Simultaneous determination of bilirubin and its glucuronides in liver microsomes and recombinant UGT1A1 enzyme incubation systems by HPLC method and its application to bilirubin glucuronidation studies

Abstract

Bilirubin, an important endogenous substances and liver function index in humans, is primarily eliminated via UGT1A1-catalyzed glucuronidation. Instability of bilirubin and its glucuronides brings substantial technical challenges to conduct in vitro bilirubin glucuronidation assay. In the present study, we developed a simple and robust HPLC method for simultaneous determination of unconjugated bilirubin (UCB) and its multiple glucuronides, i.e. bilirubin monoglucuronides (BMGs, including BMG1 and BMG2 isomers) and diglucuronide (BDG) in rat liver microsomes (RLM), human liver microsomes (HLM) and recombinant human UGT1A1 enzyme (UGT1A1) incubation systems, and applied it to study in vitro bilirubin glucuronidation. UCB, BMG1, BMG2, BDG and their isomers in the incubation mixtures were successfully separated using a C18 column with UV detection at 450nm and mobile phase consisted of 0.1% formic acid in water and acetonitrile by a linear gradient elution program. Assay linearities of bilirubin were confirmed in the range 0.01-2μM. Precision of UCB, BMG1, BMG2 and BDG (n=5) at low, medium and high concentration was within the range of RSD 0.4-3.7%, accuracy expressed in the mean assay recoveries of them (n=5) ranged from 92.8±1.5% to 104.3±2.2% for intra- and inter-day assays and the mean extraction recoveries of them (n=5) were above 91.5±1.0%. Stability of bilirubin and its glucuronides was satisfactory at 37°C in the incubation solutions during the reaction (30min), 25°C for 24h and -70°C for 7d in the processed incubation samples with methanol. Furthermore, we established stable, reliable in vitro incubation systems and optimized the incubation conditions to characterize the kinetics of bilirubin glucuronidation by RLM, HLM and UGT1A1, respectively. The kinetic parameters of formation of total bilirubin glucuronides (TBG, the sum of BMG1, BMG2 and BDG) were as follows: Km of 0.45±0.016, 0.40±0.022, 0.44±0.018μM, Vmax of 2.65±0.057, 1.86±0.029, 2.95±0.036nmol/mg/min, CLint of 5.92±0.22, 4.70±0.079, 6.72±0.27mL/mg/min by RLM, HLM and UGT1A1, respectively. Bilirubin glucuronidation obeyed the Hill equation by RLM and the Michaelis-Menten equation by HLM and UGT1A1 in the range of substrate concentration selected, respectively. In addition, the relative proportions between BDG and BMGs were in connection with enzyme sources (e.g. RLM, HLM and UGT1A1) and bilirubin concentration.

Keywords: Bilirubin; Glucuronides; HPLC; Liver microsomes; UGT1A1.