title: Protein quantification of UDP-glucuronosyltransferases 1A1 and 2B7 in human liver microsomes by LC-MS/MS and correlation with glucuronidation activities.
abstract: The aims of this study were to quantify absolute protein levels of uridine 5'-diphosphate-glucuronosyltransferases (UGTs) 1A1 and 2B7 in human liver microsomes (HLMs) and to investigate their correlation with marker activities.        A quantification method for UGT1A1 and UGT2B7 in HLMs was developed. Unique tryptic peptides of UGT1A1 and UGT2B7 in tryptically digested HLMs were simultaneously quantified by liquid chromatography (LC) equipped with tandem mass spectrometry (MS) using corresponding stable isotope-labelled peptides as internal standards. Bovine serum albumin was used as a blank matrix for calibration curve samples. Our procedure had good digestion efficiency, sensitivity, calibration curve linearity, and reproducibility of digestion to quantification.        In 16 individual HLMs, the protein levels of UGT1A1 and UGT2B7 ranged from 6.50 to 44.6 pmol/mg and 4.45 to 18.2 pmol/mg, respectively. Estradiol 3β-glucuronidation correlated strongly with the UGT1A1 level, indicating its high reliability as a reaction marker. Both morphine 3-O- and 6-O-glucuronidation significantly correlated with UGT2B7 level. However, the intercept of the linear regression clearly indicates that morphine glucuronidation was mediated by other UGT isoforms in addition to UGT2B7.
authors.affiliation: Drug Metabolism Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Osaka, Japan. yuichiro.sato@astellas.com
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Sato
authors.forename: Masanori
authors.initials: M
authors.lastname: Nagata
authors.forename: Akio
authors.initials: A
authors.lastname: Kawamura
authors.forename: Aiji
authors.initials: A
authors.lastname: Miyashita
authors.forename: Takashi
authors.initials: T
authors.lastname: Usui
mesh_terms: D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000032:analysis*; D006801:Humans; D007553:Isotope Labeling; D008862:Microsomes, Liver / Q000201:enzymology*; D053719:Tandem Mass Spectrometry; D000099148:UGT1A1 Enzyme
chemical_list: D015415:Biomarkers; D004958:Estradiol; D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme; C025482:estradiol-3-glucuronide; C491474:UGT2B7 protein, human