| Assay Method Information | |
| | CYP Activity Assay |
| Description: | Inhibition of hepatic cytochromes P450 was assessed in human liver microsomes using a substrate-specific approach of monitoring metabolites formed by each specific CYP enzyme. Metabolic reactions included phenacetin-O-deethylation (1A2), tolbutamide methylhydroxylation (2C9), S-mephenytoin 4'-hydroxylation (2C19), dextrome-thorphan-O-deethylation (2D6), and testosterone-6-β-hydroxylation (3A4). Each substrate was added at a concentration less than or equal to the Km for themetabolic pathway, and microsomal protein concentrations and assay incubation times were optimized for each reaction to ensure linear metabolite formation based on initial rates (Table 1). Microsomes were suspended in phosphate buffer and incubated at 37 °C in the presence of probe substrates. Reactions were initiated by the addition of an NADPH-regenerating buffer system and were quenched at the appropriate times using acetonitrile. Samples were centrifuged and concentrations of metabolites assessed by LC/MS. |
| Affinity data for this assay | |
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