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


CONCLUSIONS
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.
Qian
Ming-Rong
MR
Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, China.
Zeng
Su
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001711:Biotransformation; D002478:Cells, Cultured; D020719:Glucuronides / Q000096:biosynthesis / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D007099:Imipramine / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology
D020719:Glucuronides; D011994:Recombinant Proteins; C019787:bilirubin glucuronoside glucuronosyltransferase; C514870:imipramine N-glucuronide; D014453:Glucuronosyltransferase; D007099:Imipramine