Identification and Characterization of Methylated and Ring-Fission

Laboratory for Cancer Research, College of Pharmacy, and Department of ... Food Science, Rutgers, The State University of New Jersey, New Brunswick, N...
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Chem. Res. Toxicol. 2002, 15, 1042-1050

Identification and Characterization of Methylated and Ring-Fission Metabolites of Tea Catechins Formed in Humans, Mice, and Rats Xiaofeng Meng,† Shengmin Sang,‡ Nanqun Zhu,‡ Hong Lu,† Shuqun Sheng,§ Mao-Jung Lee,† Chi-Tang Ho,‡ and Chung S. Yang*,† Laboratory for Cancer Research, College of Pharmacy, and Department of Chemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, and Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901 Received December 28, 2001

(-)-Epigallocatechin gallate (EGCG), the most abundant tea catechin, has been proposed to be beneficial to human health based on its strong antioxidative and other biological activities in vitro. Inadequate knowledge regarding the bioavailability and biotransformation of EGCG in humans, however, has limited our understanding of its possible beneficial health effects. In this study, 4′,4′′-di-O-methyl-EGCG (4′,4′′-DiMeEGCG) was detected in human plasma and urine by LC/MS/MS following green tea ingestion. Both 4′,4′′-DiMeEGCG and EGCG reached peak plasma values (20.5 ( 7.7 and 145.4 ( 31.6 nM, respectively, in 4 subjects) at 2 h after the dose. The half-lives of 4′,4′′-DiMeEGCG and EGCG were 4.1 ( 0.8 and 2.7 ( 0.9 h, respectively. The cumulative urinary excretion of 4′,4′′-DiMeEGCG during a 24 h period was 140.3 ( 48.6 µg, about 5-fold higher than that of EGCG, but the excreted 4′,4′′-DiMeEGCG and EGCG in urine only accounted for about 0.1% of ingested EGCG. (-)-5-(3′,4′,5′Trihydroxyphenyl)-γ-valerolactone (M4) and (-)-5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (M6), along with another possible ring-fission metabolite, (-)-5-(3′,5′-dihydroxyphenyl)-γ-valerolactone (M6′), were detected in human urine after green tea ingestion. The cumulative excretion of M4, M6′, and M6 during a 24 h period ranged from 75 µg to 1.2 mg, 0.6 to 6 mg, and 0.6 to 10 mg, respectively. The combined excretion of all three ring-fission metabolites accounted for 1.5-16% of ingested catechins. M4, M6′, and M6 were all observed after the ingestion of pure EGCG or EGC by human subjects, whereas only M6 was produced after EC ingestion. These metabolites as well as monomethylated EGCG were detected in mice and rats after tea or EGCG administration, and the tissue levels reflected the rather low bioavailability of EGCG in rats. The presently characterized methylated EGCG metabolites and ring-fission products exist in substantial quantities and may contribute to the biological activities of tea.

Introduction Tea, a beverage produced by brewing the dried leaves and buds of the plant Camellia sinensis, has been consumed by humans for thousands of years and is one of the most popular beverages worldwide. Based on different processing methods, green tea, black tea, and oolong tea are produced as three major types of tea products. Tea polyphenols, also known as catechins, account for 30-42% of water-soluble solids in brewed green tea. There are four major tea catechins: (-)epigallocatechin-3-gallate (EGCG),1 (-)-epigallocatechin (EGC), (-)-epicatechin (EC), and (-)-epicatechin gallate * Correspondence should be addressed to this author at the Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Rd., Piscataway, NJ 08854-8020. Phone: 732-445-5360, Fax: 732-445-0687, Email: csyang@ rci.rutgers.edu. † Laboratory for Cancer Research, College of Pharmacy. ‡ Department of Food Science. § Department of Chemistry. 1 Abbreviations: EGCG, (-)-epigallocatechin-3-gallate; EGC, (-)epigallocatechin; EC, (-)-epicatechin; ECG, (-)-epicatechin-3-gallate; ESI, electrospray ionization; CID, collision-induced dissociation; MS/ MS, tandem mass spectrometry; COMT, catechol-O-methyltransferase; 4′-MeEGC, 4′-O-methyl-EGC; 4′′-MeEGCG, 4′′-O-methyl-EGCG; 4′,4′′DiMeEGCG, 4′,4′′-di-O-methyl-EGCG; TMS, tetramethylsilane.

(ECG) (1). These catechins, especially EGCG, have been reported to have various biological activities (2, 3). Tea consumption has been suggested to protect against cancer, cardiovascular diseases, and other diseases, but the results from epidemiological studies are not conclusive (2-5). The lack of sufficient information on the bioavailability and biotransformation of catechins is one of the limiting factors in understanding the biological effects of tea. In recent years, the metabolic fate of catechins is beginning to be understood. Methylation, glucuronidation, and sulfation are the major biotransformation pathways of catechins. The small intestine and liver were found to be important organ sites in the absorption and metabolism of catechins (6). Following the administration of (+)-catechin and EC to rats, the glucuronides of these two compounds and their methylated derivatives were isolated from the rat urine (7). Catechins were shown to be the substrates of rat and human catechol-O-methyltransferase (COMT) (8), and EGC and EC were better substrates than EGCG (9, 10). In humans, 4′-O-methylEGC (4′-MeEGC) was identified as one of the major metabolites after green tea and catechin consumption (11, 12). Many conjugated EGC, EC, and their metabolites

10.1021/tx010184a CCC: $22.00 © 2002 American Chemical Society Published on Web 06/27/2002

Tea Catechin Metabolites

in human urine have been characterized recently in our lab using LC/MS/MS (13). (-)-5-(3′,4′,5′-Trihydroxyphenyl)-γ-valerolactone (M4) and (-)-5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (M6), mostly in their glucuronideconjugated forms, were reported as two major ring-fission metabolites in plasma and urine after green tea ingestion by human subjects (14). Intestinal microflora might be responsible for the formation of these ring-fission products. The absorption, disposition, metabolism, and excretion of EGCG have been extensively studied in animals with tritium-labeled EGCG. After ig administration of [3H]EGCG to mice, radioactivity was widely distributed in the digestive tract, liver, lung, and many other organs. Within 24 h, about 6.5% of administrated radioactivity was excreted in the urine, and 35% radioactivity excreted in the feces (15). The chemical identities of the radioactive compounds were not determined. After iv administration of [4-3H]EGCG to rats, about 77% radioactivity was excreted in the bile within 48 h, and the biliary metabolites were identified as mono- and dimethylated EGCG. A significant amount of (-)-5-(3′,5′-dihydroxyphenyl)-γvalerolactone was also observed in rat urine and feces (16, 17). Although progress has been made in the studies on EGCG in animals, the metabolic fate of EGCG in humans still needs to be delineated. In this study, we identified mono- and dimethylated EGCG in human and rodent blood and urine samples based on LC/MS/MS analysis and characterized their apparent pharmacokinetic properties. We also identified the ring-fission products of EGCG, EGC, and EC in humans and mice. Based on these results and other information, pathways of EGCG biotransformation are proposed.

Experimental Procedures Materials. The green tea solids preparation was obtained from Thomas J. Lipton Inc. (Englewood Cliffs, NJ). One gram of green tea solids contained about 139 mg of EGCG, 110 mg of EGC, 32 mg of EC, 33 mg of ECG, 5.4 mg of caffeine, and 0.32 mg of theobromine. EGC, EC, EGCG, ECG, β-D-glucuronidase (EC 3.2.1.31), and sulfatase (EC 3.1.6.1) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemical were commercially available products of analytical or HPLC grade. Human Study. This human study was approved by the Institutional Review Board of Rutgers University (Protocol 92034). Four healthy adult volunteers (three male and one female; 25-35 years of age; weighing 66-78 kg) participated in this study. They did not smoke or drink alcoholic beverages before the study. The subjects did not ingest tea or other catechincontaining beverages for at least 2 days prior to the experiment and during the urine sample collection period. A single oral dose of green tea solids (20 mg/kg body weight) in 200 mL of warm water was given to each subject in the morning. Blood samples (5 mL each) were collected in heparin-containing tubes at 0, 0.25, 0.5, 1, 2, 3, 5, 8, 12, and 24 h. Urine samples were collected before the dose and for the time periods of 0-3, 3-8, and 8-24 h after the dose. After a wash-out period for at least 1 week, the same subjects were given a single dose of EGCG (2 mg/kg body weight) in the morning. Blood samples were collected as above. After centrifugation, the plasma samples were preserved by adding 20 µL of ascorbic acid-EDTA solution (20% ascorbic acid and 0.1% EDTA in 0.4 M sodium phosphate buffer, pH 3.6) per milliliter. To investigate the metabolic fate of individual catechins, a volunteer ingested 200 mg of pure EGCG, EGC, or EC in 200 mL of water, and urine samples were collected before the dose and at 0-3, 3-6, 6-9, 9-12, 12-18, and 18-24 h after dosing. All blood and urine samples were stored at -80 °C before

Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1043 use. Previous studies showed that catechins and their metabolites were stable under these conditions for 1 or 2 years. Animal Study. Male Sprague-Dawley rats with an average weight of about 300 g were purchased from Taconic Farm (German Town, NY). Female A/J mice were obtained from Jackson Laboratory (Bar Harbor, NY). Animals were given AIN76M diet and water ad libitum. After acclimation for 1 week, 0.6% green tea, 0.3% EGCG, or 0.1% EGCG preparation was given to mice as the sole source of drinking fluid. Mouse urine samples were collected at 9 a.m. by gently pressing the bladder, and freshly excreted fecal samples were collected at the same time. The urine and fecal samples from all five mice in one cage were pooled and preserved by adding ascorbic acid-EDTA solution. After green tea or EGCG treatment, rats and mice were anesthetized by CO2, and the blood was collected from the retroorbital plexus in the eyes with heparinized capillary tubes. The blood sample was centrifuged at 16000g for 10 min. The plasma samples were mixed with 0.1 volume of ascorbic acidEDTA solution and stored at -80 °C until use. Tissue samples (liver, kidney, and small intestine) were collected upon sacrifice of the animals. Lung and liver samples were rinsed twice with ice-cold saline, whereas kidney and small intestine samples were cut open and washed extensively to remove the content. All the tissues were blotted with tissue papers and stored at -80 °C. Urine, Feces, Blood, and Tissue Sample Preparation. Our previous method (18) was used for the preparation of plasma and urine samples. In brief, the thawed plasma was incubated with β-D-glucuronidase and sulfatase at 37 °C for 45 min. The reaction mixture was extracted with methylene chloride; the resultant aqueous layer was then extracted with ethyl acetate, and the ethyl acetate extract was dried under reduced pressure. For the preparation of urine samples, the extraction with methylene chloride was omitted. Fecal sample was mixed with 10 volumes (w/v) of 2% ascorbic acid solution. After homogenization, the sample was centrifuged at 16000g for 5 min, and 50 µL of the supernatant was processed with the same method as that for urine samples. The thawed tissue sample was homogenized with 5 volumes (w/v) of 2% ascorbic acid solution with a Polytron, and 500 µL of homogenate was then processed with the same method used for plasma samples. The samples from 5 mice in one cage were pooled for the analysis, whereas individual rat tissue was analyzed. After β-D-glucuronidase and sulfatase hydrolysis, the total amounts of each tea catechin (free plus conjugated forms) were quantitated. The amount of free (unconjugated) form was determined by preparing the samples without enzyme hydrolysis. Synthesis and Purification of Methylated EGCG. The conditions for chemical methylation of EGCG were similar to those described previously (19). In brief, EGCG (250 mg) was mixed with 0.5 mL of methyl iodide and 250 mg of K2CO3 in 10 mL of acetone. The reaction was irradiated in a ultrasonic bath for 3 h. The products were purified by Sephadex LH-20 column chromatography (eluted by 80% ethanol) and silica gel column chromatography (eluted by chloroform/methanol/water 10:1:0.1). For further purification, the crude compounds were redissolved in 10% acetonitrile aqueous solution and separated by HPLC with a Supelcosil C18 reverse-phase column (150 × 4.6 mm i.d.; particle size 5 µm; Supelco Inc., Bellefonte, PA). The isocratic mobile phase consisted of 12% acetonitrile, 3% tetrahydrofuran, and 0.1% trifluoroacetic acid at a flow rate of 1.5 mL/min and was monitored at 280 nm (11). The purified compounds were kept at -80 °C for further use. LC/MS Analysis of EGCG and Its Metabolites. LC/MS analysis was carried out with a Finnigan Spectra System which consisted of a Finnigan model P4000 pump, a model AS3000 refrigerated autosampler, a model UV6000LP photodiode-array UV detector, and a Finnigan LCQ Deca mass detector (ThermoFinigan, San Jose, CA) incorporated with an electrospray ionization (ESI) interface. A Supelco Discovery HS C18 column (75 × 2.1 mm i.d.; particle size, 3 µm) was used for separation with a flow rate of 0.2 mL/min. The column elution started with

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Table 1. 1H NMR Chemical Shiftsa of EGCG and Methylated EGCG Derivatives

EGCG H-2 H-3 H-4R H-4β H-6 H-8 H-2′ H-6′ H-2′′ H-6′′ OCH3

5.07 s 5.56 m 2.91 dd (1.6, 17.2) 3.04 dd (4.4, 17.2) 6.06 d (2.4) 6.04 d (2.4) 6.62 s 6.62 s 7.03 s 7.03 s

4′′-MeEGCG (P1)

4′,4′′-DiMe- 4′,3′′,4′′-TriMeEGCG EGCG (P2) (P3)

5.07 s 5.51 m 2.92 dd (1.8, 17.2) 3.03 dd (4.4, 17.6) 6.03 d (2.2) 6.01 d (2.2) 6.64 s 6.64 s 7.01 s 7.01 s 3.81 s

5.10 s 5.56 m 2.94 dd (2.0, 17.6) 3.04 dd (4.6, 17.4) 6.06 d (2.1) 6.04 d (2.1) 6.66 s 6.66 s 7.01 s 7.01 s 3.75 s, 3.82 s

5.11 s 5.55 m 2.93 dd (2.1, 17.7) 3.03 dd (4.0, 17.7) 6.05 d (2.7) 6.06 d (2.7) 6.66 s 6.66 s 7.03 d (1.8) 7.11 d (1.8) 3.75 s, 3.78 s, 3.83 s

a Chemical shifts are expressed in ppm downfield from the signal for TMS in acetone-d6 and coupling constants in hertz are in parentheses.

90% solvent A (10% aqueous methanol) and 10% solvent B (70% aqueous methanol). The linear gradient was changed to 31% B at 3 min, and 33% B at 17 min. It was changed to 90% B at 30 min to clean the column and then reequilibrate to 10% B from 30.1 to 40 min for the next run. The LC eluant was introduced into the ESI interface after UV scanning from 200 to 400 nm. The negative ion polarity mode was set for ESI ion source with the voltage on the ESI interface maintained at approximately -4 kV. Nitrogen gas was used as the sheath gas at a flow rate of 80 arb and the auxiliary gas at 10 arb, respectively. The heated capillary temperature and voltage were maintained at 260 °C and -24 V, respectively. The tube lens offset voltage was set at -55 V. The structural information of the EGCG and its metabolites was obtained by tandem mass spectrometry (MS/ MS) through collision-induced dissociation (CID) with a relative collision energy setting of 25%. Data Analysis. The pharmacokinetic parameters of EGCG and its metabolites in human plasma were calculated with WinNonlin (version 3.1, Pharsight Corp., CA) by using noncompartmental analysis for extravascular administration. The linear trapezoidal rule was used for the calculation of t1/2, Cmax, and AUC.

Results Structural Identification of Chemically Methylated EGCG. After reacting EGCG with methyl iodide, two major products (P1 and P2) were purified by Sephadex LH-20 column chromatography, silica gel column chromatography, and C18 reversed-phase HPLC. The yields of P1 and P2 were about 30% and 10% of EGCG, respectively. Structural analysis of these two compounds was performed by mass spectrometry and 1H NMR spectroscopy. The negative ion ESI-MS data of P1 indicated a deprotonated molecular ion peak [M-H]- at m/z 471, which was consistent with a monomethylated EGCG (MW 472). The full scan MS/MS spectrum (data not shown) of this compound exhibited the base product ion at m/z 287, which corresponded to the loss of a monomethylated galloyl moiety [(M-183)-H]-, and a product ion of m/z 183 was indeed observed in the spectrum. As shown in Table 1, the 1H NMR spectrum of P1 was very similar to that of EGCG (20) except for the presence of a methoxy signal (δ 3.81 ppm). The same proton signal (δ 7.03 ppm) from H-2′′ and H-6′′ suggested that the methylation occurred at the 4′′-position. Therefore, the chemical structure of P1 was identified as 4′′O-methyl-EGCG (4′′-MeEGCG).

Figure 1. Chemical structures of EGCG and methylated EGCG.

Compound P2 was found to have a deprotonated molecular ion at m/z 485, suggesting the formation of a dimethylated EGCG (MW 486). Full scan MS/MS was used to obtain the product ion spectrum of this compound. The product ion m/z 301 was observed as the base product ion peak of P2, which corresponded to the loss of a monomethylated galloyl moiety [(M-183)-H]-. The 1H NMR data of this compound (Table 1) were superimposable with those of 4′,4′′-di-O-methyl-EGCG (4′,4′′-DiMeEGCG) (21). The same chemical shift (δ 7.01 ppm) assigned to protons H-2′′ and H-6′′ suggested one methoxy group was located at the 4′′-position. Similarly, another methyl substitution was found to be at the 4′position, indicating that this compound was 4′,4′′-DiMeEGCG. In addition to 4′′-MeEGCG and 4′,4′′-DiMeEGCG, compound P3 was obtained with yield less than 5%. The 1H NMR data of compound P3 showed three methoxyl groups at δ 3.83, 3.78, and 3.75 ppm, indicating P3 was the trimethylated derivative of EGCG (Table 1). This was confirmed by the negative ion ESI-MS data. The molecular weight of P3 (m/z 499, [M-H]-) was 14 mass units greater than that of P2 (m/z 485, [M-H]-), indicating that P3 had one more methyl group than P2. In comparison to the 1H NMR spectrum of P2, P3 was distinguished by the downfield shift of H-2′′ and H-6′′. This suggested that the additional methoxyl group was located at position 3′′. This was confirmed by the NOE difference spectrum. When the methoxyl group at δ 3.83 was saturated, one significant positive signal at δ 7.03 ppm was observed. When the other two methoxyl groups (δ 3.78 and 3.75 ppm) were saturated, respectively, however, no positive signal was observed. Thus, δ 7.03 was assigned to H-2′′, and δ 7.11 was assigned to H-6′′. Therefore, P3 was identified to be 4′,3′′,4′′-trimethylated EGCG. The chemical structures of EGCG and some of its methylated derivatives are shown in Figure 1. Determination of EGCG and Methylated EGCG in Blood and Urine. After a single dose of green tea to human subjects, urine samples were collected and analyzed after β-D-glucuronidase and sulfatase hydrolysis. In LC/MS analysis, a major peak with retention time of 26 min was detected in the urine samples (Figure 2-P1). This peak could not be detected in the control urine samples collected before the dose. The deprotonated molecular ion (m/z 485) of this peak corresponded to a dimethylated EGCG. To identify this peak, it was analyzed together with 4′,4′′-DiMeEGCG by LC/MS/MS

Tea Catechin Metabolites

Figure 2. LC/MS/MS detection of chemically synthesized 4′,4′′DiMeEGCG and 4′,4′′-DiMeEGCG in human plasma and urine after green tea or EGCG ingestion. The chromatograms were obtained by monitoring the deprotonated molecular ion of m/z 485. P1: Representative chromatogram of human urinary 4′,4′′DiMeEGCG after green tea ingestion. P2: Product-ion mass spectrum of human urinary 4′,4′′-DiMeEGCG. P3: LC/MS/MS chromatogram of chemically synthesized 4′,4′′-DiMeEGCG. P4: Product-ion mass spectrum of chemically synthesized 4′,4′′DiMeEGCG.

under the same conditions. As shown in Figure 2-P3, chemically synthesized 4′,4′′-DiMeEGCG had the same retention time (26 min) and the same product ion mass spectrum (Figure 2-P2&P4) as the peak detected in human urine, confirming that this peak was 4′,4′′DiMeEGCG, a methylated metabolite of EGCG. 4′,4′′DiMeEGCG were also detected in human urine and plasma samples after a single dose of green tea or EGCG. Interestingly, another peak at 25.7 min showed the same molecular mass as 4′,4′′-DiMeEGCG, but with slightly different mass spectrum. This peak might represent another dimethylated EGCG metabolite. The presence of EGCG in these samples was also detected based on its retention time and mass spectrum. Time-Dependent Formation of 4′,4′′-DiMeEGCG in Humans. For developing a method to quantify EGCG and 4′,4′′-DiMeEGCG, standards were added to control plasma and urine; the samples were incubated, extracted, and analyzed by LC/MS/MS. The overall recoveries of EGCG in human plasma and urine were 80% and 90%, respectively, and similar values were obtained for the recoveries of 4′,4′′-DiMeEGCG. The detection limits of EGCG and 4′,4′′-DiMeEGCG were 5 pg. Regression analysis of the peak area versus concentration of these two compounds showed linearity over a range of 10 nM to 1 mM in plasma or urine with correlation coefficient values (r2) >0.995 (data not shown). The time-concentration profiles of EGCG and 4′,4′′DiMeEGCG in human plasma after green tea ingestion are shown in Figure 3A. Shortly after the ingestion of

Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1045

Figure 3. Plasma levels and urinary excretions vs time profiles of EGCG and 4′,4′′-DiMeEGCG following ingestion of green tea (20 mg/kg body weight). Each data point and error bar represents the mean ( SD of four human subjects. (A) Plasma level; (B) cumulative urinary excretion.

green tea (20 mg/kg body weight), 4′,4′′-DiMeEGCG appeared and reached its peak value of 20.5 ( 7.7 nM at 2 h and then started to decrease. A similar pattern was observed for its parent compound EGCG, which also peaked at 2 h with a Cmax of 145.4 ( 31.6 nM. The halflives of EGCG and 4′,4′′-DiMeEGCG in human plasma were 2.7 ( 0.9 and 4.1 ( 0.8 h, respectively, and their respective AUC0-inf values were 668.0 ( 130.1 and 77.5 ( 33.7 ng‚h/mL. The cumulative amount of EGCG excreted in the urine during a 24 h period was 26.7 ( 16.0 µg, whereas the cumulative excretion of 4′,4′′DiMeEGCG was 140.3 ( 48.6 µg (Figure 3B). When a single dose of EGCG (2 mg/kg body weight) was given to human subjects, similar plasma time-concentration profiles of EGCG (Cmax 97.3 ( 83.1 nM; t1/2 2.5 ( 1.3 h; AUC0-inf 236.6 ( 151.2 ng‚h/mL) and 4′,4′′-DiMeEGCG (Cmax 18.0 ( 9.8 nM; t1/2 2.8 ( 0.4 h; AUC0-inf 46.9 ( 22.6 ng‚h/mL) were observed. Full scan MS/MS with target deprotonated molecular ion of m/z 471 was used to detect monomethylated EGCG with chemically synthesized 4′′-MeEGCG as a standard. Only trace amounts of 4′′-MeEGCG and two other monomethylated EGCG metabolites were detected in the human plasma and urine samples after either green tea or EGCG ingestion (data not shown). Determination of Ring-Fission Products. For the identification of different ring-fission products and their possible precursors, the urine samples collected from human subjects after the ingestion of green tea, EGCG, EGC, and EC were analyzed by LC/MS/MS.

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Figure 4. LC/MS/MS detection of ring-fission products in human urine samples after ingestion of green tea or pure catechins. The human urine samples from the period of 6-9 h after ingestion of pure catechins (200 mg) and of 8-24 h after ingestion of green tea (20 mg/kg body weight) were used for the detection of ring-fission products. The LC/MS/MS chromatogram and the product ion spectra of three ring fission products, M4, M6′, and M6, are shown.

After ingestion of green tea (150 mg of green tea solids), a peak with a deprotonated molecule ion of m/z 223 and a retention time of 7.4 min was detected in the human urinary samples (Figure 4). The mass spectrum of this compound consisted of major product ion of m/z 179, which matched the mass spectrum of M4. This peak could be detected in the urine samples after EGC and EGCG ingestion, but not after EC ingestion. Similarly, another peak with a retention time of 11 min had the same mass spectrum as M6. This peak was the only ring-fission product detected in the urine sample after EC ingestion, and it also appeared in the urine sample after ingestion of EGC and EGCG. Along with M4 and M6, an extra peak (M6′) with the same molecular mass as M6 was eluted at about 10 min. This peak could also be formed after ingestion of EGCG and EGC, but not EC. The similar product ion pattern of M6′ as M6 indicated their chemical structures were closely related. The same m/z 163 [(M44)-H]- product ions in both spectra suggested they had the same lactone structure. Since the aromatic ring of M6 was ortho-substituted by two hydroxyl groups at 3′and 4′-positions, it is likely that M6′ had a metasubstituted dihydroxyphenyl structure which matched the metabolite (-)-5-(3′,5′-dihydroxyphenyl)-γ-valerolactone reported by Kohri et al. (17). In their study, (-)-5(3′,5′-dihydroxyphenyl)-γ-valerolactone existed in the urine and feces as a main metabolite after the administration of (-)-[4-3H]EGCG to rats.

Meng et al.

Figure 5. Urinary levels of ring-fission products after EGCG, EGC, or EC ingested by a human subject. Urinary levels of ringfission products after ingestion of 200 mg of (A) EGCG, (B) EGC, or (C) EC.

Time-Dependent Formation of Ring-Fission Products in Humans. For developing a method to quantify M4 and M6, pure M4 and M6 isolated from human urine after green tea ingestion were spiked into control urine and plasma samples. After incubation and extraction, the samples were analyzed by LC/MS/MS. The overall recoveries of M4 and M6 in human urine were about 90%, whereas the recoveries of both compounds in human plasma were around 80%. Since pure M6′ was not available, we assumed that its recovery and response in mass detection were the same as M6 based on their structural similarity. The time-concentration profiles of ring-fission products in human urine following EGCG, EGC, and EC ingestion are shown in Figure 5. After ingesting 200 mg of pure EGCG, the urinary level of M6 reached 4.7 µM in the sample collected at 3-6 h; similar levels of M6 was observed in the urine samples collected at the same period after ingestion of equal amounts of EGC or EC. Peak urinary level of M4 of 8.3 µM was observed at 3-6 h after EGCG ingestion or of 14 µM at 6-9 h after EGC ingestion. M6′ was observed after either EGCG or EGC ingestion, with peak urinary values of 8.3 and 6.6 µM, respectively. Following ingestion of a single dose of green tea (150 mg of green tea solids), M4 appeared in human urine and

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Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1047 Table 2. Ions of Structural Significance and Retention Time of Mouse Urinary Methylated EGCG Metabolites Observed in LC/ESI-MS2 Analysis

Figure 6. LC/MS/MS detection of mono- and dimethylated EGCG in mouse urine samples after administration of 0.3% EGCG. The mouse urine samples collected at 9 a.m. of the second day after 0.3% EGCG were given to mice as the sole drinking fluid. Urine samples from five mice in one cage were pooled for the analysis. The LC/MS/MS chromatogram with retention time indicated and product-ion spectra of urinary 4′′MeEGCG and 4′,4′′-DiMeEGCG are shown.

reached a value of 2.8 ( 1.5 µM (4 subjects) during the period of 3-8 h and did not increase, whereas the highest levels of M6′ and M6 were found in the urine collected during the 8-24 h period, showing concentrations of 36.7 ( 34.4 and 31.2 ( 26.4 µM, respectively. The cumulative excretion of M4 during a 24 h time course ranged from 75 µg to 1.2 mg. The total excretion of M6′ and M6 ranged from 0.6 to 6 mg and from 0.6 to 10 mg, respectively, which were 3-9-fold higher than that of M4. The combined amount of M4, M6′, and M6 accounted for 1.5-16% of ingested catechins. The plasma levels of M4 and M6 were described previously (14). In the present study, M4, M6, and M6′ started to appear at 5 h after green tea ingestion, and reached their peak values at about 12 h. The peak plasma levels of M6′ were around 0.36 ( 0.13 µM, which was comparable to that of M6 and much higher than that of M4. Detection and Characterization of Methylated and Ring-Fission Products in Mice and Rats. Groups of mice were given 0.6% green tea solids, 0.3% EGCG, or 0.1% EGCG for 3 days. With LC/MS/MS analysis, four monomethylated EGCG and four dimethylated EGCG were detected in mouse urine after EGCG treatment (Figure 6). Based on comparison with chemically synthesized EGCG methylated derivatives, the compounds with retention times of 17.56 and 26.05 min were identified as 4′′-MeEGCG and 4′,4′′-DiMeEGCG, respectively. The major ions and relative intensities of these metabolites observed in LC/MS/MS spectra are listed in Table 2. The mass spectrum of peak 2 (tR 19.68 min) indicated that the methylation position was on the B-ring by presenting a product ion of m/z 169 (intact gallic acid). As suggested by Miketova et al. (22), methylation of the

peak

tR (min)

ions of interest (relative intensity)

1 2 3

17.56 19.68 22.01

4 5 6 7

24.56 25.56 26.05 26.76

8

28.17

471 (2), 345 (15), 333 (10), 287 (100), 183 (20) 471 (2), 319 (100), 301 (25), 169 (20), 137 (4) 471 (80), 333 (20), 319 (100), 305 (15), 183 (15), 169 (35) 471 (80), 333 (25), 305 (35), 183 (15), 169 (20) 485 (5), 441 (100), 305 (95), 301 (70), 183 (15) 485 (3), 301 (100), 283 (30), 183 (10) 485 (10), 333 (100), 315 (25), 301 (20), 183 (5), 169 (45) 485 (100), 441 (10), 333 (80), 183 (5), 169 (10)

4′-position could block the formation of the m/z 125 ion from the A-ring; so the presence of product ion of m/z 345 [(M-126)-H]-, in which the m/z 126 ion was derived from m/z 125 by hydrogen abstraction, suggested that the chemical structure was 3′-O-methyl-EGCG. Similarly, the block of the 4′′-position was believed to retard the formation of m/z 169 incorporating the gallic acid group. The presence of m/z 183 and m/z 169 in the mass spectrum of peak 3 (tR 22.01 min) led to the elucidation of its structure as 3′′-O-methyl-EGCG. The peak with retention time 28.17 min had a methylated galloyl moiety indicated by a product ion of m/z 183. The presence of m/z 169 in the mass spectra of this peak suggested one methylation occurred at the 3′′-position since the substitution of the 4′′-position would block the formation of product ion of m/z 169 (22). The lack of m/z 305 [(M166)-H]- and the presence of m/z 319 indicated another methylation position was on the B-ring. It was likely that this peak represented 3′,3′′-O-di-methyl-EGCG. Further structural identification of these metabolites is needed. The mouse plasma, urine, and fecal levels of EGCG and 4′,4′′-DiMeEGCG are listed in Table 3. Although less than 100 nM EGGC and 4′,4′′-DiMeEGCG were observed in the plasma, much higher concentrations of EGCG and 4′,4′′-DiMeEGCG were found in the urine and feces in all three groups. The urinary levels of 4′′-MeEGCG in mouse urine seemed to be affected by the dose of EGCG. After 0.3% EGCG administration, the ratio of the levels of 4′′-MeEGCG to 4′,4′′-DiMeEGCG was 0.45 ( 0.05, whereas the ratio decreased to 0.31 ( 0.05 when 0.1% EGGC was administrated. The low levels of EGCG and 4′,4′′-DiMeEGCG in rat plasma are consistent with previous results showing poor bioavailablity of EGCG in rats (23, 24). The tissue levels of EGCG and 4′,4′′-DiMeEGCG observed in rat and mice are shown in Table 4. When 0.6% green tea preparation was given as the drinking fluid, the levels of EGCG and 4′,4′′-DiMeEGCG observed in rat liver and kidney were much lower than those in mouse tissues. Although rather high levels of EGCG were found in both rat and mouse small intestine, the level of 4′,4′′-DiMeEGCG in rat small intestine was much lower than that of mouse small intestine. M4, M6′, and M6 were also observed in mouse urine after green tea or EGCG administration. When 0.1% or 0.3% EGCG was administrated to mice, only 0.2-3.2 µM M6′ was observed along with small amounts of M4 and M6. Following 0.6% green tea administration, M4, M6′, and M6 were found in mouse urine at levels ranging from 1.5 to 8.3 µM, from 3.1 to 26.5 µM, and from 1.2 to 6.6 µM, respectively.

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Meng et al.

Table 3. Levels of EGCG and 4′,4′′-DiMeEGCG in Mouse Plasma, Urine, and Feces plasmaa (nM)

urineb (µM)

fecesb (µg/g wet weight)

treatment

EGCG

4′,4′′-Di-MeEGCG

EGCG

4′,4′′-Di-MeEGCG

EGCG

4′,4′′-Di-MeEGCG

0.6% tea 0.3% EGCG 0.1% EGCG

63.3 ( 39.3 90.6 ( 12.5 22.4 ( 9.6

31.0 ( 10.5 36.7 ( 11.5 15.5 ( 5.8

11.6-46.4 26.9-98.6 13.4-40.3

9.1-21.0 34.7-57.2 7.2-22.0

56-110 18-89 24-53

13-25 45-48 4-35

a Each value represents the mean ( SD of five mice. b The data are the range of urinary (or fecal) levels of the three daily samples collected in the 3 days of treatment; each sample was pooled from five mice.

Table 4. Levels of EGCG and 4′,4′′-DiMeEGCG in Rat and Mouse Tissue Samples liver (ng/g wet weight) animal rata mouseb

kidney (ng/g wet weight)

small intestine (ng/g wet weight)

treatment

EGCG

4′,4′′-DiMeEGCG

EGCG

4′,4′′-DiMeEGCG

EGCG

4′,4′′-DiMeEGCG

0.6% tea 0.6% tea 0.3% EGCG 0.1% EGCG

6.7 ( 3.7 42.5 42.9 13.4

1.3 ( 0.3 26.8 59.4 12.8

1.7 ( 0.4 8.9 14.3 4.8

0.9 ( 0.2 2.4 6.6 2.3

230.2 ( 164.9 112.7 114.4 14.8

11.1 ( 6.4 90.3 107.5 6.8

a Each value represents the mean ( SD from five rats. b Each value represents the tissue level (mean of a duplicated assay) of a pooled sample from five mice.

Free vs Conjugated EGCG and 4′,4′′-DiMeEGCG in Plasma, Tissue, and Urine Samples. It was reported recently that after ingestion of EGCG, more than 90% of the EGCG in human plasma existed in its free form (25). In the present study, the plasma samples collected at 1 h after a single dose of green tea were analyzed with and without β-D-glucuronidase and sulfatase for the total and free forms of each catechin, respectively. The percentage of the free form of EGCG was 77.6 ( 28.3%, whereas 60.6 ( 3.1% of 4′,4′′-DiMeEGCG existed in the free form. To investigate the tissue levels of free EGCG and 4′,4′′-DiMeEGCG, livers were homogenized in the presence of D-saccharic acid 1,4lactone to inhibit glucuronidase. Approximately 68.4% EGCG and 51.7% 4′,4′′-DiMeEGCG existed in the free form in mouse liver. In human and animal urine, both EGCG and 4′,4′′-DiMeEGCG were predominantly in the glucuronide-conjugated form. The direct detection of 4′,4′′-DiMeEGCG mono-glucuronides (MW 662) in mouse urine sample was accomplished by monitoring the deprotonated molecular ion of m/z 661. A major peak (tR 10.4 min) was detected along with three other small peaks which all gave product ions of m/z 485 in their MS/MS spectra, suggesting the glucuronidation occurred at four positions of 4′,4′′-DiMeEGCG (data not shown). Their exact chemical structures need further investigation.

Discussion In the present study, several methylated and ringfission products of EGCG were detected after ingestion of green tea or pure EGCG by humans and rodents. The observation of 4′,4′′-DiMeEGCG as a prominent metabolite in humans, rather than the monomethylated EGCG, is quite surprising. 4′,4′′-DiMeEGCG and EGCG reached peak levels in the plasma at about the same time (2 h), suggesting that EGCG was readily methylated after absorption. The urinary excretion of 4′,4′′-DiMeEGCG was much higher than that of EGCG, even though the latter had a higher plasma level. This phenomenon could be due to high COMT activity in human kidney (26). The cumulative excretion of EGCG and 4′,4′′-DiMeEGCG during a 24 h period accounted for about 0.1% of ingested EGCG. The urinary unmethylated EGCG was at such a low level, which was not consistently detected in our previous study

(27). It is likely that substantial amounts of EGCG and methylated EGCG are excreted through the bile into feces. Only a trace amount of 4′′-MeEGCG was found in human plasma or urine samples, whereas a significant amount of 4′′-MeEGCG and two other monomethylated EGCG metabolites appeared in mouse urine. This could be due to either the species differences or the different doses used in human and animal studies. Enzymatic studies indicated that 4′′-MeEGCG effectively competed with EGCG in vitro and was further methylated to 4′,4′′DiMeEGCG (unpublished data). When EGCG was given at low concentration, 4′′-MeEGCG would be further methylated to form 4′,4′′-DiMeEGCG. When higher doses of green tea or EGCG were administrated, formation of monomethylated EGCG would be favored. For instance, the ratio of mouse urinary 4′′-MeEGCG to 4′,4′′-DiMeEGCG was lower (0.31 ( 0.05) with 0.1% of EGCG than (0.45 ( 0.05) with 0.3% of EGCG. The detection of EGCG and its metabolites in major tissues of mice and rats confirms that EGCG and its metabolites are distributed to various organ sites (15, 23). The blood level of 4′,4′′-DiMeEGCG was about one-third to one-half of that of EGCG in both species. The levels of EGCG and 4′,4′′-DiMeEGCG were low in rat liver and kidney, but the level of EGCG was rather high in the small intestine which had direct contact with ingested EGCG, which is consistent with the reported poor bioavailability of EGCG in rats (23). In comparison to rats, higher levels of EGCG were observed in mouse liver and kidney, reflecting the higher bioavailability of EGCG in mice. Large quantities of EGCG and 4′,4′′-DiMeEGCG were also found in mouse feces, which was consistent with the results reported by Suganuma et al. (15). In their study, 35% of the administrated radioactivity of [3H]EGCG was found in mouse feces during a 24 h period. After green tea ingestion, in addition to the previously identified catechin metabolites, M4 and M6, another metabolite (M6′) with possible structure (-)-5-(3′,5′dihydroxyphenyl)-γ-valerolactone was observed. In our previous study, the ring-fission products from EGCG were not investigated. The present study showed that all three metabolites (M4, M6, and M6′) were formed after either EGCG or EGC ingestion, whereas the ingestion of EC only produced M6. ECG might also contribute to

Tea Catechin Metabolites

Figure 7. Proposed metabolic pathways of EGCG. SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine; UGT, UDP-glucuronosyltransferase.

the formation of M6 since it contains the same B-ring structure as EC. The biological activities of these ringfission products might be interesting since they preserve the di- or trihydroxyphenolic structures and have a lactone moiety. Based on the present results and previous information, we propose the metabolic pathways for EGCG as shown in Figure 7. After absorption, EGCG undergoes methylation, glucuronidation, and sulfation in the small intestine, liver, kidney, and other organs. Since all the enzymes that catalyze these reactions coexist in the tissues, the sequence of these reactions and the activity of these enzymes may affect the structures and levels of the final products. For example, the glucuronidation at the 3′-position will inhibit the subsequent methylation at the 4′-position; if methylation occurs at the 4′-position first, glucuronidation at the 3′-position will also be affected (unpublished data). The formed conjugates can also be hydrolyzed by other enzymes, such as glucuronidase, to yield their free forms in certain tissues. The major route of excretion of EGCG and methylated EGCG conjugates is through the bile into the colon, where they are likely to be metabolized by colonic microflora. EGC may be an intermediate during the degradation of EGCG by microbial esterases. Only a small amount of the EGC formed can be reabsorbed into systemic circulation and further metabolized. The major products of EGCG degradation are M4, M6′, and M6, in which M6′ and M6 appear to be the products of the dehydroxylation of M4. Some of these ring-fission products are reabsorbed into systemic circulation, and significant amounts appear in the plasma and urine. Methylated ring-fission products can be formed by either the metabolism of methylated catechins or the methylation of ring-fission products, such as M4 and M6, which retain the catechol structures from catechins. These ringfission products can also be further metabolized in the colon into simple phenolic acids, such as 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid, and 3-methoxy-4-hydroxybenzoic acid, which were detected in human urine after tea ingestion (28). It is commonly believed that the ortho-dihydroxyl structure in the B-ring is important in exhibiting antioxidative activities and methylation on the B-ring will attenuate the antioxidative activities. Methylated EC derivatives and their glucuronides were reported to have decreased activities in scavenging superoxide anion radical (7), whereas methylation at the 3′′- position of EGCG

Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1049

did not affect the antioxidative activity (29). Oral administration of 3′′-O-methyl-EGCG and 4′′-MeEGCG were shown to have stronger activities than EGCG in inhibiting type I and IV allergic reactions in mice (30, 31). 3′O-Methyl-EC was reported to have similar activity as EC in abrogating cell death induced by hydrogen peroxide (32). In our laboratory, 4′′-MeEGCG was shown to have similar activities to EGCG in scavenging 2,2-diphenyl1-picrylhydrazyl (DPPH) radical (33), whereas 4′,4′′DiMeEGCG and 4′,3′′,4′′-TriMeEGCG only had about one-tenth of the activities of EGCG. However, 4′′-MeEGCG, 4′,4′′-DiMeEGCG, and 4′,3′′,4′′-TriMeEGCG exhibited stronger activities than EGCG in inhibiting the spontaneous release of arachidonic acid in human colon cancer HT-29 cell (unpublished results). The biological activities of these methylated derivatives need further elucidation. In this study, we identified and characterized the pharmacokinetic properties of several methylated EGCG and ring-fission metabolites in humans and animals after green tea or EGCG administration. To our knowledge, this is the first report on the detection and kinetics of EGCG metabolites in humans and animals; it is also the first time a thorough profile of ring-fission products in humans and animals has been reported. These metabolites exist in substantial quantities and may have interesting biological activities. Further studies on these compounds will enhance our understanding of the health effects of tea and tea polyphenol consumption.

Acknowledgment. We thank Dr. Pius Maliakal and Dr. Joshua Lambert for their comments on the manuscript. The LC/MS analysis was conducted in the analytical center (directed by Dr. Brian Buckley) at the Environmental and Occupational Health Sciences Institute. This study was supported by NIH Grant CA 56673.

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