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

The O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC) by live r 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 m icrosomes the deethylated product was confirmed as the main metabolite , the reaction rate was linear with respect to both time and microsoma l protein concentration and was independent of small changes in the ad ded co-factors. A linear formation rate for the deethylated metabolite was also confirmed with dog and human microsomes. The intra-assay pre cision for rat, dog and human microsomes was 3, 5 and 4%, respectively . Hanes transformations of the dog and human data showed two phases, i n contrast to a linear decline seen for the rat. Hybrid parameters for V(max) and K(m), calculated from the apparently linear portions of th ese curves. gave inter-day 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 sp ecies. 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 muM. respectively. Dire ct evidence that at least two isoenzymes (cytochrome P450 1A2 and 2E1) metabolize EFC was obtained by experiments with competitive, suicide and immuno-inhibitors. Compared with ethoxycoumarin, the involvement o f P450 2E1 in O-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.