Imipramine N-glucuronidation in human liver microsomes: biphasic kinetics and characterization of UDP-glucuronosyltransferase isoforms.

A method for the direct determination of imipramine N-glucuronidation in human liver microsomes by high-performance liquid chromatography with UV detection was developed. Imipramine was incubated with human liver microsomes and UDP-glucuronic acid. The Eadie-Hofstee plots of imipramine N-glucuronidation in human liver microsomes were biphasic. For the high-affinity component, the K(m) was 97.2 +/- 39.4 microM and the V(max) was 0.29 +/- 0.03 nmol/min/mg of protein. For the low-affinity component, the K(m) was 0.70 +/- 0.29 mM and the V(max) was 0.90 +/- 0.28 nmol/min/mg of protein. The imipramine N-glucuronosyltransferase activities were not detectable in two samples of human jejunum microsomes. Among recombinant UDP-glucuronosyltransferases (UGTs) in baculovirus-infected insect cells (Supersomes or Bacurosomes) or human B-lymphoblastoid cells tested in the present study (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15), only UGT1A4 showed imipramine N-glucuronosyltransferase activity. The activity in UGT1A4 Supersomes was higher than that in recombinant UGT1A4 expressed in human B-lymphoblastoid cells at all imipramine concentration tested. The kinetics of imipramine N-glucuronidation in UGT1A4 Supersomes did not fit the Michaelis-Menten plot, showing a K(m) of >1 mM. In contrast, in UGT1A4 expressed in human B-lymphoblastoid cells, K(m) was 0.71 +/- 0.36 mM and the V(max) was 0.11 +/- 0.03 nmol/min/mg of protein. Interindividual differences in the imipramine N-glucuronidation in liver microsomes from 14 humans were at most 2.5-fold. The imipramine N-glucuronosyltransferase activities in 11 human liver microsomes were significantly (r = 0.817, P < 0.005) correlated with the glucuronosyltransferase activities of trifluoperazine, a typical substrate of UGT1A4. This is the first report of the biphasic kinetics of imipramine N-glucuronide in human liver microsomes.