The present 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 3 transitions optimized on 1 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 10 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 5fmol, and was successfully applied for the absolute and relative quantification of the CYP3A4 in both the whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation is 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. Conclusion: The established method provides a sensitive tool to evaluate the enzymatically active is cytochrome P450 protein in patients with normal or chronic/severe hepatic injury.

Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using 3 transitions optimized on 1 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 study group consisted of human liver homogenates from 10 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).