A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man: Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin

Abstract

The O-deethylation of 7-ethoxy-4-trinuoromethylcoumarin (EFC) by liver microsomes has been assessed as a method for monitoring the activity of cytochrome P450. The principle advantage of this substrate is the formation of a fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC) which can be assayed directly in the reaction medium. For rat microsomes the deethylated product was confirmed as the main metabolite, the reaction rate was linear with respect to both time and microsomal protein concentration and was independent of small changes in the added co-factors. A linear formation rate for the deethylated metabolite was also confirmed with dog and human microsomes. The intraassay precision for rat, dog and human microsomes was 3, 5 and 4%, respectively. Hanes transformations of the dog and human data showed two phases, in contrast to a linear decline seen for the rat. Hybrid parameters for V max and K m , calculated from the apparently linear portions of these curves, gave interday SD for the V max of rat, dog and man of 2. 14 and 4%, respectively, and approximately 15% for the K m, in all species. The V max, in rat, dog and human microsomes was 1.4 ± 0.2, 4.3 ± 1.5 and 0.9 ± 0.5 nmol HFC min nmol P450, respectively. The K m , was 11.0 ± 3.1, 67 ± 19 and 6.8 ± 2.5 μM, respectively. Direct evidence that at least two isoenzymes (cytochrome P450 1A2 and 2E1) metabolize EFC was obtained by experiments with competitive, suicide and immuno-inhihitors. Compared with ethoxycoumarin, the involvement of P450 2E1 in 0-deethylation seemed similar in the rat. In conclusion, EFC provides a straightforward and reproducible assay for microsomal enzyme activity, requiring at most 25 pmol mL of cytochrome P450.