Biosynthesis of imipramine glucuronide and characterization of imipramine glucuronidation catalyzed by recombinant UGT1A4
Abstract
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=VmaxdotS/(Km+S+S2/Ki) was used to calculate the parameters, using MATLAB software. The values of apparent Km, Ki and Vmax for imipramine glucuronidation via UGT1A4 were 1.39±0.09 mmol/L, 6.24±0.45 mmol/L and 453.81±32.12 pmol/minper mgcell 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.
Keywords:
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=VmaxdotS/(Km+S+S2/Ki) was used to calculate the parameters, using MATLAB software. The values of apparent Km, Ki and Vmax for imipramine glucuronidation via UGT1A4 were 1.39±0.09 mmol/L, 6.24±0.45 mmol/L and 453.81±32.12 pmol/minper mgcell 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.