Masanori Hasegawa

Sex determination of forensic samples by dual PCR amplification of an X-Y homologous gene

Sex determination by polymerase chain reaction (PCR) analysis of the X-Y homologous amelogenin gene is highly reliable since the detection of an X-specific amplified fragment validates the procedure. Previously, we reported that 250 ng of template DNA are required for sex determination by this method. We report here a refinement of the technique to include dual PCR. Dual PCR using two sets of primers results in the detection of X- and Y-specific amplified fragments from as little as 0.005 ng of template DNA. This is a powerful technique for the analysis of trace forensic samples and its application is discussed.

Purification of Forensic Specimens for the Polymerase Chain Reaction (PCR) Analysis

Purification methods of deoxyribonucleic acid (DNA) from degraded and contaminated forensic samples were investigated for polymerase chain reaction (PCR) analysis. DNA extracted from putrefied tissue or bloodstains sometimes contained the copurified contaminant, that was identified as the porphyrin compound (hematin). When contaminated but less degraded DNA was analyzed by PCR, it was necessary to eliminate the impurity by anion exchange column chromatography or chelating resin preparation, and ultrafiltration using Centricon microconcentrators. When highly degraded DNA was analyzed, trace amounts of high molecular weight DNA was recovered by electroelution method, and then further purified by both column chromatography and ultrafiltration. From thus purified samples, the amelogenin gene for sex determination could be amplified by dual PCR technique.

Stereoselective analyses of selegiline metabolites: possible urinary markers for selegiline therapy.

The stereoselective analysis of selegiline metabolites in human urine and plasma by gas chromatography using the chiral column with the non-chiral reagent was investigated for the differentiation of selegiline therapy from the methamphetamine (MA) abuse. This method gave clear separations of MA and amphetamine (AM) isomers without any artifactual optical-opposite peaks due to the reagent. After the administration of selegiline tablets, desmethylselegiline (DMS), MA and AM were observed as (-)-isomers in the urine and plasma. Within the first 48 h after dosing, approximately 40% of selegiline administered was excreted in urine as these three metabolites. The parent drug, selegiline, was not detected in any urine or plasma samples. On the other hand, MA and AM were observed only as (+)-isomers in the urine of MA abusers. For the distinction of selegiline users from street MA abusers in urinalysis, (-)-DMS, a specific metabolite of selegiline, was not a suitable marker. (-)-DMS rapidly disappeared from urine and was excreted only 1% of the given dose. By the moment analysis with the trapezoidal integration, the mean residence times of (-)-DMS in plasma and urine were 2.7 and 3.8 h, respectively, which were 5-20 times shorter than those of (-)-MA or (-)-AM. The values of AM/MA in the urine increased from 0.24 to 0.67 (r = 0.857) along with time after the selegiline administration. This ratio was not a sufficient marker to differentiate selegiline users from MA abusers, although the values of AM/MA in 74% of MA abusers were less than 0.24. The present GC technique improved the chiral analyses of MA and AM. This chiral analysis is the most useful technique to avoid the misinterpretation in the discrimination between clinical selegiline therapy and illicit MA use.

A fatal disaster case based on exposure to hydrogen sulfide - an estimation of the hydrogen sulfide concentration at the scene.

Four adult men fell into an artificial lake which was being used to raise flatfish, after a water pipe had been connected to a tube allowing seawater to flow into the lake. Forensic autopsies were carried out on three of the four men, who died soon after the incident. From autopsy findings, the cause of death was diagnosed to be suffocation after aspirating seawater in the three victims. To clarify why the men fell into the lake, a chemical analysis for hydrogen sulfide was carried out using the extractive alkylation technique combined with gas chromatography/mass spectrometry. The sulfide was detected as its derivative, bis(pentafluorobenzyl)sulfide, in body tissues taken from all the victims, and the concentration of hydrogen sulfide gas at the scene was estimated as having been nearly fatal.

Detection and measurement of S-benzyl-N-acetylcysteine in urine of toluene sniffers using capillary gas chromatography

We examined the urinary excretion of S-benzyl-N-acetylcysteine (SBAC) of toluene sniffers using capillary gas chromatography. SBAC was extracted from 10 ml urine with chloroform and backextracted into 1 M sodium bicarbonate solution. After acidification, the aqueous solution was reextracted with ethyl acetate, and then derivatized to its methyl ester (ME). The peak appearing in the gas chromatogram was identified as SBAC-ME by mass spectrometry. The calibration curve was constructed by plotting the peak height ratio of SBAC-ME and internal standard (S-phenethyl-N-acetylcysteine)-ME against analyte concentration using 10 ml toluene unexposed urine. It showed good linearity over the range of 0.05–3.0 mg/l (r = 0.99). We have applied this technique to urine samples from toluene sniffers. SBAC was detected in all urinary samples of sniffers (n = 30, 0.11–47.13 mg/l), but not at all in the urine of toluene unexposed subjects (n = 60). These results prove that SBAC is also formed from toluene by human metabolism, and detection of SBAC is considered a useful marker for inhalation of toluene.

A case of drowning lacking typical autopsy findings of carbon monoxide poisoning despite the high CO concentration

Carbon monoxide (CO) is the leading cause of poisoning death in Japan [1, 2]. In a forensic autopsy, the measurement of blood carboxyhemoglobin (CO-Hb) is important to determine the cause of death of victims of fire, suicide, and/ or accident. Moreover, CO-Hb measurement is important in the examination of victims of large-scale disasters, such as forest fires [3]. We present here one case in which a routine CO analysis was helpful in determining the circumstances related to the cause of death. The victim was found in a bathtub with no characteristic findings of CO poisoning. The detection of a high level of CO-Hb at autopsy led to a reinvestigation by the police and prevented a secondary incident in which a member of the victim’s family could have succumbed to CO poisoning. A woman aged in her fifties (156 cm tall and 45.9 kg body weight) was found dead in a bathroom of her home. The victim was sitting in the bathtub with her face under water. Her family called an ambulance, but postmortem rigidity was observed by the emergency personnel upon arrival. The victim was a smoker but not a drinker and did not have any past history of disease. About 12 h after the woman’s death, an autopsy was performed. Although no signs of external trauma were present, the sclera and palpebral conjunctivae showed some petechiae. Any findings suggesting putrefaction were not recognized. Magenta lividity was noted on the victim’s back but faded under finger pressure. The internal examinations revealed that the heart weighed 414 g and was slightly hypertrophic, containing 150 ml of dark-red blood without coagulum. Marked atherosclerosis in the aorta and coronary arteries was observed. Aspirated liquid was in the upper airways, and the lungs were over-distended. The lungs were congested and edematous; the right and left lungs weighed 550 and 420 g, respectively. The other viscera showed signs of flaccid congestion. Histologically, acute lung emphysema with lacerations of the septa, capillary congestion, and moderate intraalveolar edema were observed. Based on circumstantial details, autopsy, histology, and toxicological findings, drowning was considered the most probable cause of death. A drug screening test using a Triage DOA plus TCA (Biosite Diagnostic, San Diego, CA, USA) panel was negative. Postmortem blood samples were collected for subsequent investigation. CO-Hb was measured using the AVOXimeter 4000 (AVOX; International Technidyne, NJ, USA) [4], which yields results within 10 s. The CO-Hb level, as measured using AVOX, was 57.2 % in the right cardiac blood and 55.9 % in the left cardiac blood. CO-Hb was also measured by the conventional double-wavelength (or spectrophotometric) method [5]. It was 55.7 % in the right and left cardiac blood. Quantitation of ethanol was performed using headspace gas chromatography, according to our previous method [6], with slight modifications. No blood ethanol was detected. A reinvestigation of the scene was conducted about 1 week after the victim’s autopsy. The victim had been J. Fujihara H. Takeshita (&) Department of Legal Medicine, Shimane University School of Medicine, Izumo 693-8501, Japan e-mail: htakeshi@med.shimane-u.ac.jp

Carrier diagnosis by RFLP analysis in a family affected with infantile hypophosphatasia: Case report

Carrier diagnosis by RFLP analysis in a family affected with infantile hypophosphatasia: Case report