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 Km 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.
Footnotes
This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM063215] (to T.S.T.); and an investigator-initiated grant from Bristol-Myers Squibb (to R.P.R.).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033829.
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ABBREVIATIONS:
- BMG
- bilirubin monoglucuronide
- BDG
- bilirubin diglucuronide
- HEK
- human embryonic kidney
- UDPGA
- uridine-diphosphate glucuronic acid
- HPLC
- high-performance liquid chromatography
- DMSO
- dimethyl sulfoxide
- LOQ
- limit of quantification.
- Received April 7, 2010.
- Accepted July 28, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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