Biosynthesis of imipramine glucuronide and characterization of imipramine glucuronidation catalyzed by recombinant UGT1A4

Acta Pharmacol Sin. 2006 May;27(5):623-8. doi: 10.1111/j.1745-7254.2006.00314.x.


Aim: To study the profile of imipramine N+-glucuronidation using homogenates of recombinant uridine-5'-diphosphoglucuronosyltransferase 1A4 (UGT1A4) from baculovirus-infected sf9 cells.

Methods: Recombinant UGT1A4 was obtained from sf9 cells infected with recombinant baculovirus. Imipramine N(+)-glucuronide was biosynthesized by incubating imipramine with recombinant UGT1A4 and then purified with solid-phase cartridges. A reversed phase-high pressure liquid chromatography (RP-HPLC) assay method was used to directly measure the concentration of imipramine and its metabolite, imipramine N(+)-glucuronide, with p-nitrophenol as the internal standard. The validated method was used to characterize the activity of recombinant UGT1A4 and carry out kinetic studies on imipramine glucuronidation in vitro.

Results: The high concentration of imipramine inhibited glucuronide conjugation, so the formula V=V(max).S/(Km+S+S(2)/K(i)) was used to calculate the parameters, using MATLAB software. The values of apparent K(m), K(i), and V(max) for imipramine glucuronidation via UGT1A4 were 1.39+/-0.09 mmol/L, 6.24+/-0.45 mmol/L and 453.81+/-32.12 pmol/min per mg cell homogenate (n=3), respectively.

Conclusion: As a specific substrate of UGT1A4, imipramine was used as a convenient method to characterize the activity of recombinant UGT1A4 by using HPLC. Furthermore, the profile of imipramine glucuronidation was evaluated by using recombinant UGT1A4 in vitro.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Biotransformation
  • Cells, Cultured
  • Glucuronides / biosynthesis
  • Glucuronides / metabolism*
  • Glucuronosyltransferase / genetics
  • Glucuronosyltransferase / metabolism*
  • Imipramine / analogs & derivatives*
  • Imipramine / metabolism*
  • Kinetics
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spodoptera / cytology


  • Glucuronides
  • Recombinant Proteins
  • bilirubin glucuronoside glucuronosyltransferase
  • imipramine N-glucuronide
  • Glucuronosyltransferase
  • Imipramine