The aim of this study was to optimize methods for quantifying 13 uridine 59-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide-based quantification using liquid chromatography-tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37°C, 60°C, and 95°C) followed by tryptic digestion for 2-20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal.

ABSTRACT:The mass balance and metabolite profiles of 2-(2- total of 10 metabolites were found in urine. On the basis of the metabolites found in urine, major primary metabolic reactions of mirabegron were estimated to be amide hydrolysis (M5, M16, and M17), accounting for 48% of the identified metabolites in urine, followed by glucuronidation (M11, M12, M13, and M14) and N-dealkylation or oxidation of the secondary amine (M8, M9, and M15), accounting for 34 and 18% of the identified metabolites, respectively. In feces, the radioactivity was recovered almost entirely as the unchanged form. Eight of the metabolites characterized in urine were also observed in plasma. These findings indicate that mirabegron, administered as a solution, is rapidly absorbed after oral administration, circulates in plasma as the unchanged form and metabolites, and is recovered in urine and feces mainly as the unchanged form.amino

ABSTRACT:The aims of this study were to develop a robust method for simultaneous quantification of carboxylesterases (CESs) 1 and 2 and to quantify those absolute protein levels in human liver tissue fractions. Unique peptide fragments of CES1 and CES2 in tryptically digested human liver microsomes (HLMs) and cytosol (HLC) were simultaneously quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using corresponding stable isotope-labeled peptides as internal standards. Bovine serum albumin was used as a blank matrix for the calibration curve samples. Our procedure showed good digestion efficiency, sensitivity, linearity of calibration curve, and reproducibility. The protein levels of CES1 and CES2 in 16 individual HLMs varied 4.7-fold (171-801 pmol/mg) and 3.5-fold (16.3-57.2 pmol/mg), respectively, that are approximately 10 times higher than the expression levels in HLC. The CES1/CES2 level ratio varied substantially from 3.0 to 25, and the correlation between the protein levels of CES1 and CES2 was negative, indicating significant interindividual variability and independence in their expression levels. CES1 levels significantly correlated with hydrolysis of the CES1 substrates, clopidogrel (5 M) and oxybutynin (10 M), whereas CES2 levels correlated strongly with hydrolysis of the CES2 substrate, irinotecan (1 M), indicating that quantified protein levels are highly reliable. This is the first report to demonstrate the absolute protein levels of CESs quantified by LC-MS/MS.

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.