Absolute quantification of UGT1A1 in various tissues and cell lines using isotope label-free UPLC-MS/MS method determines its turnover number and correlates with its glucuronidation activities

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• 作者：Beibei Xu ; Song Gao ; Baojian Wu ; Taijun Yin ; Ming Hu
• 关键词：UGT ; uridine 5&prime ; -diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase) ; CYP ; cytochrome P450 ; UPLC ; ultra performance liquid chromatography ; MRM ; multiple reaction monitoring ; DP ; declustering potential ; CE ; collision energy ; CXP ; collision cell exit potential ; LLOQ ; lower limit of quantification ; HLM ; human liver microsome ; RLM ; rat liver microsome ; PTM ; post translational modification
• 刊名：Journal of Pharmaceutical and Biomedical Analysis
• 出版年：25 January, 2014
• 年：2014
• 卷：88
• 期：Complete
• 页码：180-190
• 全文大小：2244 K

文摘

Uridine 5鈥?diphosphate-glucuronosyltransferase (UGT)1A1 is a major phase II metabolism enzyme responsible for glucuronidation of drugs and endogenous compounds. The purpose of this study was to determine the expression level of UGT1A1 in human liver microsomes and human cell lines by using an isotope label-free LC-MS/MS method. A Waters Ultra performance liquid chromatography (UPLC) system coupled with an API 5500Qtrap mass spectrometer was used for the analysis. Two signature peptides (Pep-1, and Pep-2) were employed to quantify UGT1A1 by multiple reaction monitoring (MRM) approach. Standard addition method was used to validate the assay to account for the matrix effect. 17尾-Estradiol was used as the marker substrate to determine UGT1A1 activities. The validated method has a linear range of 200-0.0195 nM for both signature peptides. The precision, accuracy, and matrix effect were in acceptable ranges. UGT1A1 expression levels were then determined using 8 individual human liver microsomes, a pooled human liver microsomes, three UGT1A1 genotyped human liver microsomes, and four cell lines (Caco-2, MCF-7, Hela, and HepG2). The correlations study showed that the UGT1A1 protein levels were strongly correlated with its glucuronidation activities in human liver microsomes (R² = 0.85) and in microsomes prepared from cell lines (R² = 0.95). Isotope-labeled peptides were not necessary for LC-MS/MS quantitation of proteins. The isotope label-free absolute quantification method used here had good accuracy, sensitivity, linear range, and reproducibility, and were used successfully for the accurate determination of UGT1A1 from tissues and cell lines.