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.

Cryosurgery for chronic injuries of the cutaneous nerve in the upper limb. Analysis of a new open technique.

We have treated six patients with chronic pain following nerve injury using a cryosurgical probe. All had a significant return of hand function and improvement of pain during a mean follow-up of 13.5 months. Open visualisation of the injured nervous tissue is essential for patients undergoing this technique. Four patients regained normal sensation in the dermatome of the previously injured nerve.

Percutaneous cervical cordotomy for the control of pain in patients with pleural mesothelioma.

BACKGROUND: Severe chest pain is common in mesothelioma. Percutaneous cervical cordotomy, which interrupts the spinothalamic tract at the C1/C2 level causing contralateral loss of pain sensation, is particularly appropriate in mesothelioma as the tumour is unilateral and systemic analgesia may be ineffective and is limited by harmful side effects. METHOD: A retrospective review was performed to determine the effectiveness and complication rate of this procedure. RESULTS: Fifty two patients were using opioids prior to cordotomy. The median daily dose of morphine before and after cordotomy was 100 mg (range 0-1000 mg) and 20 mg (range 0-520 mg), respectively (p < 0.001). Forty three patients (83%) had a reduction in pain such that their dose of opioid could be at least halved. Twenty patients (38%) were able to stop completely. Recurrence of pain requiring an increase in opioid medication was recorded in 18 patients at a median time of nine weeks (range 0.7-26 weeks). Four patients developed mild weakness, two had troublesome dysaesthesia. The median time from cordotomy to death was 13 weeks (range 0.3-52 weeks). Six early deaths within two weeks of cordotomy occurred early in the series and reflect postoperative chest infection and poor selection as the patients were in the terminal stages of mesothelioma. CONCLUSIONS: Percutaneous cervical cordotomy is successful in treating pain from mesothelioma. There was a low complication rate in this series. Referral to a unit experienced in cordotomy is recommended as soon as pain from chest wall invasion is suspected.

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.