Katsuyoshi Kurihara

Case report: Etizolam and its major metabolites in two unnatural death cases

A simultaneous analytical method for etizolam and its main metabolites (alpha-hydroxyetizolam and 8-hydroxyetizolam) in whole blood was developed using solid-phase extraction, TMS derivatization and ion trap gas chromatography tandem mass spectrometry (GC-MS/MS). Separation of etizolam, TMS derivatives of alpha-hydroxyetizolam and 8-hydroxyetizolam and fludiazepam as internal standard was performed within about 17 min. The inter-day precision evaluated at the concentration of 50 ng/mL etizolam, alpha-hydroxyetizolam and 8-hydroxyetizolam was evaluated 8.6, 6.4 and 8.0% respectively. Linearity occurred over the range in 5-50 ng/mL. This method is satisfactory for clinical and forensic purposes. This method was applied to two unnatural death cases suspected to involve etizolam. Etizolam and its two metabolites were detected in these cases.

Distribution of neuropeptide-containing nerve fibers in the human submandibular gland, with special reference to the difference between serous and mucous acini.

Abstract.Distribution of neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)-, galanin (GAL)-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the human submandibular gland was examined by the peroxidase-antiperoxidase method with attention to high-quality fixation and the condition of patients. NPY-, VIP-, and GAL-immunoreactive varicose fibers were densely distributed around the acini and ducts. Some of these fibers extended between acinar cells. The density of SP- and CGRP-immunoreactive fibers was relatively low. The number of NPY-, VIP-, and GAL-immunoreactive fibers around the mucous acini was significantly higher than around the serous acini. In the perivasculature, NPY-immunoreactive fibers were more numerous than other immunoreactive fibers. No somatostatin-, leucine-, or methionine-enkephalin-immunoreactive fibers were detected. Our findings suggest that a large number of periacinar VIP-, NPY-, and GAL-immunoreactive fibers may participate in regulating the synthesis of saliva and its secretion. Since the VIP-, NPY-, and GAL-immunoreactive fibers are more numerous around the mucous acini than around the serous ones, these fibers may take part more actively in regulating the secretory mechanisms in the mucous acini than in the serous ones. The relatively low number of CGRP- and SP-immunoreactive fibers suggests that they are less involved in the function of the human submandibular gland. Perivascular peptidergic fibers, especially NPY-immunoreactive fibers, may be involved in controlling local blood flow in this gland.

Analysis of the sarcomere protein gene mutation on cardiomyopathy - Mutations in the cardiac troponin I gene

Developments in the molecular genetic studies of cardiomyopathy (CM) have led to discovery of a large number of mutations in the genes encoding the sarcomeric proteins. In this study, comprehensive screening of TNNI3 was performed in 36 consented autopsy cases diagnosed as CM, in order to evaluate the prevalence of gene mutations in sudden death caused by CM. In DCM cases, a new missense mutation Pro16Thr was detected. A single nucleotide polymorphism at -8 position of intron 3 (IVS 3 -8 T>A) was identified, which had a significant difference in allele frequency between DCM and control cases. From these results, it was indicated that this study contribute to genetic based diagnosis, risk stratification and prevention of sudden death caused by CM.

Dural Sinus Thrombosis with Severe Hypernatremia Developing in a Patient on Long-Term Lithium Therapy

Dural sinus thrombosis has not been described in a patient with hypernatremia resulting from lithium-induced nephrogenic diabetes insipidus. A 63-year-old man on chronic lithium therapy for schizoaffective disorder was transferred to the Emergency Department with dehydration and signs of central nervous system dysfunction after a 3-week isolation in a room in a psychiatric hospital due to exacerbation of psychiatric disorder, during which he refused to eat. Laboratory examination revealed hypertonic hypernatremia (osmolality, 359 mOsm/kg and Na, 171 mEq/L) and hyposthenuria (specific gravity, 1.010 and osmolality, 249 mOsm/kg), with normal serum endogenous vasopressin concentration (2.3 pg/mL). The serum lithium concentration was within the therapeutic range (0.94 mEq/L). Cranial computed tomography demonstrated subarachnoid hemorrhage and suggested dural sinus thrombosis. Although treatment with indomethacin (25 mg parenterally at 8-hour intervals) was somewhat effective in restoring renal concentrating capacity, he died of massive hemorrhagic infarction on the sixth hospital day, probably secondary to dural sinus thrombosis. The clinical diagnosis was confirmed by postmortem examination. Physicians should be alert for the possibility of dural sinus thrombosis as a complication of hypernatremia resulting from lithium-induced nephrogenic diabetes insipidus.

Experimental study on external contamination of hair by synthetic cannabinoids and effect of hair treatment

Abstract We recently reported a fatal case of poisoning by N-1-naphthalenyl-1-pentyl-1H-indole-3-carboxamide (NNEI). In this case, NNEI was detected from 13 sections of a 40 cm length of hair after washings. The concentrations of NNEI were generally similar among the hair segments. These results strongly suggested that externally contaminated NNEI could not be removed from the hair by washings. The aim of the present study is to demonstrate the adsorption and removal by washings for NNEI and [1-(5-fluoropentyl)-1H-indol-3-yl]-(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using liquid chromatography–tandem mass spectrometry. NNEI and MAM-2201 were detected in black hair even after a single immersion in NNEI and MAM-2201 aqueous solutions, followed by washings. However, their adsorption seemed less pronounced for dyed or bleached hair. Therefore, hair analysis cannot be considered effective in testing of synthetic cannabinoid abuse, because the external synthetic cannabinoid(s) can be easily adsorbed to hair of a non-user by passive exposure.

ABO genotyping by TaqMan assay and allele frequencies in a Japanese population

ABO genotyping have become common tools for forensic casework. We developed a new rapid ABO genotyping method using a fast real-time PCR system with the TaqMan® Sample-to-SNP™ Kit. Eight single nucleotide polymorphism (SNP) sites in the ABO gene (nt 261, 297, 467, 657, 703, 829, 930 and 1061) were selected to determine the ABO genotypes. ABO genotypes were easily determined by examining allelic discrimination patterns. This method enabled analyses to be completed in about 1h per plate with no postmortem change influences. The detection limit in each SNP site was examined as 100pg per reaction. ABO genotyping from 1000 Japanese individuals was also examined to determine the distribution of ABO genotypes and allele frequencies. Thus, 31 genotypes were clearly identified, and these were controlled by four common and seven rare alleles. The power of discrimination, heterozygosity and polymorphism information contents were 0.913, 0.775 and 0.812, respectively. Therefore, selecting these eight SNP sites could be useful for high specific ABO genotyping. This rapid, sensitive and accurate genotyping method is useful for forensic casework.

Simultaneous determination of 5 psychotropic drugs of various types in an autopsy case of acute multiple drug poisoning.

We attempted the simultaneous determination of 5 drugs, mirtazapine, sertraline, chlorpromazine, amoxapine and zolpidem, detected in a gas chromatography-mass spectrometry screening test in an autopsy case. The solid-phase extraction of the analytes from biological samples was achieved using Oasis(®)HLB cartridges (Waters, Milford, MA, USA). Gas chromatography was performed on a HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent Technologies). The mass spectrometer was operated with an electron energy of 70 eV in electron impact mode. The qualitative and quantitative analyses were performed in full-scan mode and the selected ion monitoring mode, respectively. The total ion chromatogram showed good separation of these drugs. Linear graphs were obtained with good correlation coefficients for these drugs from 0.001 to 2.0 μg/mL (r(2)=0.9909-0.9986) using imipramine-d6 as an internal standard. The recoveries of these drugs were found to be 62.8-88.0% in spiked whole blood. Mirtazapine, sertraline, chlorpromazine, amoxapine and zolpidem were found in post-mortem samples of the deceased at concentrations of 2.67, 0.07, 0.25, 0.32 and 0.68 μg/mL, respectively. The concentration of mirtazapine was within the lethal level and those of amoxapine and zolpidem were within the toxic level. We diagnosed that the cause of death was acute multiple drug poisoning. The simple and practical procedure used in this study is useful for the simultaneous determination of psychotropic drugs of various types in post-mortem biological samples.

A case of V-A shunt catheters migration into the pulmonary artery

A man in his sixties, who developed CPA at home, was transferred to the emergency center. Since CT images revealed a tube-shaped foreign body in the pulmonary artery, pulmonary embolism was initially suspected; however, this did not lead to a definite diagnosis. Autopsy revealed that the foreign body in the cadaver was a fragment of a V-A shunt catheter implanted about 30 years previously for the treatment of hydrocephalus. Although fibrous adhesion of a part of the catheter to the pulmonary artery wall was seen, suggesting that a fracture of the catheter had occurred a long time before, it was not known when the fracture had occurred. Since no pulmonary arterial obstruction secondary to the catheter or new thrombi, which had been initially suspected, were observed, the cause of death was determined to be ischemic cardiac failure. A fracture of a shunt catheter may be typically associated with some clinical manifestations, which are often found and treated. In this case, however, no symptoms appeared and the fracture of the shunt catheter remained untreated for a long time. This case was therefore considered to be extremely rare, and is an example of how a serious iatrogenic disease could occur.

Fatal diphenhydramine poisoning with increased vascular permeability including late pulmonary congestion refractory to percutaneous cardiovascular support

Background. Fatal adult cases of acute diphenhydramine poisoning are extremely rare. Case Report. Transiently awakened by a roommate, a 39-year-old man admitted to massive ingestion of an over-the-counter drug containing diphenhydramine salicylate. On admission the patient was semicomatose and developed circulatory collapse with severe dehydration and metabolic acidosis, followed by status epilepticus. Despite extensive life support measures including percutaneous cardiopulmonary support, vascular permeability progressively increased, with pulmonary congestion as well as peripheral vasodilation evident as rubedo. The patient died without improvement of cardiac function. Subsequent diphenhydramine assays in serum specimens obtained at the time of delayed congestive symptoms indicated decreases in drug concentration to a sublethal amount. Discussion. We suspect that metabolites of diphenhydramine with histamine-agonist actions contributed to the development of fatal delayed symptoms.

Ontogeny of regulatory neuropeptides in the bullfrog taste organ

During metamorphic stages (stages XX-XXV), the first appearance of nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), galanin (GAL), and neuropeptide Y (NPY) in the bullfrog taste organs was different for each substance. CGRP fibers appeared first in association with the immature taste organs at stage XX. Up to stage XXV, the taste organs, epithelial disks, are close to their adult form, and SP, VIP and GAL fibers appeared within them. Throughout these stages, NPY fibers were absent, and no taste cells had immunoreactivity of the five neuropeptides. The present findings and previous physiological studies suggest that the immature taste organs in metamorphic stages already function as chemical and mechanical receptors and that these sensory mechanisms are under the control of peptidergic innervation.