Materials and Methods
- Enzymology of Methylation of Tea Catechins and Inhibition of Catechol-O-methyltransferase by ()-Epigallocatechin Gallate

Chemicals and Reagents. EGCG was a gift from Unilever-Bestfoods (Englewood Cliffs, NJ). EGC and (-)-epicatechin gallate (ECG) were purified by Dr. Chi-Tang Ho of Rutgers University (New Brunswick, NJ). 4'-MeEGC, 3'-MeEGC, 4"-MeEGCG, 4',4"-DiMeEGCG, and 4',3",4"-tri-O-methyl-EGCG were synthesized and purified in our laboratory (Meng et al., 2001, 2002). (-)-EGCG-4"-O-glucuronide (EGCG-4"-Gluc), (-)-EGCG-3"-O-glucuronide (EGCG-3"-Gluc), (-)-EGCG-3'-O-glucuronide (EGCG-3'-Gluc), (-)-EGCG-7-O-glucuronide (EGCG-7-Gluc), (-)-EGC-3'-O-glucuronide (EGC-3'-Gluc), and (-)-EGC-7-O-glucuronide (EGC-7-Gluc) were biosynthesized; their structures were identified by NMR and mass spectrum in our laboratory (Lu et al., 2003). SAM, dithiothreitol, (-)-epicatechin, L-DOPA, L-3-O-methyl-Dopa (3-MeDOPA), UGP-glucuronic acid (UDPGA), porcine liver COMT (EC 2.1.16), and 1,1-diphenyl-2-picrylhydrazyl were purchased from Sigma-Aldrich (St. Louis, MO). The protein assay kit was obtained from Bio-Rad Labs (Hercules, CA). Other reagents and HPLC-grade solvents were purchased from VWR Scientific Products (West Chester, PA). Pooled human liver cytosol samples were obtained from BD Biosciences (Woburn, MA) in a frozen well preserved state.

Treatment of Animals and Preparation of Cytosol. Eight-week-old female CF-1 mice and male Sprague-Dawley rats were purchased from Jackson Laboratory (Bar Harbor, ME). All mice and rats were fed Purina Laboratory Chow 5001 (Purina, St. Louis, MO) diet and allowed one-week acclimation. Afterwards, eight mice and five rats were sacrificed. Liver and intestine were promptly removed, washed with ice-cold saline, and samples were pooled for preparation of microsomes and cytosol by differential ultracentrifugation (Hong et al., 1989). The protein content was determined according to the instruction of the Bio-Rad protein assay kit.

Methylation of Catechins and Their Glucuronides by Cytosolic COMT. The methylation of substrates by cytosolic COMT was conducted at conditions similar to our previous studies (Meng et al., 2001). Incubation mixture contained 0.1 mg hepatic or intestinal cytosolic protein, different concentrations of catechins or their glucuronides, 10 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol, 1.2 mM MgCl2, and 0.05-0.2 mM SAM in a total volume of 0.1 ml. To determine the IC50 of COMT inhibition, 0.1 mg rat hepatic cytosolic protein, 10 µM EGC, 0.05 mM SAM, and different concentrations (0.05-25.6 µM) of COMT inhibitors were used in a 10-min incubation. For studies on the mechanism of inhibition of COMT, 0.1-mg rat hepatic cytosolic protein, 0.2 mM SAM and different concentrations (10, 20, 40, 60, 100, and 160 µM) of EGC were used as substrates in a 5-min incubation; alternatively, 1.0 mM EGC and different concentrations of SAM (12.5, 25, 50, 100, 200, and 400 µM) were used. The reaction was started by the addition of SAM and stopped by adding 0.1 ml of ice-cold methanol containing 1% ascorbic acid after incubation at 37°C for the stated length of time. After centrifugation, 20 µl of the supernatant was used for the analysis of methylation products with HPLC or LC/MS/MS.

Methylation of L-DOPA by Porcine Liver COMT. Similar conditions with slight modifications were used for enzymatic methylation of L-DOPA. The reaction mixture consisted of 100 µM L-DOPA, 25 units porcine liver COMT, 60 µM SAM, 1.2 mM MgCl2, and 1.0 mM dithiothreitol in a final volume of 100 µl of 10 mM Tris-HCl buffer (pH 7.4). The reaction was stopped by adding 20 µl of perchloric acid after incubation for 30 min at 37°C. After vortexing and centrifugation, 50 µl of aqueous phase was injected onto HPLC to analyze the conversion of L-DOPA to 3-MeDOPA.

Glucuronidation of Methylated Catechins in Liver Microsomes. The reaction mixture consisted of 0.2 mg of mouse liver microsomal protein, different concentrations of methylated catechins, 1 mM UDPGA, 0.15 mM ascorbic acid, 2 mM MgCl2, 0.02% Triton-X-100, 1 mM saccharic acid-1,4-lactone, and 40 mM Tris-HCl buffer (pH 7.5) in a final volume of 100 µl. After incubation for 30 min at 37°C, the reaction was stopped by 100 µl of ice-cold methanol containing 1% ascorbic acid. After centrifugation at 10,000g for 10 min, 160 µl of the supernatant was vortexed with 200 µl of methylene chloride to remove Triton-X-100 and lipids. After centrifugation, the supernatant (10 µl) was analyzed by LC/MS/MS.

HPLC Analysis of Methylated Catechins and 3-MeDOPA. Our previous HPLC method (Meng et al., 2001) was used with modifications. The eluent started with 4% buffer B and 96% buffer A from 0 to 7 min, and then the binary linear gradient was changed by increasing buffer B to 22% at 25 min, 38% at 31 min, 41% at 37 min, and 98% at 38 min. It was maintained at 98% buffer B from 38 to 42 min and changed back to 4% at 43 min. The total running time was 54 min. A coulochem electrode array system with potential settings of -100, 100, 300, and 500 mV in the four channels was used to analyze catechins and their metabolites simultaneously. EGCG, EGC, 4"-MeEGCG, and their glucuronides were detected in channel 2, whereas 4',4"-DiMeEGCG, 4'-MeEGC, 3'-MeEGC, and their glucuronides could only be detected in channel 3 and 4 (Fig. 1). The detection limits (S/N = 3) of catechins and methylated catechins were 5 to 10 ng/ml. 3-MeDOPA was detected with retention times of 7.5 min in channel 3. The detection limit was also 5 to 10 ng/ml. The formations of 4"-MeEGCG, 4',4"-DiMeEGCG, 4'-MeEGC, 3'-MeEGC, and 3-MeDOPA were quantified with standard curves of these compounds.

LC/MS/MS Analysis of Methylated Catechins. The HPLC conditions were similar to previously described (Li et al., 2001) with modifications. The LC/MS/MS system consisted of a Finnigan SpectraSystem separation module equipped with a AS3000 refrigerated autosampler, a P4000 gradient pump, and a UV6000LP UV/Vis detector, followed by a Finnigan LCQDeca mass detector (San Jose, CA) fitted with an electrospray ionization source. A 3-µm Supelcosil HS C18 column (75 mm × 2.1 mm i.d.; Supelco, Bellefonte, PA) was used, and the flow rate was maintained at 0.2 ml/min. The binary gradient was 100% A (0-3 min), 90% A to 67% A (3-17 min), 67% A to 10% A (17-30 min), and 100% A (30-40 min). Solvent A was methanol/water (5:495, v/v), and Solvent B was methanol/water (450:50, v/v). The LCQDeca ion trap mass detector was operated in negative ion polarity mode, and the acquisition time was set for 40 min. EGCG or EGC-7-Gluc was used for the tune-up to select optimal settings for the MS detector to detect methylated catechins or methylation products of catechin glucuronides, respectively. The deprotonated molecules exhibiting the same molecular mass as the target catechin conjugates were selected and dissociated with 30% relative collision energy to form product ions. If a deprotonated aglycone ion of tea catechins or their metabolites appeared in the product-ion mass spectrum, the assigned molecule was identified as a certain methylation product or glucuronide of catechins. The chemical identity of the methylated catechins was determined by comparing their retention time and fragment patterns with those of the standards. The deprotonated aglycone ions for EGCG, EGC, 4'-MeEGC, 4"-MeEGCG, and 4',4"-DiMeEGCG were at m/z 457, 305, 319, 471, and 485, respectively.

Data Analyses. The kinetic parameters (Km and Vmax) of methylation were calculated with GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). The values obtained represent the best-fit values ± standard error. Two-way analysis of variance was performed with Microsoft Excel software (Microsoft, Redmond, WA) for evaluating statistical differences between different groups of data. Differences were considered significant when P 

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