Methylation of catechins and procyanidins by rat and human catechol-O-methyltransferase: metabolite profiling and molecular modeling studies.
After oral administration of (-)-epicatechin to rats, three kinds of metabolites (M-1, M-2, and M-3) were detected in the urine. After isolation of the compounds by preparative high-performance liquid chromatography, structural analysis was carried out by mass spectrometry and NMR. As a result, two compounds, M-1 and M-2, were identified as (-)-epicatechin and 3'-O-methyl-(-)-epicatechin, respectively. M-3 was suggested to be a monomethylated (-)-epicatechin, but definitive elucidation was not possible because of its small quantity. Methylation of (-)-epicatechin with rat liver homogenates and subsequent structural analysis showed that M-3 was 4'-O-methyl-(-)-epicatechin. Identification of conjugated forms of the urinary metabolites also was attempted. Two conjugates in the urine were purified and analyzed by mass spectrometry and NMR. These conjugates were shown to be (-)-epicatechin-5-O-beta-glucuronide and 3'-O-methyl-(-)-epicatechin-5-O-beta-glucuronide, respectively. Metabolism and excretion of (-)-epicatechin were examined. (-)-Epicatechin and its methylated derivatives in the free forms were detected in plasma and urine, but not in bile. Significant differences in the excretion ratio of the conjugated forms of (-)-epicatechin and 3'-O-methyl-(-)-epicatechin were observed between urine and bile. Time-course analysis of (-)-epicatechin metabolites showed that the most predominant metabolites in plasma and urine were the conjugates of (-)-epicatechin and 3'-O-methyl-(-)-epicatechin, respectively, and the cumulative amount of the urinary metabolites excreted during the 24-h period was about 8% of the administered (-)-epicatechin.