THE ROLE OF S-MEPHENYTOIN 4'-HYDROXYLASE IN IMIPRAMINE METABOLISM BY HUMAN LIVER-MICROSOMES - A 2-ENZYME KINETIC-ANALYSIS OF N-DEMETHYLATION AND 2-HYDROXYLATION

1 The metabolism of imipramine (N-demethylation and 2-hydroxylation) w as studied in relation to the activity of S-mephenytoin 4'-hydroxylase in human liver microsomes . 2 Eadie-Hofstee plots for the formation o f despiramine and 2-hydroxyimipramine were biphasic, suggesting that a t least two enzymes are involved in both the N-demethylation and 2-hyd roxylation of imipramine by human liver microsomes. 3 The respective m ean (+/- s.d.) kinetic parameters for the N-demethylation and 2-hydrox ylation of imipramine derived from a two-enzyme kinetic analysis were: K,1 = 1.1 +/- 0.4 and 1.6 +/- 0.6 mu M, V(max)1 = 0.11 +/- 0.03 and 0 .15 +/- 0.07 nmol mg(-1) min(-1) and V(max)1/K(m)1 = 0.10 +/- 0.02 and 0.09 +/- 0.04 ml mg(-1) min(-1); K(m)2 = 214 +/- 84 and 257 +/- 148 m u M, V(max)2 = 2.22 +/- 0.69 and 0.53 +/- 0.15 nmol mg(-1) min(-1), an d V(max)2/ K(m)2 = 0.011 +/- 0.001 and 0.003 +/- 0.002 ml mg(-1) min(- 1) 4 With regard to imipramine N-demethylation and 2-hydroxylation at 2 mu M (representing high-affinity reactions) and at 400 mu M (represe nting low-affinity reactions), only N-demethylation at 2 mu M showed a close correlation with the 4'-hydroxylation of S-mephenytoin (r(s) = 0.952, P < 0.01; n = 10 livers). 5 Concentrations up to 250 IJ M S-mep henytoin inhibited the N-demethylation of imipramine (2 mu M), but no further inhibition was observed using concentrations from 250 to 750 m u M. 6 Imipramine inhibited S-mephenytoin 4'-hydroxylation competitive ly with a K-i value of 12.5 mu M. 7 The results suggest that the cytoc hrome P450 responsible for the 4'-hydroxylation of S-mephenytoin is pa rtially involved in the metabolism of imipramine as one of the high-af finity components of its N-demethylation.