Derrick J. Pounder

Post-mortem drug redistribution — A toxicological nightmare

Detailed human case data is presented to illustrate the dramatic extent of the phenomenon of post-mortem drug redistribution. The data suggests that there is a post-mortem diffusion of drugs along a concentration gradient, from sites of high concentration in solid organs, into the blood with resultant artefactual elevation of drug levels in blood. Highest drug levels were found in central vessels such as pulmonary artery and vein, and lowest levels were found in peripheral vessels such as subclavian and femoral veins. In individual cases, in multiple blood samples obtained from ligated vessels, concentrations of doxepin and desmethyldoxepin ranged from 3.6 to 12.5 mg/l and 1.2 to 7.5 mg/l, respectively; amobartital, secobarbital and pentobarbital from 4.3 to 25.8 mg/l, 3.9 to 25.3 mg/l and 5.1 to 31.5 mg/l respectively; clomipramine and desmethylclomipramine from 4.0 to 21.5 mg/l and 1.7 to 8.1 mg/l, respectively and flurazepam 0.15 to 0.99 mg/l; imipramine and desipramine from 4.1 to 18.1 mg/l and 1.0 to 3.6 mg/l, respectively. We conclude that this poorly studied phenomenon creates major difficulties in interpretation and undermines the reference value of data bases where the site of origin of post-mortem blood samples is unknown.

POSTMORTEM DIFFUSION OF DRUGS FROM GASTRIC RESIDUE : AN EXPERIMENTAL STUDY

Postmortem drug diffusion from gastric residue was assessed in a human cadaver model. Fifty milligrams of amitriptyline (Ami) and 5 g of paracetamol (Par) suspended in 350 ml of 10% methanol, 0.1 N HCl, and 50 ml urograffin with 5 g lithium carbonate (alkaline model) or without lithium (acidic model) was instilled into the stomach through an esophageal tube via a neck dissection. Multiple samples were obtained after 48 h at room temperature (range in mean hourly room temperature: 15.6-20.7 degrees C, n = 9). The pH of the gastric contents (alkaline model range = 8.3-8.9, n = 5; acidic model range = 3.4-3.8, n = 5) had no significant effect. Drug diffusion was most marked in the left lung base, with drug concentrations (micrograms/g) of 0.1-13.9 for Ami, 65-524 for Par, and 13-161 for lithium. Similarly affected were the left lobe of the liver (Ami, 0.1-54.9; Par, 7-218; lithium, 7-39), the spleen (Ami, 0.6-24.3; Par, 104-663; lithium, 27-106), and pericardial fluid (Ami, 0-4.5; Par, 48-641; lithium, 12-56). Diffusion into gallbladder bile, cardiac blood, aortic blood, and blood of the inferior vena cava was less severe. The left kidney and left lung were more severely affected than the right kidney and lung, and similarly the left and right psoas muscles. Least affected was the right anterior lobe of the liver and the lung apexes. This phenomenon may significantly influence drug concentrations in liver and in blood samples obtained from the torso, and consequently liver/blood drug ratios. To circumvent the problem of postmortem drug diffusion from the stomach, it is recommended that blood be sampled from a peripheral vessel, skeletal muscle from a limb, liver from deep within the right lobe, and lung from the apex rather than the base.

Postmortem diffusion of alcohol from the stomach.

We explored postmortem ethanol diffusion from the stomach using a human cadaver model with multiple blood site sampling. In all, 400 ml of alcohol solution (5%, 10%, 20%, or 40% methanol and ethanol weight/volume in water) was introduced into the stomach by oesophageal tube. Blood methanol concentrations correlated with ethanol concentrations (methanol range, 1–676 mg%; ethanol range, 1–531 mg%; r = 0.9973). The pattern of ethanol diffusion showed marked between-case variability. Typically, concentrations were highest in pericardial fluid and, in decreasing order, in left pulmonary vein, aorta, left heart, pulmonary artery, superior vena cava, inferior vena cava, right heart, right pulmonary vein, and femoral vein. Diffusional flux was broadly proportional to the concentration of ethanol used. It was time-dependent (as assessed by 24-h and 48-h sampling) and markedly inhibited by refrigeration at 4°C. After gastric instillation of 400 ml of 5% solution for 48 h at room temperature in paired cadavers, ethanol concentrations (mg%) were as follows: pericardial fluid 135, 222; aorta 50, 68; left heart 77, 26; right heart 41, 28; femoral vein 0. Using a 10% solution, ethanol concentrations (mg%) were as follows: pericardial fluid 401, 255; aorta 129, 134; left heart 61, 93; right heart 31, 41; femoral vein 5, 7. Introducing 50 ml of 10% alcohol solution into the oesophagus after oesophagogastric junction ligation produced similar aortic blood ethanol concentrations. This suggests that postmortem gastro-oesophageal reflux and diffusion from the oesophagus is the mechanism of artefactual elevation of aortic blood ethanol. Introducing 150 ml of 10% alcohol solution directly into the lesser sac resulted in marked reduction of diffusion into the pericardial sac. This finding suggests that close apposition of the gastric fundus against the diaphragm and a large gastric volume are significant factors that influence diffusional flux. Deflation and anchoring of the left lung at the apex had little effect, suggesting that diffusion into the left pulmonary vein is via the pericardial fluid rather than the lung substance. In nine fatalities with known alcohol consumption shortly before death, the highest observed stomach contents ethanol concentration was 8.7%. Two cases showed marked variations in blood ethanol concentrations in 10 samples with ranges (mg%) of 97–238 and 278–1395; pericardial fluid 1060 and 686; vitreous humour 34 and 225; and stomach contents 300 ml at 5.5% and 85 ml at 1.9%, respectively. We conclude that postmortem diffusion of ethanol from the stomach into the blood is a potentially significant artefact in a small minority of cases. Both aortic and cardiac chamber blood may be affected by this artefact. Between-case and within-case variability causes unpredictability. For postmortem ethanol analysis, the optimum sampling technique appears to be aspiration by needle puncture of the femoral vein (or external iliac vein) after cross-clamping proximal to the sampling site.

Alcoholic Ketoacidosis at Autopsy

Total ketone bodies (acetone, acetoacetate, and beta-hydroxybutyrate) were measured in 105 medicolegal autopsies (71 non-alcoholics, 22 chronic alcoholics, and 12 diabetics) using a coupled enzymatic head-space gas chromatographic method. Samples included vitreous humour, pericardial fluid, and blood from the femoral vein, inferior vena cava (IVC), superior vena cava (SVC), and aorta. Vitreous ketone levels showed good correlation with blood and pericardial fluid levels, suggesting that vitreous could be used as an alternative autopsy specimen for this analysis. This opens up the possibility of using simpler clinical laboratory methodologies which cannot be applied to autopsy blood due to hemolysis. In 71 non-alcoholics (age 18 to 96, median 67) total ketones (mM/L) were: vitreous 0.19 to 3.35, median 0.49; pericardial fluid 0.02 to 1.54, median 0.35; femoral blood 0.23 to 8.08, median 1.00; aortic blood 0.25 to 9.96, median 0.90; IVC blood 0.30 to 6.49, median 1.27; SVC blood 0.32 to 6.00, median 1.07. Eleven outliers (> 2.5 mM/L in femoral blood) mostly had prolonged illness prior to death. The 22 alcoholics (age 36 to 83, median 62) included four extreme outliers with femoral blood total ketone levels of 129.9 (also diabetic), 39.4 (no anatomical cause of death), 38.5 (suicidal hanging), and 18.6 (hypothermia), suggesting that while alcoholic ketoacidosis may be a previously overlooked potential cause of death, interpretation must be guarded and made within the total case context. The other 18 alcoholics had ketone levels not statistically different from non-alcoholics, suggesting that ketoacidosis is a significant factor in at most a small minority of alcoholic deaths. Three of 12 diabetics had extreme elevations of femoral blood ketone bodies: 87.5, 20.4, and 17.4 mM/L. Measurement of ketone bodies in vitreous humour or pericardial fluid using clinical laboratory methodologies is recommended in unexplained deaths in chronic alcoholics as well as diabetics.

Shaken adult syndrome

A 30-year-old Palestinian collapsed when under interrogation by the Israeli General Security Service and was declared brain dead 3 days later. Information on the circumstances and interrogation methods was denied on security grounds. Autopsy disclosed extensive anterior chest and shoulder bruising and acute subdural haemorrhage but no other trauma. On this evidence, violent shaking was postulated as the mechanism of injury. Later, this was admitted by Israeli investigators and corroborated by histopathologically proved diffuse axonal injury and retinal haemorrhages. This is the first reported case of fatal shaken adult syndrome.

Site to site variability of postmortem drug concentrations in liver and lung

We evaluated postmortem diffusion of gastric drug residue into tissues and blood in eight suicidal overdoses. Analyses were performed on liver (five sites), lung (four sites), spleen, psoas muscle and kidney (left and right), blood (peripheral and torso), vitreous, pericardial fluid, bile and, urine as well as residual gastric contents. Standard analytical techniques and instrumentation gas chromatograph/mass spectrometer and high performance liquid chromatography (GC-MS and HPLC) were used throughout. These case studies confirm previous studies of an animal and human cadaver model of gastric diffusion, in that in several instances there was drug accumulation in the left posterior margin of the liver and, to a lesser extent, the left basal lobe of the lung. Uncontrollable variables, such as postmortem interval, refrigeration before autopsy, and position of the body appear to influence significantly drug accumulation in a specific site. We suggest that autopsy sampling techniques should be standardized on blood taken from a ligated peripheral (preferably femoral or external iliac) vein, and liver from deep within the right lobe.

Zopiclone poisoning: tissue distribution and potential for postmortem diffusion

Zopiclone is the first cyclopyrrolone hypnotic and is chemically unrelated to any existing drug. The authors studied the tissue distribution and postmortem redistribution of zopiclone in a fatal suicidal overdose. A 29-year-old female weighing 64 kg had cardiac blood ethanol 153 mg% and zopiclone blood concentrations in the range 0.9-2.0 microgram/ml in 10 distinct sampling sites. After 40 h at room temperature the range was 0.9-1.8 micrograms/ml in 15 samples. Portal venous blood and urine concentrations were 3.0 and 10.5 micrograms/ml, respectively. Tissue concentrations (microgram/g) were spleen 5.8, peri-renal fat 5.0, psoas muscle 3.3, brainstem 2.8, gastrocnemius muscle 1.9, myocardium 1.6, and kidney 1.7. Eight liver samples had concentrations in the range 0.5-4.9 micrograms/g, with highest concentrations in the left lobe and adjacent to the gallbladder, probably reflecting postmortem diffusion from gastric residue (700 ml, 55.1 micrograms/ml) and bile (14.1 micrograms/ml). Of six lung samples, paired upper and middle samples had concentrations in the range 2.1-2.3 micrograms/g, the right postero-basal 1.3 micrograms/g and the left postero-basal 3.4 micrograms/g. Drug concentration in putrefactive pleural fluid was also higher on the left (2.1 micrograms/ml) than the right (1.4 micrograms/ml), probably reflecting postmortem diffusion from gastric residue. The authors conclude that zopiclone showed little preferential concentration in solid organs and consequently has relatively stable postmortem blood concentrations, with little drug redistribution artefacts. Postmortem diffusion from gastric drug residue elevates drug levels in the left lobe of the liver and left lung lower lobe.

Drug accumulation and elimination in Calliphora vicina larvae

Calliphora vicina larvae were fed on drug-laden muscle from three suicides involving amitriptyline, temazepam and a combination of trazodone and trimipramine; triplicate daily harvestings were analysed. The limit of detection for all four drugs was 0.01 micrograms drug/g larvae. Mean drug concentrations (microgram/g) in the initial muscle were:amitriptyline, 2.68; temazepam, 4.04; trazodone, 21.56; and trimipramine, 19.58. Larval rearings for days 4-8 (15 larval samples per drug) had mean and ranges of drug concentrations (microgram/g) of 0.10 (r, 0.02-0.24) for amitriptyline; 0.52 (r, 0.26-0.78) for temazepam; 0.13 (r, 0.05-0.32) for trazodone; and 0.28 (r, 0.10-0.59) for trimipramine. After day 8 there was a precipitous fall in larval drug concentrations associated with pupariation. At day 11 ranges of drug concentrations (microgram/g) were: amitriptyline, < 0.01-0.01; temazepam, 0.01-0.08; trazodone, < 0.01-0.01; and trimipramine, 0.04-0.04. Day 16 pupae had corresponding ranges (microgram/g) of < 0.01, 0.01-0.01, < 0.01 and < 0.01-0.02. Transfer to drug-free food at day 5 led to similar falls in drug concentrations (microgram/g) from day 5 to day 6: 0.08-0.03 for amitriptyline, 0.61-0.09 for temazepam, 0.13-0.01 for trazodone, and 0.30-0.02 for trimipramine. The results show considerable variation in larval drug concentrations, both at the same developmental stage and at different stages of the life cycle, under conditions which closely reflect case situations. In practice, the precipitous decrease in drug concentrations in non-feeding larvae and at pupariation make it desirable to sample only larvae actively feeding on a corpse.

OPRM1 and CYP2B6 Gene Variants as Risk Factors in Methadone‐Related Deaths

Methadone is a medication valued for its effectiveness in the treatment of heroin addiction; however, many fatal poisonings associated with its use have been reported over the years. We have examined the association between CYP2B6 and µ‐opioid receptor (OPRM1) gene variations and apparent susceptibility to methadone poisoning. Genomic DNA was extracted from postmortem whole blood of 40 individuals whose deaths were attributed to methadone poisoning. The presence of CYP2B6*4,*9, and *6 alleles and the OPRM1 A118G variant was determined by SNP genotyping. CYP2B6 *4, *9, and *6 alleles were found to be associated with higher postmortem methadone concentrations in blood (P ≤ 0.05). OPRM1 A118G was also associated with higher postmortem methadone concentrations in blood but not to a level of statistical significance (P = 0.39). In these methadone‐related deaths, OPRM1 118GA was associated with higher postmortem benzodiazepine concentrations (P = 0.04), a finding not associated with morphine‐related deaths. The risk of a methadone‐related fatality during treatment may be evaluated in part by screening for CYP2B6*6 and A118G.

Postmortem absorption of drugs and ethanol from aspirated vomitus — An experimental model

Using human cadavers an experimental model was developed to simulate the agonal aspiration of drug- and alcohol-laden vomitus. By needle puncture, an acidified (N/20 HCl) 60-ml slurry of drugs (paracetamol 3.25 g, dextropropoxyphene 325 mg) and ethanol 3% w/v was introduced into the trachea. After 48 h undisturbed at room temperature, blood samples were obtained from ten sites. Ethanol and drug concentrations were highest in the pulmonary vessels in all five cases studied. Pulmonary vein mean ethanol was 58 mg% (range 13-130), paracetamol 969 mg/l (range 284-1934), propoxyphene 70 mg/l (range 11-168). Pulmonary artery mean ethanol was 53 mg% (range 10-98), paracetamol 476 mg/l (range 141-882), propoxyphene 29 mg/l (range 7.6-80). Ethanol and drug concentrations in aortic blood were higher than in the left heart and concentrations in the superior vena cava were higher than in the right heart, suggesting direct diffusion into these vessels rather than diffusion via the pulmonary and cardiac blood. Potential interpretive problems arising from this phenomenon can be avoided by using femoral vein blood for quantitative toxicological analysis.