title: 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 5'-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 physiologically-based 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). Abundances were comparable to previously reported values, including CYP2C9 (40.0 ± 26.0 pmol mg(-1)), CYP2D6 (11.9 ± 13.2 pmol mg(-1)), CYP3A4 (68.1 ± 52.3 pmol mg(-1)), UGT1A1 (33.6 ± 34.0 pmol mg(-1)), and UGT2B7 (82.9 ± 36.1 pmol mg(-1)), expressed as mean ± 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.
authors.affiliation: Manchester Pharmacy School (B.A., M.R.R., J.B., A.R-H.), University of Manchester, Manchester, United Kingdom; Simcyp Limited (A.R-H.), a Certara Company, Sheffield, United Kingdom.
authors.forename: Brahim
authors.initials: B
authors.lastname: Achour
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Russell
authors.forename: Jill
authors.initials: J
authors.lastname: Barber
authors.forename: Amin
authors.initials: A
authors.lastname: Rostami-Hodjegan
mesh_terms: D000328:Adult; D000368:Aged; D000428:Alcohol Drinking / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005260:Female; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008875:Middle Aged; D015999:Multivariate Analysis; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D012727:Sex Characteristics; D012907:Smoking / Q000235:genetics / Q000378:metabolism
chemical_list: D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase