Stability investigations of cytochrome P450 (CYP) enzymes immediately after death in a pig model support the applicability of postmortem hepatic CYP quantification

Abstract

Quantification of drug‐metabolizing cytochrome P450 (CYP) isoforms using LC–MS/MS has been proposed as a potential way of estimating antemortem CYP levels using postmortem tissue, but the postmortem stability of CYP proteins is incompletely investigated. If one can use data obtained from the analysis of postmortem specimens to inform physiologically based pharmacokinetic (PBPK) models this greatly increases the access to rare specimens among special subpopulations. In this study, we developed and validated an LC–MS/MS method for targeted CYP protein quantification in a porcine animal model to study postmortem stability. We measured 19.9–28.3 pmol CYP1A2, 50.3–66.2 pmol CYP2D25, 132.9–142.7 pmol CYP2E1, and 16.8–48 pmol CYP3A29 protein per mg PLM in nondegraded tissue. In tissue stored at 4°C, we found that the CYP protein levels were unaffected by degradation after 72 h. At 21°C CYP1A2, CYP2D25, and CYP2E1 protein levels were nearly unaffected by degradation after 24 h, whereas a loss of approximately 50% was seen after 48 h. At 21°C CYP3A29 had a loss of 50% at 24 h and 70% at 48 h exhibiting less postmortem stability. In vitro enzyme activity measurements in the same tissue stored at 21°C showed a 50% decrease after 24 h and a complete loss of enzyme activity after 48 h. When stored at 4°C, the in vitro enzyme activity decreased to 50% activity after 96 h. In conclusion, measuring CYP levels by an LC–MS/MS approach was clearly less affected by postmortem changes than an activity‐based approach. The found postmortem stability for 24 h at 21°C for 3 out of 4 CYP isoforms supports the use of properly stored postmortem tissue to inform PBPK models.

Sex- and Lifestyle-Related Factors are Associated with Altered Hepatic CYP Protein Levels in People Diagnosed with Mental Disorders

“…Both studies found PMI and temperature to be very important for the protein stability of the CYP isoforms, and low MPPGL has been associated with postmortem degradation (Hansen et al, 2019;Pedersen et al, 2021). In the study by Pedersen et al 2021, both MPPGL and the concentration of CYP isoforms (pmol/mg microsomal protein) decreased as the liver was putrefied. In the current investigated population, there was a clear positive correlation between MPPGL (using a rising MPPGL cut-off level) and CYP levels (Figure 2), which is in contrast to results by Zhang et al 2015(Zhang et al, 2015 who found no association between MPPGL content and CYP activities.…”

“…Another major limitation of this study is the likely presence of postmortem degradation in our dataset. The postmortem stability of CYP1A2 and CYP3A4 was previously investigated in a postmortem population (Hansen et al, 2019), and using a porcine animal model (Pedersen et al, 2021). Both studies found PMI and temperature to be very important for the protein stability of the CYP isoforms, and low MPPGL has been associated with postmortem degradation (Hansen et al, 2019;Pedersen et al, 2021).…”

“…The postmortem stability of CYP1A2 and CYP3A4 was previously investigated in a postmortem population (Hansen et al, 2019), and using a porcine animal model (Pedersen et al, 2021). Both studies found PMI and temperature to be very important for the protein stability of the CYP isoforms, and low MPPGL has been associated with postmortem degradation (Hansen et al, 2019;Pedersen et al, 2021). In the study by Pedersen et al 2021, both MPPGL and the concentration of CYP isoforms (pmol/mg microsomal protein) decreased as the liver was putrefied.…”

“…The liver samples were stored at -80 °C until further use. HLM was isolated as previously described (Pedersen et al, 2021). In brief, 500 mg of hepatic tissue was minced with a scalpel and homogenized together with cold homogenization buffer (0.1 M potassium phosphate buffer with 0.125 M potassium chloride and 1 mM EDTA, pH 7.5) in a glass homogenizer (Potter-Elvehjem type glass homogenizer, VWR) using a motor-driven (RW16 motor, IKA) Teflon pestle.…”

“…Previous studies have shown that quantification of CYP-enzymes can be performed in postmortem hepatic tissue by LC-MS/MS measurement of CYP isoform-specific peptides. Depending on the postmortem interval and temperature, CYP enzymes will degrade over time, but in many cases, meaningful quantification is possible and will reflect antemortem levels (Hansen et al, 2019;Pedersen et al, 2021).…”