Studies on the metabolic N- and O-demethylation of [6-³H]codeine

We have modified our radiometric assay for ethylmorphine N-demethylase to examine the metabolism of codeine. We find that the current assay gives excellent separation of metabolites with a zero time activity of 0.08%. The N- and O-demethylations are linear for up to 30 min with up to 1 mg/ml of microsomal protein. The pH profiles show slightly different maxima (N-demethylation, pH 8.0; O-demethylation, pH 7.8). The kinetic parameters for N-demethylation were markedly higher (V(max) = 5.6 nmol/min/mg protein; K(M) = 714 μM) than those for O-demethylase (V(max) = 0.75 nmol/min/mg protein; K(M) = 149 μM). These data suggest that the HCHO results primarily from the N-demethylase. Further, the differences in the kinetic parameters and the pH profile suggest that these two activities are catalyzed by different enzymatic systems.