A large interhemispheric glioependymal cyst associated with partial defect of the corpus callosum in an elderly man.

Interhemispheric cysts are congenital, and usually present symptoms during childhood. However, they are occasionally detected in adults. These cystic lesions are sometimes associated with defects of the corpus callosum. Although defects of the corpus callosum by themselves do not present clinical symptoms, they are often accompanied by other brain malformations. A man in his late 60s was found dead at the scene of a fire. At autopsy, his brain weighed 1223 g and had a large interhemispheric cyst, measuring 5.5 × 4.5 × 4.0 cm in size. The cyst contained clear fluid but was not connected to the ventricular system. On slices of the cerebrum, the corpus callosum did not connect the right and left cerebral hemispheres, and the right lateral ventricle was dilated. By the existence of the cyst, compressed by the cyst, the hemispheres were displaced on either side. Histologically, the cerebral parenchyma around the cyst was slightly edematous but structurally normal. Immunohistochemically, both glial fibrillary acidic protein and podoplanin were expressed in the cystic components. Thus, the cystic lesion was diagnosed as a glioependymal cyst. In this case, because the cyst was located at the interhemispheric space between the right and left frontal lobes, the individual experienced no obvious symptoms, despite its large size. The individual's brain malformations included the partial defect of the corpus callosum and the cyst. The dilation of the right lateral ventricle was considered to result from the location of the cyst. Under the influence of the cyst, the third ventricle was displaced downward, and one or both of the interventricular foramen were obstructed. The decedent had burns over his whole body. Burns to the epiglottis and soot in the airway were also observed. Volatile hydrocarbons, such as benzene and styrene, were detected in the blood. The percentage of carboxyhemoglobin levels in a total of hemoglobin levels were 19-25%. Therefore, the individual's cause of death was established as death by fire.

Giant intracranial arteriovenous malformation as the focus of epileptic seizures.

A man in his late thirties was found in a supine position in the hallway of his house. He had been diagnosed with epilepsy at approximately 20 years old. Since stopping treatment, epileptic events occurred more frequently and his condition deteriorated in the past 2 years. Autopsy revealed that head injuries were found on the left side of his head. A fracture from the left parietal bone to the anterior cranial fossa was also detected. A subdural hemorrhage (hematoma) spanned a wide range. A subarachnoid hemorrhage was also identified in the left parietal region. His brain weighed 1603 g, was edematous, and showed right uncal herniation. In the right cerebral hemisphere, a thick, enlarged blood vessel ran from the sagittal sinus. An egg-sized tumorous lesion of blood vessels was found on the bottom of the frontal lobe. This vascular lesion had formed between the sagittal sinus and right anterior cerebral artery. Pathologically, veins and arteries were found together, and, thus, this case was diagnosed as an arteriovenous malformation (AVM). No other pathological and toxicological findings were observed. Subdural hematoma, the cause of death, occurred from the fall to the floor. An epileptic seizure may have been the cause of the fall. AVM on his brain was considered to be the focal lesion of epileptic seizures.

Postmortem genetic analysis of sudden unexpected death in infancy: neonatal genetic screening may enable the prevention of sudden infant death.

Tandem mass screening has recently been started in Japan, but genetic screening has yet to be widely performed in neonates and many unexpected deaths are still being reported. We previously reported two cases of sudden infant death that may have been prevented had newborn screening been performed. In this study, we retrospectively reviewed 71 cases of sudden infant death for 66 arrhythmia- and 63 metabolic disease-related genes to identify how many cases of sudden infant death may have been prevented had mass screening been performed. Next-generation sequencing revealed that six cases had arrhythmia-related gene variants and five cases had metabolic disease-related gene variants. Had genetic screening been performed in addition to biochemical and physiological screening during the neonatal period to identify those at risk of arrhythmia or metabolic disease, these infants could have been diagnosed and treated, preventing their deaths. As such, screening of newborns may prevent sudden infant death.

Basal subarachnoid hemorrhage by rupture of arteriovenous malformation at the cerebellopontine angle.

A man in his late forties had lived as a recluse for more than ten years. He was found dead in his room. At autopsy, subarachnoid hemorrhage (SAH) was detected at the base of the brain, which weighed 1333 g. The cerebellar tonsil was swollen. The cerebral ventricle was enlarged and filled with blood. A hematoma was observed in the upper part of the left side of the cerebellar hemisphere. The location and size of SAH in this case indicated that the rupture of a cerebral aneurysm (CA) had occurred; however, CA was not detected. A mass of blood vessels buried in the hematoma was observed at the left cerebellopontine angle (CPA). The vascular lesion showed round-shaped blood vessels as well as flat-shaped vessels with the appearance of veins, but with elastic fibers indicative of arteries. The lesion was considered to be the nidus and was 5-8 mm in size. Feeding arteries appeared to be from the anterior inferior cerebellar artery (AICA). However, the draining vein and anastomotic parts of the artery and vein were not confirmed. Based on these histopathological features, this vascular lesion was diagnosed as arteriovenous malformation (AVM). A differential diagnosis between AVM at CPA and CA is needed in order to identify the source of non-traumatic SAH.

An autopsy case of caffeine intoxication related by energy drink.

An autopsy case of caffeine intoxication related to the consumption of caffeinated products, such as energy drinks and caffeine contained drugs, was reported. Case report: A male in his early twenties was working a night shift job. After work one morning he came home and was not feeling well. He was vomiting a great deal and could not move, so his family had him lay down to rest. That afternoon they discovered his death. Although the decedent was examined by postmortem computed tomography, his cause of death could not be determined. A forensic autopsy was performed to investigate his cause of death. Autopsy findings: There were no obvious injuries on his whole body. Only slight brain edema and congestion of other organs were observed, but no significant lesions were found in his organs. A yellowish granular substance was observed in the stomach, duodenum, and small intestine. Alcohol concentration was 0.01mg/ml in the blood and urine. GC-MS and LC-MS/MS analyses did not detect any chemical substance except caffeine from his blood and urine. By LC-MS/MS analysis, the caffeine level (p g/mL) was 182, 71, and 10700 in the blood, urine, and gastric contents, respectively. Results and discussion: The fatal level of caffeine in blood has been reported as >100 u g/ mL. Thus, the cause of death was diagnosed as caffeine intoxication. It was revealed that he had consumed many energy drinks to stay awake and alert. Because he had a history of feeling ill and vomiting, it is suspected that he had an excessive intake of caffeine. Energy drinks, unlike most medicines, are able to be consumed repeatedly, and caffeine intoxication is a possible result. The reporting of this case will enlighten the danger of repetitious and chronic consumption of caffeinated products, such as energy drinks and caffeine contained drugs.

An autopsy case of cerebral hemorrhage in an HIV-infected patient with suspected HIV-associated cerebrovascular disease.

A male in his late 30s was found dead in his home. He was diagnosed with human immunodeficiency virus (HIV) about six years prior. The HIV infection was well controlled before his death. He was 166 cm in height and 75 kg in weight. Aside from discoloration of the skin on the right lower patellar, there were no obvious injuries. His brain weighed 1456 g. A cut surface of the brain revealed left thalamic hemorrhage. Histologically, infiltration of phagocytic cells was observed in the bleeding site. Thalamic hemorrhage was considered to be his cause of death in this case. Due to the effects of anti-HIV therapy, the mortality rate from HIV infection has decreased and the causes of death of HIV-infected persons have changed. HIV-infected persons have been suggested to be associated with cerebrovascular disease, especially juvenile ischemic stroke. Patients with acquired immunodeficiency syndrome (AIDS) have an increased risk of cerebrovascular disease. Possible mechanisms of cerebrovascular disease in HIV-infected individuals include coagulopathy, secondary effects of embolism and central nervous system infection, and direct vascular disease due to HIV. At the time of autopsy, his post-mortem interval was estimated to be approximately two weeks. Therefore, it was difficult to clarify histologically the cerebrovascular disorder that caused his cerebral hemorrhage. In recent years, anti-HIV therapy has reduced the number of AIDS-related deaths, but deaths in HIV-infected people from cardiovascular disease are increasing. This case is considered to be a valuable forensic autopsy case of an HIV-infected patient who actually died due to cerebral hemorrhage in Japan.

Association between serum catecholamine levels and VNTR polymorphism in the promoter region of the monoamine oxidase A gene in forensic autopsy cases.

Monoamine oxidase A (MAOA) catalyzes oxidative deamination of catecholamines. A functional variable number tandem repeat (VNTR) polymorphism in the promoter region of the MAOA gene has been previously reported. In the present study, we measured serum adrenaline (Adr), noradrenaline (Nad), and dopamine (DA) levels in 90 male and 34 female Japanese autopsy cases in which amphetamines or psychotropic drugs were not detected.We examined the frequencies of MAOA-uVNTR alleles in these cases and investigated the effects of the MAOA-uVNTR polymorphism on serum Adr, Nad, and DA levels. Evaluation indicated no significant association between MAOA-uVNTR polymorphism and serum Adr, Nad, or DA levels in males, although a significant association between MAOA-uVNTR polymorphism and serum Adr and DA levels were observed in females. Females with the 3/3 genotype had higher serum Adr and DA levels than those with a 4-repeat allele (3/4 and 4/4 genotypes) (p = 0.048 and 0.020, respectively). There was no significant association between MAOA-uVNTR polymorphism and serum Nad levels in females. The present study indicates that MAOA-uVNTR polymorphism influences serum Adr and DA levels only in females.

The effect of tea catechins on the forensic identification of urine: Urine camouflage to evade drug tests.

BACKGROUND: We encountered a urine sample suspected of being mixed with tea, submitted by a suspect attempting to camouflage illegal drugs. Although urine should turn reddish-pink during a urea test with p-Dimethylaminocinnamaldehyde (DAC), this suspect's sample exhibited a blue coloration when tested with DAC. AIM: Our aim was to examine the influence and mechanism of green tea on various urine identification tests. RESULTS: Our examination revealed that DAC forms a compound with the urea in urine, resulting in a reddish pink coloration with a molecular weight of 217. However, it has been reported that DAC binds to polyphenols such as catechin. In the case of catechin, DAC binds to the C8 position, forming a compound that exhibits the highest absorption at 640 nm and appears blue. we investigated the effect of urine from volunteers who had consumed a large amount of catechin on the urea test with DAC. Additionally, we carried out quantitative analysis of catechin in urine by LC-MS/MS after enzymatic treatment with ß-glucuronidase. The concentration of urinary excreted catechin reached its peak approximately 3 to 4 h after ingestion. During the DAC test, urine samples collected 3 to 4 h after catechin ingestion displayed a bluish pink color, but not the blue color observed in the original suspect sample. CONCLUSION: This study investigated the impact of catechin on urine tests, revealing that a blue color in the DAC test indicates a high likelihood of camouflage by the suspect.

Urinary phenylacetylglutamine as a possible biomarker for central nervous system disorders in forensic autopsy cases.

Phenylacetylglutamine (PAG) is a metabolite that is excreted in human urine. Phenylalanine is metabolized to phenylacetic acid, which is then amide-bonded to glutamine to form PAG. We are currently studying PAG as a urinary biomarker in forensic autopsy cases. MATERIALS AND METHODS: Urine samples were collected from 188 forensic autopsy cases and the urinary PAG concentration was analyzed quantitatively using GC-MS. Urinary creatinine (Cr) concentration was also analyzed by GC-MS. For statistical analysis, the JMP Pro 15.0.0 software program was used. The relationship between urine PAG/Cr (the ratio of each concentration), sex, age, postmortem interval (PMI), survival duration, and cause of death was statistically analyzed. RESULTS AND DISCUSSION: The median (range) of PAG/Cr was 0.12 (0.002-3.26). The PAG/Cr ratio showed no significant relationship to sex or survival duration. Regarding the cause of death, traumatic brain injuries had a significantly higher ratio than intoxication (p=0.023). Cerebrovascular disease, such as cerebral hemorrhage and subarachnoid hemorrhage, did not differ significantly from any cause of death group. However, when traumatic brain injuries and cerebrovascular accidents are combined as one cause of death group, the PAG/Cr value of CNS damages was significantly higher than that of intoxication (p=0.062). CONCLUSION: Urinary PAG/Cr might be a biomarker not only for traumatic brain injuries but also for antemortem CNS damages.

A validated method for the separation of ethyl glucoside isomers by gas chromatography-tandem mass spectrometry and quantitation in human whole blood and urine.

Ethyl glucoside (EG) is present in Japanese sake in high concentrations, and can be found in other alcoholic beverages like beer and wine in varying amounts. EG exists as alpha (α) and beta (ß) isomers, and the concentrations and ratios of these isomers differ depending on the alcoholic beverage. Herein, we report a validated analysis method for the separation of EG isomers in human whole blood and urine, by GC-MS/MS. Whole blood and urine samples were deproteinized and interferences removed by weak cation exchange cartridges. The target analytes were acetylated using acetic anhydride and pyridine by microwave-accelerated derivatization. Separation was performed using tandem columns, with detection in the multiple reaction monitoring (MRM) mode. The MRM transitions for all compounds were m/z 157.0 > 115.1 for the quantifying transition, and m/z 157.0 > 73.1 and m/z 141.0 > 81.0 for the qualifying transitions. Assay validation included linearity, LOD and LLOQ, bias, within-run and between-run precision, stability, and dilution integrity. Baseline separation of the 2 isomers was achieved with linear calibration (r2 > 0.99) across the calibration range 0.625 to 50 µg/mL for both α- and ß-EG in both whole blood and urine. The validated method was then applied to actual human whole blood and urine samples collected at autopsy, as well as relevant alcoholic beverage samples. The quantitation of EG isomers could benefit the forensic toxicology community by acting as markers for recent alcoholic beverage consumption.

Giant intracranial arteriovenous malformation as a possibility of epileptic seizures in a case of drowning.

A male in his late 50s had been complaining of headaches and dizziness for 25 years. He also had episodes of losing consciousness, but had not sought treatment because of financial hardship. He was found in the ocean. Autopsy revealed foamy liquid leaking from his nose and mouth, and pleural effusions. The trachea and bronchi contained the same foamy liquid. The lungs were swollen and edematous, and leaked a large amount of foamy liquid. His cause of death was diagnosed as drowning. In the brain, the veins on the frontal lobe and the temporal pole, each on the right cerebral hemisphere, were dilated. A vascular lesion measuring 5 × 5 × 8 cm was found on the bottom of the right frontal lobe, and was located between the right middle cerebral artery and those veins. This vascular lesion extended to the brain parenchyma, and the basal ganglia of the right cerebrum was displaced outward and upward. The vascular lesions in the brain showed blood vessels of various sizes and shapes, and some of the vessel walls were thickened. The vascular lesion on the right frontal lobe was diagnosed as an arteriovenous malformation (AVM). According to the police investigation, the harbor where his body was found was a place he often came for fishing and walking. The possibility of suicide cannot be ruled out. Moreover, it was considered that his AVM might have rendered him unconscious, causing him to fall into the ocean.

Diagnostic meaning of urinary ethyl glucoside concentrations in relationship to alcoholic beverage consumption.

Incidents and accidents often involve the drinking of alcoholic beverages. We investigated compounds that indicate the consumption of alcoholic beverages even after ethanol (EtOH) becomes undetectable in blood and urine. Ethyl glucoside (EG) has been isolated as a possible drinking marker, and a GC-MS/MS method for EG isomers has been developed. EG isomers in several alcoholic beverages were analyzed. In sake, only αEG was observed in high concentrations. In wine and beer, both α and ßEG were detected. Whisky, however, did not contain EG. EtOH and EG concentrations were analyzed in urine up to 48 h after ingestion. Maximum EtOH concentrations were reached in 1-2 h and was mostly eliminated in 6 h. Maximum EG concentrations were reached in 3-6 h, gradually decreased, and remained low after 24 h. After drinking sake, the αEG concentrations were much higher than that of other alcoholic beverages. After drinking wine or beer, ßEG was detected, but lower than αEG. Also, αEG was detected in urine after drinking whisky that contained no EG. This suggested that αEG may be synthesized in vivo. Disaccharide-degrading enzymes such as α-glucosidase are present in the human small intestine. It was considered that αEG was synthesized when alcohol was consumed with certain foods, such as carbohydrates. In actual forensic autopsy cases, EtOH and EG isomer analysis provided useful information regarding drinking history. In conclusion, it is considered that urinary EG isomers can be used as drinking markers that complement EtOH analysis.

A comparative study of pleural effusion in water area, water temperature and postmortem interval in forensic autopsy cases of drowning.

Japan is surrounded by the sea and is also a mountainous country with many rivers. Japan has the second- highest rate of deaths caused by drowning in the world. Pleural effusion (PE) is one of the major findings at autopsy. It is found in approximately 80% of drowning mortalities and is observable for a relatively long postmortem interval (PMI). We focused on the amount of pleural fluid in drowning cases, discussed the relationship of PE with the drowning environment, water temperature, and postmortem interval, and established more simple and practical criteria for the diagnosis of drowning. We measured the weight of the lungs, PE, and their sum as the intrathoracic (IT) weight (total weight of lungs + pleural effusion), and calculated the PE ratio [(PE weight/IT weight) × 100]. A total of 130 drowning deaths diagnosed through forensic autopsies were investigated in this study. The cases were classified by drowning environment (freshwater, brackish water, and seawater), water temperature (under 20 °C, more than 20 °C), and postmortem interval (less than 1 day, 1-3 days, more than 3 days). The present study demonstrated that the PE ratio may be more effective for the diagnosis of drowning. Moreover, the accumulation of PE is affected by drowning environment, water temperature, and PMI. Collectively, it is important to assess the PE ratio and consider these factors in autopsy cases of victims found in water.

Distinct breakpoints in two cases with deletion in the Yp11.2 region in Japanese population.

The amelogenin gene on the Y chromosome (AMELY) is a homolog of the X chromosome amelogenin gene (AMELX), and the marker is employed for sexing in forensic casework. Deletion of the sequences in the Yp11.2 region containing the AMELY locus has been found in males from various ethnic populations. Two cases of AMELY null males found in the Japanese population had different Y haplogroups and deletion mapping. Proximal and distal breakpoints of a sample of haplogroup D2* were located in TSPYA and TSPYB arrays, respectively, suggesting that the deletion mechanism was non-allelic homologous recombination (NAHR). On the other hand, a sample of haplogroup O3a3c* had the distal breakpoint in the TSPYB array and the proximal breakpoint at position 7.94 Mb, not in the TSPYA array. The likely deletion mechanism is non-homologous end-joining. High-resolution STS mapping in the TSPYB array showed the distal breakpoints differed according to the haplogroups. The deletion length was estimated as 3.1-3.7 Mb and 1.6-1.7 Mb for the sample of haplogroup D2* and O3a3c*, respectively. These deletion events should have occurred independently.

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.

Organ distribution of endogenous p-cresol in hemodialysis patients.

BACKGROUND: p-Cresol concentrations are high in the blood of hemodialysis (HD) patients. However, its organ distribution has not yet been investigated in detail. We herein report the distribution of p-cresolin HD patients from forensic autopsy cases. METHODS: p-Cresol was measured in the blood, urine, lungs, liver, and kidneys from 4 HD and 4 non-HD cases. Samples were extracted with p-cresol-d8 (internal standard), derivatized,and injected on the GC-MS. RESULTS AND DISCUSSION: The total urinary p-cresol/Cr was 79.73 ng/ml in HD cases,which was 16-fold higher than that in non-HD cases. p-Cresol in the blood and kidneys were 30-fold higher or more at 11.92 and 13.08 µg/mL(g), respectively. p-Cresol in the liver and lungs were approximately 20-fold higher at 4.82 and 9.99 µg/g, respectively. p-Cresol was markedly increased in not only the blood, but also the urine and organs of HD cases. The distribution of p-cresol in the blood, urine, and organs differed between HD and non-HD cases. In HD cases, the percentages of conjugated (C) and protein-bound conjugated (PC) urinary p-cresol were 57 and 41%, respectively. C and PC p-cresol was 66% and 25% in the kidneys, respectively, and similar results were obtained in the lungs. J. Med. Invest. 66 : 81-85, February, 2019.

VNTR polymorphism in the monoamine oxidase A promoter region and cerebrospinal fluid catecholamine concentrations in forensic autopsy cases.

Monoamine oxidase A (MAOA) plays important roles in the metabolism of catecholamines and modulates adrenergic, noradrenergic, and dopaminergic signaling. A polymorphic promoter variable number tandem repeat (VNTR) locus (MAOA-uVNTR) is located approximately 1.2 kb upstream from MAOA exon 1. Functional studies revealed that MAOA-uVNTR affects gene expression. In the present study, we examined the frequencies of MAOA-uVNTR alleles in Japanese autopsy cases, in which amphetamines or psychotropic drugs were not detected. In total, 87 males and 35 females were evaluated and investigated for the possible effect of MAOA-uVNTR polymorphisms on cerebrospinal fluid (CSF) catecholamine concentrations. In males, there was no significant association between MAOA-uVNTR polymorphisms and CSF adrenaline (Adr), noradrenaline (Nad), or dopamine (DA) levels. In contrast, females who were homozygous for the 3-repeat allele (i.e., 3/3 genotype carriers) had higher CSF levels of Adr (p = 0.024) and DA (p = 0.035) than individuals who were heterozygous or homozygous for the 4-repeat allele (3/4 and 4/4, respectively). We found no significant association between MAOA-uVNTR polymorphisms and CSF Nad levels in females. Thus, the results of the present study indicated that MAOA-uVNTR polymorphism influences CSF Adr and DA levels in females.

Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine.

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.

Effects of PCR inhibitors on mRNA expression for human blood identification.

Detection of body fluid-specific mRNAs, such as those specific for blood, using real-time polymerase chain reaction (PCR) has become a useful tool in forensic science. Blood stains often contain PCR inhibitors that may be co-extracted with RNA and adversely affect PCR. The effects of inhibitors on the detection of mRNA markers for blood identification, namely, hemoglobin beta (HBB) and actin beta, were examined herein. Inhibitors were added to a real-time PCR mix, reverse transcription mix, and blood samples before RNA extraction, and the following parameters: Ct, delta Ct (dCt), and melting temperature (Tm) values, were monitored. Hematin, humic acid, indigo carmine, and tannic acid were used as PCR inhibitors. The results showed that Ct values for HBB in samples containing inhibitors in their real-time PCR mix increased in a concentration-dependent manner, and were undetectable at higher concentrations. Moreover, Ct values for HBB in tannic acid-spiked samples reached a maximum once, and inhibition decreased at higher concentrations. dCt values increased in hematin-spiked samples, but decreased in tannic acid-spiked samples. Tm values decreased following the addition of each inhibitor. The reverse transcription reaction was scarcely inhibited at concentrations that markedly affected real-time PCR. The complete removal of inhibitors added to blood was difficult. However, the observed inhibitory effects were weaker than those when inhibitors were added to the PCR cocktail. PCR inhibition was effectively reduced by repurification of complimentary DNA with DNA extraction kits. These results will assist examiners in deducing contaminating inhibitors and selecting an appropriate method to remove them.