Mio Takayama

GC-PCI-MS/MS and LC-ESI-MS/MS databases for the detection of 104 psychotropic compounds (synthetic cannabinoids, synthetic cathinones, phenethylamine derivatives).

Designer psychotropic compounds continue to be a major problem in Japan and all around the world. Electron impact mass spectrometry (GC-EI-MS) and liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data on these compounds have been widely reported. In this report, we present a detection method that has been rarely utilized to analyze these types of compounds, gas chromatography with positive chemical ionization and tandem mass spectrometry (GC-PCI-MS/MS). We report on the development of GC-PCI-MS/MS and LC-ESI-MS/MS databases of 104 psychotropic compounds, including 32 cannabinoid derivatives, 29 cathinone derivatives, 34 phenethylamine derivatives, and several other designer compounds. Using this database, we were able to detect 5 psychotropic compounds in an actual forensic autopsy case. If GC-PCI-MS/MS is used together with the more established methods of GC-EI-MS and LC-ESI-MS/MS, we believe the forensic toxicology community could be better prepared to deal with the challenges of these ever-changing compounds.

Development of a preparation method to produce a single sample that can be applied to both LC-MS/MS and GC-MS for the screening of postmortem specimens

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC-MS and LC-MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC-MS and LC-MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratorys daily routine for screening many different compounds from postmortem samples.

Tissue Distribution of Suvorexant in Three Forensic Autopsy Cases

Suvorexant (Belsomra®) is a relatively new insomnia medication that has been available in USA and Japan since 2014. It is a dual orexin receptor antagonist that promotes sleep by inhibiting the binding of orexin neurons to the OX1R and OX2R receptors. In this report, we describe the detection and quantitation of suvorexant from the postmortem specimens of three separate autopsy cases handled by our department. Suvorexant was identified by fast gas chromatography/mass spectrometry during routine screening, and quantitated by a fully validated liquid chromatography-tandem mass spectroscopy method. Quantitation was achieved by positive electrospray ionization in the selected reaction monitoring mode. Monitored transitions were m/z 451 > 186 for quantitation and m/z 451 > 104 for qualification. To our knowledge, this is the first instance of suvorexant being quantitated from actual autopsy cases. It is likely that this compound will be encountered more often by the forensic toxicology community going forward.

The global distribution of the p.R1193Q polymorphism in the SCN5A gene

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations.

An unexpected death due to massive ascites and a giant mucinous ovarian cystadenoma

A female in her thirties fell face down in her room. She was motionless when her sister found her. She was transported to the hospital by ambulance and was in a state of cardiopulmonary arrest on admission. She did not respond to resuscitation. Her abdomen had started to swell 3years before her death. An autopsy was performed to clarify the decedents cause of death. She was 172cm tall and weighed 146kg. Her maximum abdominal girth was 172.1cm. A subcutaneous hemorrhage measuring 4.5cm in diameter was observed in the epigastric region. The abdominal cavity contained brownish ascites (54.1L). The left ovary was markedly swollen, and the combined weight of the uterus and right ovary was more than 13.0kg. A left ovarian tumor consisting of serous and mucinous cysts was detected. There were no metastatic lesions in the peritoneum or other organs. She might have suffered circulatory disturbance caused by the ascites and ovarian tumor. Moreover, being in a prone position would have resulted in an increase in intra-abdominal pressure, further exacerbating her circulatory problems. Therefore, her cause of death was considered to be circulatory failure caused by significant ascites and a large ovarian tumor.

Quantification of neuropathological findings by image data for the diagnosis of dementia in forensic autopsy cases

The aim of the present study was to quantify neuropathological findings using image analysis software for the diagnosis of dementia in deceased who underwent forensic autopsy. Of the autopsies performed within 48 hours of death and excluding those of patients with head injury, thermal injury, heat stroke, or intracranial lesions, 8 were of autopsy cases clinically diagnosed with dementia and thus included in the dementia group (D). The non-dementia group (non-D) consisted of 6 deceased without dementia. To compare the D and non-D groups, 6 regions and 7 types of pathological findings were observed semi-quantitatively using 4 conventional stainings. Quantitative analysis of collected image data was performed using image analysis software. Semiquantitative analysis of senile plaques and neurofibrillary tangles was performed with Bielschowsky-Hiranos silver staining image data. An easy, simple, and effective quantification method of the pathological findings was achieved. However, no significant differences were observed between the two groups, and diagnosis of dementia by the quantification of pathological findings was not successful. Diagnosis of dementia using image data may be possible in future studies with an increased number of autopsies, and by utilizing staining techniques with higher specificity and sensitivity, such as immunohistochemical staining.

Quantification of immunohistochemical findings of neurofibrillary tangles and senile plaques for a diagnosis of dementia in forensic autopsy cases

We report the quantification of immunohistochemical findings for a diagnosis of dementia in autopsy cases among older decedents. Autopsy cases were selected with the following requirements: >65yo; no head injuries, thermal injuries, or heat stroke; no intracranial lesions; and within 48h of death. Among cases that met all requirements, 10 had a clinical diagnosis of dementia were included in dementia group. Non-dementia group consisted of 38 cases without any record of dementia. To compare these groups, immunohistochemically, beta-amyloid, tau protein, gephyrin, and IL-33 were examined in five regions. Quantitative analysis was performed by collecting with image data analyzed using analysis software. Image data on tau-immunopositive neurofibrillary tangles (NFT) and beta-amyloid-positive senile plaques (SP) were photographed. Criteria for dementia were made by counting and measuring NFT and SP from image data using software. Differences in SP and NFT were effective for discriminating between the two groups. These criteria may reveal the presence and progression of dementia. Total of tau-positive NFT in Ammons horn (AH) may be useful for diagnosing dementia. When the total is more than 41 in approximately 6mm(2) of AH, the possibility of dementia is considered. Total of beta-amyloid-positive SP in the parahippocampal gyrus (PHG) may be useful for diagnosing dementia. When the total in approximately 5mm(2) of PHG is more than 47, the possibility of dementia is considered. Immunohistochemical staining may be more useful for obtaining image data for quantification than conventional staining techniques, such as Bielschowsky-Hiranos silver staining.

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.

Diagnostic meaning of blood p-cresol concentration in forensic autopsy cases

In some forensic autopsy cases there are high concentrations of p-cresol in the blood. In vivo, p-cresol is the only isomer yielded by intestinal bacteria and is excreted into urine. We investigated the diagnostic meaning of p-cresol in the blood of forensic autopsy cases. Blood samples from 110 autopsy cases within 48 h of the postmortem interval (PMI) and 10 healthy adults were examined. Blood with p-cresol-d8 as an internal standard was analyzed using a GC-MS/MS method. Using acid and heat deproteinization, free (F) and conjugated (non-protein bound: C; protein bound: PC) p-cresol were individually analyzed. The p-cresol concentrations were 1.39 ± 0.86 µg/ml [mean ± SD] and 1.18 (0.19-18.80) µg/ml [median (range)] in healthy adults and autopsy cases, respectively. The p-cresol showed no significant relationship to age, sex, fasting duration, survival duration, or PMI. No significant differences were found between causes of death. Significantly higher levels of C p-cresol were found in cases with atherosclerosis in the basilar or renal arteries, or stenosis in the coronary artery. Significantly higher levels of p-cresol except F were found in cases with hyalinosis of the kidney. Cases with low BMI also showed significantly higher p-cresol concentrations. The 22 cases of abnormally high total p-cresol were investigated. It was considered that high concentrations of p-cresol could be an indicator of certain diseases and physical conditions that effect the production, absorption, metabolism, circulation, and excretion of p-cresol. Measuring the levels of p-cresol may provide valuable information about the antemortem physical conditions.

DRD2/ANKK1 gene polymorphisms in forensic autopsies of methamphetamine intoxication fatalities

Dopamine D2 receptor/ankyrin repeat and kinase domain containing 1 (DRD2/ANKK1) gene polymorphisms have been associated with responses to psychotropic drugs and addiction. We analyzed two DRD2/ANKK1 polymorphisms, Taq1A and -141C Ins/Del, in 37 fatal methamphetamine (MA) intoxication cases and 235 control cases in which MA and psychotropic drugs were not detected. The association among polymorphism, cause of death, and cerebrospinal fluid (CSF) dopamine concentration was evaluated. The Taq1A polymorphism distribution in the fatal MA intoxication cases differed from in the controls (P = 0.030) with a significantly high A1/A1 + A1/A2 genotype frequency. No significant associations were observed between -141C Ins/Del polymorphisms and MA intoxication cases or between DRD2/ANKK1 polymorphisms and CSF dopamine concentrations. Our findings suggest that the DRD2/ANKK1 Taq1A polymorphism is associated with susceptibility to fatal MA intoxication.