Postmortem redistribution of amphetamines and benzodiazepines in humans: Important variables that might be influencing the central blood / peripheral blood ratio.

INTRODUCTION: The primary objective of postmortem forensic toxicology is to determine if toxicological substances detected in bodily material of victims have contributed to the death of the victim. Interpretation of postmortem drug concentrations is hindered by the fact that time and site dependent variations in postmortem drug concentrations occur, as a result of postmortem redistribution (PMR). An often-used marker for the occurrence of PMR, is the cardiac blood concentration/peripheral blood concentration ratio (C/P ratio) of a drug. In this study, we investigated the relationship between 13 variables and the C/P ratios of amphetamines and benzodiazepines. METHOD: Toxicological results of all postmortem cases that were positive for amphetamines (amphetamine, MDMA, MDA) and/or benzodiazepines (diazepam, desmethyldiazepam, temazepam, oxazepam, midazolam, α-hydroxymidazolam) investigated by the Netherlands Forensic Institute between January 1 2010 and July 31 2020 were reviewed. A total of 112 amphetamine positive cases (224 paired specimen) and 179 benzodiazepine positive cases (358 paired specimen) were selected. The C/P ratios were determined for all selected cases. Ratios were compared between subgroups by performing either a Mann-Whitney U test or a Kruskal-Wallis test followed by post-hoc Mann-Whitney U test. RESULTS: After dividing cases in quartiles based on their amphetamine concentration in femoral blood, the amphetamine C/P ratio was significantly lower in cases with a high amphetamine concentration (quartile 4) compared to cases with a low amphetamine concentration (quartiles 1 and 2) with median C/P ratios of 1.6, 2.4 and 2.2, respectively (p-value<0.001 and p-value=0.001, respectively). The MDA C/P ratio was significantly higher in cases where trauma was the cause of death compared to cases where intoxication was the cause of death with median C/P ratios of 3.3 and 1.6, respectively (p-value<0.001). The MDA C/P ratio was also significantly lower in cases where resuscitation was attempted compared to cases where no resuscitation was attempted with median C/P ratios of 1.6 and 2.4, respectively (p-value=0.003). However, a significant dependency between the variables cause of death and attempted resuscitation was observed. No significant differences in benzodiazepine C/P ratios were observed between subgroups of any of the investigated variables. However, the low p-value of BMI suggests a potential difference in midazolam C/P ratio between BMI subgroups (p-value=0.027). CONCLUSION: When interpreting postmortem toxicological results, it might prove useful to take the above-mentioned variables into account.

A fatal mono-intoxication with 4-fluoroisobutyrylfentanyl: Case report with postmortem concentrations.

We report on a case of a 35-year-old man who died suddenly and unexpectedly due to a 4-fluoroisobutyrylfentanyl (4-FIBF) mono-intoxication. Pathological, toxicological and chemical investigations were conducted at the Netherlands Forensic Institute. A full three-cavity forensic pathological examination was performed according to international guidelines. Biological samples obtained during autopsy were comprehensively investigated for the presence of toxic substances using headspace gas chromatography (GC) with flame ionization detection, liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS), GC-MS, high-performance LC with diode array detection and LC-tandem MS (LC-MS-MS). The seized crystalline substance found next to the body was investigated using a presumptive color test, GC-MS, Fourier-transform infrared spectroscopy and nuclear magnetic resonance. Pathological investigation identified minor lymphocytic infiltrates in the heart, considered irrelevant for the cause of death. Toxicological analysis of the victims' blood indicated the presence of a fluorobutyrylfentanyl (FBF) isomer, with no other compounds detected. The FBF isomer was identified in the seized crystalline substance as 4-FIBF. 4-FIBF concentrations were quantified in femoral blood (0.030 mg/L), heart blood (0.12 mg/L), vitreous humor (0.067 mg/L), brain tissue (>0.081 mg/kg), liver tissue (0.44 mg/kg) and urine (approximately 0.01 mg/L). Based on the outcomes of the pathological, toxicological and chemical investigations, the cause of death of the deceased was attributed to a fatal 4-FIBF mono-intoxication. The presented case underlines the added value of a combined bioanalytical and chemical investigative approach to identify and subsequently quantify fentanyl isomers in postmortem cases. Furthermore, it demonstrates the importance of investigating the postmortem redistribution of novel fentanyl analogs to establish reference values and to subsequently allow for correct interpretation of cause of death analysis in future casework.

Postmortem redistribution of cocaine and its metabolites, benzoylecgonine and ecgonine methyl ester in humans: Important variables that might be influencing the central blood / peripheral blood ratio.

INTRODUCTION: A big challenge in forensic toxicology is the correct interpretation of the results of quantitative analyses in postmortem cases. Postmortem drug concentrations not necessarily reflect the drug concentrations at the time of death, due to postmortem changes in drug concentrations caused by postmortem redistribution (PMR). Cardiac blood is more prone to PMR related concentration changes than peripheral blood. Because of this difference in susceptibility to PMR related concentration changes, the ratio of cardiac blood concentration/peripheral blood concentration (C/P) of a drug is an often-used marker of PMR. In this study, we investigated the relationship between different potentially significant variables and the C/P ratios of cocaine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in humans. The aim was to elucidate the mechanisms involved in PMR of these substances and potentially provide guidelines aiding forensic toxicologists in the interpretation of postmortem quantitative results of cocaine and its metabolites. To differentiate between postmortem concentration changes due to redistribution versus degradation of cocaine, the relationships between these variables and metabolite/cocaine ratios were investigated as well. METHOD: Toxicological results of all postmortem cases that were positive for cocaine, BE and/or EME investigated by the Netherlands Forensic Institute between January 1st 2010 and July 31st 2020 were reviewed. The C/P ratios, BE/cocaine ratios and EME/cocaine ratios were determined for all selected cases. Cocaine, BE and/or EME were quantified in both femoral blood and cardiac blood in a total of 148 cases. Ratios were compared between subgroups by performing either a Mann-Whitney U test or a Kruskal-Wallis test followed by post-hoc Mann-Whitney U test. RESULTS: A statistically significant difference in C/P ratio of EME was observed between trauma and non-trauma cases with median C/P ratios of 2.03 and 1.57, respectively (p value=0.001). A statistically significant difference in EME/cocaine ratio was observed between the BMI subgroups 18.5 - 25.0 kg/m2 and> 25 kg/m2 with median EME/cocaine ratios of 3.79 and 1.58, respectively (p-value<0.001). CONCLUSION: Postmortem cocaine concentrations should be interpreted with caution, considering the occurrence of both PMR and postmortem degradation. When interpreting postmortem toxicological results in cocaine-related fatalities, it might prove useful to take the above-mentioned variables into account.

Postmortem redistribution of morphine in humans: Important variables that might be influencing the central blood/peripheral blood ratio.

INTRODUCTION: In the field of forensic toxicology, many unexpected deaths are investigated as to whether toxicological substances may have caused or contributed to someone's death. One of the factors that makes interpretation of the results of quantitative analysis in postmortem toxicology challenging, is that measured postmortem drugs levels may vary according to the sampling site and the interval between death and specimen collection. These site- and time-dependent variations are caused by 'postmortem redistribution' (PMR). Literature shows that there are several factors that determine the degree of PMR, such as cell and tissue changes after death, decomposition and the physicochemical characteristics of drugs. Blood from peripheral sites seems to be less affected by PMR than cardiac blood. Therefore, the ratio of cardiac blood concentration/peripheral blood concentration (C/P) of a drug is often used as a marker of the extent of postmortem redistribution. In this study, we investigated the relationship between different potentially important variables and the C/P ratio of morphine in humans in order to provide new insights that might assist in the interpretation of quantitative results in forensic casework. METHOD: Toxicological results of all morphine positive postmortem cases investigated by the Netherlands Forensic Institute between January 1, 2010 and July 31, 2020 were reviewed. Morphine was quantified in both femoral and cardiac blood in a total of 103 cases. The C/P ratios were determined for all selected cases. To collect data for this study, all corresponding files were reviewed. C/P ratios were compared between subgroups by performing either a Mann-Whitney U test or a Kruskal-Wallis test, followed by a post-hoc Mann-Whitney U test. Bonferroni correction was performed to correct for the likelihood of a significant result by chance due to multiple testing. After Bonferroni correction, a p-value< 0.004 was considered statistically significant. RESULTS: The data suggests a relationship between grade of decomposition at autopsy, position of the corpse at discovery, route of administration, attempted resuscitation and the C/P ratio of morphine with p-values of 0.010, 0.026, 0.035 and 0.046, respectively. CONCLUSION: Grade of decomposition at autopsy, position of the corpse at discovery, route of administration and attempted resuscitation seem to be influencing the C/P ratio of morphine. Of these four variables, the route of administration seems to have the greatest impact.

A hypomorphic Cbx3 allele causes prenatal growth restriction and perinatal energy homeostasis defects.

Mammals have three HP1 protein isotypes HP1 beta (CBX1), HP1 alpha (CBX3) and HP1 alpha (CBX5) that are encoded by the corresponding genes Cbx1, Cbx3 and Cbx5. Recent work has shown that reduction of CBX3 protein in homozygotes for a hypomorphic allele (Cbx3hypo) causes a severe postnatal mortality with around 99 percent of the homozygotes dying before weaning. It is not known what the causes of the postnatal mortality are. Here we show that Cbx3hypo/hypo conceptuses are significantly reduced in size and the placentas exhibit a haplo-insufficiency. Late gestation Cbx3hypo/hypo placentas have reduced mRNA transcripts for genes involved in growth regulation, amino acid and glucose transport. Blood vessels within the Cbx3hypo/hypo placental labyrinth are narrower than wild-type. Newborn Cbx3hypo/hypo pups are hypoglycemic, the livers are depleted of glycogen reserves and there is almost complete loss of stored lipid in brown adipose tissue (BAT). There is a 10-fold reduction in expression of the BAT-specific Ucp1 gene, whose product is responsible for nonshivering themogenesis. We suggest that it is the small size of the Cbx3hypo/hypo neonates, a likely consequence of placental growth and transport defects, combined with a possible inability to thermoregulate that causes the severe postnatal mortality.

Prolonged niacin treatment leads to increased adipose tissue PUFA synthesis and anti-inflammatory lipid and oxylipin plasma profile.

Prolonged niacin treatment elicits beneficial effects on the plasma lipid and lipoprotein profile that is associated with a protective CVD risk profile. Acute niacin treatment inhibits nonesterified fatty acid release from adipocytes and stimulates prostaglandin release from skin Langerhans cells, but the acute effects diminish upon prolonged treatment, while the beneficial effects remain. To gain insight in the prolonged effects of niacin on lipid metabolism in adipocytes, we used a mouse model with a human-like lipoprotein metabolism and drug response [female APOE*3-Leiden.CETP (apoE3 Leiden cholesteryl ester transfer protein) mice] treated with and without niacin for 15 weeks. The gene expression profile of gonadal white adipose tissue (gWAT) from niacin-treated mice showed an upregulation of the "biosynthesis of unsaturated fatty acids" pathway, which was corroborated by quantitative PCR and analysis of the FA ratios in gWAT. Also, adipocytes from niacin-treated mice secreted more of the PUFA DHA ex vivo. This resulted in an increased DHA/arachidonic acid (AA) ratio in the adipocyte FA secretion profile and in plasma of niacin-treated mice. Interestingly, the DHA metabolite 19,20-dihydroxy docosapentaenoic acid (19,20-diHDPA) was increased in plasma of niacin-treated mice. Both an increased DHA/AA ratio and increased 19,20-diHDPA are indicative for an anti-inflammatory profile and may indirectly contribute to the atheroprotective lipid and lipoprotein profile associated with prolonged niacin treatment.

Comprehensive gas chromatography-electron ionisation mass spectrometric analysis of fatty acids and sterols using sequential one-pot silylation: quantification and isotopologue analysis.

RATIONALE: Fatty acids and sterol lipids play crucial roles in several biological processes and several biological facts underline the interconnection between these lipid classes. Therefore, it is of interest to develop a comprehensive method analysing both classes in the form of their most favourable derivatives suitable for quantification and isotopologue analysis. METHODS: Lipids were derivatised by a sequential one-pot procedure using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). No clean-up or concentration steps were necessary. The prepared samples were directly available for gas chromatography-electron ionisation mass spectrometric (GC-EI-MS) analysis on a standard column. For quantification, the SIM mode was used and for isotopologue analysis scheduled scan mode was applied. RESULTS: Development of a sequential one-pot derivatisation for GC-EI-MS allowing comprehensive analysis of fatty acids and sterols as their most favourable derivatives. Validation carried out using human plasma, comparison with certified NIST plasma. LLOQ of usually 3.3 ng/mL achieved. Isotopologue analysis of 2-[(13)C]-acetate incorporation in HL-60 cells proving feasibility of method. CONCLUSIONS: The presented method successfully combines two consecutive silylation reactions in one pot, enabling the analysis of both fatty acids and sterols in a comprehensive analytical method. The method has great potential for the quantification of lipids as well as the comprehensive study of both biochemical pathways, using [(13)C]-flux analysis.

Evaluation of different column chemistries for fast urinary metabolic profiling.

Fast analytical methodologies are mandatory for large scale metabolic profiling. Here, we present a thorough evaluation of different column chemistries in combination with different mobile phases for fast LC-MS urinary metabolic profiling. Three porous HILIC materials were investigated, next to core-shell C18-, XB-C18- and PFP-RPLC material. The performance of the selected column chemistries was tested in a non-targeted manner with pooled urine samples and in a targeted manner with a set of 54 common urinary metabolites. In order to evaluate the differential behaviour of the tested columns in a targeted manner, we applied a peak scoring algorithm. This algorithm takes into account several quality criteria such as retention time, dead time, peak height and peak shape. In general, HILIC columns generate more retention for polar metabolites. Our results show that the diol-HILIC column outperforms the RPLC columns. However, because of their opposite nature, comprehensive behaviour is observed as well, which was shown by investigating gender differences in a small urinary sample set. All applied column chemistries enabled sufficient peak capacity within a short gradient time.

Mild and selective labeling of malondialdehyde with 2-aminoacridone: assessment of urinary malondialdehyde levels.

Malondialdehyde (MDA) has become a well-established biomarker for oxidative stress. The most commonly used way to determine urinary MDA levels is the thiobarbituric acid (TBA) assay, which suffers from several drawbacks. In this manuscript, we describe a novel derivatization strategy for the highly sensitive and selective fluorescence-based determination of MDA in urinary samples. The methodology is based on the mild labeling of MDA with 2-aminoacridone, which can be carried out in aqueous citrate buffer at 40 °C, yielding a highly fluorescent substance. No further sample preparation than mixing with the necessary chemicals is necessary. The formed MDA derivative can conveniently be separated from the label itself and matrix constituents by gradient LC in less than 5 minutes on a cyano-based reversed-phase material. The method was validated with respect to matrix effects, linearity, selectivity and sensitivity (values as low as 1.8 nM for the LOD and 5.8 nM for the LOQ could be achieved). Standard addition quantitation was applied for the determination of MDA in human urine samples. Additionally, the protocol was applied to the measurement of a stability indicating analysis of MDA in urine at different storage conditions.