Lack of low Km diazepam N-demethylase in livers of poor metabolizers for S-mephenytoin 4'-hydroxylation

Pharmacogenetics. 1994 Dec;4(6):323-31. doi: 10.1097/00008571-199412000-00005.


Metabolism of diazepam was studied in vitro to identify the forms of cytochrome P450 (CYP) responsible for N-demethylation (nordazepam formation) and 3-hydroxylation (temazepam formation), using liver microsomes obtained from extensive (EM) and poor metabolizers (PM) for S-mephenytoin 4'-hydroxylation. Involvement of at least two P450 forms in diazepam N-demethylation was suggested by a biphasic pattern in Lineweaver-Burk and Eadie-Hofstee plots from the EM, whereas a monophasic pattern was observed from the PM liver microsomes. The kinetic parameters for the N-demethylation in the EM group were: Km 1, 19.4 +/- 0.4 microM; Vmax 1, 0.27 +/- 0.04 nmol min-1 per mg protein; Km 2, 346 +/- 34 microM; Vmax2, 1.82 +/- 0.63 nmol min-1 per mg protein (n = 3, mean +/- SD). The PM group showed the mean values of Km and Vmax (Km, 319 +/- 30 microM; Vmax, 1.49 +/- 0.62 nmol min-1 per mg protein) (n = 3) similar to those of Km2 and Vmax2 in the EM group. An antibody raised against CYP2C9 (anti-human CYP2C) strongly inhibited diazepam N-demethylation in EM liver microsomes at a low substrate concentration (20 microM). However, the anti-human CYP2C showed no clear inhibition of N-demethylation in EM liver microsomes at a high substrate concentration (200 microM). Diazepam N-demethylation in PM liver microsomes was not clearly inhibited by the anti-human CYP2C at either the low or high substrate concentrations. These data suggest that different P450 forms mediated diazepam N-demethylation in EM and PM liver microsomes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies
  • Aryl Hydrocarbon Hydroxylases*
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2E1
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / immunology
  • Cytochrome P-450 Enzyme System / metabolism*
  • Diazepam / metabolism
  • Humans
  • Hydroxylation
  • In Vitro Techniques
  • Kinetics
  • Mephenytoin / metabolism*
  • Microsomes, Liver / metabolism*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / immunology
  • Mixed Function Oxygenases / metabolism
  • Oxidoreductases, N-Demethylating / genetics
  • Oxidoreductases, N-Demethylating / immunology
  • Oxidoreductases, N-Demethylating / metabolism
  • Phenotype


  • Antibodies
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Cytochrome P-450 CYP2E1
  • Aryl Hydrocarbon Hydroxylases
  • CYP2C19 protein, human
  • Cytochrome P-450 CYP2C19
  • Oxidoreductases, N-Demethylating
  • diazepam N-demethylase
  • Diazepam
  • Mephenytoin