title: Estimating the Differences of UGT1A1 Activity in Recombinant UGT1A1 Enzyme, Human Liver Microsomes and Rat Liver Microsome Incubation Systems in Vitro.
abstract: Estimating the extent to which drugs inhibit uridine 5'-diphosphate-glucuronosyltransferases1A1 (UGT1A1) enzyme activity is important for predicting hepatotoxicity and neurotoxicity. UGT1A1 enzyme activity is commonly evaluated by detecting the elimination of bilirubin substrate or the generation of bilirubin glucuronides. However, the present methods are inadequate for accurately assessing bilirubin metabolism, selecting incubation conditions, and comparing different systems. Therefore, in our study, we first established a ultra-performance liquid chromatography (UPLC)-MS/MS method to identify bilirubin and bilirubin glucuronides. To ensure the reaction was linear, we performed assays to optimize the protein concentration and incubation time. Finally, we measured UGT1A1 activity using three different systems. Data revealed the optimum incubation conditions were 10 min with 0.5 mg/mL human liver microsomes (HLM), recombinant human UGT1A1 (rUGT1A1), and rat liver microsomes (RLM). Bilirubin glucuronidation obeyed Michaelis-Menten kinetics in all three systems. The diversity in bilirubin metabolism among species was revealed. rUGT1A1 had the strongest binding affinity for bilirubin, but the lowest metabolism velocity. Compared with the other systems, RLM exhibited a significant difference. It has the lowest CLint and the highest Km. The difference in parameters between three systems may be attributable to the species differences. In conclusion, these in vitro studies provide useful information regarding drug interactions and the prediction of toxicity for future studies.
authors.affiliation: National Institutes for Food and Drug Control.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Zhong
authors.initials: Z
authors.lastname: Dai
authors.forename: Binyu
authors.initials: B
authors.lastname: Wen
authors.forename: Shuangcheng
authors.initials: S
authors.lastname: Ma
authors.forename: Yujie
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001663:Bilirubin / Q000031:analogs & derivatives / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004347:Drug Interactions; D064420:Drug-Related Side Effects and Adverse Reactions; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D010601:Pharmacology, Clinical; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D000099148:UGT1A1 Enzyme
chemical_list: D001663:Bilirubin; D020719:Glucuronides; D014453:Glucuronosyltransferase; D011994:Recombinant Proteins; D000099148:UGT1A1 Enzyme; C016266:bilirubin glucuronate