findings may indicate that catechins are degraded in the colon or that they are absorbed, but rapidly metabolized or quickly sequestered in the tissues. This calculation of apparent catechin retention did not take into account possible metabolism and tissue sequestration of the catechins. Further studies are required to resolve this issue.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS Study design RESULTS DISCUSSION REFERENCES

 
This study demonstrates a significant increase in levels of EGC, EC and EGCG in plasma, EGC and EC in urine and EGC, EC, EGCG and ECG in fecal samples after ingestion of black tea. In contrast to studies of humans drinking green tea (23 24 25) , the average peak concentration of EGCG in plasma for this study was much lower than the average peak plasma concentration of EGC and EC, even though the concentration of EGCG in the tea was higher than EGC and EC. The same was true for ECG. This suggests differences in the patterns of absorption, metabolism and excretion of gallated forms of catechins vs. nongallated forms. Consistent with this hypothesis, an investigation of catechin absorption, distribution and elimination kinetics in rats established that only 0.1% of ingested EGCG is bioavailable compared with 13.7 and 31.2% for EGC and EC, respectively (28) .

Gallated forms may also be converted to nongallated forms or to other phenolic and valerolactone metabolites both before and after absorption. Okushio et al. (29 ,30) found that catechins are absorbed from the gastrointestinal tract of rats into the portal blood. In addition, they confirmed that tannase converts EGCG and ECG to gallic acid and the respective nongallated forms, EGC and EC. Nemoto et al. (31) isolated tannase-producing organisms from the oral cavity of humans, and Yang et al. (32) demonstrated that EGCG was converted to EGC in the oral cavity. They attributed this to a catechin esterase activity in the saliva and speculated that the activity was from an enzyme of human origin; however, they were unable to confirm their hypothesis. A number of different catechin metabolites have been isolated from plasma, urine and feces. Das (33) confirmed the presence of phenolic acid and valerolactone metabolites in the urine of rats, guinea pigs, rabbits, monkeys and humans after administration of (+)-catechin. Suganuma et al. (34) studied the disposition of [³H] EGCG in rats after administration directly into the stomach and found 6.4% of the radioactivity in the urine, including [³H] EGCG and at least five metabolites. These studies support the finding of disproportionately lower concentrations of EGCG and ECG in the plasma, urine and feces vs. those in the black tea preparation.

The nature of the tea preparation and the way in which it is administered may also influence the bioavailability of the gallated catechins. The tea preparation used for this study contained no additives (i.e., sugar, whitener or vanilla flavoring) but was administered with cookies. Food can influence absorption of many nutrients; thus, it is possible that the ingestion of cookies along with the tea preparation inhibited the absorption of the gallated catechins.

Plasma ECG levels increased in a linear fashion over the 24-h period, suggesting that the plasma response of ECG may not be due solely to absorption processes. In addition, an as yet undefined metabolic pathway for ECG in the body after absorption or in the gastrointestinal tract may produce this effect.

As expected, the peak concentrations of EGC, EC and EGCG found in the plasma and urine were lower than those previously reported for studies of green tea (23 24 25) . This result is consistent with the lower concentrations of catechins in black tea.

In agreement with similar studies of [³H] EGCG in mice (34) , EGC, EC, and EGCG continued to rise in plasma with the administration of each successive portion of tea, reaching a steady state at between 5 and 8 h after the first dose. This produced a dose-response curve similar to that seen for therapeutic drugs. This information will be useful for designing future investigations aimed at correlating specific plasma levels of EGC, EC, EGCG and ECG with the total antioxidant capacity of plasma as well as with biomarkers of oxidative damage such as lipid peroxides, protein carbonyls, DNA adducts, DNA strand breaks and F₂-isoprostanes.

Although micromole amounts of EGC, EC, EGCG and ECG were excreted in the feces, the quantity represented a very small percentage of the ingested dose. These results are consistent with data presented previously by He and Kies (21) for total polyphenolics in feces. There could be a number of reasons for the low recovery of catechins in the feces in this study.

The gut bacteria may metabolize a large percentage of the ingested EGC, EC, EGCG and ECG before fecal excretion. The wide variability in fecal catechin concentrations from subject to subject may be due to differences in number and species of those normal gut flora. Li et al. (35) found (-)-5-(3', 4', 5'-trihydroxyphenyl)--valerolactone and (-)-5-(3', 4'-dihydroxyphenyl)--valerolactone in the plasma, urine and feces of healthy humans consuming green tea. Because these two valerolactones were not formed when incubated with human liver microsomes, they hypothesized that anaerobic bacteria in the intestines produced them. Along with a lag time between tea consumption and renal excretion for these metabolites compared with EGC, this suggests that these metabolites are formed in the colon, absorbed and then excreted. Clifford et al. (36) administered a strong black tea preparation to habitual tea-drinkers for 2 d. ¹H NMR (proton nuclear magnetic resonance) and HPLC profiles of 24-h urine specimens collected during the consumption of black tea demonstrated a significant increase in the presence of hippuric acid. The authors proposed that the hippuric acid was produced by the metabolism of black tea polyphenols by the gut microflora to 3-phenylproprionic acid followed by endogenous ß-oxidation and conjugation to glycine. In vitro fermentation studies of human fecal microbiota similar to those conducted by Thompson et al. (37) for lignans would help to clarify these findings by assessing the degree of stability of the catechins in the presence of the normal gut flora.

Breakdown of the catechins in the gastrointestinal tract as a result of normal digestive processes could also explain the low percentages of EGC, EC, EGCG and ECG found in the plasma and excreted in the urine and feces. These results are supported by the study of Das and Griffiths of (+)-catechin metabolism in rats (38) , which detected only a very small percentage of ingested (+)-catechin (1.3–1.5% for [U-¹⁴C] catechin and 0.56–0.61% for [A-¹⁴C] catechins) in the rat feces.

Alternatively, the catechins may be quickly absorbed by the digestive tract and either rapidly metabolized or distributed to the tissues. This would explain the low overall recovery of catechins from the plasma, urine and feces observed in this study. In fact, metabolism of the catechins may begin in the enterocytes during absorption. Zhu et al. (39) reported low systemic availability of EC, ECG and EGCG in rats after oral administration of a decaffeinated catechin fraction extracted from tea leaves as opposed to the intravenous (i.v.) administration of the same extract. An oral dose 100 times the i.v. dose was required to produce the same systemic levels of catechins. The authors concluded that a high first-pass effect, wide tissue distribution and incomplete absorption were responsible for the low oral bioavailability of catechins. Similarly, Terao (40) reported that metabolic conversion of (-)-epicatechin by 5'-UDP-glucuronyl transferase begins in the intestinal mucosa of rats and that (-)-epicatechin conjugates contribute to the antioxidant activity of rat plasma. Kuhnle et al. (41) studied the absorption and metabolism of catechin and epicatechin in the small intestines. During perfusion of isolated jejunum with flavanols, they observed 3-O- and 4-O-methylation and O-methyl glucuronidation of the catechins in the course of transfer across the enterocytes to the serosal side. This was not true for the ileum in which largely unmetabolized flavanols appeared on the serosal side. Harada et al. (42) studied the catechin conjugates produced by rats after oral administration of wine or tea. (+)-Catechin-5-O-ß-glucuronide and (-)-epicatechin 5-O-ß-glucuronide were the major metabolites recovered in the plasma and excreted in the urine and bile. Like the parent compounds, these metabolites exhibited strong radical-scavenging properties. Pietta et al. (43) studied the formation of catechin metabolites after green tea consumption by healthy humans. They found detectable amounts of 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3-methoxy-4-hydroxybenzoic acid in plasma and urine. They determined that free plasma catechins were responsible for only 20% of the increase in total radical-trapping antioxidant capacity of the plasma, providing support for the hypothesis that catechin metabolites and conjugates explain the remaining 80% increase.

Therefore, our finding of very low concentrations of unmetabolized catechins in the plasma, urine and feces supports previous investigations. These results suggest that there are many possible pathways for the metabolism and degradation of catechins both before and after absorption as has been previously surmised (44) .

For this study, the ACR is very likely overstated because the formula made no correction for metabolic and degradation by-products. Although similar formulas have been useful for calculating the retention of other nutrients, the undefined complexities of catechin metabolism greatly limit its usefulness in this case. Future studies to define catechin metabolic and degradation pathways in humans are necessary before formulas can be developed to better describe the true bioavailability of these compounds in humans.

Nevertheless, we have established that the HPLC-coularray method can be applied to the analysis of EGC, EC, EGCG and ECG in plasma, urine and feces after the administration of a black tea preparation. In addition, we have demonstrated a significant increase in plasma, urine and fecal concentrations over baseline levels after multiple doses of black tea. Moreover, we found only 1.68% of the total catechins consumed (400 mg) in the plasma urine, and feces (Table 4 ), providing further evidence to support the hypothesis that catechins undergo considerable metabolism and/or degradation either in the gastrointentinal tract or in the body after absorption. Future studies should focus on identifying, characterizing and defining catechin metabolic pathways, their end products and the antioxidant properties of those end products.

	FOOTNOTES

 
¹ Presented at the minisymposium, Bioavailability of Nutrients, at Experimental Biology 2000, April 15–19, San Diego, CA [Warden, B., Smith, L., Beecher, G., Clevidence, B. & Balentine, D. 2000 A balance study of the prominent catechins in black tea. FASEB J. 14: A298 (abs.)].

² Supported in part by USDA/ARS, Beltsville Human Nutrition Research Center and Lipton.

⁴ Abbreviations used: ACR, apparent catechin retention; CHD, coronary heart disease; EC, (-)-epicatechin; ECG, (-)-epicatechin gallate; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin gallate; i.v., intravenous; THF, tetrahydrofuran; Vc-EDTA, vitamin C-EDTA.

Manuscript received January 22, 2001. Initial review completed February 8, 2001. Revision accepted March 30, 2001.

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