Genetic analysis of the rhabdomyolysis-associated genes in forensic autopsy cases of methamphetamine abusers.

Methamphetamine (MA) use sometimes causes rhabdomyolysis, which has been associated with mortality. We analyzed potential rhabdomyolysis-susceptibility genes from autopsy samples of 18 methamphetamine abusers. We examined mutations in the ryanodine receptor 1 (RYR 1), carnitine palmitoyltransferase II (CPT II), very long-chain acyl-CoA dehydrogenase (VLCAD), and cytochrome P450 (CYP) 2D6 genes. Different RYR1 mutations that caused amino acid substitutions ((612)Ala>Thr and (4295)Ala>Val) were identified in 2 cases. In the CPT II gene, there was a new mutation ((545)Glu>Ala) in 1 case and there were mutations that did not change activity in 17 cases. In the VLCAD gene, there were mutations that did not change activity in 6 cases. In the CYP2D6 gene, homozygosity for CYP2D6∗10, which is associated with significantly reduced metabolic activity, was found in 3 cases, while 2 cases carried a different previously unreported missense mutation ((344)Arg>Gln and (48)His>Tyr). RYR1 mutations and the new CPT II mutation identified in this study were not observed in a control group. Eighteen cases that were genetically analyzed were also investigated immunohistochemically to diagnose the possibility of rhabdomyolysis. However, there were no significant mutations that reduced enzyme activity in the suspected cases of rhabdomyolysis. These data suggested no obvious relationship between the genetic mutations observed in this study and rhabdomyolysis.

Diagnostic approach to drug-screening tests for fatal diabetic ketoacidosis: forensic autopsy of a methamphetamine abuser.

To diagnose the cause of death in autopsy cases, systematic examinations, such as macroscopic, pathological, biochemical, and toxicological are important. In this case report, drug examinations also gave very useful information to diagnose the cause of death, fatal diabetic ketoacidosis (DKA). A female methamphetamine abuser in her forties was found dead lying on a hotel bed. Diagnosing her cause of death was difficult only from the macroscopic findings because there was no fatal and/or serious injury or disease. On toxicological examination, acetone was detected at a high concentration (682 microg/mL in blood, 887 microg/mL in urine) using gas chromatography (GC). Using gas chromatography-mass spectrometry (GC-MS), methamphetamine was detected in the blood, urine, hair, and visceral organs; however, these concentrations were low. At the same time, GC-MS examination revealed a high glucose peak. From the results of the biochemical examination of urine, acetoacetic acid was 1940 micromol/L, beta-hydroxybutyric acid was 14,720 micromol/L, and glucose was 4620 mg/dL. Histologically, Langerhans' islets in the pancreas were fibrotic and atrophic, and no insulin-immunoreactive cells were observed. The subsequent police investigation also revealed that she had contracted diabetes mellitus type 1; therefore, we concluded that her cause of death was DKA, due to a lack of insulin injection.

An autopsy case of rhabdomyolysis related to vegetamin and genetic analysis of the rhabdomyolysis-associated genes.

We report an autopsy case of a man who died 2 days after taking an overdose of vegetamin. The autopsy findings were as follows: the epidermis on the axillary fossa and the inguinal skin had become macerated. Skeletal muscle was discolored. Concentrations of urea nitrogen, creatinine and urine myoglobin were 1.95 g/day, 0.66 g/day and 1100 ng/mL, respectively. Immunohistochemically, myoglobin was strongly stained at the Bowman's capsule, and tubular lumen and epithelium. 8-OH-dG was strongly stained in renal tubular epithelium in which cell nuclei were strongly stained. ORP-150 was observed in intraglomerular cells and renal tubular epithelium. The concentrations of phenobarbital, promethazine and chlorpromazine ranged from therapeutic to toxic levels, from toxic to lethal levels and toxic level, respectively. His cause of death was considered to be vegetamin-induced rhabdomyolysis. In genetic analysis of this subject, there were two heterozygous silent mutations in the three hot-spot regions in the RYR1 gene. In the CPT II gene, the subject was found to be heterozygous for an amino acid substitution in exon 4, (1203)G>A causing a (368)Val>Ile amino acid substitution. There was no mutation in the VLCAD gene or CYP2C19 gene. The subject was heterozygous for CYP2D6*1 and CYP2D6*2.

Application of the drowning index to actual drowning cases.

The drowning index (DI) was devised to diagnose drowning deaths, and is the weight ratio of the lungs and pleural effusion to the spleen. Among drowning (94 cases), mechanical asphyxia (47 cases), and acute cardiac (42 cases) deaths, within 2 weeks postmortem we compared six markers, the weight of each lung, pleural effusion weight, total weight of the lungs and pleural effusion, spleen weight, heart weight, and the DI. Statistical analysis revealed that the total weight was heavier, while spleen weight was lighter, and the DI was significantly larger in the drowning group (p<0.05). We examined the relation between the postmortem time and these markers. We divided 94 drowning cases into three groups according to the postmortem duration, group A (0-3 days; 43 cases), group B (3-7; 29 cases), and group C (7-14; 22 cases). The cut-off point of the DI was analyzed using the receiver operating characteristic (ROC) curve. As a result, the DI cut-off point was 14.1 in cases within two postmortem weeks. Drowning is still a difficult autopsy diagnosis, but in our experience, DI is a valuable indicator.

An autopsy case of suicide by acetylene explosion: a case report.

We report an autopsy case of a male welder in his thirties who was found dead in an exploded truck cabin. The roof, windows and doors of the cabin had been blown up to 50 metres away. An oxygen cylinder and an acetylene cylinder, both unexploded, were found in the back of the truck. The deceased was lying on the driver's seat. His entire body was burnt, carbonised and partially skeletonised. There was a small amount of soot in his oesophagus and stomach and a large volume of bloody fluid in the trachea and bronchi. There was an extensive haemorrhage in the posterior thoracic wall. No drugs were detected in the blood. Hardly any carbon monoxide and combustion-related gases were detected in the blood, therefore he was not considered to have died from the fire. Acetylene was detected in his blood (21.5 microg/ml in the femoral vein blood) and urine (7.49 microg/ml), with marked haemorrhaging in his back. We therefore concluded that the victim died because of an acetylene explosion in the cabin and also that this was a suicide.

Analysis of acetylene in blood and urine using cryogenic gas chromatography-mass spectrometry.

A method for quantitative analysis of acetylene in blood and urine samples was investigated. Using cryogenic gas chromatography-mass spectrometry (GC-MS), acetylene was measured with isobutane as the internal standard in the headspace method, which revealed a linear response over the entire composite range with an excellent correlation coefficient, both in blood (R = 0.9968, range = 5.39-43.1 microg/ml) and urine (R = 0.9972, range = 2.16-10.8 microg/ml). The coefficients of variation (CV) for blood ranged from 2.62 to 11.6% for intra-day and 4.55 to 10.4% for inter-day. The CV for urine ranged from 2.38 to 3.10% for intra-day and 4.83 to 11.0% for inter-day. The recovery rate as an index of accuracy ranged from 83 to 111%. The present method showed good reliability, and is also simple and rapid. In actual samples from a charred cadaver due to acetylene explosion, the measured concentrations of acetylene by this method were 21.5 microg/ml for femoral vein blood, 17.9 microg/ml for right atrial blood, 25.5 microg/ml for left atrial blood and 7.49 microg/ml for urine. Quantification of acetylene provides important information, because the acetylene concentration is a vital reaction or sign. For example, when acetylene is filled in a closed space and then explodes, in antemortem explosion, the blood acetylene concentration of the cadaver might be significant. On the other hand, in postmortem explosion, acetylene is not detected in blood. Furthermore, when several victims are involved in one explosion, comparison of the sample concentrations can also provide useful information to establish the conditions at the accident scene; therefore, the present method is useful in forensics.

Genetic analysis of ryanodine receptor 1 gene and carnitine palmitoyltransferase II gene: an autopsy case of neuroleptic malignant syndrome related to vegetamin.

We report an autopsy case of a man in his forties who died 2 days after taking an overdose of vegetamin. The autopsy findings were as follows: externally, the upper epidermis of some parts of the body had become loosened. The epidermis was easily detached from the dermis using the fingers. Viscous fluid adhered around the nose and mouth. The brain was edematous and weighed 1520 g. Skeletal muscle was discolored. The urine was a slightly red-tinged yellow. The organs showed congestion. Urine tests: urea nitrogen: 1.95 g/day; creatinine: 0.66 g/day; urine myoglobin: 1100 ng/mL. Blood level of drugs: phenobarbital: 38.2 microg/ml; promethazine: 2.22 microg/ml; chlorpromazine: 0.96 microg/ml. Immunohistochemistry identified myoglobin in the kidney. From these findings, his cause of death was considered to be vegetamin-induced neuroleptic malignant syndrome and rhabdomyolysis. Mutation of the ryanodine receptor 1 gene is associated with malignant hyperthermia. However, there was no mutation which causes amino acid substitution in the three hot-spot regions of the ryanodine receptor 1 gene. Partial deficiency of carnitine palmitoyltransferase II is the commonest cause of recurrent rhabdomyolysis in adults. The subject was found to be heterozygous for an amino acid exchange in exon 4, (1203)G-->A causing a (368)Val-->Ile amino acid substitution. It is necessary to examine other candidate gene mutations.

[Review of the drug analysis system accompanied by forensic autopsy in Finland].

In Japan, drug analyses for forensic autopsies have been traditionally carried out at each laboratory of the Department of Forensic Medicine. However, it is difficult to maintain a high quality of drug analysis in each department due to an insufficient number of staff and lack of equipment. Therefore, the establishment of more advanced toxicology centers which can handle all drugs associated with forensic autopsies is essential. In addition, a systematic system for requesting drug analyses from each department and dealing with the results from the center is needed. The number of forensic autopsies carried out in Finland is as high as that in Japan although the population is 1/24th that of Japan, and toxicological analyses for the entire country are centralized in one place, the Laboratory of Toxicology, Department of Forensic Medicine, University of Helsinki. Since the autopsies and drug analyses are carried out at a University as in Japan, the drug analysis system in Finland can be a good model when considering the future system in Japan. Therefore, a review of the drug analysis system accompanied by forensic autopsy in Finland was carried out with the collaboration of the Departments of Forensic Medicine, University of Helsinki and University of Turku. Based on the above studies and the present situation in Japan, we discuss the future drug analysis system needed in Japan.

Rapid GC-MS analysis of methamphetamine and its metabolites in urine--application of a short narrow-bore capillary column to GC-MS.

A rapid analysis of methamphetamine and its metabolites in urine was performed by gas chromatography-mass spectrometry (GC-MS) using a short narrow-bore capillary column (NBC) (5 m x 0.1 mm I.D.). For detection, selected ion monitoring (SIM) was performed for the characteristic ions of each of the compounds. The analytes were independently detected within 2 min. Linearity was demonstrated over a range from 25-2500 ng/ml. As an application of this study, a urine sample from a drug-abuse suspect was analyzed. The analytes from the actual sample were detected with reasonable reproducibility. The results indicate the possibility of rapid analysis using a conventional GC-MS with a short NBC at a relatively low inlet pressure.

[Continuous challenges in Japanese forensic toxicology practice: strategy to address specific goals].

In this paper, the status quo of forensic toxicology in Japan and the West is surveyed and a strategy to address future goals of Japanese forensic toxicology is proposed. Forensic toxicology in the West consists of three main areas--post-mortem forensic toxicology, human-performance forensic toxicology and forensic urine drug testing. In Japan, post-mortem forensic toxicology is practiced in university forensic medicine departments while most of the human-performance forensic toxicology is carried out in police laboratories. However, at least at present, strictly controlled workplace urine drug testing is not being performed, despite the abuse of drugs even by uniformed members of the National Defence Forces and police. For several years, the author has been introducing Western forensic toxicology guidelines and recommendations, translated into Japanese with the help of Western forensic toxicologists, to Japanese forensic toxicologists. Western forensic toxicology practice is at an advanced stage, whereas Japanese practice is in a critical condition and holds many problems awaiting solution, as exemplified by the urine drug testing in police laboratories. There is never any sample left for re-examination by the defence in all cases, though the initial volume of the urine sample available for examination is 30-50 ml. Only one organisation carries out everything from sampling to reporting and, in addition, the parent drug and its metabolites are not quantified. It is clear that the police laboratories do not work within good laboratory practice guidelines, nor do they have quality manuals or standard operating procedures manuals. A basic change in Japanese forensic toxicology practice is now essential. The author strongly recommends that, first of all, Japanese toxicologists should prepare forensic toxicology guidelines based on the Western models. The guidelines would progress the following objectives for forensic toxicology laboratories: 1) to have documented good laboratory practice standards; 2) to have a quality control system including a quality manual and standard operating procedures manual; 3) to have some degree of compulsion to implement quality assurance both through their own internal efforts and by appropriate remedial actions based on the results of an external proficiency testing scheme. For forensic toxicologists, the implications are that they should be: 1) responsible for ensuring that laboratory practices are performed under satisfactory conditions and 2) required to be certified as a forensic toxicology specialist in order to prove their forensic toxicology ability. For their part, governments should: 1) carry out administrative reforms related to forensic toxicology; 2) simplify the procedure for obtaining certified reference materials; 3) introduce a strict workplace urine drug testing programme for government employees, at least for those related to law enforcement. When all of these objectives have been realised, the specific goal will be achieved through which Japanese forensic toxicology is able, in practice, to fulfill its responsibility to society.