Chikatoshi Maseda

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.

Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

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.

Improved gas chromatography with electron-capture detection using a reaction pre-column for the determination of blood cyanide: A higher content in the left ventricle of fire victims

We developed a head-space method for the determination of blood cyanide by gas chromatography with electron-capture detection. In this technique, a reaction pre-column, packed with chloramine-T, was used for the conversion of hydrogen cyanide into cyanogen chloride. Since the reaction pre-column eliminated the necessity for trapping hydrogen cyanide from the biological samples, blood cyanide was quickly analysed by acidification only. The reaction pre-column was durable for at least several months. The calibration curve gave good linearity when dichloromethane was used as the internal standard, and the lower detection limit taken from this plot was ca. 0.05 micrograms/ml. The relative standard deviation of spiked blood samples was in the range 0.6-3.9%. We determined blood cyanide levels at autopsy in victims who had died from fire using this method. A significantly higher cyanide content was detected in the left ventricular blood than in the right. There was a positive correlation between blood cyanide and carboxylhaemoglobin contents. This simple and sensitive technique could be very useful for the determination of cyanide in various samples.

Gas chromatographic determination for forensic purposes of petroleum fuel inhaled just before fatal burning

The determination of petroleum fuel in the blood of burned bodies was carried out by three different gas chromatographic procedures. Seven components of gasoline (isopentane, n-pentane, 2-methylpentane, benzene, 2-methylhexane, 3-methylhexane and toluene) and five of kerosene (xylene, C9H20, mesitylene, pseudocumene and C11H24) were chosen as indicators with a coefficient of variation of 5-24%. The methods were applied to four autopsy cases with a relatively low carboxyhaemoglobin (HbCO) content. When gasoline exposure had occurred, the blood concentrations determined were almost identical whatever the components selected. Great variations in the components determined were found after kerosene exposure, and hydrocarbons greater than or equal to C14 were hardly inhaled by the victims. A higher content of fuel in the left than in the right ventricular blood observed in the autopsy cases suggests fuel inhalation just before death. The same phenomenon was also observed in the content of blood HbCO. Determinations of petroleum fuel and HbCO in both the right and left ventricular blood would be useful for the forensic diagnosis on burned bodies with a low HbCO content.

Rapid and sensitive quantitation of cyanide in blood and its application to fire victims

We developed a head-space method for the determination of blood cyanide by gas chromatography with electron-capture detection. In this technique, a reaction precolumn packed with chloramine-T was used for the conversion of hydrogen cyanide into cyanogen chloride. Since the reaction precolumn eliminated the necessity of trapping hydrogen cyanide from biological samples, blood cyanide could be analyzed quickly by acidification only. Using this method, blood cyanide levels of fire victims were determined at autopsy. The serum values of cyanide ranged from 0.11 micrograms/ml to 18.12 micrograms/ml. However, a significantly higher cyanide content was detected in the left ventricular blood than in the right. This indicates that death was caused by the fire and suggests that the collecting point of the blood sample is an important factor in the determination of inhaled cyanide. There was a positive correlation between blood cyanide and carboxyhemoglobin contents.

Genotyping of 38 insertion/deletion polymorphisms for human identification using universal fluorescent PCR

Short insertion/deletion (Indel) polymorphisms of approximately 2-6 bp are useful as biallelic markers for forensic analysis, and the application of Indel genotyping as a supplementary tool would improve human identification accuracy. We examined the allele frequencies of 37 autosomal Indels in the Japanese population and developed a novel dual-color genotyping method for human identification on the basis of universal fluorescent PCR, including the sex-typing amelogenin locus. Target genomic fragment sizes for 38 Indels were 49-143 bp. We analyzed these Indels in 100 Japanese individuals using the M13(-47) sequence as a universal primer. For dual-color genotyping, we designed a novel universal primer with high amplification efficiency and specificity. Using FAM-labeled M13(-47) and HEX-labeled modified M13(-47) primers, fluorescent signals at all loci were clearly distinguished in two independent multiplex PCRs. Average minor allele frequency was 0.39, and accumulated matching probability was 2.12 × 10(-15). Complete profiles were successfully amplified with as little as 0.25 ng of DNA. This method provides robust, sensitive, and cost-effective genotyping for human identification.

Universal fluorescent labeling of amplification products using locked nucleic acids.

Amplification/hybridization‐based genetic analyses using primers containing locked nucleic acids (LNAs) present many benefits. Here, we developed a novel design for universal fluorescent PCR using LNAs. Universal fluorescent PCR generates intermediate nonlabeled fragments and final fluorescent fragments in a two‐step amplification process that uses locus‐specific primers with universal tails and universal fluorescent primers. In this study, a few standard nucleotides were replaced with LNAs only in the fluorescent universal primers. The sequence of the fluorescent universal primer significantly affected the amplification efficiency. For primers with three LNAs, the fluorescent primers with stable M13(‐47) sequences provided the most efficient signal (approximately tenfold higher than the primers with M13(‐21) sequences at lower Tm values). Moreover, AT‐rich LNA substitutions in the fluorescent primers produced much lower amplification efficiencies than GC‐rich substitutions. GC‐rich LNAs produced greater differences in Tm values among primers, and resulted in the preferential production of fluorescently labeled amplicons. The specificity and sensitivity of LNA‐containing fluorescent primers were assessed by genotyping eight STRs in Japanese individuals, and full STR profiles could be generated using as little as 0.25 ng of genomic DNA. The method permitted clear discrimination of alleles and represents sensitive STR genotyping at a reduced cost.

Multiplex PCR-based Alu insertion polymorphisms genotyping for identifying individuals of Japanese ethnicity.

Discrimination of Alu insertions is a useful tool for geographic ancestry analysis, and is usually performed by Alu element amplification and agarose gel electrophoresis. Here, we have developed a new fluorescence-based method for multiple Alu genotyping in forensic identification. Allele frequencies were determined in 70 Japanese individuals, and we selected 30 polymorphic Alu insertions. Three primers were designed for each Alu locus to discriminate alleles using the 3-6 bp differences in amplicon sizes. Furthermore, we classified the amplification primers for the 30 loci into three different sets, and PCR using each set of primers provided 10 loci fragments ranging from 50 to 137 bp. Based on population data, the probability of incorrectly assigning a match was 3.7×10(-13). Three independent amplifications and subsequent capillary electrophoresis enabled the sensitive genotyping of small amounts of DNA, indicating that this method is suitable for identifying individuals of Japanese ethnicity.

Liquid chromatography-tandem mass spectrometry method for the determination of thiosulfate in human blood and urine as an indicator of hydrogen sulfide poisoning

Being a stable metabolite of hydrogen sulfide, thiosulfate has been utilized as an index for hydrogen sulfide poisoning (HSP). Thiosulfate analysis is mainly performed using gas chromatography/mass spectrometry (GC-MS) due to its high sensitivity and specificity. The GC-MS analysis requires two-step derivatizations of thiosulfate, and the derivative is not stable in solution as it has a disulfide moiety. To resolve this stability issue, we developed a novel analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for monitoring the pentafluorobenzyl derivative of thiosulfate (the first reaction product of the GC-MS method) in this study. The established method exhibited high reproducibility despite being a more simplified and rapid procedure compare to the GC-MS method. Phenyl 4-hydroxybenzoate was used as an internal standard because 1,3,5-tribromobenzene which had been used in the GC-MS method was not suitable compound for LC-MS/MS with Electrospray ionization (ESI) negative detection. The linear regression of the peak area ratios versus concentrations was fitted over the concentration ranges of 0.5-250μM and 0.25-250μM in blood and urine, respectively. The validation results satisfied the acceptance criteria for intra- and inter-day accuracy and precision. Blood and urine samples from 12 suspected HSP cases were tested using this method. The thiosulfate concentration detected in the sample coincided well with that determined at the scene of each HSP accident.