Screening of six UGT enzyme activities in human liver microsomes using liquid chromatography/triple quadrupole mass spectrometry

Abstract

As a screening technique for inhibitory interactions of these six human liver UGT enzymes, this method will be useful for advancing mechanistic understanding of drug interactions.

Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5′-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

“…The method was modified from that of Joo et al [29]. Each incubation mixture was prepared in a final volume of 100 µL, as follows: pooled human liver microsomes (0.2 mg/mL), 5 mM UDPGA, 10 mM MgCl 2 , 50 mM Tris buffer (pH 7.4), various concentrations of aschantin in DMSO (final concentrations of 0.1-200 µM, DMSO less than 1% [v/v]), and the UGT enzyme-specific substrate of the cocktail set (A set: 0.5 µM SN-38, 2 µM chenodeoxycholic acid, and 0.5 µM trifluoperazine; B set: 1 µM N-acetylserotonin, 0.2 µM mycophenolic acid, and 1 µM naloxone).…”

Screening of non‐steroidal anti‐inflammatory drugs for inhibitory effects on the activities of six UDP‐glucuronosyltransferases (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) using LC‐MS/MS

“…The UGT enzyme activity in the individual incubation was in accordance with the results from cocktail incubations [8]. In addition, the IC 50 values of the known inhibitors for each UGT enzyme in each cocktail set were comparable to those obtained from the incubation of the individual substrates [8]. Briefly, HLMs were diluted in the incubation buffer to a concentration of 0.25 mg/ml microsomal protein and then activated by incubation in the presence of 25 μg alamethicin/ml for 15 min on ice.…”

“…This cocktail method has already been validated by comparing data obtained from the incubation of each individual probe substrate alone with data from the cocktail method. The UGT enzyme activity in the individual incubation was in accordance with the results from cocktail incubations [8]. In addition, the IC 50 values of the known inhibitors for each UGT enzyme in each cocktail set were comparable to those obtained from the incubation of the individual substrates [8].…”

“…SN-38 glucuronidation, chenodeoxycholic acid 24-acylglucuronidation, trifluoperazine N-glucuronidation, Nacetylserotonin glucuronidation, mycophenolic acid glucuronidation and naloxone 3-glucuronidation were determined as the probe activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively, using cocktail incubation and tandem mass spectrometry, as described previously [8]. This cocktail method has already been validated by comparing data obtained from the incubation of each individual probe substrate alone with data from the cocktail method.…”

“…Aliquots of the supernatants were analysed by LC-MS/MS. All glucuronide metabolites of the UGT isoform-specific substrates were measured by tandem mass spectrometry as described previously [8]. All incubations were performed in triplicate and mean values were used for analysis.…”