Popliteal Vein Blood Sampling and the Postmortem Redistribution of Diazepam,Methadone, and Morphine

Postmortem redistribution (PMR) refers to the site‐ and time‐related blood drug concentration variations after death. We compared central blood (cardiac and subclavian) with peripheral blood (femoral and popliteal) concentrations of diazepam, methadone, and morphine. To our knowledge, popliteal blood has never been compared with other sites. Intracardiac blood (ICB), subclavian blood (SB), femoral blood (FB), and popliteal blood (PB) were sampled in 30 cases. To assess PMR, mean concentrations and ratios were compared. Influence of postmortem interval on mean ratios was also assessed. Results show that popliteal mean concentrations were lower than those for other sites for all three drugs, even lower than femoral blood; mean ratios suggested that the popliteal site was less subject to PMR, and estimated postmortem interval did not influence ratios except for diazepam and methadone FB/PB. In conclusion, our study is the first to explore the popliteal site and suggests that popliteal blood is less prone to postmortem redistribution.     

Site‐, Technique‐, and Time‐Related Aspects of the Postmortem Redistribution of Diazepam,Methadone, Morphine,and their Metabolites: Interest of Popliteal Vein Blood Sampling

Sampling site, technique, and time influence postmortem drug concentrations. In 57 cases, we studied drug concentration differences as follows: subclavian vein‐dissection/clamping versus blind stick, femoral vein‐dissection/clamping versus blind stick, right cardiac chamber, and popliteal vein‐dissection and clamping only. Cases were distributed in group #1 (all cases with both techniques), group #2 (dissection/clamping), and group #3 (blind stick). Sampled drugs were diazepam, methadone, morphine, and their metabolites. To assess PMR, mean concentrations and ratios were calculated for each group. Time‐dependent variations of blood concentrations and ratios were also assessed. Results indicate that site, method, and time may influence postmortem distribution interpretation in different ways. Popliteal blood seems less subject to PMR. In conclusion, our study is the first to evaluate concurrently three main aspects of PMR and confirms that the popliteal vein may represent a site that is more resistant to the changes seen as a result of PMR.     

Postmortem Blood Concentrations of R‐ and S‐Enantiomers of Methadone and EDDP in Drug Users: Influence of Co‐Medication and P‐glycoprotein Genotype

Abstract: We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity using postmortem samples. R‐ and S‐methadone were measured in femoral blood from 90 postmortem cases, mainly drug users. The R‐enantiomer concentrations significantly exceeded that of the S‐enantiomers (Wilcoxon’s test, p < 0.001). The samples were divided into four groups according to other drugs detected (methadone only, methadone and strong analgesics, methadone and benzodiazepines, or methadone and other drugs). There was no significant difference in any of the R‐methadone/total methadone ratios among the four groups. The median R/S ratio was 1.38, which tends to be higher than that reported for the plasma of living subjects. In addition, we investigated whether small nucleotide polymorphisms in the MDR1 gene that encode the drug transporter P‐glycoprotein were associated with the concentrations of R‐ and S‐methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine. No significant association was detected.     

Distribution of morphine and morphine glucuronides in body tissue and fluids--postmortem findings in brief survival

An intoxication following administration of morphine, tramadol and atracurium in a suicide case is reported. The route of administration and the amount of the particular drug were known from the investigation of the death scene and the findings of the postmortem examination. Tramadol was present in the gastric contents as well as in blood, liver, kidney and brain samples, whereas the drug could not be detected in muscle. All body fluids and tissues investigated contained morphine as well as its 3- and 6-glucuronides with the exception of muscle tissue. The concentrations of morphine and its glucuronide metabolites were determined by LC/MS following solid phase extraction. Interestingly, the concentration of M6G in brain, liver and kidney were close to the concentration of M3G in the particular tissue. This phenomenon might be explained by a preferential hydrolysis of M3G or by a preferential formation of M6G postmortem. Measurement of morphine and M6G in femoral blood and cerebrospinal fluid may be a useful indicator in rapid deaths.     

Postmortem diffusion of drugs from the bladder into femoral venous blood.

We describe significantly elevated drug concentrations in the femoral venous blood due probably to postmortem diffusion from the bladder. A 16-year-old deceased male was found in a shallow ditch in winter. The estimated postmortem interval was 9 days and putrefaction was not advanced. The cardiac chambers contained fluid and coagulated blood and a small amount of buffy coat clots. Diffused hemorrhages were found in the gastric mucosa. The bladder contained approximately 600 ml of clear urine. Gas chromatographic-mass spectrometric analysis of the urine disclosed allylisopropylacetylurea (a fatty acid ureide sedative), diphenhydramine, chlorpheniramine and dihydrocodeine. The cause of death was considered to be drowning due to a drug overdose and cold exposure. The concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine in the femoral venous blood (1.89, 3.27 and 3.30 microg/ml, respectively) were much higher than those in blood from the right cardiac chambers (0.294, 0.237 and 0.240 microg/ml, respectively). Urine concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine were 22.6, 37.3 and 43.1 microg/ml, respectively. The stomach contained negligible amounts of diphenhydramine, free dihydrocodeine and total dihydrocodeine (0.029, 0.018 and 0.024 mg, respectively); concentrations of these drugs in the femoral muscle were 0.270, 0.246 and 0.314 microg/g, respectively. These results indicate that postmortem diffusion of diphenhydramine and dihydrocodeine from the bladder resulted in the elevated concentrations of these drugs in the femoral venous blood. Not only high urinary drug concentrations but also a large volume of urine in the bladder might accelerate the postmortem diffusion.     

Medicolegal studies on alcohol detected in dead bodies — alcohol levels in skeletal muscle

An experiment was carried out on rats to determine whether or not a skeletal muscle sample was suitable for the determination of ethanol concentration in a carcass. Gas chromatography was used to estimate the ethanol and n-propanol concentrations in the femoral muscle and intracardial blood. The ethanol concentration of each sample was corrected according to the moisture ratio of circulating blood, viz., 78.5%.The ethanol concentration ratio of blood to muscle was 1.03 two hours after ethanol administration. When the carcasses of rats pre-treated with ethanol were stored at 15 °C and 25 °C, respectively, the ethanol concentrations in muscle and blood increased with time. At all times the concentration was higher in blood than in muscle, and also higher in samples collected from the carcass stored at 25 °C than at 15 °C.When the control carcass was stored in the same manner, the postmortem production of ethanol was noticed in both blood and muscle. As in the experimental rats, the control rats exhibited a higher blood ethanol than muscle ethanol level. Again, the ethanol concentration was higher in samples collected from the carcass stored at 25 °C than at 15 °C. The ratio of ethanol to n-propanol was less than 20:1 in blood and less than 10.1 in muscle.These results suggest that skeletal muscle may be a suitable tissue for the postmortem detection of ethanol.     

Postmortem Concentration and Redistribution of Diazepam,Methadone, and Morphine with Subclavian and Femoral Vein Dissection/Clamping

Postmortem redistribution (PMR) concerns blood drug concentration variations after death, depending on many factors such as sampling site and technique. In our study, we focused on sampling method. 30 cases were sampled, each at cardiac, subclavian, femoral, and popliteal sites. Targeted substances were diazepam, methadone, and morphine. Blind stick and dissection/clamping techniques were concomitantly performed at subclavian and femoral sites. Subclavian and femoral concentrations were compared according to technique used. To assess the influence of sampling technique on PMR, central/peripheral ratios were calculated depending on sampling method. Results show that drug concentrations tend to be lower when drawn from a clamped subclavian or femoral vein whereas ratios including subclavian and/or femoral blood concentration are influenced according to the technique used. In conclusion, clamping a subclavian or femoral vessel before sampling tends to result in lower drug concentrations and may influence ratios, suggesting the importance of isolating vessels from thoraco‐abdominal viscera.     

Distribution of Venlafaxine,O‐desmethylvenlafaxine,and O‐desmethylvenlafaxine to Venlafaxine Ratio in Postmortem Human Brain Tissue

Venlafaxine (VEN) and its metabolite O‐desmethylvenlafaxine (ODV) inhibit reuptake of serotonin and norepinephrine. This study examines whether VEN is differentially distributed in postmortem brain and examines relationships between brain and femoral blood concentrations from donors prescribed VEN for treatment of depression. Using high‐pressure liquid chromatography‐ultraviolet detection, VEN and ODV concentrations were measured in temporal, occipital, and cerebellar cortex of six postmortem brains. The ODV/VEN ratio was calculated as a relative measure of drug metabolism within each region where higher ratios indicated a greater conversion of VEN to ODV. Compared to the other regions examined, the cerebellum showed decreased VEN (p = 0.056), ODV (p = 0.006), and ODV/VEN (p = 0.027) ratios. In parts per million, VEN was higher in temporal and occipital cortex, but not cerebellum, as compared to femoral blood concentration. These observations suggest that VEN and ODV are differentially distributed in the brain, and metabolism of VEN to ODV may vary across brain regions.     

Parent and Metabolite Opioid Drug Concentrations in Unintentional Deaths Involving Opioid and Benzodiazepine Combinations,,

Effects of benzodiazepines on postmortem opioid parent and parent/metabolite blood concentration ratios were determined for fentanyl‐, hydrocodone‐, methadone‐, or oxycodone‐related accidental deaths. These opioids are partially metabolized by the CYP3A4 enzyme system, which is also affected by diazepam and alprazolam. Opioid/metabolite combinations examined were as follows: fentanyl/norfentanyl, hydrocodone/dihydrocodeine, methadone/EDDP, and oxycodone/oxymorphone. Parent opioid concentrations were analyzed for 877 deaths. Parent/metabolite concentration ratios were analyzed for 349 deaths, excluding cases with co‐intoxicants present known to interfere with opioid elimination. Alprazolam in combination with diazepam significantly decreased median hydrocodone concentrations by 48% (p = 0.01) compared to hydrocodone alone. The methadone parent/metabolite concentration ratio was reduced by 35% in the presence of diazepam compared to methadone alone (p = 0.03). Benzodiazepines did not statistically significantly affect fentanyl or oxycodone concentrations. Possible factors affecting opioid concentrations and possible toxicity development, including any differential effects on specific opioids, should continue to be explored.     

Postmortem redistribution of morphine and its metabolites

The postmortem redistribution of morphine, morphine-3-glucuronide, morphine-6-glucuronide and total morphine was assessed in 40 heroin-related deaths. In blood taken from subclavian, heart, and femoral regions, concentrations of morphine and its metabolites were similar. While there was a trend for higher concentrations in heart blood, when compared with femoral or subclavian blood, this was not significant. There was also no significant difference in concentrations between admission and autopsy blood in which the postmortem interval was on average 59 h. From our observations, significant postmortem redistribution of morphine and its metabolites seems unlikely.     

The effect of lethal electrical shock on postmortem serum myoglobin concentrations

Postmortem serum myoglobin concentrations in blood from the femoral vein (peripheral withdrawal) and the heart (central withdrawal) of nine electrical fatalities were compared with those of 74 individuals who had died of other causes. Independent of the cause of death or topographical site, serum myoglobin concentrations rose dramatically with the passage of postmortem time (maximum concentrations in the control group: 975,100 micrograms/l). In 59% of the total sample (electrical fatalities plus controls), serum myoglobin concentrations were higher in the central blood, in the other 41% the concentrations were higher in the peripheral blood. The differences in concentrations between the peripheral and the central withdrawal area correlated with neither the postmortem interval nor the cause of death. Up to the second day postmortem there was a statistically significant difference in serum myoglobin concentrations between electrical fatalities and controls. The individual values within each group, however, varied widely and overlapped between groups. Controls who had also suffered muscle injury (polytrauma, myocardial infarction) did not have significantly higher serum myoglobin concentrations than controls without muscle injury. Myoglobin concentrations appear to be greatly influenced by the extent and duration of the muscle cramps induced by the electrical current. Correct interpretation of serum myoglobin concentrations depends on the knowledge of events surrounding the lethal electrical shock. Postmortem determination of serum myoglobin concentrations alone is, therefore, not sufficient to establish intravital exposure to electrical current and can aid the diagnosis only in special cases.     

Postmortem Distribution of 3‐Beta‐Hydroxybutyrate

The concentrations of 3‐beta‐hydroxybutyrate (3HB) in femoral blood, urine, vitreous humor as well as pericardial and cerebrospinal fluids were retrospectively examined in a series of medico‐legal autopsies, which included cases of diabetic ketoacidosis, hypothermia fatalities without ethanol in blood, bodies presenting mild decompositional changes, and sudden deaths in chronic alcoholics. Similar increases in 3HB concentrations were observed in blood, vitreous, and pericardial fluid, irrespective of the cause of death, suggesting that pericardial fluid and vitreous can both be used as alternatives to blood for postmortem 3HB determination. Urine 3HB levels were higher than blood values in most cases. Cerebrospinal fluid 3HB levels were generally lower than concentrations in blood and proved to be diagnostic of underlying metabolic disturbances only when significant increases occurred.     

Methadone‐Related Deaths – Epidemiological,Pathohistological, and Toxicological Traits in 10‐year Retrospective Study in Vojvodina,Serbia

The number of methadone‐related deaths (MRDs) during a 10‐year period (2002–2011) in the region of Vojvodina, Serbia, was increased. The cases were evaluated according to epidemiological parameters, pathohistological findings, and toxicological screening. The majority of victims were men, aged from 20 to 38. Pathohistologically, the signs of acute focal myocardial damage were present in the heart of victims with drug abuse history shorter than 2 years, while both signs of recent and chronic focal myocardial damage were developed among victims with longer drug abuse history (2–5 years). In postmortem blood samples of 54.84% of victims, methadone was detected in combination with diazepam, both in therapeutic range. Alcohol was absent in most cases. Other detected drugs were antipsychotics and antidepressants in therapeutic concentrations. These findings raise the attention to the concomitant use of methadone and benzodiazepines with the need for further studies to clarify the mechanism of death in such cases.     

Comparison of results for quantitative determination of morphine by radioimmunoassay, enzyme immunoassay, and spectrofluorometry.

The quantitative results (accuracy and precision) for determination of opiates by radioimmunoassay (RIA), enzyme immunoassay (EMIT), and spectrofluorometry on split samples are compared. A variety of physiological samples were studied, including random urine from a methadone maintenance clinic and postmortem urine, blood, bile, brain, and lung tissue from heroin-induced or heroin-related deaths. The opiate concentrations detected by the two immunoassay methods were in good agreement with each other in the absence of interfering substances which are believed to react with the antimorphine antibodies. The immunoassay results were in agreement within the relative standard deviation with the fluorometry results in 55% of the urine samples and 80% of the blood samples. The immunological methods are superior to fluorometry for quantitation of morphine in urine samples due to quenching interferences in fluorometry from urine. They were comparable to fluorometry for quantitation of morphine in blood samples.     

Interpretation of a 3,4-methylenedioxymethamphetamine (MDMA) blood level: discussion by means of a distribution study in two fatalities

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy" is a currently used or abused designer drug and fatalities are frequently encountered in forensic practice. However, the question remains open whether an MDMA blood level can be toxic or even potentially lethal. In order to provide insight in the interpretation of a detected MDMA concentration, the distribution of MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) in various body fluids and tissues was studied and discussed in two different fatalities. Apart from peripheral blood samples (such as femoral and subclavian blood), various blood samples obtained centrally in the human body and several body fluids (such as vitreous humour) were examined. In addition, various tissues such as cardiac muscle, lungs, liver, kidneys, and brain lobes were analysed. In contrast to the peripheral blood levels, high MDMA and MDA levels were found in cardiac blood and the majority of the organs, except for the abdominal adipose tissue. The high concentrations observed in all lung lobes, the liver and stomach contents indicate that post-mortem redistribution of MDMA and MDA into cardiac blood can occur and, as a result, blood sampled centrally in the body should be avoided. Therefore, our data confirm that peripheral blood sampling remains "the golden standard". In addition, a distinct difference in peripheral blood MDMA concentrations in our two overdose cases was established (namely 0.271 and 13.508 microg/ml, respectively). Furthermore, our results suggest that, if a peripheral blood sample is not available and when putrefaction is not too pronounced, vitreous humour and iliopsoas muscle can be valuable specimens for toxicological analysis. Finally, referring to the various mechanisms of death following amphetamine intake, which can result in different survival times (e.g. cardiopulmonary complications versus hyperthermia), the anatomo-pathological findings and the toxicological results should be considered as a whole in arriving at a conclusion.     

Postmortem disposition of morphine in rats

The antemortem and postmortem distribution of morphine was studied in rats for the purpose of establishing whether drug distribution is altered after death. Samples were examined for free and total morphine concentration, pH and water content at 0-96 h after death. Morphine was administered antemortem at various intervals. All groups of rats studied showed a significant (P less than 0.05) increase in postmortem cardiac blood morphine concentrations. These changes, which are detectable within 5 min after death are likely to be related to an observed, rapid decrease in cardiac blood pH from 7.34 +/- 0.02 to 6.74 +/- 0.05. Significant increases in free morphine levels were, also, observed 24 and 96 h after death in liver, heart and forebrain while urine morphine levels decreased. The liver showed the greatest increase (20-fold) in free morphine levels 96 h after death, while hindbrain levels did not significantly change. Bacterial hydrolysis of morphine glucuronides accounted only in part for the observed increase in free morphine concentration. Postmortem fluid movement and pH-dependent drug partitioning was detected. It would appear that several mechanisms are responsible for postmortem drug distribution. Understanding the mechanisms and patterns responsible may eventually lead to better choices of postmortem tissue which may better represent antemortem drug levels.     

Detection of Acetaminophen–Protein Adducts in Decedents with Suspected Opioid–Acetaminophen Combination Product Overdose

Acetaminophen overdose is a leading cause of drug‐induced liver failure in the United States. Acetaminophen–protein adducts have been suggested as a biomarker of hepatotoxicity. The purpose of this study was to determine whether protein‐derived acetaminophen–protein adducts are quantifiable in postmortem samples. Heart blood, femoral blood, and liver tissue were collected at autopsy from 22 decedents suspected of opioid–acetaminophen overdose. Samples were assayed for protein‐derived acetaminophen–protein adducts, acetaminophen, and selected opioids found in combination products containing acetaminophen. Protein‐derived APAP‐CYS was detected in 17 of 22 decedents and was measurable in blood that was not degraded or hemolyzed. Heart blood concentrations ranged from 11 ng/mL (0.1 μM) to 7817 ng/mL (28.9 μM). Protein‐derived acetaminophen–protein adducts were detectable in liver tissue for 20 of 22 decedents. Liver histology was also performed for all decedents, and no evidence of centrilobular hepatic necrosis was observed.     

氯胺酮在急性中毒大鼠体内的死后再分布研究

目的探索氯胺酮在大鼠体内的死后再分布变化规律及温度对再分布的影响。方法48只雄性SD大鼠随机分为2个实验组（室温组24只、冷藏组18只）和1个对照组（6只）,实验组大鼠以氯胺酮290mg／kg灌胃,45min后缺氧处死,分别置于室温（24℃）和冷藏（4℃）条件下,于死后不同时间（0、12、24、48h）取心血、外周血、肝、肺、肾、心肌、大脑,检测其中氯胺酮含量;对照组大鼠以生理盐水灌胃,各对应组织器官样品为空白对照。血和组织样品中加入内标物SKF。。后碱化,乙酸乙酯萃取,GC／MS全扫描定性,内标法、工作曲线法气相色谱定量分析。结果室温条件下,大鼠死后48h内随着死亡时间延长,心血、肺、肝中氯胺酮的浓度呈升高趋势（P〈0．05）,肾脏中氯胺酮的浓度先升高后下降（P〈0．05）,外周血、心肌和脑中氯胺酮的浓度无显著性变化（P〉0．05）。冷藏条件下,血液及组织中氯胺酮浓度变化无显著性差异（P〉0．05）,除心肌外,各样本浓度均低于相应时段室温条件保存的样本。结论氯胺酮在大鼠体内存在死后再分布现象。温度对大鼠死后血液及组织中氯胺酮浓度变化有较明显的影响。     

Fatal methanol intoxication with different survival times--morphological findings and postmortem methanol distribution

Three corresponding cases of fatal methanol intoxication with different survival times were investigated ante-mortem and postmortem. Ante-mortem serum methanol concentrations were determined during treatment in hospital for 4 days. Furthermore, postmortem distribution of methanol in various tissues and fluids was measured after autopsy. Morphological and toxicological findings are discussed based on the literature. The morphological findings correlated with the different survival times. The results of the toxicological analyses were partly in keeping with previously published data. Interestingly, very high methanol levels were determined in brain with very low concentrations in femoral venous blood. These results may have implications for postmortem toxicological analysis, brain death diagnosis and organ explanation for transplantation.