Reaction phenotyping substrate concentration, selecting

Prior to P450 reaction phenotyping or inhibition experiments, it is important to determine enzyme kinetic parameters such as Km and Umax for the formation of selected metabolites that are subjected to quantitative analysis by LC-MS. For example, -warfarin is catalyzed by CYP2C9 to a specific metabolite, 7-hydroxy-5 -warfarin (Fig. 15.13). Thus, a CYP2C9 inhibition assay is developed based on the reaction. In the assay, Y-warfarin is incubated with HLM in the presence of a test compound, followed by quantification of 7-hydroxy-5 -warfarin by LC—MS (Zhang et ah, 2001). To set up this assay in our lab, enzyme kinetics for the formation of 7-hydroxy-iS-warfarin in HLM was determined. In this experiment, warfarin was incubated at concentrations from 0 to 250 >M with HLM at optimized conditions. Rates of 7-hydroxy-S -warfarin formation at various substrate concentrations were determined as shown in Figure 15.13a, from which Km and Umax values were calculated. The warfarin assay represented an analytical challenge since the turnover of warfarin in the HLM system was extremely low. To be able to quantitatively determine low concentrations of 7-hydroxy-5 -warfarin in the incubations, a very sensitive LC—MS method that used MRM with a 4000 QTRAP has been developed (Fig. 15.13a). 

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