Bilirubin glucuronidation revisited: proper assay conditions to estimate enzyme kinetics with recombinant UGT1A1

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 ∼0.2 μM. 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 μM), with the monoglucuronide being the predominant species (∼70%). 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.

Fig. 1.

Chromatograms for bilirubin glucuronidation in the presence (A) or absence (B) of UDPGA. Incubations were conducted with 50 μM bilirubin at 0.5 mg/ml protein for 30 min in the presence or absence of UDPGA. Twenty microliters of the incubation supernatant were injected onto HPLC for analysis.

Fig. 2.

Substrate-concentration versus rate plots for total bilirubin glucuronide formation. A, incubations with 0.05 mg/ml protein for 5 min; B, incubations with 0.5 mg/ml protein for 6 min. The bars indicate the range of triplicate measurements. The embedded figures are Eadie-Hofstee plots for the same data. The Michaelis-Menten equation was fit to data in A, and the Hill equation was fit to data in B.

Fig. 3.

Effects of bilirubin concentration on the proportions of BMGs or BDG. A, incubations with 0.05 mg/ml protein for 5 min; B, incubations with 0.5 mg/ml protein for 6 min. ● represents BDG and ♦ represents BMG.

Fig. 4.

Kinetic scheme for bilirubin glucuronidation. k₁, k_-1, k₂, k₃, k₄, k_-4, k₅, and k_-5 refer to the rate constants of the corresponding reaction steps.