A case of death due to outbreak of fire during thinner abuse.

A 22-year-old male, who could not escape from a residential fire, was found dead at the scene, although the other three persons who had been in the same room escaped safely. At the medico-legal autopsy, the burned body presented the second degree level of body destruction (T. Nagano. Jpn J Legal Med., 1982) and the pugilistic attitude caused by fire. Soot was observed not only in the trachea but also in the bronchi or even the alveoli. Forensic toxicological analyses were performed using the intratracheal gas, left and right ventricular blood, urine, stomach contents, cerebrum, cerebellum, left and right lungs, liver, spleen, kidney, skeletal muscle and adipose tissue. The carboxyhemoglobin concentration was 61.4% in the left ventricular blood and 59.5% in the right ventricular blood. Ethanol and toluene were qualitatively detected in the intratracheal gas by gas chromatography-mass spectrometry. The ethanol and toluene concentration was 0.48 mg/g and 20.4 microgram/g, respectively, in the blood, and 0.40 mg/g and 28.7 microgram/g, respectively, in the cerebrum. Moreover, the urinary hippuric acid concentration was 1.76 mg/ml. The ethanol concentrations were not at the toxic level, while the toluene concentrations in the blood and cerebrum were almost at the lethal level. However, since the preservation of the capacity for vital reactions was apparent at the autopsy in the form of soot in the air passages and the formation of CO-Hb in the blood, it was surmised that the victim was still alive when the fire broke out, but subject to severe disturbance of the central nervous system function. The cause of death in the present case was diagnosed as death due to fire.

Postmortem degradation of administered ethanol-d6 and production of endogenous ethanol: experimental studies using rats and rabbits.

Deuterium-labeled ethanol-d6 was employed to study the metabolism and postmortem change of ethanol in putrefied organ tissues. First, 4 ml/kg body weight of 25% (w/v) solution of ethanol-d6 was administered orally to each of 15 rats. The heart blood and organs were collected 15-90 min after the administration and the ethanol-d6 was analyzed by head space gas chromatography/mass spectrometry. The ethanol-d6 concentration in the organ tissues reached its maximum at 15 min after the administration and then gradually declined, showing the same pattern as human ethanol metabolism. Ethanol-d6 (3 ml of the same solution/kg body weight) was injected into the vein of a rabbit's ear (total of 12 rabbits). The rabbit was killed with carbon monoxide 30 min after the administration and the carcass was allowed to stand for 1-4 days at 30 degrees C in a moist chamber. The concentration of ethanol-d6 decreased moderately. Postmortem ethanol and 1-propanol concentrations, in contrast, showed marked increases 2.5 days and more after sacrifice in line with the degree of putrefaction of each organ tissue including skeletal muscle. This suggests the postmortem activation of micro-organism activity. These results indicate that ethanol concentrations in cadaver tissues must be carefully assessed with due consideration of postmortem degradation and production.

Experimental studies on postmortem diffusion of ethanol-d6 using rats.

In an investigation of postmortem ethanol diffusion deuterium-labeled ethanol-d6 was instilled by peroral gavage immediately after death by CO into the stomach of rat carcasses which were subsequently kept for 12-72 h at 5 or 30 degrees C. The heart blood, abdominal fluid and several tissues were collected and analyzed by head space gas chromatography-mass spectrometry. Rat carcasses showed no macroscopic changes until at least 72 h at 5 degrees C, and 12 h at 30 degrees C. At 30 degrees C, slight macroscopic change was observed after 24 h, moderate change after 48 h and marked change after 72 h. In the abdomen ethanol-d6 diffused gradually into neighboring organs (hepatic left lobe, left kidney and spleen) at 5 degrees C, with ethanol-d6 reaching a peak concentration of 0.75-2.38 mg/g at 24 h. At 30 degrees C, ethanol-d6 was also detected in neighboring organs and reached a peak concentration of 1.06-2.61 mg/g at 12 h. Thereafter, the ethanol-d6 concentration in the liver, kidney and spleen decreased, with concentrations ranging from 0.30 to 0.61 mg/g at 30 degrees C and 0.05 to 1.47 mg/g at 5 degrees C at 48 h. In the femoral skeletal muscle, ethanol-d6 was not detected until 24 h or more storage at 30 degrees C and never detected at 5 degrees C. In the brain and the organs in the thoracic cavity ethanol-d6 was detected after 12 h or more at 5 or 30 degrees C. Comparison of these results of direct peroral gastric instillation with those when ethanol-d6 was injected into the stomach through a laparotomy incision suggest that the brain and thoracic cavity changes were a result of diffusion from the mouth and esophagus. After 24 h at 30 degrees C, the postmortem ethanol production (0.33-0.85 mg/g) was comparable to those in previous reports. These results indicate that the assessment of ethanol concentration in the heart blood and organs in autopsy cases must be carefully conducted in comparison with the alcohol content of the stomach.

Sex determination by polymerase chain reaction on mummies discovered at Taklamakan desert in 1912.

Sex determination was performed by the polymerase chain reaction (PCR) on eight adult mummies and one child mummy which were discovered at Taklamakan desert in 1912 and now belong to the Lüshun Museum in China. Archaeologically, these mummies were humans living in the seventh century, that is, more than 1300 years ago. Putative sex determination was performed based on external morphology for six of the eight adults, but it was impossible for the other two adults and one child mummy due to marked destruction on the external morphology. Hair, muscle and skin samples were then collected from each adult mummy, and skin and rib samples from the child mummy. Forty PCR cycles were performed as follows: denaturation at 94 degrees C for 40 s, annealing at 55 degrees C for 30 s and extension at 72 degrees C for 1 min. The primer and PCR reaction mixture were prepared according to the report by Witt and Erickson (M. Witt and R. P. Erikson, A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction. Hum. Genet., 82 (1989) 271-274)). Two different pairs of primer were used. One was X1, X2 (X1: 5'-AATCATCAAATGGAGATTTG-3'; X2: 5'-GTTCAGCTCTGTGAGTGAAA-3') to flanking the 170 bp fragment of the alphoid repeats on the human X chromosome, and the other was Y11, Y22 (Y11: 5'-ATGATAGAAACGGAAATATG-3'; Y22: 5'-AGTAGAATGCAAAGGGCTC-3') to flanking the 130 bp fragment of the alphoid repeats on the human Y chromosome. Extracted DNA solutions from mummy samples was purified using a spin column (T. Yoshii, K. Tamura, T. Taniguchi, K. Akiyama and I. Ishiyama, Water-soluble eumelanin as a PCR-inhibitor and a simple method for its removal. Jpn. J. Legal Med., 47 (1993) 323-329 (in Japanese with English abstract) for removing PCR-inhibitors, and bovine serum albumin (BSA) was employed to inhibit the remaining impurities even after the purification with the column. In six adult cases where the putative sex was determined from external morphology, the sex in five cases was consistent with that by PCR using hair, muscle, and skin samples, but the other one was inconsistent. In two adult cases where sex estimation was externally impossible, the sex was determined to be male because both X-specific and Y-specific bands were clearly detected. The child mummy was definitely male. This study shows that the sex determination was possible by the PCR method even with very ancient human samples > 1300 years old, that spin column was useful for removing impurities in the DNA solution from ancient human samples and that the BSA of optimum concentration suppressed the action of the PCR-inhibitory factors.

Fatal injuries of the brain stem and/or upper cervical spinal cord in traffic accidents: nine autopsy cases.

Nine forensic autopsy cases were studied. All had injuries of the brain stem and/or upper cervical spinal cord due to traffic accidents. Among the nine subjects, eight were pedestrians and one was a left, front seat occupant of a vehicle. Examination of these 9 cases revealed that three had ponto-medullary avulsion, two had medullary avulsion and the other four had laceration of the upper cervical spinal cord. Atlanto-occipital dislocation was observed in five cases, and a ring fracture around the foramen magnum in the other two cases. Intraventricular haemorrhage, probably due to tears of the choroid plexus caused by hyperextension or hyperflexion of the head, was found in seven cases. In just one of these seven cases, both of the lateral ventricles were filled with dark red haemocoagulum. Hyperextension is considered to occur more commonly in road trauma cases where the victim is alcoholically intoxicated.

Screening of volatile substances and determination of toluene (a thinner component) in the blood and urine in emergency medical care and autopsy cases by the pulse heating method.

Screening of volatile substances was performed by pulse heating gas chromatography - mass spectrometry (GC-MS) using a GS-Q column in 211 emergency medical care and 342 autopsy cases. At least 36 standard substances could be separately detected. Six kinds of volatile substances were screened in a total of 553 cases. Toluene and/or hippuric acid were detected in the blood and/or urine in respectively, 4 emergency medical care and 8 autopsy cases. There were 11 abusers (9 males and 2 females) in these 12 positive cases. The ages of the abusers ranged from 13-26 years. There was no particular pattern to the monthly frequency distribution of identification of thinner (toluene) abuse cases, which occurred throughout the year. It is believed that these data at least partly reflect the present status of thinner/glue abuse in Japan. We conclude that pulse heating GC-MS is useful in the screening and quantitative determination of volatile substances including toluene and other thinner/glue components.

Determination of the volatile anesthetics halothane, enflurane, isoflurane, and sevoflurane in biological specimens by pulse-heating GC-MS.

Four kinds of volatile anesthetics (halothane, enflurane, isoflurane, and sevoflurane) that were dissolved in 3 microL of experimental plasma samples were examined by the pulse-heating gas chromatographic-mass spectrometric method, and this approach was found to be reliable for qualitative and quantitative analysis. The analytical results also showed good recovery and accuracy. This method was then applied to real blood specimens taken from patients during surgery. The same blood specimens were also analyzed simultaneously by the conventional headspace method for comparison. The data for the clinical blood specimens examined by these two methods showed reasonable correlation coefficients of 0.914 (enflurane) and 0.937 (sevoflurane). These results indicate that the pulse-heating method is applicable for toxicological and clinical analysis of several kinds of volatile anesthetics.

Screening and determination of methamphetamine and amphetamine in the blood, urine and stomach contents in emergency medical care and autopsy cases.

Methamphetamine (MA) and amphetamine (AMP) were screened and their levels were determined using the Toxi-Lab thin-layer chromatography system and gas chromatography-mass spectrometry, respectively, in the blood, urine and stomach contents from 211 emergency medical care and 417 autopsy cases. MA and AMP were detected in 5 emergency medical cases, and the blood MA and AMP concentrations ranged from 0.697-0.041 micromol/100 g and from 0.0944-0.0003 micromol/100 g, respectively. MA and AMP were detected in 19 autopsy cases, in which blood MA and AMP concentration ranged from 14.3-0.123 micromol/100 g and from 0.256-0.0017 micromol/100 g, respectively. The autopsy cases included 5 cases of sudden death with blood MA concentration of less than 3 micromol/100 g. MA and AMP screening and determination in emergency medical care and autopsy cases provide useful information and are indispenable in clarifying the dimensions of MA abuse in Japan.

Toxicological analysis of the psychotropic drugs chlorpromazine and diazepam using chemically fixed organ tissues.

Toxicological analysis for chlorpromazine and diazepam was performed using chemically fixed organ tissue specimens. After chlorpromazine and diazepam had been injected into rabbits, organ tissues (brain, lung, liver, kidney and skeletal muscle) were collected and fixed in 3 fixative solutions: buffered 10% formalin solution (pH 7.4, 10% BF), non-buffered 10% formalin solution (pH 5.1, 10% non-BF), buffered 4% paraformaldehyde solution (pH 7.4, 4% BPA). Chlorpromazine and diazepam were determined by GC-MS (gas chromatography-mass spectrometry) after 5 different fixation periods, and were detected even after 28 days of fixation. Recoveries of chlorpromazine and diazepam in 10% BF were within the range 48-86% and 68-171%, respectively after 28-day fixation, those in 10% non-BF were 22-54% and 48-78%, respectively, and those in 4% BPA solution were 13-59% and 14-50%, respectively. Thus, 10% BF was found to be the most suitable fixation medium for analysis of chlorpromazine and diazepam.

Toxicological analysis for drugs and poisons using the formalin-fixed organ tissues. 1. Methamphetamine.

Methamphetamine (MA) was analyzed using formalin-fixed organ tissues from experimental animals and an autopsied cadaver. The brain, lung, liver, kidney and skeletal muscles of rabbits that had been administered MA were collected, and then fixed with a non-buffered 10% formalin solution at room temperature (10-20 degrees C) for five different fixation periods (1, 3, 7, 14 and 28 days). After the Extrelut column extraction, MA was derivatized using anhydrous trifluoroacetic acid (TFA) and analyzed by GC-MS using a DB-17 column. MA-TFA in the eluates from the organ tissues after the formalin-fixation could be identified by GC-MS. Even one day after the formalin-fixation, concentrations of MA in the fixed organ tissues already significantly decreased to 1.3-3.1% when compared with those of MA in the non-fixed tissues, and to 0.04-0.4% in all of the fixed organ tissues at 28 days after the fixation. Moreover, MA in the organ eluates from the autopsied male cadaver after one-month formalin-fixation was qualitatively detected. These results obviously show the usefulness of MA detection using formalin-fixed organs in the practice of forensic medicine and police science.

Toxicological analysis of drugs and poisons in formalin-fixed organ tissues. 2. Volatile substances.

Diethylether, chloroform and toluene were administered by inhalation and ethanol intravenously to rabbits. As soon as possible after death, tissue specimens were collected from the brain, lung, liver, kidney and skeletal muscle and fixed in non-buffered 10% formalin at room temperature (10-20 degrees C) for 4 different periods (1, 2, 5 and 14 days). The volatile substances were analyzed and identified by gas chromatography/mass spectrometry (GC-MS). The measured concentrations of ethanol, diethylether, chloroform and toluene in the brain tissue 1 day after fixation decreased to 8, 23, 73 and 84% respectively compared with those in the non-fixed brain tissue (100%). The rank order of the rate of decrease in the fixed state was: ethanol > diethylether >> chloroform > toluene. These volatile substances could be detected clearly in all the tissue specimens, even after a 14-day fixation period. These results provide useful toxicological information that will help to differentiate whether volatile substances have been administered antemortem or postmortem.

[Forensic toxicological application of TOXI-LAB screening for biological specimens in autopsy cases and emergency cares].

Using the TOXI-LAB drug detection system, emergency toxicological screening was performed in autopsy cases and emergency cares. In 280 autopsy cases (male 182 cases 65%, female 98 cases 35%), drug positive cases were 28 cases of male (15%) and 24 cases of female (24%). The age groups which showed higher rate of drug positive cases were 10s-40s in male (approximately 20%) and 20s in female (67%). In the 238 cases of emergency care (male 129 cases 54%, female 104 cases 44%, unknown 5 cases 2%), drugs were positive in 29 cases of male (22%) and 32 cases of female (30%). The age groups which showed relatively higher rate of drug positive cases were 40s in male (64%), 20s (71%) and 30s (89%) in female. Forty-four different kinds of drugs were detected in TOXI-LAB positive cases, in which the psychotropic drugs and the sedative-hypnotic drugs amounted to approximately 70%. Methamphetamine and amphetamine, which were the main abused drugs showing a socially important problem, were detected in total 15 cases. TOXI-LAB was based on thin-layer chromatography (TLC); however, from the extraction to development, coloration and detection have been accelerated (about 50 min) and simplified. In order to perform the forensic toxicological practice in Japan, it becomes more useful that TOXI-LAB is used in autopsy cases and emergency cares, if the drugs, which have caused poisoning cares in Japan, are added to TOXI-LAB. The present study describes the advantage and problem of TOXI-LAB drug detection system through demonstrating the practical cases of autopsy cases and emergency cares.

[A fatal case due to intoxication with seven drugs detected by GC-MS and TDx methods].

A twenty-year-old woman was suspected to ingest large amounts of 4 kinds of over-the-counter analgesic and antipyretic drugs, and was found dead. Drugs and poisons were screened by TOXI-LAB drug detection system in the serum and urine, and analyzed by GC-MS and TDx system in the blood, urine, organ tissues and contents of stomach and small intestine. The concentrations (microgram/g) of bromisovalum, apronalide, ibuprofen, ethenzamide, acetaminophen and caffeine in the heart blood were 36.5, 7.58, 43.1, 16.9, 1.22 and 177, respectively. Salicylic acid concentration in the serum was 82.1 micrograms/ml. The concentrations of bromisovalum and caffeine are high enough to be lethal levels. Neither fatal pathological findings nor traumatic wounds were seen. The overdose of both bromisovalum and caffeine and synergistic, additive or combined effects of other 5 drugs above are considered to be her cause of death.

Application of hemoglobin analysis by CO-oximeter to medico-legal practice with special reference to diagnosis of asphyxia.

Hemoglobin analysis by CO-oximeter is more easily and rapidly available method than other spectrophotometric or GC methods. In the present study, the possibility of diagnosis of asphyxia by CO-oximeter was examined experimentally using rabbits. When measured value of oxyhemoglobin (O2Hb) was lower than about 20% and that of deoxyhemoglobin (HHb) was, on the other hand, higher than about 80%, being accompanied by low measured values of both methemoglobin (MetHb) and CO-hemoglobin (COHb) (< 5%), asphyxia should be firstly considered as the cause of death. To the corpses found at the scene of fire, three chief factors causing death such as burns, suffocation due to oxygen deficiency and intoxication by poisonous gases may have occurred simultaneously, and through measuring the values of O2Hb, COHb, MetHb and HHb, the main lethal factor could be selected out among these three factors above and the cause of death could be examined much more closely. Because of easy and rapid analysis, it could be considered that CO-oximeter was useful for forensic practices and police activities.

[An autopsy case of fatal nicotine poisoning].

A fatal case of nicotine poisoning is reported in which a 44-year-old female committed suicide in a short time by taking orally the eluate from tobacco. External examination showed no abnormal findings except for markedly dark red-purple postmortem lividity, and internal examination demonstrated no pathological changes but the signs of sudden death. Through the toxicological investigation by GC and GC-MS, however, nicotine was detected in the solution which she had taken orally and in the blood, urine and the contents of the stomach and small intestine. The nicotine concentrations of the blood, urine and contents of stomach and small intestine were 6.3 micrograms/ml, 1.5 micrograms/ml, 30 micrograms/ml and 71 micrograms/g respectively, and enough to be lethal.