Interindividual variability in the prevalence of OPRM1 and CYP2B6 gene variations may identify drug-susceptible populations.

Methadone is used worldwide for the treatment of heroin addiction; however, fatal poisonings are increasingly reported. The prevalence of CYP2B6 and µ-opioid receptor (OPRM1) gene variations were examined between a postmortem population where the deaths were associated with methadone and a live nondrug-using control population using Taqman™ SNP Genotyping assays. The CYP2B6*6 allele was higher in the postmortem population, but the difference was not significant (P = 0.92). The CYP2B6 T750C promoter variation was similar in frequency for both populations. Linkage between T750C and CYP2B6*6 was identified for both populations (P < 0.01). The prevalence of the OPRM1 A118G variation was significantly higher in the control population (P = 0.0046), which might indicate a protective mechanism against opioid toxicity. Individual susceptibility to methadone may be determined by screening for CYP2B6*6.

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 micro-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 < or = 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.

A chloroform-related death: analytical and forensic aspects.

Chloroform is still encountered occasionally in clinical and forensic toxicology, hence knowledge of the special problems presented in the detection and measurement of this compound in biological specimens may be required. The aim of this paper is to review the available documentation on this topic in the context of a chloroform-related death. Early one morning in February 1999 a 34-year-old female was found dead fully clothed on a path near to a neighbour's garden. Amfetamine intoxication combined with hypothermia was accepted as the cause of the death in the absence of any other identifiable cause. Further investigation 17 months later revealed a blood chloroform concentration of 31 mg/L and the cause of death was revised to chloroform poisoning. A murder trial ensued, the indictment specifying forced inhalation as the route of exposure. The liver chloroform concentration measured 38 months after collection was reported as 1064 mg/kg and opinions were offered at trial that the autopsy findings, which included a gastritis, but no evidence of injury to the inside of the mouth and oesophagus, excluded the possibility of ingestion of a toxic dose of chloroform. It was asserted that the explanation for the high liver concentration was that the liver had concentrated chloroform from blood after death against a concentration gradient. At appeal against conviction 7 years later the conviction was quashed. It was found that the liver concentration should have been reported at trial as 1 mg/kg. Moreover, chloroform found in the stomach contents (162 mg/kg) 86 months after collection was irrefutable evidence that some, if not all, of the chloroform had been ingested. Screening for volatile poisons should always be considered if a cause of death is not immediately obvious, especially in young people and in known substance abusers. If the presence of an unstable or volatile analyte is suspected then sample collection, transport, and storage must be performed with the analysis in mind. Quantitative analysis of all available specimens should proceed forthwith once the presence of an unstable analyte is established if the cause of death is in doubt or if prosecution may follow. In the case of chloroform especial precautions are needed: (i) headspace analysis should be performed at 35 degrees C to preclude the possibility of artefactual formation from trichloroacetic acid, (ii) precautions to prevent cross-contamination of biological samples in the laboratory must be taken, and (iii) interpretation of analytical results must take account of the widespread presence of chloroform in the environment on the one hand, and that the toxicity of chloroform varies greatly depending on the circumstances and intensity of exposure on the other.

Detection of anabolic steroids in head hair.

We developed a gas chromatography/mass spectrometry method for detection and quantitation of anabolic steroids in head hair. Following alkaline digestion and solid-phase extraction, the MO-TMS derivatives gave a specific fragmentation pattern with EI ionization. For stanozolol, the TMS-HFBA derivative showed several diagnostic ions. For androstanolone, mestanolone (methylandrostanolone), and oxymetholone two chromatographic peaks for cis and trans isomers of derivatives were seen. Recoveries were 35 to 45% for androstanolone, oxymetholone, chlorotestosterone-acetate, dehydromethyltestosterone, dehydrotestosterone, fluoxymesterone, mestanolone, methyltestosterone, and nandrolone; 52% for mesterolone, trenbolone; 65% for bolasterone; 24% for methenolone and 17% for stanozolol. Limits of detection were 0.002 to 0.05 ng/mg and of quantitation were 0.02 to 0.1 ng/mg. Seven white male steroid abusers provided head hair samples (10 to 63 mg) and urine. In the hair samples, methyltestosterone was detected in two (confirmed in urine); nandrolone in two (also confirmed in urine); dehydromethyltestosterone in four (but not found in urine); and clenbuterol in one (but not in urine). Oxymethalone was found in urine in one, but not in the hair. One abuser had high levels of testosterone: 0.15 ng/mg hair, and 1190 ng/mL urine. We conclude that head hair analysis has considerable potential for the detection and monitoring of steroid abuse.

Stability of tricyclic antidepressants in formalin solutions.

We investigated the stability of the secondary amines, desipramine (DP) and nortriptyline (NRT), and the tertiary amines, imipramine (IP) and amitriptyline (AT), in formaldehyde (F) and paraformaldehyde (PF) aqueous solutions. NRT showed little instability in 0.37 to 37% F and PF solutions, but AT formation was detected and increased, up to 0.46 to 2.7%, in parallel with rising F and PF concentrations. DP was unstable and levels decreased to 74 to 96% with increasing F concentrations, and fell only to 96% in 10% PF solution. IP formation increased in the same manner as AT to 2.9 to 3.5% of the initial DP. When AT and IP were stored in F and PF solutions, concentrations of AT and IP did not change. DP in F pH 3 to 11 phosphate buffer (PB) solutions showed high recovery in the order: pH 5 > pH 7 > pH 9 > pH 3 and pH 11. DP in PF buffered solutions decreased slightly only at pH 3 (3.5%). By contrast, IP did not change at any pH (pH 3 to 11) of the F or PF solutions. During storage for 21 days at room temperature in 3.7% F and PF solutions, IP and DP degradation was accelerated when compared with the values in pH 3 and 7 PB solutions. However, IP detected in DP F or PF solution was only 0.2% of the initial DP 21 days after storage. Thus, AT, NRT, IP and DP degraded gradually in F and PF solutions during storage at room temperature. TCAs may first react nucleophilically with formaldehyde to form hemiaminals. DP in 3.7% formaldehyde aqueous solution formed little of its methylated product, IP, at room temperature.

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.

Electrolyte concentration differences between left and right vitreous humor samples.

Between-eye differences in electrolyte concentrations were studied in 200 medico-legal autopsies using an ion-specific electrode system. Taking the highest of the paired vitreous potassium concentrations, cases < 15 mM/L were classified as biochemically nonputrefied (Cat.1, n = 124), cases 15 to 20 mM/L as early putrefaction (Cat.2, n = 51), and cases > 20 mM/L as biochemically putrefied (Cat.3, n = 25). Mean paired vitreous sodium for all cases (n = 200) was 112 to 173 mM/L (mean 148, standard deviation (SD) = 8.9); between-eye differences were 0 to 8 mM/L (0% to 5.1% of mean), averaging 1.5 mM/L (1%) and with only one case (in Cat.3) outside instrument accuracy (+/- 3 mM/L). Mean paired vitreous chloride for all cases was 73 to 124 mM/L (mean 109, SD = 7.8); between-eye differences were 0 to 9 mM/L (0% to 8.8% of mean), averaging 1.7 mM/L (1.5%) and with 5 of 200 cases outside instrument accuracy (+/- 3 mM/L). Thus between-eye concentration differences of sodium and chloride are tolerable using this methodology. Previous reports of greater variability likely reflect errors introduced by sample manipulation prior to analysis. By contrast, between-eye differences in potassium in Cat.1 cases were 0 to 2.34 mM/L (0% to 21.8% of mean) averaging 0.37 mM/L (3.3%). Significant and erratic between-eye differences in potassium undermine the usefulness of vitreous potassium in estimation of time of death.

Drug concentration in selected skeletal muscles.

We evaluated the homogeneity of drug concentrations in muscle in 14 cadavers, comprising 11 drug overdoses and three cases of chronic therapeutic drug use. Analyses were performed on samples from twelve named muscles and femoral venous blood. Standard analytical techniques and instrumentation were used throughout. There was marked within-case variability in drug concentrations with highest:lowest concentrations ranging up to 21.7. Overall highest concentrations were found in the diaphragm and mean diaphragm:blood ratios ranged from 1.1 (temazepam, two cases) and 1.2/1.3 (paracetamol, six cases) up to 6.5/13.5 (amitriptyline, three cases) and 5.3/21.3 (propoxyphene, four cases). Excluding the diaphragm, mean muscle:blood ratios ranged from 0.4 (prothiaden), 0.5 (temazepam), and 0.7 (paracetamol) up to 3.7 (temazepam), 4.3 (propoxyphene) and 5.7 (amitriptyline). We suggest that muscle is suitable for qualitative analysis but not for quantitative corroboration of a blood sample or as a quantitative alternative to blood.

Forensic medicine in China.

Although China has a long history of forensic medicine, with the first standard text published in 1247, modern practices appeared only in the 1930s under Professor Lin Ji. After the founding of the People's Republic of China in 1949, there was a period of rapid development, which was later interrupted by the Cultural Revolution of 1966-1976. Today, China has about 10,000 experts in forensic medicine organized within the separate agencies of police, prosecutor's offices, courts, universities, and the Justice Ministry. Eight medical colleges, the Institute of Forensic Sciences of the Ministry of Justice in Shanghai which publishes the Journal of Forensic Medicine, and the Forensic Medicine Association of China which publishes the Chinese Journal of Forensic Medicine are key organizations.

Who takes alcohol prior to suicide?

Alcohol abuse is strongly associated with suicide. Alcoholics are at a high risk of suicide, and studies of case series of suicide show that alcoholics account for between 20% and 40% of all suicides. What is less clear is the role of alcohol in the events leading up to the suicide. This study reviews the characteristics of individuals who consumed alcohol prior to suicide. All cases of suicide assessed by the Department of Forensic Medicine, Dundee University between 1988 and 1995 were reviewed. Data were obtained on blood-alcohol levels of 349 cases, together with the method and circumstances of the suicide, demographic variables and reports of past psychiatric history. Forty-five per cent of suicide cases had consumed alcohol and 19% were drunk (BAC > 150 mg/dl) at the time of the suicide. Consumption of alcohol was not associated with a particular method of suicide, nor with social factors such as employment status, marital status or social class. However, alcohol use was more common among those with no previous psychiatric history. This study confirms that alcohol consumption is a common precursor to suicide. It suggests that alcohol may play a more important role in the events leading to suicide amongst individuals with no previous psychiatric history.

Fatal air embolism occurring during consensual intercourse in a non-pregnant female.

Death from air embolism during pregnancy has been reported following sexual activity, particularly vaginal insufflation. We report a death from air embolism in a non-pregnant woman during consensual penile intercourse, in a position with the pelvis elevated above heart level. Air is thought to have entered the veins via a vaginal laceration, which occurred during digital foreplay.

Site-to-site variability of drug concentrations in skeletal muscle.

The homogeneity of drug concentrations in skeletal muscle was assessed in eight fatal overdoses. Ten to 30 random samples were taken from leg muscle weighing 1,650 to 7,985 g. For cases involving paracetamol the mean muscle-to-blood ratio ranged from 0.1 to 1.1 (n = 4) for amitriptyline 1.1 to 3.6 (n = 3), and for dothiepin 0.8 to 2.1 (n = 2). The coefficient of variance was large for all drugs, ranging from 10.5 (carbamazepine) to 50 (thioridazine). Skeletal muscle is not homogeneous with respect to drug concentrations in fatal overdose cases. Of 16 instances of drug detection in blood 2 (nortriptyline and promethazine) were not detected in muscle. Muscle-to-blood drug ratios varied significantly among cases, possibly influenced by survival time after drug ingestion. Quantitative interpretations of muscle drug levels present significant difficulties. However, skeletal muscle can be used for qualitative corroboration of blood analyses and is a suitable specimen for drug detection where none other is available.

Detection of white restorative dental materials using an alternative light source.

We assessed the value of an alternative light source for detecting white composite dental materials in burned and unburned teeth. Teeth filled with 18 different restorative materials (composite, glass ionomer or hybrid composite), were viewed with a Polilight. Between 415 nm and 555 nm, the glass ionomers showed distinctly different optical properties from the other materials: they either fluoresced or appeared darker. Wavelengths 415 nm to 530 nm gave a general enhancement in composite detection (17 of 18 materials). Light above 590 nm was of little value, enhancing detection in only 2 of 18 materials. After simulated burning of the teeth, there was enhanced visibility of 8 of 18 materials at wavelengths under 350 nm. Burning destroyed the previously distinct optical properties of the glass ionomers. Overall, this alternative light source aids the identification of white composite dental materials and could be used in routine forensic odontology practice.

Alcohol estimation at necropsy: epidemiology, economics, and the elderly.

AIMS: To gather data on blood alcohol concentrations in a forensic necropsy population and to analyse the information on trends that may predict where alcohol testing is going to prove cost-effective. METHODS: Alcohol assays were performed on blood, urine, and vitreous samples in 1620 consecutive medicolegal necropsy examinations. RESULTS: Alcohol was detected in only 7% of natural deaths from all causes and in four of 40 deaths categorised as unknown/obscure. Alcohol concentrations > or = 350 mg/100 ml were found in nine drug/alcohol abuse deaths (range 362-506 mg/100 ml), five accidental deaths (356-504 mg/100 ml), and one homicide victim (400 mg/100 ml). Those categorised as alcohol abusers were represented in all but one category of death (unknown/obscure deaths in males), showing that many true alcoholics die with their alcoholism rather than of it; 39% of males and 34% of females with histories of alcohol abuse had alcohol present in their blood at necropsy at concentrations > or = 50 mg/100 ml, v only 9% (male) and 6% (female) without such history. CONCLUSIONS: The study highlights the problems of elderly and "hidden" alcoholics and illustrates cases where routine assays would provide additional significant information. Routine alcohol testing is useful in all cases of suspected unnatural death but universal testing of forensic necropsies is not cost-effective.

Possible markers for postmortem drug redistribution.

The possibility that postmortem biochemical changes in blood might parallel drug redistribution and thus serve as markers was explored in a detailed case study. Eighteen blood and 14 tissue and fluid samples were taken at autopsy 16 h after the death of a 34-year-old female from amitriptyline overdose. Ranges of drug concentrations in blood were amitriptyline 1.8 to 20.2 micrograms/mL, nortriptyline 0.6 to 7.3 micrograms/mL, levels were lowest in femoral vein and highest in pulmonary vein blood. Corresponding levels of 17 amino acids showed markedly different patterns of site-to-site variability. There was a strong positive correlation between individual amino acid and drug concentrations in pulmonary blood samples (n = 5), particularly for glycine, leucine, methionine, serine, and valine. In blood samples from the great veins and right heart (n = 10), the correlation was less strong (r = 0.6 to 0.7). Methionine showed a strong positive correlation in pulmonary samples (r = 0.93), and negative correlation in great veing samples (r = -0.68). Lactic acid showed a strong negative correlation in pulmonary samples (r = -0.93) but a positive correlation in great vein samples (r = 0.71). Alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyl transferase, glucose, and bilirubin had a weak positive correlation with drug levels in great vein samples but not pulmonary samples. The results suggest that hepatic enzymes are relatively poor markers for postmortem hepatic drug shifts but that amino acids, particularly methionine, may be useful markers for pulmonary drug shifts.

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.