Simultaneous Quantification of the Abundance of Several Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzymes in Human Liver Microsomes Using Multiplexed Targeted Proteomics

Abstract

Cytochrome P450 (P450) and uridine 59-diphospho-glucuronosyltransferase (UGT) enzymes mediate a major proportion of phase I and phase II metabolism of xenobiotics. In vitro-in vivo extrapolation (IVIVE) of hepatic clearance in conjunction with physiologicallybased pharmacokinetics (PBPK) has become common practice in drug development. However, prediction of xenobiotic kinetics in virtual populations requires knowledge of both enzyme abundances and the extent to which these correlate. A multiplexed quantification concatemer (QconCAT) strategy was used in this study to quantify the expression of several P450 and UGT enzymes simultaneously and to establish correlations between various enzyme abundances in 24 individual liver samples (ages 27-66, 14 male ), expressed as mean 6 S.D. Previous reports of correlations in expression of various P450 (CYP3A4/CYP3A5*1/*3, CYP2C8/CYP2C9, and CYP3A4/CYP2B6) were confirmed. New correlations were demonstrated between UGTs [including UGT1A6/UGT1A9 (r s = 0.82, P < 0.0001) and UGT2B4/UGT2B15 (r s = 0.71, P < 0.0001)]. Expression of some P450 and UGT enzymes were shown to be correlated [including CYP1A2/UGT2B4 (r s = 0.67, P = 0.0002)]. The expression of CYP3A5 in individuals with *1/*3 genotype (n = 11) was higher than those with *3/*3 genotype (n = 10) (P < 0.0001). No significant effect of gender or history of smoking or alcohol use on enzyme expression was observed; however, expression of several enzymes declined with age. The correlation matrix produced for the first time by this study can be used to generate more realistic virtual populations with respect to abundance of various enzymes.

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…Details of methodologies used by these laboratories are provided in the respective reports (Fallon et al, 2013b;Achour et al, 2014a). Differences in methodological workflows are highlighted in Table 1.…”

“…All other reagents and solvents used were from standard suppliers and were of reagent or HPLC grade with all purities as defined by the manufacturer. Materials and chemicals used for the quantification of UGT abundances were as described previously (Fallon et al, 2013b;Achour et al, 2014a).…”

“…Sample preparation and LC-MS analysis were performed as described previously (Russell et al, 2013;Achour et al, 2014a). Briefly, protein concentrations in samples were estimated based on Bradford assay.…”

“…Several studies published recently reported different proteomic methodologies driven by advances in LC-MS technology (Fallon et al, 2008;Ohtsuki et al, 2012;Achour et al, 2014a;Vildhede et al, 2014;Harwood et al, 2015;Fallon et al, 2016). These methodologies focused on obtaining expression values from a range of mammalian tissues/organs (e.g., liver, intestine, kidneys) and in vitro systems (e.g., hepatocytes, Caco-2 cell lines), with a view to providing systems data for in vitro-in vivo extrapolation (IVIVE) of pharmacokinetic profiles using computerized physiologically based pharmacokinetic (PBPK) models (Rostami-Hodjegan, 2012;Bosgra et al, 2014).…”

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…It has rapidly advanced and made tremendous impact on a variety of biological fields. Recently, mass spectrometry (MS)-based targeted proteomics with selected reaction monitoring (SRM) has become a promising tool for relative and absolute quantification of proteins (6)(7)(8)(9)(10)(11)(12)(13). Traditionally, this SRM technique was used in conjunction with liquid chromatography (LC) to analyze small molecules such as drugs and their metabolites (14).…”