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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSStudy designRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • bioavailability • catechins • human study • HPLC-coularray • black tea
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSStudy designRESULTSDISCUSSIONREFERENCES |
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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.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSStudy designRESULTSDISCUSSIONREFERENCES |
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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)
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Study design |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSStudy designRESULTSDISCUSSIONREFERENCES |
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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:
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSStudy designRESULTSDISCUSSIONREFERENCES |
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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)
.
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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
).
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSStudy designRESULTSDISCUSSIONREFERENCES |
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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.
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FOOTNOTES |
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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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