Masato Funayama

Polymorphism of 17 STRs by multiplex analysis in Japanese population

Genotype and distribution of allele frequencies at 17 STRs were studied in 526 unrelated Japanese individuals using the PowerPlex 16 system and the AmpFlSTR Identifiler.

Rapid drug extraction from human whole blood using a modified QuEChERS extraction method

A modified QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of forensically important drugs and poisons (more than 90 compounds) in human whole blood. Because the QuEChERS method is commonly used for the analysis of pesticide residues in foods, we customized the QuEChERS method for forensic use. This extraction method consists essentially of two steps: (1) extraction/partitioning and (2) dispersive-solid phase extraction. In step 1, three-fold diluted blood was mixed with an internal standard (D5-diazepam for basic drugs or D5-phenobarbital for acidic drugs) solution, a QuEChERS pre-packed extraction kit (containing magnesium sulfate and sodium acetate) and a stainless steel bead, then partitioned into three layers by centrifugation. In step 2, the top layer (acetonitrile) was transferred into a centrifuge tube containing a dispersive-solid phase sorbent (containing primary secondary amine, end-capped octadecylsilane, and magnesium sulfate) and mixed for purification. After the centrifugation, supernatant was injected into LC-MS/MS. The QuEChERS method was applied in an autopsy case and we confirmed that this method can easily extract various types of drugs and metabolites from human whole blood. The combination of the modified QuEChERS method and LC-MS/MS could enable technicians inexperienced in forensic toxicological analysis to acquire reliable data quickly and easily.

A fatal case of infantile scurvy

Abstract We report a case of infant death due to scurvy, which is very rare in Japan. We initially had little knowledge of the disease and suspected that the bleeding in the body was caused by domestic violence. The case did not fall under the category of the battered child syndrome but the death was caused by ignorance with respect to child care. In addition the parents usually locked the child alone in a room during the day and this is probably a case of neglect.

Rapid determination of disulfoton and its oxidative metabolites in human whole blood and urine using QuEChERS extraction and liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of disulfoton and five of its oxidative metabolites (disulfoton-sulfoxide, disulfoton-sulfone, demeton-S, demeton-S-sulfoxide and demeton-S-sulfone) in human whole blood and urine. Extraction was undertaken using a QuEChERS method, which is commonly used in food analysis. D10-Disulfoton was used as the internal standard. Separation was carried out using a CAPCELL-PAK MG II column (35×2.0 mm i.d., 5 μm, Shiseido) with a mobile phase of 10 m mol/L ammonium formate and methanol. This method was applied in an autopsy case, and disulfoton and its oxidative metabolites were successfully detected in both blood and urine. The concentrations of disulfoton in the blood and urine were 360 and 23.8 ng/mL, respectively. There was a relatively low concentration of demeton-S in both the blood (4.0 ng/mL) and urine (45.7 ng/mL). To date, there have been no reported cases of detection of demeton-S in human samples.

Detection of diatoms in blood by a combination of membrane filtering and chemical digestion

An improved method for detecting diatom in blood is reported. Blood of cadavers was obtained by cardiac puncture at inquest or from the left atrium directly at autopsy. The blood was hemolyzed by sodium dodecyl sulfate (SDS) and filtered through membrane filter (47 mm in diameter and 5 microns in pore size). When the blood was putrefied, two or three pieces of membranes filters were needed because of choking membrane pore. The membrane filters were digested with fuming nitric acid and diluted with distilled water. The diluted solution was filtered through membrane filter (25 mm in diameter) again. After drying the membrane filter was immersed in oil. Diatoms on the membrane filter were clearly observed microscopically.

Regional differences in homicide patterns in five areas of Japan

This article describes regional differences in the homicide patterns which occurred in Sapporo City and the surrounding area, and in Akita, Ibaraki, Chiba and Toyama prefectures in Japan. Information collected from each case of homicide included factors such as age, sex of the victim and assailant, causes of death, disposition of the offender, relationship between assailant and victim, reasons for criminal action, et al. The statistical features of homicidal episodes among the five different regions showed considerable variation, as follows. The mean death rates for homicide (number of victims per 100,000 of population) during the period 1986-1995 were 0.44 (Sapporo), 0.8 (Akita), 0.58 (Toyama), 0.7 (Ibaraki) and 0.75 (Chiba), respectively. Close family relationship between the victim and assailant was observed in the homicidal acts which occurred in Sapporo, Akita and Toyama. Assailants relationship to victim was commonly extra-familial in Ibaraki and Chiba-neighboring megalopolis Tokyo, where some events of murder by a foreigner occurred. Homicide by female assailant, murder by mentally abnormal killers and homicide-suicide events were closely associated with family members. And these factors contributed to the considerable number of victims in Sapporo, Akita and Toyama. But, this close family relationship of the victim to the assailant did not correspond with the elevation in the number of deaths, and it was rather inversely related to the higher death rates recognized in Ibaraki and Chiba. This comparative study suggested that rapid urbanization considerably affects regional differences in homicide patterns.

Asphyxial death caused by postextraction hematoma

A 71-year-old man was admitted to an emergency room because of postextraction hemorrhage and died of asphyxia caused by airway obstruction. About 40 days earlier, he had had 11 carious teeth removed without postextraction bleeding. At autopsy, liver cirrhosis was found, but examination of his previous extractions and postmortem external findings did not show a general hemorrhagic tendency. The surgical incision in the gingiva had been sutured, and no damage to the bony socket or large vasculature was found. We could not determine the etiologic source of the decedents rapidly increasing hematoma. Postextraction hemorrhage or hematoma is a common complication in routine dental extraction, but marked hematoma formation around the airway may cause critical respiratory problems in the short run. In such cases, the maintenance of the airway, including control of hemorrhage, is necessary at an early stage.

Population genetics of 17 Y-chromosomal STR loci in Japanese

Haplotypes and allele frequencies of 17 Y-chromosomal short tandem repeat (Y-STR) markers were examined using the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems) in a population sample of 1166 Japanese male volunteers in 6 prefectures: Miyagi, Yamagata, Osaka, Tottori, Fukuoka, and Okinawa. A total of 1058 haplotypes were observed from 1166 males, and the most common haplotype detected in 12 males had a frequency of 1.03% and the discrimination capacity was 0.907. The R(ST) analysis showed statistically significant differences between Okinawa and the other subpopulations.

Diagnosis of drowning using post-mortem computed tomography based on the volume and density of fluid accumulation in the maxillary and sphenoid sinuses.

Recent studies have reported that drowning victims frequently have fluid accumulation in the paranasal sinuses, most notably the maxillary and sphenoid sinuses. However, in our previous study, many non-drowning victims also had fluid accumulation in the sinuses. Therefore, we evaluated the qualitative difference in fluid accumulation between drowning and non-drowning cases in the present study. Thirty-eight drowning and 73 non-drowning cases were investigated retrospectively. The fluid volume and density of each case were calculated using a DICOM workstation. The drowning cases were compared with the non-drowning cases using the Mann-Whitney U-test because the data showed non-normal distribution. The median fluid volume was 1.82 (range 0.02-11.7) ml in the drowning cases and 0.49 (0.03-8.7) ml in the non-drowning cases, and the median fluid density was 22 (-14 to 66) and 39 (-65 to 77) HU, respectively. Both volume and density differed significantly between the drowning and non-drowning cases (p=0.001, p=0.0007). Regarding cut-off levels in the ROC analysis, the points on the ROC curve closest (0, 1) were 1.03ml (sensitivity 68%, specificity 68%, PPV 53%, NPV 81%) and 27.5 HU (61%, 70%, 51%, 77%). The Youden indices were 1.03ml and 37.8 HU (84%, 51%, 47%, 86%). When the cut-off level was set at 1.03ml and 27.5HU, the sensitivity was 42%, specificity 45%, PPV 29% and NPV 60%. When the cut-off level was set at 1.03ml and 37.8HU, sensitivity was 58%, specificity 32%, PPV 31% and NPV 59%.

Simultaneous determination of 11 aconitum alkaloids in human serum and urine using liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of four aconitines (aconitine, mesaconitine, hypaconitine, jesaconitine) and seven of their hydrolysis products (benzoylmesaconine, benzoylhypaconine, 14-O-anisoylaconine, benzoylaconine, aconine, mesaconine, hypaconine) in human serum and urine samples. Extraction was undertaken using a mixed-mode cation-exchange cartridge (OASIS MCX), and D(5)-aconitine was used as an internal standard. Separation of aconitum alkaloids was carried out using an L-column ODS with the mobile phase consisting of 10mM ammonium formate and methanol. The intra- and inter-day precisions were 0.3% to 9.9% and 3.2% to 12.8%, respectively. Intra- and inter-day accuracies were -14.1% to 7.3%, and -10.6% to 8.3%, respectively. The limit of detection and limit of quantification of analytes were 0.04-0.38 ng/mL and 0.12-1.15 ng/mL respectively. This method was applied in an autopsy case and successfully detected aconitines and their metabolites as well as some anti-psychiatric drugs.