Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

Abstract

ABSTRACT:The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDPglucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

Hepatic Basolateral Efflux Contributes Significantly to Rosuvastatin Disposition I: Characterization of Basolateral Versus Biliary Clearance Using a Novel Protocol in Sandwich-Cultured Hepatocytes

“…Therefore, the effects of these changes may have minimal overall impact on RSV CL Bile . Available data suggest that expression of MRP3 and MRP4 in rat and human SCH may be relatively stable over days in culture (Swift et al, 2010a;Ohtsuki et al, 2012;Schaefer et al, 2012). However, robust quantitative proteomics data for transport proteins that mediate CL BL are not yet available.…”

The emerging role of transport systems in liver function tests

“…Future studies should be designed such that they yield a clear definition and grading of posthepatectomy liver failure, which is currently not classified by a generally accepted scoring system (Rahbari et al, 2011). In all instances, future clinical studies should certainly involve longitudinal studies in individual patients, e.g., those listed for transplantation to overcome inherent limitations such as interindividual differences in transporter expression (Meier et al, 2006;Ohtsuki et al, 2011).…”

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

“…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).…”