Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1

Abstract

ABSTRACT:Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono-and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a K m of ϳ0.2 M. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05-2 M), with the monoglucuronide being the predominant species (ϳ70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system.

Correlation between Bilirubin Glucuronidation and Estradiol-3-Gluronidation in the Presence of Model UDP-Glucuronosyltransferase 1A1 Substrates/Inhibitors

“…Methods for quantitation of bilirubin monoglucuronides and diglucuronide were as described previously (Zhou et al, 2010a). Estradiol-3-glucuronide was quantified either through an LC-MS method with an LC-10ADVP system (Shimadzu, Columbia, MD) or through a capillary LC-MS/MS method (for estradiol and bilirubin interaction experiments only) on a TSQ Quantum Discovery Max MS system (Thermo Fisher Scientific) coupled with an Agilent 1100 capillary HPLC system (Agilent Technologies, Santa Clara, CA).…”

“…Supernatant from each sample (5 l) were injected onto the HPLC system for quantification. Detailed incubation conditions for bilirubin glucuronidation were as described previously (Zhou et al, 2010a). Bilirubin incubations were conducted with 0.05 mg/ml protein for 5 min.…”

Estimating the Differences of UGT1A1 Activity in Recombinant UGT1A1 Enzyme, Human Liver Microsomes and Rat Liver Microsome Incubation Systems <i>in Vitro</i>

“…Peaks were assigned based on the lipophilicity of the glucuronides and retention times reported previously. [12][13][14]16,20) Bilirubin and all metabolites were also identified using MS in the present study (Figs. 3, 4).…”

“…The parameters V max and K m were defined as the maximum velocity and the substrate concentration at which the velocity equaled half of the maximum velocity, respectively. 13,14,[18][19][20] The intrinsic clearance (CL int ) was calculated as V max /K m when the reaction exhibited Michaelis-Menten kinetics. …”

“…Because commercial products to measure bilirubin glucuronidation are unavailable, these studies only estimated the levels of bilirubin and its main metabolites using UV absorption without further mass spectra-based identification. [12][13][14] As the two-phase bilirubin metabolism reaction involves glucuronidation and sulfation, its glucuronides and sulfides have the same molar extinction coefficient as does bilirubin; therefore, bilirubin metabolism cannot be confirmed using only UV. Because bilirubin metabolism is a sequential reaction that produces two monoglucuronides and a diglucuronide, it is important to optimize the incubation conditions, such as protein concentration and incubation time, to guarantee the initial rate.…”