Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases

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• 作者：Hakala ; Kati S. ; Suchanova ; Bohumila ; Luukkanen ; Leena ; Ketola ; Raimo A. ; Finel ; Moshe ; Kostiainen ; Risto
• 关键词：UDP-glucuronosyltransferases ; LC/ESI/MS/MS ; n-in-one cocktails ; Intrinsic clearance
• 刊名：Analytical Biochemistry
• 出版年：2005
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：June 1, 2005
• 卷：341
• 期：1
• 页码：105-112
• 文件大小：445 K

摘要

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl_int) as the slope of the linear portion of the Michaelis–Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17β-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl_int values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V_max/K_m values determined by estimating the enzyme kinetic parameters V_max and K_m from the Michaelis–Menten curve. When substrate concentrations were well below their K_m values, Cl_int values determined as the slope of the linear part of the Michaelis–Menten fitting correlated well with the values determined as V_max/K_m ratios from the Michaelis–Menten curve. The correlation between Cl_int values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl_int values as the slope of the linear part of the Michaelis–Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.