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Postmortem computed tomography (PMCT) is a useful method to identify various causes of death and measure the volume of internal organs and gases. The purpose of this study was to investigate postmortem changes as measured by PMCT, and the relationship between the volume of organs and gases and postmortem interval (PMI). Forty-six cadavers (22 men, 24 women) were examined by CT before autopsy. The volumes of the lungs, intrahepatic gas, and intrarectal gas were measured by CT using a workstation. A stepwise regression analysis was used to establish a predictive equation to ascertain the measured volume using factors including sex, age, height, body mass index (BMI), body surface area (BSA), and PMI. For estimation of PMI, stepwise regression analysis was used. In the equations for each measured volume, height, diaphragmatic height, and BSA were adopted for the left lung; height and diaphragmatic height were adopted for the right lung; PMI was adopted for intrahepatic gas; and sex and PMI were adopted for intrarectal gas. In the PMI equations, left lung volume, intrahepatic gas, and intrarectal gas were adopted together with sex, weight, and BMI. Values of intrahepatic gas decreased with postmortem changes, while intrarectal gas increased. This may be useful in investigation of postmortem changes. It will be necessary to include other parts of the intestine and to analyze volume changes in gases from these parts after death., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Acute carbon dioxide (CO2) poisoning causes no specific features that are revealed upon autopsy, and the pathophysiological mechanism of this syndrome is unclear. To address this issue, in the present study, we exposed rats to CO2 concentrations ranging from 10% to 60% and determined the effects on mRNA expression. According to the results of Gene Ontology (GO) and cluster analyses of microarrays data, we selected the following genes for further analysis: alkylglycerone phosphate synthase (Agps), hypocretin (Hcrt), tyrosine hydroxylase (Th), heat shock protein beta 2 (Hspb2), and opioid receptor delta 1 (Oprd1) expressed in the frontal cortex and renin (Ren), pancreatic polypeptide (Ppy), corticotropin releasing hormone receptor 2 (Crhr2), carbonic anhydrase 1 (Car1), and hypocretin receptor 1 (Hcrtr1) expressed in the hypothalamus. We found significant differences between the expression levels of Agps and Hspb2 mRNAs in the frontal cortex and that of Ppy, Crhr2 mRNAs in the hypothalamus in the presence of high concentrations of CO2. Further investigation of these genes may clarify the pathophysiology of acute CO2 poisoning and facilitate the development of novel forensic tests that can diagnose the cause of death., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

A woman in her thirties was found dead on a bed. Considerable amounts of "aroma liquid" and "bath salt" products and hypnotic drug tablets were scattered beside the bed. Autopsy showed pulmonary congestion and edema. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses of "aroma liquid" and "bath salt" products showed the presence of new cathinone designer drugs, 4-methoxy PV8 (4-methoxy PHPP), PV9 (α-POP), and 4-methoxy PV9 (4-methoxy α-POP), and a dissociative agent, diphenidine. Drug screening in stomach contents, blood and hydrolyzed urine of the woman by GC-MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the presence of the above 4 types of drugs and 3 types of benzodiazepines, triazolam, flunitrazepam, and nitrazepam, and their metabolites. The above 7 drugs and 3 benzodiazepine metabolites were simultaneously determined by LC-MS/MS after modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction using diazepam-d5 as the internal standard. The concentrations of 4-methoxy PV8, PV9, 4-methoxy PV9, and diphenidine in the femoral blood were 2.69, 0.743, 0.261, and 1.38μg/ml, respectively, which were significantly higher than concentrations reported in previous cases. Alcohol concentration in the femoral blood was 1.52mg/ml. Based on the pathological and toxicological findings, the cause of death was determined to be 3 types of cathinone drugs, 4-methoxy PV8, PV9, and 4-methoxy PV9, and diphenidine poisoning under the influence of 3 benzodiazepines and alcohol., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Pesticide poisoning is one of the most common causes of death by poisoning in Japan, and various kinds of pesticides including organophosphates, carbamates and pyrethroids are listed as causative substances. The purpose of our study was to develop a rapid and reliable screening method for various kinds of pesticides in whole blood by using a unique calibration-locking database and gas chromatography-mass spectrometry. A database of 70 pesticides was constructed using NAGINATA™ software with parameters such as mass spectrum, retention time and qualifier ion/target ion ratio (QT ratio) and calibration curve. Diazepam-d(5) was used as the internal standard for construction of each calibration curve within the range of 0.01-5.0 μg/ml. We examined the applicability of the constructed database by analyzing whole blood samples spiked with 70 pesticides. The pesticides in blood were extracted with hexane under acidic conditions or with an enhanced polymer column (Focus™), subjected to GC-MS, and screened by the pesticides database. Among the 70 pesticides examined, 66 and 62 were successfully identified at the level of 1 and 0.1 μg/ml, respectively, by hexane and 63 and 51 were identified by the Focus column without the use of standard compounds. The time required for data analysis was significantly reduced. Since the established method can produce qualitative and semi-quantitative data without the need for standard substances, this new screening method using NAGINATA™ should be useful for confirming the presence of pesticides in blood in future clinical and forensic cases., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Diagnosis of fatal hypothermia is considered to be difficult in forensic practice because of the lack of any specific pathological findings. The mechanism that induces abnormal behavior such as undressing or hiding during the state of hypothermia has not been clarified. In order to solve these problems, we made a rat model of fatal hypothermia and investigated the expression of some mRNA within the hypothalamus and the frontal cortex. The expression of aldehyde dehydrogenase 6 family, member A1 (ALDH6A1), cocaine- and amphetamine-regulated transcript peptide (CARTPT), desmin (DES), heat shock 70kDa protein 4 (HSPA4), serotonin receptor 2A (HTR2A), opioid receptor, delta 1 (OPRD1) and transthyretin (TTR) supposedly related to fatal hypothermia was determined using quantitative real-time PCR. The expression of OPRD1 in the hypothalamus of fatal hypothermia was significantly increased, while the expression of TTR within the frontal cortex was significantly decreased compared to that in the control. These findings suggest that OPRD1 and TTR may be involved in thermoregulation at a low ambient temperature., (2011 Elsevier Ireland Ltd. All rights reserved.)

We present two cases where a single locus mismatch was found in the locus D19S433 using the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems) (Identifiler Kit) during paternity and maternity tests. This mismatch differed from the mismatch pattern where there is usually a one repeat difference. We designed forward and reverse primers so that they were positioned further away from the primer set contained in the Identifiler Kit. The results showed the existence of a silent allele 13 in both families, due to a point mutation that changed guanine to adenine at 32 nucleotides downstream from the 3' end of the AAGG repeat sequences in all four members. A single locus mismatch due to a silent allele may occur in any locus using any kit. Accordingly, we should pay attention to this silent allele when carrying out human identification and parentage analysis., ((c) 2009 Elsevier Ireland Ltd. All rights reserved.)

The color of postmortem lividity and control skin in 21 adult cadavers whose postmortem interval was within 72h, was measured by spectrophotometry in order to estimate the postmortem interval, objectively. The L *a *b * system, which has been widely used for the digital expression of skin color, was used and linear regression analysis was performed to determine the relationship between the postmortem interval and 31 color factors including L * (Value), a * and b * (Chroma and Hue) and C * (Chroma). The difference in Chroma between postmortem lividity and control skin (DeltaC * and DeltaC( *)/C(c)( *)) was only weakly correlated with the postmortem interval. We propose a new equation for calculating the postmortem interval using several color factors obtained by measurement of postmortem lividity, together with some autopsy findings that are known to affect the formation of postmortem lividity. The new equation makes it possible to estimate the postmortem interval within +/-4.76h.

Time-of-flight mass spectrometry coupled with liquid chromatography (TOF-MS) has been developed for screening and determination of benzodiazepines with an exact mass database. Benzodiazepines display similar chemical structures and molecular weights, and thus show similar mass spectra and protonated molecule ions. Discrimination of mass spectrometry at low resolving power using single liquid chromatography mass spectrometry (LC-MS) is commonly difficult. TOF-MS analysis was performed using a 1100 TOF (Agilent Technologies) equipped with a Zorbax C18 Extend column. Purine and fluorine compound solution was always introduced into the ion source, and real-time mass adjusting was performed. Specimens were prepared utilizing the liquid-liquid extraction procedure with 1-chlorobutane. Benzodiazepines are widely used in medical practice in Japan, and data acquired from TOF-MS measurements of 41 benzodiazepines, including active metabolites, were used to create an exact mass database. This database comprised molecular formulae, calculated exact masses and retention times. Calibrations were also included in a database. Precision for the 41 drugs was considered sufficient for quantitative analysis. In analysis of samples from patient who had taken > or =2 benzodiazepines, selectivity was improved using the TOF-MS exact mass database. TOF-MS is effective for forensic toxicology in discriminating between benzodiazepines with similar structure and metabolites.

The aim of this study was to examine whether the detection of pulmonary fat embolization is valid as a significant indicator of heat exposure in forensic autopsies. In 54 cases where there was no evidence of fracture, burn or pancreatitis, 25 cases (46.3%) showed pulmonary fat embolization, the degree of which was pathohistologically classified as slight in all cases. Among the 25 cases where the pulmonary fat embolization was detected, the individual had died under a high ambient temperature in 14 cases (56%). Based on logistic regression analysis, pulmonary fat embolization was found to be associated with a high ambient temperature, but not associated with coronary arteriosclerosis, fatty infiltration in the liver, severe infectious diseases, intracranial hemorrhage or the detection of methamphetamine in the blood. Further investigations are necessary before these findings can be applied in forensic autopsy cases; however, it is likely that the detection of pulmonary fat embolization is valuable as one of the diagnostic findings indicating antemortem heat exposure.

A 43-year-old woman was found dead in a car in the supine position. She had been suffering from depression for 2 years and hesitation wounds on the left forearm and wrist were observed. On microscopic examination, pulmonary congestion and edema were observed with heart failure cells in many alveoli, thereby suggesting not only acute but also chronic heart failure. Drug screening in the blood by gas chromatography-mass spectrometry (GC-MS) revealed the presence of amoxapine and levomepromazine, and their concentrations in tissues were determined by GC-MS with three-step solvent extraction followed by acetylation. The concentration of amoxapine in the blood and liver was 0.86-1.77 and 18.76microg/ml, respectively; the levels were much higher than the therapeutic level but did not reach the lethal level. The concentrations of levomepromazine in tissues were within the therapeutic level. Based on the pathological and toxicological findings, the cause of death was determined to be amoxapine poisoning on the basis of chronic heart failure due to the chronic use of psychotropic drugs.

We previously demonstrated that pulmonary fat embolism was induced by elevation of the core body temperature, in rats with a fatty liver. The aim of the present examination was to investigate the core body temperature at which pulmonary fat embolism developed capillaries through exposure to a high temperature, in rats with a fatty liver. Following heat stress, pulmonary fat embolism was observed to a slight degree at a core body temperature of 41 and 42 degrees C, whereas the severity of pulmonary fat embolism was greatly increased and was classified as severe at a core body temperature of 43 degrees C. Moreover, the concentrations of aspartate aminotransferase and alanine aminotransferase within plasma were significantly increased at a core body temperature of 43 degrees C. These results clearly indicate that the development of pulmonary fat embolism could be related to hyperthermia at above 42 degrees C following heat stress, and that fat emboli may be derived from the fatty liver itself. It is thus likely that pulmonary fat embolism can be considered as one form of evidence of hyperthermia in an individual with a fatty liver.

We reviewed 32 cases where a forensic autopsy detected methamphetamine in the blood, and all of these autopsies were performed at two institutes between 1991 and 2003. In accordance with several criteria, the blood concentration in 11 cases was classified as above the toxic level, and 10 of these cases were diagnosed as methamphetamine poisoning. In 20 cases (62.5% of total cases), the blood concentration was of a 'toxic level', and 10, 2 and 1 of these cases were diagnosed as methamphetamine poisoning, cardiomyopathy and intracerebral hemorrhage, respectively. Since it is unclear how the effects of methamphetamine may contribute to the death of an individual, a diagnosis of the exact cause of death is often difficult to make in cases where the blood concentration of methamphetamine was of a 'toxic level'. Therefore, a diagnosis has to be carefully made in consideration of the pathological findings, the pharmacological effects of methamphetamine and the process until death in such cases. Additionally, the mechanism of methamphetamine-related death needs to be more fully studied to enable an appropriate diagnosis to be made easily.

In anaesthetized, mechanically ventilated rats with brain death induced by epidural balloon inflation, we assessed the levels of cerebral myo-inositol in three brain areas 4 h after brain death had been confirmed. The levels were measured using HPLC, along with the water content. Myo-inositol levels were significantly increased in the cerebellum (P<0.05) and decreased in the brainstem (P<0.001), compared to findings in controls. Such changes can serve as hallmarks of brain death.

In medico-legal autopsies for drowned bodies, the location of drowning needs to be determined. To investigate the usefulness of electrolyte analysis in pleural effusion as an indicator of the location where the deceased has drowned, we determined the concentrations of electrolytes in the pleural effusion of rats drowned in four kinds of water. The concentrations of sodium and chloride ions in the pleural effusion of rats that drowned in seawater were significantly greater than those of rats that drowned in freshwater at both 1 day and 3 days after drowning. The concentration of potassium ions in pleural effusion 1 day after drowning showed no difference between each group, although it then increased from 1 to 3 days after seawater drowning, whereas it decreased from 1 to 3 days after freshwater drowning. The concentration of total protein in pleural effusion increased from 1 to 3 days after drowning, however, there was no significant difference in the concentration of total protein in pleural effusion between each group at either 1 day or 3 days after drowning. These results suggest that analysis of electrolytes in pleural effusion may be useful for determining whether drowning has occurred in seawater or in freshwater.

An adult Japanese man (A) entered a pit to remove sludge in a drainage pipe at a dye works in Japan. When he took off a joint of the pipe, the sludge in the pipe flowed into the pit. As he suddenly lost consciousness, three colleagues (B, C, D) entered the pit to rescue him. All of these (A, B, C and D) lost consciousness in the pit, and died soon after the accident. Since hydrogen sulfide gas was detected in the sludge of the pit, gas poisoning was suspected. Toxicological analyses of sulfide and thiosulfate, a metabolite of sulfide, in blood and urine of the victims were made, using the extractive alkylation technique combined with gas chromatography/mass spectrometry. Sulfide and thiosulfate were detected in whole blood of the four workers at levels of 0.32-9.36 mg/l and 0.11-0.23 mmol/l, respectively. These concentrations were at least 6-187 times higher in sulfide and 37-77 times higher in thiosulfate than those in healthy persons, and were similar to values found in fatal cases of hydrogen sulfide poisoning. Thiosulfate was not detected in the urine of four workers, which indicated acute death. Based on these results, all four patients were victims of hydrogen sulfide poisoning, who died soon after the exposure.

Determining the time of brain death is one of the critical issues in forensic examinations. Few authors have attempted to determine the time of brain death using pharmacokinetic approaches. We investigated cerebral concentrations of mannitol of which a single dose (1 g/kg) was administered in the course of brain death. The inflation of an epidural balloon was adopted as a rodent model of brain death. Brain death was determined using ordinary tests. Specimens were collected 4 h after brain death. Brain water content was higher in brain dead (BD) groups than those in control groups. Cerebral concentrations of mannitol in the BD group were significantly higher than those in the control group (P<0.01). In all areas of brain the concentration was the highest at the time when mannitol was administered during balloon inflation. Interhemispheric difference in the cerebrum was observed, followed by balloon inflation (P<0.05). Significant differences were observed in the average concentration of administered mannitol before and after brain death in the contralateral hemisphere (P<0.01) and in the brainstem (P<0.01). As the concentrations of mannitol in the brain are affected by cerebral trauma and brain death follows, mannitol can be used to determine the time of brain death at forensic examinations.

We examined the cardiac conduction system of three alcohol abusers who died suddenly. Cases 1 and 2 showed mitral valve disorder (mitral valve prolapse and rheumatic valvular disease), while Case 3 showed mild Ebstein's anomaly. On examination of the conduction system, Cases 1 and 2 showed severe fibrofatty infiltration into the conduction system, and we conclude that these findings were probably a complication of alcohol abuse. Both cases also demonstrated severe small artery disease. The conduction system of Case 3 showed an anomalous location of the bundle of His with its fragmentation. These three cases suggest that such considerable conduction system abnormalities may be significant findings in alcohol abusers with valvular disease. We consider that alcohol intake may be one of the direct accelerating factors for arrhythmogenic potential to the abnormal conduction system in alcohol abusers who have asymptomatic valvular disease.

A 56-year-old Japanese woman died after being given 10 mg of tetracaine to induce spinal anesthesia in preparation for orthopedic surgery. Autopsy findings and microscopic analysis revealed no external injuries or disease, and further toxicological examinations were carried out to determine the cause of death. For this we used gas chromatography-mass spectrometry. Tetracaine was not detected in most tissue samples, but the metabolite, p-butylaminobenzoic acid, was clearly detected in all samples. The concentration of the metabolite in the brain stem (234.7 ng/g) was higher than in the cerebrum (30.5 ng/g), cerebellum (36.7 ng/g), whole blood of the left heart (164.8 ng/ml) and whole blood of the left femoral vein (84.0 ng/ml). These distribution patterns were exactly same as rabbits sacrificed by high spinal anesthesia. Our findings suggest that tetracaine spread to the high regions of cerebrospinal nerves and acted directly on the central nervous system. Therefore, the cause of her death was high spinal anesthesia induced by tetracaine. This is apparently the first toxicologically proven case in which spinal anesthesia affected high brain centers.

A method to identify reddish alcoholic beverages used for dissolving stimulant drugs was devised using aroma components as indicators. Thirteen brands of beverages including red wines, blueberry liquors, raspberry liquors, strawberry liquor, a mixture of red wine and blueberry juice and a mixture of red wine and grape juice were examined. Aroma components in each sample were effectively concentrated with a porous polymer beads column (Porapak Q) and were analyzed by gas chromatography/mass spectrometry (GC/MS). Specific aroma components were detected in each beverage and reddish alcoholic beverages were successfully classified into five groups using five aroma components as indicators. The present method should prove to be useful in criminal investigations.

The authors developed a simple, reliable and sensitive method for the determination of pentazocine in human solid tissues using high-performance liquid chromatography, combined with a three-step liquid-liquid extraction procedure. Levallorphan tartrate served as the internal standard. The extract was evaporated to dryness and dissolved in the mobile phase of acetonitrile/10 mM phosphate buffer (pH 4.0). The eluent was pumped at a flow rate of 0.4 ml/min through a Spherisorb Ph (2.1 mm I.D. x 150 mm) column. A fluorescence detector with excitation at 247 nm and emission at 320 nm was used. The lower limit of detection was about 0.5 ng/g. The calibration curve was linear over the concentration ranges from 1 to 500 ng/g in each tissue examined and could be determined up to at least 10.0 microg/g by means of reduction of injected volumes. Using this method, the concentrations of pentazocine could be determined in the tissues of an autopsied individual for toxicological evaluation.

A 29-year-old woman, who had been suffering from left facial palsy, died 3.5 hours after undergoing stellate ganglion block (SGB). Autopsy revealed subcutaneous emphysema of the body, bilateral pneumothorax and a huge post-tracheal hematoma. The lower half of the trachea was markedly flattened by pressure from this hematoma. The cause of death was certified as airway obstruction due to a post-tracheal hematoma following SGB. The doctor cut both the anterior and the posterior wall of the trachea, because the trachea was flattened by the hematoma. He then intubated and sent air into the post-tracheal space. We consider that the patient could have recovered if she had received the correct tracheotomy. We therefore conclude that there was professional error on the part of the doctor in the form of medical malpractice.

A 29-year-old man, possibly a schizophrenic patient, was found dead 9 h after admission to a hospital. Autopsy revealed neither significant injuries nor diseases except for congestion of all organs. Microscopic examination revealed severe edema in the lung and slight centrilobular necrosis in the liver. Since amobarbital and levomepromazine were detected by drug screening, the concentrations of these drugs in the victim's body fluids and tissues were determined using gas chromatography-mass spectrometry. The whole blood concentrations of amobarbital and levomepromazine were 9.02 microg/ml and 231 ng/ml, respectively. These levels exceeded therapeutic ranges, but are not so toxic or fatal. However, on the basis of the findings in the literature and of the severe lung edema and centrilobular necrosis in the liver, the cause of his death was judged to be amobarbital and levomepromazine intoxication.

A man was arrested for violating the Stimulant Control Law. Twelve small plastic containers each containing 1-3 ml of reddish purple alcoholic beverage in which methamphetamine had been dissolved, and a bottle of blueberry liquor of a similar colour, were found in his house. He admitted that the blueberry liquor belonged to him, but strongly insisted that he had been asked to keep the containers by another person and that he had no knowledge of their contents. The alcoholic beverage and the blueberry liquor were analyzed in order to test whether he had dissolved methamphetamine in the blueberry liquor. The aroma components found in the alcoholic beverage in each of the plastic containers were identical, but differed from those found in the blueberry liquor. Based on these results and further police investigations, the suspect's allegation was supported and he was not prosecuted.

A sensitive and selective method has been developed for simultaneous determination of triazolam and its major metabolites, alpha-hydroxytriazolam and 4-hydroxytriazolam, in human whole blood and urine. The drugs were effectively extracted using a 3-step solvent extraction procedure followed by tert- butyldimethylsilyl derivatization, and subjected to gas chromatography-mass spectrometry in the negative ion chemical ionization mode. Estazolam was used as an internal standard. The calibration curve for each compound was linear in the range from 0.5 to 100 ng/g, and the lower limit of detection was 0.1 ng/g for whole blood and 0.2 ng/g for urine. Within-day precision of this method was evaluated in whole blood and urine samples at the concentration of 10 ng/g; the coefficient of within-day variation ranged from 2.7 to 10.8%.

We examined ethanol distribution in the brain of a 62-year-old male, who had died of acute subdural hemorrhage 4 days after injury. His blood ethanol concentration was 2.70 mg/g on admission. Blood pressure suddenly decreased and spontaneous respiration ceased 7 h after admission. He was artificially ventilated and died 4 days later. Ethanol was detected in the subdural hematoma, cerebellum, medulla oblongata and all cerebral lobes. Ethanol level in the right parietal lobe was highest; the levels in the right hemisphere of the cerebrum were higher than those in the left. Ethanol levels in the right parietal lobe were almost the same as the blood level calculated 7 h after admission; while the levels in the left parietal lobe and cerebellum were much lower. Thus, it was judged that cerebral blood flow first ceased in the right parietal lobe at 7 h after admission and completely ceased in other regions 11-12 h after. Assessment of ethanol distribution in multiple brain regions seems useful for estimating the time and progression profile of brain death.