Rapid conversion of tea catechins to monomethylated products by rat liver cytosolic catechol-O-methyltransferase

Article abstract of DOI:10.1080/00498250110079798

1. The metabolic O-methylation of several catechol-containing tea polyphenols by rat liver cytosolic catechol-O-methyltransferase (COMT) has been studied. 2. When (-)-epicatechin was used as substrate, its O-methylation showed dependence on incubation time, cytosolic protein concentration, incubation pH and concentration of S-adenosyl-L-methionine. The O-methylation of increasing concentrations of (-)-epicatechin followed typical Michaelis-Menten kinetics, and the apparent K_m and V_max were 51 μM and 2882 pmol mg protein^-1 min^-1, respectively, at pH 7.4, and were 17 μM and 2093 pmol mg protein^-1 min^-1, respectively, at pH 10.0. 3. Under optimized conditions for in vitro O-methylation, (-)-epicatechin, (+)-epicatechin and (-)-epigallocatechin were rapidly O-methylated by rat liver cytosol. In comparison, (-)-epicatechin gallate and (-)-epigallocatechin gallate were O-methylated at significantly lower rates under the same reaction conditions. 4. COMT-catalysed O-methylation of (-)-epicatechin and (-)-epigallocatechin was inhibited in a concentration-dependent manner by S-adenosyl-L-homocysteine, a demethylated product of S-adenosyl-L-methionine. The IC50 was ca. 10 μM. 5. In summary, the results showed that several catechol-containing tea polyphenols were rapidly O-methylated by rat liver cytosolic COMT. These observations raise the possibility that some of the biological effects of tea polyphenols may be exerted by their O-methylated products or may result from their potential inhibition of the COMT-catalysed O-methylation of endogenous catecholamines and catechol oestrogens.

Full text of DOI:10.1080/00498250110079798

xenobiotica, 2001, vol. 31, no. 12, 879±890
Rapid conversion of tea catechins to
monomethylated products by rat liver cytosolic
catechol-O-methyltransferase
BAO TING ZHU *, USHMA K. PATEL , MAY XIAOXIN CAI ,
{
{
ANTHONY J. LEE and ALLEN H. CONNEY
{
{
{
{ Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of
South Carolina, Columbia, SC 29208, USA
{ Laboratory for Cancer Research, Department of Chemical Biology, College of
Pharmacy, Rutgers University, Piscataway, NJ 08854, USA
Received 28 February 2001
1. The metabolic O-methylation of several catechol-containing tea polyphenols by rat
liver cytosolic catechol-O-methyltransferase (COMT) has been studied.
2. When (¡)-epicatechin was used as substrate, its O-methylation showed dependence
on incubation time, cytosolic protein concentration, incubation pH and concentration of
S-adenosyl-l-methionine. The O-methylation of increasing concentrations of (¡)-
epicatechin followed typical Michaelis±Menten kinetics, and the apparent Km and Vmax
were 51mm and 2882 pmol mg protein¡1 min¡1, respectively, at pH 7.4, and were 17 mm
and 2093 pmol mg protein¡1 min¡1, respectively, at pH 10.0.
3. Under optimized conditions for in vitro O-methylation, (¡)-epicatechin, (‡)-
epicatechin and (¡)-epigallocatechin were rapidly O-methylated by rat liver cytosol. In
comparison, (¡)-epicatechin gallate and (¡)-epigallocatechin gallate were O-methylated
at signi®cantly lower rates under the same reaction conditions.
4. COMT-catalysed O-methylation of (¡)-epicatechin and (¡)-epigallocatechin was
inhibited in a concentration-dependent manner by S-adenosyl-l-homocysteine, a
demethylated product of S-adenosyl-l-methionine. The IC50 was 10mm.
5. In summary, the results showed that several catechol-containing tea polyphenols
were rapidly O-methylated by rat liver cytosolic COMT. These observations raise the
possibility that some of the biological e€ects of tea polyphenols may be exerted by their O-
methylated products or may result from their potential inhibition of the COMT-catalysed
O-methylation of endogenous catecholamines and catechol oestrogens.
Introduction
Catechol-O-methyltransferase (COMT), an enzyme ubiquitously present in
high activity in rodents and humans, catalyses the metabolic O-methylation of
endogenous catecholamines and catechol oestrogens (Axelrod and Tomchick 1958,
Axelrod 1966, Guldberg and Marsden 1975, Thakker and Creveling 1990, Zhu
and Conney 1998). In addition to the endogenous catechol substrates, COMT
also catalyses metabolic O-methylation of many catechol-containing xenobiotics
(Axelrod and Tomchick 1958, Axelrod 1966, Guldberg and Marsden 1975,
Thakker and Creveling 1990, Zhu et al. 1994). Zhu et al. (1994) reported that
quercetin and ®setin, two catechol-containing dietary ¯avonoids, are rapidly O-
methylated by cytosolic COMT, with rates two to three orders of magnitude
higher than for endogenous catecholamines.
* Author for correspondence; e-mail: BTZhu@cop.sc.edu
#
Xenobiotica ISSN 0049±8254 print/ISSN 1366±5928 online
2001 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/00498250110079798

880
Bao Ting Zhu et al.
Tea polyphenols
Stereochemistry
R1
R2
(¡)-Epicatechin
(‡)-Epicatechin
(¡)-Epigallocatechin
2R, 3R
2S, 3S
2R, 3R
ÐH
ÐH
ÐOH
ÐH
ÐH
ÐH
(¡)-Epicatechin gallate
2R, 3R
2R, 3R
ÐH
(¡)-Epigallocatechin gallate
ÐOH
Figure 1. Chemical structures of several catechol-containing dietary tea polyphenols used.
Like dietary quercetin and ®setin, several of the common tea polyphenols, such
as (¡)-epicatechin, (‡)-epicatechin, (¡)-epigallocatechin, (¡)-epicatechin gallate
and (¡)-epigallocatechin gallate (®gure 1), also contain a catechol structure and
share certain degrees of structural similarity to them. These catechol-containing
tea components constitute a major fraction of the water-extractable polyphenols
contained in the tea leaves (Balentine 1992). Recent studies showed that several of
these tea polyphenols (e.g. (¡)-epigallocatechin and epicatechins) can be readily
absorbed in human subjects after drinking tea, and some of them can reach
submicromolar peak concentrations (ranging from 0.1 to 0.4 mm) in the plasma
(Lee et al. 1995, Unno et al. 1996, Yang et al. 1998, van het Hof et al. 1998, Li et al.
2000). Another earlier study using 3H-labelled (¡)-epigallocatechin gallate showed
that this tea catechin is widely distributed in mouse tissues (Suganuma et al. 1998).
We recently reported that the catechol-containing tea polyphenols are rapidly
metabolized to monomethyl ethers by the cytosolic COMT present in human term
placenta (Zhu et al. 2000). We describe here our additional results on the rapid O-
methylation of tea catechins by rat liver cytosol. These observations raise the
possibility that some of the biological e€ects of tea polyphenols may be caused by
their methylated products or may result from their potential inhibition of COMT-
catalysed O-methylation of endogenous catecholamines and catechol oestrogens.
Materials and methods
Chemicals
(¡)-Epicatechin, (‡)-epicatechin, (¡)-epicatechin gallate, (¡)-epigallocatechin, (¡)-epigallocatechin
gallate, S-adenosyl-l-methionine, dithiothreitol and Tris-HCl were purchased from the Sigma Chemi-
cal Co. (St Louis, MO, USA). The purities for the tea polyphenols used were 95±98% as determined by

COMT-catalysed O-methylation of tea polyphenols
881
HPLC analysis. [Methyl-3H]S-adenosyl-l-methionine (speci®c activity 11.2±13.5Ci mmol¡1) was
obtained from Perkin Elmer Life Science Products (Boston, MA, USA). All organic solvents used in
this study were of HPLC grade and obtained from the Fisher Scienti®c Co. (Spring®eld, NJ, USA).
Preparation of rat liver cytosol
Six-week-old female Sprague±Dawley rats (body weight 125±150g) were purchased from Harlan
Sprague±Dawley Laboratory (Houston, TX, USA). After arrival, they were allowed to acclimatize for 1
week before use. The animals were euthanized under CO2 inhalation, and the livers from ®ve female
rats were collected and pooled. Rat livers were immediately minced in 3 vols ice-cold 0.1m Tris-HCl/
KCl solution (pH 7.4) and homogenized with a Polytron homogenizer for 2±3 min. Tissue homogenates
were centrifuged at 9000g for 15 min, and the supernatants were pooled and ®ltered through two layers
of cheesecloth to remove lipid clots. The ®ltrates were recentrifuged at 105 000g (48C) for 90 min. The
supernatants (cytosolic preparations) were stored in 1-ml aliquots at ¡808C. The protein concentration
was determined by using the BioRad protein assay kit with bovine serum albumin (BSA) as a standard.
Rat liver COMT-catalysed O-methylation of catechol-containing tea polyphenols
The reaction mixture was prepared at 4 8C and consisted of the desired concentration of a tea
polyphenol substrate, 0.5mg rat liver cytosolic protein ml¡1, 50 mm S-adenosyl-l-methionine (contain-
ing 0.25 mCi [methyl-3H]S-adenosyl-l-methionine), 1.2 mm magnesium chloride, and 1.0 mm dithio-
threitol in a ®nal volume of 1 ml Tris-HCl bu€er (10 mm, pH 7.4). The reaction was initiated by
addition of rat liver cytosolic protein and was carried out at 37 8C for 20min under constant mild
shaking. The reaction was arrested by placing the tubes on ice followed immediately by addition of
0.5 ml ice-cold water and extraction of the 3H-containing methylated catechin metabolites with 5 ml
ethyl acetate. After centrifugation for 20 min at 3000g, portions of the upper ethyl acetate phase (2 ml)
were removed for measurement of radioactivity in a scintillation counter (Beckman Instruments, model
LS 5000TD). Blank values obtained from incubations without rat liver cytosolic protein were
determined in each individual assay and subtracted. The blank radioactivity counts were usually
<20% of the values obtained from incubations in the presence of rat liver cytosolic protein. By using the
3H-labelled monomethylated products of (¡)-epicatechin and (¡)-epigallocatechin (isolated by HPLC),
the extraction eciency for the O-methylated (¡)-epicatechin and (¡)-epigallocatechin was >94%. The
rate (velocity) for the O-methylation of various tea polyphenol substrates by rat liver cytosolic COMT
was expressed as pmol methylated tea polyphenol formed per mg protein per¡1 min¡1
.
Liquid chromatography-mass spectrometry analysis of methylated tea polyphenols
To determine the structures of the methylated tea polyphenols, 50 mm (¡)-epicatechin or (¡)-
epigallocatechin was ®rst incubated in vitro for 30 min with 1.0 mg rat liver cytosolic protein ml¡1
,
100 mm S-adenosyl-l-methionine, 1.2mm magnesium chloride, and 1.0 mm dithiothreitol in a ®nal
volume of 1ml Tris-HCl bu€er (10 mm, pH 7.4). The reaction mixture was extracted twice with
10 ml ethyl acetate. The organic solvent extracts were combined and dried under a stream of nitrogen,
and the residues were redissolved in 100ml 20% acetonitrile in water by vortex mixing. The mixture
was then centrifuged at 15000g for 10 min, and a 50-ml aliquot of the supernatant was injected into
the HPLC for analysis of methylated tea polyphenol metabolites according to the method for tea
catechins described by Lee et al. (1995). The incubations described above were ®rst done with tea
catechins and [methyl-3H]-S-adenosyl-l-methionine to determine the retention times of the radioactive
metabolites. Parallel incubations of the catechins with only non-radioactive S-adenosyl-l-methionine
were performed for collection of metabolites for mass spectrometric analysis.
The peaks corresponding to methylated metabolites of (¡)-epicatechin or (¡)-epigallocatechin were
collected from the HPLC column and were dried under a stream of nitrogen. They were then analysed
by liquid chromatography-mass spectrometry (Finnegan LCQ; Thermoquest Corp., San Jose, CA,
USA) using electrospray-mass spectrometry with an ion trap operated in the negative-ion mode.
Results
Con®rmation by mass spectrometry of metabolic formation of monomethylated tea
catechins
Incubation of (¡)-epigallocatechin with rat liver cytosol in the presence of
[methyl-3H]S-adenosyl-l-methionine (0.25m Ci) resulted in formation of a less
polar radioactive metabolite peak with a retention time of 22.0 min (®gure 2A, B).
Similarly, incubation of (¡)-epicatechin with rat liver cytosol in the presence of

882
Bao Ting Zhu et al.
Figure 2. HPLC separation of the methylated metabolites of (¡)-epicatechin and (¡)-epigallocatechin
formed by rat liver cytosol. (A) Representative UV trace for authentic S-adenosyl-l-methionine
(SAM), (¡)-epigallocatechin (EGC) and (¡)-epicatechin (EC). (B, C) Representative radioactive
traces for the methylated metabolites of (¡)-epigallocatechin and (¡)-epicatechin, respectively.
The enzymatic formation and analysis of methylated tea catechins were as described in the
Materials and methods.
[methyl-3H]S-adenosyl-l-methionine (0.25 mCi) resulted in formation of two less
polar radioactive metabolite peaks, with retention times of 27.2 and 30.0 min,
respectively (®gure 2A, C). The ratio of the two radioactive peaks was 5:1.
For mass spectrometric analysis of the methylated metabolites of tea catechins,
we ®rst used the HPLC to isolate sucient amount of the 22.0-min metabolite
peak formed from (¡)-epigallocatechin and the 27.2-min metabolite peak formed
from (¡)-epicatechin. Analysis by liquid chromatography-mass spectrometry (LC/
MS) of the metabolite derived from (¡)-epigallocatechin (MW ˆ 306) revealed a

COMT-catalysed O-methylation of tea polyphenols
883
base peak (deprotonated ion) with a mass of 319, consistent with the identity of this
metabolite as a monomethylated metabolite of (¡)-epigallocatechin. Similarly,
analysis of the major metabolite derived from (¡)-epicatechin (MW ˆ 290) re-
vealed a base peak (deprotonated ion) with a mass of 303, consistent with the
identity of this major metabolite as a monomethylated metabolite of (¡)-
epicatechin.
Kinetic studies on the O-methylation of catechol-containing tea polyphenols by
rat liver cytosol
When (¡)-epicatechin (30 mm) was used as substrate for metabolic O-methyla-
tion by rat liver cytosol, formation of methylated products was dependent on
incubation time (®gure 3, upper) and cytosolic protein concentration (®gure 3,
lower). The dependence of O-methylation of a tea catechin on the concentrations
Figure 3. Dependence of hepatic O-methylation of (¡)-epicatechin on incubation time (upper) and
cytosolic protein concentration (lower). The incubation conditions and HPLC analyses were as
described in the Materials and methods. Data are the mean of triplicate determinations.

884
Bao Ting Zhu et al.
Figure 4. Double-reciprocal plot of increasing concentrations of S-adenosyl-l-methionine (SAM)
versus the rate of O-methylation of (¡)-epicatechin. The incubation conditions and HPLC
analyses were as described in the Materials and methods. Each point is the mean of triplicate
determinations.
of S-adenosyl-l-methionine follows typical Michaelis±Menten kinetics, with a
Km
ˆ
13 mm as calculated from a double-reciprocal plot (®gure 4).
We also determined the pH dependence (from 4.5 to 13) for metabolic O-
methylation of (¡)-epicatechin by rat liver cytosol (®gure 5). Rates of (¡)-
epicatechin O-methylation by rat liver cytosol were highest at pH 8.0. This
>
pH dependence pattern is very di€erent from that when human placental cytosol
was used as the enzyme source, which showed a characteristic narrow pH optimum
at 7.4 (data not shown).
Under optimized conditions for in vitro O-methylation, (¡)-epicatechin, (‡)-
epicatechin and (¡)-epigallocatechin at a 10 or 100 mm concentration were all
rapidly O-methylated by rat liver cytosol (table 1). Note that similar rates observed
for the enzymatic O-methylation of (¡)-epicatechin and (‡)-epicatechin suggest
that di€erent stereochemistry (2R,3R versus 2S,3S; ®gure 1) between the two
isomers does not signi®cantly a€ect the rate of their enzymatic O-methylation in
vitro. In comparison, (¡)-epicatechin gallate and (¡)-epigallocatechin gallate were
O-methylated at markedly lower rates under the same reaction conditions (table 1).
We also determined the kinetic parameters (apparent Km and Vmax) for the
O-methylation of (¡)-epicatechin by rat liver cytosolic COMT. The rate of meta-
bolic O-methylation of increasing concentrations of (¡)-epicatechin at pH 7.4
or 10.0 showed typical Michaelis±Menten curve patterns (®gure 6). Double-
reciprocal plotting of this data showed the apparent Km and Vmax were 51 mm

COMT-catalysed O-methylation of tea polyphenols
885
Figure 5. pH dependence of the O-methylation of (¡)-epicatechin by rat liver cytosol. The indicated
pH was the original pH of the 20 mm Tris-HCl stock bu€er solution. The incubation conditions
and HPLC analyses were as described in the Materials and methods. Each point is the mean of
duplicate determinations.
Table 1. Rates of O-methylation of several catechol-containing tea polyphenols by rat liver cytosolic
COMT at pH 7.4 and 10.0.
Rate of O-methylation
(pmol mg protein¡1 min¡1
)
Concentration
(mm)
Tea polyphenol substrate
pH 7.4
pH 10.0
870 14
(¡)-Epicatechin
10
100
713 19
1530 29
1230
6
(‡)-Epicatechin
10
100
401 13
1261
777
1226 10
8
3
(¡)-Epigallocatechin
(¡)-Epicatechin gallate
(¡)-Epigallocatechin gallate
10
100
793 18
580
927
9
7
1296
246
5
9
10
100
145
170
1
6
243 11
10
100
262
285
3
8
160
160
2
3
Incubation mixture consisted of 10 or 100 mm of a tea polyphenol substrate, 50 mm [3H-methyl]S-
adenosyl-l-methionine (containing 0.2 mCi), 0.5 mg ml¡1 rat liver cytosolic protein, 1 mm dithiothreitol
and 1.2 mm MgCl2 in a ®nal volume of 1.0 ml Tris-HCl bu€er (10 mm, pH 7.4 or 10.0). Incubations
were carried out at 378C for 20 min. Each point is the mean SD of triplicate determinations.
Measurements for most of the tea polyphenols substrates were repeated three times and similar results
were observed.
and 2882 pmol mg protein¡1 min¡1, respectively, at pH 7.4, and 17 mm and
2093 pmol mg protein¡1 min¡1, respectively, at pH 10.0. Similarly, the rate of
metabolic O-methylation of (¡)-epigallocatechin by rat liver cytosol also followed
typical Michaelis±Menten kinetics (data not shown). The apparent Km for the

886
Bao Ting Zhu et al.
Figure 6. Double-reciprocal plots of the O-methylation of (¡)-epicatechin by rat liver cytosol.
The left inset depicts the relationship between the increasing concentrations of (¡)-
epicatechin and the rate of its enzymatic O-methylation in vitro. Open circles represent the
data obtained at pH 7.4; solid circles at pH 10.0. The incubation conditions and HPLC
analyses were as described in the Materials and methods. Each point is the mean of duplicate
determinations.
O-methylation of (¡)-epigallocatechin at pH 7.4 was 64 mm, and the Vmax was
1832 pmol mg protein¡1 min¡1
.
Inhibition of rat liver O-methylation of tea polyphenols by S-adenosyl-l-
homocysteine
When 5 mm (¡)-epicatechin or (¡)-epigallocatechin was used as substrate, their
O-methylation by rat liver cytosol was inhibited by S-adenosyl-l-homocysteine
(the demethylated product of S-adenosyl-l-methionine) in a concentration-
dependent manner, with an IC50
10 mm. Kinetic analysis showed that S-
adenosyl-l-homocysteine inhibited the COMT-catalysed O-methylation of (¡)-
epicatechin with a mixed (competitive plus non-competitive) mechanism (data not
shown).
Discussion
It is known that polyphenolic dietary compounds (such as tea catechins and
bio¯avonoids) undergo extensive metabolism in the body to form more water-
soluble conjugates, largely glucuronides and sulphates. Recently, we reported that
the catechol-containing tea polyphenols could also be rapidly metabolized to
monomethyl ethers by the cytosolic COMT present in human term placenta
(Zhu et al. 2000). In the present study, it was found that several tea polyphenols

COMT-catalysed O-methylation of tea polyphenols
887
were rapidly O-methylated by rat liver cytosolic COMT. The results showed that
(¡)-epicatechin, (‡)-epicatechin, (¡)-epigallocatechin, (¡)-epicatechin gallate and
(¡)-epigallocatechin gallate are all substrates for COMT-mediated O-methylation.
Under the same conditions for in vitro O-methylation, (¡)-epicatechin, (‡)-
epicatechin and (¡)-epigallocatechin were rapidly O-methylated by rat liver
cytosol, whereas (¡)-epicatechin gallate and (¡)-epigallocatechin gallate were O-
methylated at signi®cantly lower rates.
When (¡)-epicatechin was used as a representative substrate, its O-methylation
by rat liver cytosolic COMT showed characteristic dependence on the incubation
pH, with maximal velocity observed at pH 8±12 (®gure 5). Notably, when the same
incubation conditions and the same substrate were used, human placental cytosolic
COMT showed a narrow pH optimum at 7.4 (Zhu et al. 2000), which is very
di€erent from the pH dependence observed in this study with rat liver cytosolic
COMT. The reasons for the observed di€erences in pH dependence are not clear
at present.
The results showed that the apparent Km of S-adenosyl-l-methionine (the
methyl donor) for the in vitro O-methylation of (¡)-epicatechin is 13mm (®gure
2). Since this S-adenosyl-l-methionine concentration is within the range of its
tissue levels as previously reported for animals (Ueland 1982, Wagner et al. 1984,
Zhu and Liehr 1996), it suggests that the COMT-catalysed rapid O-methylation
of tea catechins is a biochemically feasible reaction in vivo.
During the metabolic O-methylation of catechol substrates (such as tea
catechins), S-adenosyl-l-methionine is converted to S-adenosyl-l-homocysteine
in equimolar amounts with the monomethyl products formed. It is known that S-
adenosyl-l-homocysteine is an important feedback inhibitor for COMT-catalysed
O-methylation of a variety of catechol substrates (Ueland 1982). The present
results also showed that the rapid metabolic O-methylation of tea polyphenols is
subject to the inhibitory regulation by S-adenosyl-l-homocysteine. This potent
inhibition of the O-methylation of tea catechols by S-adenosyl-l-homocysteine
may e€ectively prevent the endogenous S-adenosyl-l-methionine pool from being
readily depleted, which would be important for maintaining the homeostasis of the
universal methyl donor S-adenosyl-l-methionine during metabolic O-methylation
of excess amounts of endogenous and/or exogenous catechols.
Regarding the possible structures of the methylated tea polyphenols, earlier
studies with norepinephrine and quercetin (a catechol-containing ¯avonoid)
showed that COMT at pH 7.4 predominantly methylates the meta-hydroxyl
group of these substrates (Creveling 1970, Creveling et al. 1972, Zhu et al.
1994). Our HPLC analysis of the methylated products of (¡)-epicatechin showed
two less polar peaks with 5:1 ratio (®gure 2). Further LC/MS analysis con®rmed
that the quantitatively major metabolite of (¡)-epicatechin was a monomethylated
derivative. It is thus suspected that the major monomethylated metabolite of (¡)-
epicatechin likely is the 30-O-methyl isomer, with the 40-O-methyl isomer a minor
metabolite (see ®gure 1 for the 30 and 40 positions). Similarly, it is suspected that
the major methylated metabolite of (¡)-epigallocatechin would be the 30-O-methyl
isomer. This suggestion is consistent with our chromatographic observation
showing that one major monomethylated metabolite (likely the 30-O-methyl
metabolite) was detected on HPLC (®gure 2). The largely undetectable formation
of 40-O-methyl metabolite from (¡)-epigallocatechin as substrate might be, in
part, due to the following two reasons: (1) (¡)-epigallocatechin has two identical

888
Bao Ting Zhu et al.
meta-hydroxyl groups (i.e. two 30-hydroxyl groups) that could be O-methylated at
pH 7.4, and this would quantitatively favour the formation of 30-O-methyl product
relative to the 40-O-methyl product when compared to (¡)-epicatechin as sub-
strate; and (2) the 40-hydroxyl group of (¡)-epigallocatechin might have lower
accessibility by the enzyme than that of (¡)-epicatechin.
Several recent studies showed that many common tea polyphenols such as (¡)-
epigallocatechin and (¡)-epicatechin could be readily absorbed in human subjects
after drinking tea, and some of them can reach submicromolar concentrations (0.1±
0.4 mm) in the plasma (Lee et al. 1995, Unno et al. 1996, Yang et al. 1998, van het
3
Hof et al. 1998, Li et al. 2000). A recent study using H-labelled (¡)-epigalloca-
techin gallate also showed that this tea catechin is widely distributed in the mouse
tissues (Suganuma et al. 1998). It is thus expected that a signi®cant fraction of the
ingested catechol-containing tea polyphenols would be O-methylated in the body
(Hackett and Griths 1981, 1982, Kuhnle et al. 2000). It will be of considerable
interest to determine whether there are any important biological actions that are
associated with methylated tea polyphenols. Noteworthily, Sano et al. (1999)
reported that two quantitatively minor methylated catechins isolated from oolong
tea showed a stronger anti-allergic activity than an unmethylated catechin. Like-
wise, it will also be of interest to determine whether tea polyphenols can e€ectively
inhibit the metabolic O-methylation of endogenous catecholamines and catechol
oestrogens, and to determine whether some of tea’s e€ects are related to inhibition
of the methylation metabolism of these endogenous catechols. E€ective inhibition
of the O-methylation of endogenous catecholaimes by tea components would be
expected to alter their physiological functions as neurotransmitters/neurohor-
mones, and inhibition of COMT-catalysed methylation metabolism of levodopa
(the most e€ective agent for treatment of Parkinson’s disease) would markedly
increase its bioavailability in the central nervous system and reduce its peripheral
side-e€ects. In the case of catechol oestrogens, inhibition of COMT-catalysed O-
methylation of endogenous 2- and 4-hydroxylated oestrogens by tea polyphenols
may lead to elevated tissue levels of the procarcinogenic 4-hydroxyoestradiol
(Liehr 2000) plus decreased tissue levels of the anticarcinogenic 2-methoxyoes-
tradiol (Zhu and Conney 1998). Although our earlier studies showed that chronic
administration of dietary quercetin (a strong inhibitor of catechol oestrogen O-
methylation in vivo) enhanced oestradiol-induced kidney tumour formation in
male Syrian hamsters (Zhu et al. 1994, Zhu and Liehr 1994), the e€ects of tea
polyphenols on hormonal carcinogenesis remain to be determined.
In summary, several catechol-containing tea polyphenols are very rapidly O-
methylated by rat liver cytosolic COMT. This metabolic O-methylation of tea
polyphenols is subject to inhibition by S-adenosyl-l-homocysteine. The results of
the present study call for further research to determine whether some of the
important biological actions of tea polyphenols may be caused by their methylated
products. Studies are also needed to determine the potential inhibitory e€ects of
tea polyphenols on the COMT-catalysed O-methylation of endogenous catecho-
lamines and catechol oestrogens.
Acknowledgements
The authors thank Mao-Jung Lee and Chung S. Yang for advice on the HPLC
separation of tea catechins, and Chuan Li, Bozena Winnik and Brian Buckley for

COMT-catalysed O-methylation of tea polyphenols
889
help with the liquid chromatography-mass spectrometry analysis of the O-methy-
lated catechins.
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