title: Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry.
abstract: This study aimed at establishing a sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM-MS measurement were also determined. The method was applied to liver samples from ten non-cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM-based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM-MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.
authors.affiliation: Laboratory of Molecular Pharmacology, CHU de Québec Research Centre and the Faculty of Pharmacy, Laval University, Québec, Canada.
authors.forename: Anna
authors.initials: A
authors.lastname: Cieślak
authors.affiliation: Proteomics Platform of the Québec Genomics Center, CHU de Québec Research Centre, Québec, Canada.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Kelly
authors.affiliation: Laboratory of Molecular Pharmacology, CHU de Québec Research Centre and the Faculty of Pharmacy, Laval University, Québec, Canada.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Trottier
authors.affiliation: Laboratory of Molecular Pharmacology, CHU de Québec Research Centre and the Faculty of Pharmacy, Laval University, Québec, Canada.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Verreault
authors.affiliation: Liver Research Laboratories, Pomeranian Medical University, Szczecin, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Wunsch
authors.affiliation: Liver Research Laboratories, Pomeranian Medical University, Szczecin, Poland.|Liver and Internal Medicine Unit, Department of Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Milkiewicz
authors.affiliation: Proteomics Platform of the Québec Genomics Center, CHU de Québec Research Centre, Québec, Canada.
authors.forename: Guy
authors.initials: G
authors.lastname: Poirier
authors.affiliation: Proteomics Platform of the Québec Genomics Center, CHU de Québec Research Centre, Québec, Canada.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Droit
authors.affiliation: Laboratory of Molecular Pharmacology, CHU de Québec Research Centre and the Faculty of Pharmacy, Laval University, Québec, Canada. olivier.barbier@crchul.ulaval.ca.
authors.forename: Olivier
authors.initials: O
authors.lastname: Barbier
mesh_terms: D051544:Cytochrome P-450 CYP3A / Q000145:classification / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D006801:Humans; D008099:Liver / Q000293:injuries / Q000378:metabolism; D008862:Microsomes, Liver / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A
keywords: Alcoholic liver disease; Biomedicine; Cytochrome P450 (CYP)3A4; MRM-MS quantification; Primary biliary cholangitis; Primary sclerosing cholangitis