Determination of Absolute Protein Content of Hepatic CYP4F Enzymes in Human Liver Microsomes Using LC/MS/MS Methodology and Comparison with Immunoquantification and Enzyme Activity.

摘要

The purpose of the work presented herein was to determine the absolute protein content of multiple cytochrome P450 (CYP) enzymes in individual donor human liver microsome (HLM) samples. The CYP4F subfamily of enzymes have recently been identified to be involved in the metabolism of endogenous compounds (arachidonic acid, leukotriene B4), nutrients (vitamin K1 and vitamin E), and xenobiotics (parfuramidine (DB289), DB1230, fingolimod). The CYP3A subfamily of enzymes are known to metabolize a wide variety of physiologically and pharmaceutically important substances. The determination of the absolute enzyme protein content and the inter-individual variability in the expression of these enzymes is important in understanding the effects they may have on the disposition of both endogenous and exogenous substances. Therefore, the absolute enzyme protein content in 20 individual donor HLM was determined by LC/MS/MS for CYP4F2, CYP4F3B, CYP3A, CYP3A4, and CYP3A5. The contribution of CYP4F enzymes to the overall hepatic CYP "pie" was also determined. The observed enzyme protein values were then correlated with immunoquantified protein content and enzyme activity. The enzyme protein contents determined by LC/MS/MS were well correlated with immunoquantification results (r 2 ≥ 0.60) for both CYP4F and CYP3A. The CYP4F enzymes displayed significantly less (∼2- to 4-fold) variability than did the CYP3A enzymes (∼7- to 20-fold). The CYP4F protein contents did not correlate with primary metabolite formation rates for DB289 or DB1230. Chemical inhibition experiments were performed which provided additional evidence for the metabolism for DB289 and DB1230 by enzymes other than CYP4F enzymes. However, a lack of improved correlation for the chemical inhibition experiments suggest that the poor correlation observed for CYP4F protein content and metabolic activity is not solely due to the contributions of additional enzymes. The LC/MS/MS methodology used for the current study has thus been shown to provide a rapid and reproducible (CV ≤ 24%) method for quantitation of enzyme protein expression level that obviates the need for specific antibodies for immunoquantification, a major problem for the less commonly studied enzymes that share significant sequence homology such as the CYP4F enzymes.