Imipramine N-Glucuronidation in Human Liver Microsomes: Biphasic Kinetics and Characterization of UDP-Glucuronosyltransferase Isoforms

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Abstract
A method for the direct determination of imipramineN-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 imipramineN-glucuronidation in human liver microsomes were biphasic. For the high-affinity component, theKm was 97.2 ± 39.4 μM and theVmax was 0.29 ± 0.03 nmol/min/mg of protein. For the low-affinity component, theKm was 0.70 ± 0.29 mM and theVmax 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 imipramineN-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 aKm of >1 mM. In contrast, in UGT1A4 expressed in human B-lymphoblastoid cells,Km was 0.71 ± 0.36 mM and theVmax was 0.11 ± 0.03 nmol/min/mg of protein. Interindividual differences in the imipramineN-glucuronidation in liver microsomes from 14 humans were at most 2.5-fold. The imipramineN-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.