Investigating the correlation between genotypes and hepatic protein expression of CYP2C9, CYP2C19, CYP2D6, and CYP3A5 using postmortem tissue from a Danish population.

The cytochrome (CYP) P450 family of enzymes plays a central role in the metabolism of many drugs. CYP genes are highly polymorphic, which is known to affect protein levels, but for some low frequent CYP genotypes the correlation between genotype and CYP protein expression is less established. In this study, we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes of 250 Danish individuals included in a postmortem study. For 116 of the individuals, the hepatic CYP protein levels were investigated by a proteomics approach. Overall, we found the postmortem genetic and proteomic data to be in agreement with those of other studies performed on fresh hepatic tissue, showing the usability of postmortem hepatic tissue for this type of investigation. For less investigated genotypes we could corroborate previously found results: 1) statistically significantly lower levels of hepatic CYP2C9 protein in individuals carrying the CYP2C9*3 variant compared to individuals with two wild type (wt) alleles, 2) comparable levels of CYP2C19 in CYP2C19*2/*17 and CYP2C19*1/*2 individuals, 3) reduced CYP2D6 protein levels in heterozygous individuals with the CYP2D6*3, CYP2D6*4, and CYP2D6*5 gene deletion variants, and 4) significantly lower levels of CYP3A5 protein in CYP3A5*3 homozygous individuals compared to individuals that were heterozygous for the CYP3A5*3 allele or homozygous individuals for the wt alleles. In conclusion, the use of postmortem tissue significantly increases the access to human specimens for research purposes, and postmortem proteomics can be used to investigate the link between CYP genotypes and hepatic protein expression. Significance Statement In tissue from a large postmortem cohort (n=250) we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes. Hepatic CYP protein levels were investigated in 116 individuals using a proteomics approach. For common genotypes, we found results similar to previous knowledge, pointing towards the usability of postmortem tissue. For the less investigated genotypes, we were able to corroborate genotype / protein expression correlations. It is a novel approach to use a large postmortem cohort to investigate genetic / protein expression correlations.

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

In this study, we used human postmortem tissue to investigate hepatic protein expression levels of cytochrome P450 (CYP) 1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 by LC-MS/MS in a population of people suffering from mental disorders (n = 171). We report hepatic protein levels of these six CYP isoforms in 171 individuals in total, and define a focused population dataset of 116 individuals after excluding 55 samples due to low microsomal protein per gram of liver (MPPGL) yield. Postmortem decay was most likely the reason for the low MPPGL yield in the 55 samples. In the focused population, we found women to have significantly higher protein levels of CYP3A4 than men in addition to decreased CYP3A4 protein levels among obese individuals. Furthermore, MPPGL was negatively correlated with body mass index (BMI). An increase in CYP1A2 protein levels was observed among smokers, and increased CYP2E1 protein levels were observed among individuals with a history of alcohol abuse. Finally, individuals who received phenobarbital (CYP3A4 inducer) had significantly higher CYP3A4 levels. In conclusion, lifestyle-related factors prevalent among people suffering from mental disorders are associated with altered CYP protein levels, which may alter drug metabolism and affect the efficacy of commonly prescribed drugs. Furthermore, this investigation demonstrates that postmortem hepatic tissue can be used to study how lifestyle and effectors affect hepatic CYP-levels in a large cohort of patients. SIGNIFICANCE STATEMENT: Using a large number of postmortem hepatic tissue specimens (n=116) originating from the autopsy of individuals diagnosed with mental disorders, we were able to show that hepatic CYP-levels were affected by alcohol, smoking, BMI, and sex and that MPPGL was affected by BMI. These lifestyle-related changes may alter drug metabolism and affect the efficacy of commonly prescribed drugs. It is a novel approach to use a large postmortem cohort to investigate how lifestyle and effectors affect hepatic CYP-levels.

Stability of Lisdexamfetamine in Sampled Whole Blood-Implications of Sampling Tube Additives and Storage Temperature for Interpretation of Impairment.

Lisdexamfetamine (LDX) is a prodrug that is enzymatically converted into dextroamphetamine (d-AMP), a central nervous system stimulant. The stability of LDX in sampled whole blood is an important issue that may be crucial in the assessment of impaired driving caused by d-AMP. This study investigated the stability of LDX in whole blood collected in two different tubes containing a fluoride oxalate (FX) mixture and a fluoride citrate (FC) mixture. Without additives, LDX was unstable. LDX was also unstable in FX blood stored at ambient temperature or 4°C. After 3 days of storage at ambient temperature, an initial LDX concentration of 47 ± 1 ng/g (mean ± SD) was no longer detectable in the samples (n = 3). Instead, 19 ± 0.6 ng/g d-AMP was formed. The stability was improved at 4°C. After 7 days of storage at 4°C, 88 ± 5% of an initial LDX concentration of 50 ± 0.4 ng/g was recovered and 3.8 ± 0.3 ng/g d-AMP was formed. The stability of LDX was greater in FC blood than in FX blood; 79 ± 10% and 93 ± 4% of initial LDX concentrations of 48 ± 2 and 51 ± 0.5 ng/g were recovered from FC blood after 7 days of storage at ambient temperature and 4°C, respectively, and the corresponding formation of d-AMP was 5.8 ± 0.6 and 0.5 ± 0.3 ng/g, respectively. When FX and FC blood were stored at -20°C or -80°C, no detectable degradation of LDX or formation of d-AMP was observed after 3 weeks of storage.

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

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.

Distribution of Eight QT-Prolonging Drugs and Their Main Metabolites Between Postmortem Cardiac Tissue and Blood Reveals Potential Pitfalls in Toxicological Interpretation.

Femoral blood concentrations are usually used in postmortem toxicology to assess possible toxic effects of drugs. This includes QT-prolongation and other cardiac dysrhythmia, which could have been the cause of death. However, blood concentration is only a surrogate for the active site concentration, and therefore cardiac tissue concentration may provide a more accurate toxicological interpretation. Thus, cardiac tissue and femoral and cardiac blood concentrations were examined for eight frequently used QT-prolonging drugs (QTD) and their metabolites in a mentally ill population. In total, 180 cases were included from the Danish autopsy-based forensic study SURVIVE. The concentrations were analyzed using ultra-performance liquid chromatography coupled with tandem mass spectrometry utilizing stable isotopically labeled internal standards. The results showed that the cardiac tissue concentrations were significantly higher compared to femoral and cardiac blood concentrations, with two exceptions. The median cardiac tissue-to-femoral blood concentration ratio (Kb) ranged from 2.2 (venlafaxine) to 15 (nortriptyline). The inter-individual fold difference between the minimum and maximum Kb ranged from 2.6-fold (Z-hydroxynortriptyline) to 61 (venlafaxine). For 12 compounds, postmortem redistribution appeared to be minimal, whereas four compounds displayed some degree of postmortem redistribution. Citalopram and quetiapine were selected for in-depth analysis of the relation between the toxicological interpretation and femoral blood/cardiac tissue concentrations. Within this dataset, citalopram displayed a wide overlap in cardiac tissue concentrations (~50%) between non-toxic and toxic citalopram cases, as estimated from femoral blood concentrations. In contrast, quetiapine displayed no overlap in cardiac tissue concentrations between non-toxic and toxic quetiapine cases based on femoral blood concentrations. The implication of the citalopram finding is that possible intoxications can be overlooked when only considering femoral blood concentrations. Based on the present findings, non-toxic cardiac tissue 10th-90th percentile concentration ranges were estimated for citalopram (0.93-4.4 mg/kg) and quetiapine (0.0073-0.60 mg/kg).

Quantification of 16 QT-prolonging Drugs and Metabolites in Human Postmortem Blood and Cardiac Tissue Using UPLC-MS-MS.

QT-prolonging compounds present a treatment risk in mentally ill patients. Knowledge of the concentration in the heart compared with blood is necessary to assess the cardiac toxicity of QT-prolonging compounds. To address this issue, this article presents a validated analytical method for the quantification of 16 QT-prolonging drugs (QTD) and metabolites in postmortem whole blood and postmortem cardiac tissue. Samples were prepared by protein precipitation and quantified using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Deuterated internal standards were used. Validation results showed that the bias was ±15% and precision was ≤15% for all compounds in both matrices. The recovery ranged from 78.8 to 127.4%, and the matrix effect ranged from 61.0 to 128.7% across both matrices. The limit of detection and the lower limit of quantification were below the therapeutic concentrations of the prescription drugs. No noteworthy degradation during storage of the extracts was detected. The method was applied in five authentic cases of mentally ill patients. In conclusion, an analytical method was successfully developed and validated for the quantification of QTD in postmortem whole blood and cardiac tissue. To the best of the authors' knowledge, this article presents the first fully validated method for quantification of QTD in cardiac tissue.