Trimethadione N-demethylation by rat liver CYP2E1 in vitro.

: Trimethadione (TMO) is a model drug utilized for estimation of hepatic metabolism in clinical studies, and it was reported that TMO N-demethylase activity was inhibited by CYP2E1 inhibitors and substrates in rat in vivo. This study was performed to investigate the involvement of the CYP2E1 subfamily on TMO N-demethylation in vitro and to clarify these inhibitory mechanisms. The effects of acetone (AC), imidazole (IM) and N-nitrosodimethylamine (NDA) on TMO N-demethylation were studied in vitro. Rat hepatic microsomal fractions were employed as the enzyme source of TMO N-demethylase and the activity was determined by the production of dimethadione (DMO). DMO was analyzed by a GC/FTD equipped with a narrow-bore capillary column. TMO N-demethylation was biphasic by the graphic analysis of Eadie-Hofstee plots; this suggests the involvement of at least two enzymes in TMO metabolism in the rat. The kinetic parameters for the formation of DMO were analyzed graphically using double-reciprocal plots. The apparent K(m1), K(m2) and Vmax1, Vmax2 values for DMO formation were 4, 20 mM and 182, 595 pmol/mg protein/min, respectively. AC and IM inhibited TMO N-demethylase activity competetively. However, mixed inhibition kinetics was observed by NDA. Furthermore, TMO N-demethylase activity was inhibited by antiserum to CYP2E1 by 62% and CYP3A2 by 46%. These results indicate that the CYP2E1 subfamily is the major enzyme involved in TMO N-demethylation in rat in vitro although the CYP3A2 is also involved in this transformation.