Postmortem protein stability investigations of the human hepatic drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP3A4 using mass spectrometry.

Variability in expression and activity of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes can play a causal role in fatal intoxication cases and is thus of forensic interest. We investigated the feasibility of LC-MS/MS based quantification and in vitro enzyme activity measurements of two major drug-metabolizing enzymes CYP1A2 and CYP3A4 in postmortem human liver microsomes (HLM). In autopsy cases (postmortem interval 24-36 h) we found CYP1A2 and CYP3A4 protein levels similar to that measured in a non-decayed reference HLM pool, whereas CYP1A2 and CYP3A4 enzyme activities were absent or severely decreased. Stability studies showed that CYP1A2 and CYP3A4 protein abundances were relatively stable in tissue stored in vitro for up to seven days at 4 °C. When tissue was stored for more than one day at 21 °C variable and case-specific decay patterns were observed, and CYP abundances declined especially after 3-4 days storage. Investigations of 50 autopsy cases revealed mean CYP1A2 and CYP3A4 levels of 49 and 47 pmol per mg HLM protein and inter-individual variabilities similar to those reported in other studies. This study supports postmortem quantification of CYP proteins in autopsy hepatic tissue by mass spectrometry. SIGNIFICANCE: This study indicates that MS-based detection of drug-metabolizing cytochrome P450 (CYP) proteins is achievable in postmortem hepatic tissue and that acceptable quantification data are obtainable but dependent on the storage conditions and postmortem sampling time. CYP abundance data could contribute to a conceivable way of assessing individual CYP activity phenotypes in a postmortem context.

Serum Amyloid P Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels.

The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.