Studies on the n-demethylation and O-de-ethylation of ethylmorphine[6-³H] by male rat hepatic microsomes

A sensitive and convenient radioassay for the in vitro determination of ethylmorphine N-demethylase and O-de ethylase activity has been developed. Ethylmorphine[6-³H] was prepared by reduction of the corresponding morphinone in nearly quantitative yield. After incubation with hepatic microsomes from male rats, the reaction was terminated by the addition of 5 ml of acetone. The sample was saturated with potassium acetate and extracted twice with acetone giving complete extraction of the radiolabeled ethylmorphine and its metabolites. After the combined organic phases were evaporated. the samples were dissolved in methanol and applied to a Silica Gel GF plate with subsequent development in ethyl acetate-methanol-concentrated NH₄OH. The amount of radioactivity detected for the morphine and norethylmorphine bands at zero time was approximately 0.05% of the original amount of labeled ethylmorphine added to the incubation media. Similarly the K(m) values were 52 and 250 μM for the O- and N-dealkylation respectively, while the V(max) values were 5.0 and 1.8 nmol/mg of protein per min. Finally, with this assay we have observed constant specific activity for both the N- and O-dealkylation of ethylmorphine[6-³H] with as little as 10 μg of microsomal protein per ml of incubation media.