濵島 誠

DNA methylation in gene promoter regions influences gene expression. Circadian clock genes play an important role in the formation of a biological clock and aberrant methylation of these genes contributes to several disorders. In this study, we examined forensic autopsy specimens to determine whether DNA methylation status in the promoter regions of nine circadian clock genes (Per1, Per2, Per3, Cry1, Cry2, Bmal1, Clock, Tim, and Ck1e) is related to a change in acquired diathesis and/or causes of death. Methylation-specific PCR and direct sequencing methods revealed that the promoters of Per1, Cry2, Bmal1, Clock, and Ck1e were unmethylated in all the forensic autopsy specimens, while the promoters of Per2, Per3, Cry1, and Tim were partially methylated. Methylation status varied between individuals and between tissues in the same patient. A detailed analysis of methylation patterns in the Cry1 promoter region revealed that the patterns also varied between individuals and the Cry1 promoter had highly methylated patterns in two cases that had been exposed to methamphetamine. These results suggest that the methylation status of clock gene promoters varies between individuals. Methamphetamine use may influence methylation in the Cry1 gene promoter region and disturb circadian rhythmicity. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

A number of reports are available in the literature that describe liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS) analysis of amanitins, very toxic mushroom toxins, in biological samples. However, the extractive pretreatment methods and LC separation column materials vary remarkably according to the different reports. This communication presents a very simple and sufficiently sensitive method for LC-MS analysis of amanitins. A plasma sample was diluted with distilled water and buffer solution, and applied to a Discovery DSC 18 column (500 mg packing material), followed by washing with distilled water and elution with methanol. The extract, after evaporation and reconstitution in mobile phase solution, was subjected to LC-MS analysis with a conventional octadecyl LC separation column. The selected ion monitoring of alpha-amanitin and beta-amanitin at m/z 919-921 and m/z 920-922, respectively, gave symmetrical peaks and good separation of both amanitin peaks. Using an external calibration method, linearity, detection limits, recovery rates, and precision were tested; they were all satisfactory. To our knowledge, the present method gives the simplest LC-MS analysis for amanitins among those so far reported. We recommend the method for use in actual forensic and clinical toxicological analysis of amanitins in biological samples.

A detailed procedure has been established for simultaneous analysis of aconitine, mesaconitine, hypaconitine, and jesaconitine in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The method uses a new unique polymer column (Shodex ODP2 HP-4B) for separation, which enabled the injection of relatively crude organic extracts without complicated pretreatments. Quantitation was made by mass chromatography with each product ion referenced against dextromethorphan as internal standard. Aconitine and its three analogues showed good linearity over the range of 1.25-40 ng/ml; the detection limits were 0.3-0.5 ng/ml. Validation data including accuracy, precision, and recovery rates are presented. To further validate the present method, the mixture of aconitine, mesaconitine, and hypaconitine (0.2 mg each) was orally administered to rats, and the concentrations of the three compounds in whole blood specimens were measured 15, 30, and 45 min after dosing; their concentrations in rat whole blood were 0.74-18.3 ng/ml. The present LC-MS-MS method is very effective for simple simultaneous analysis of aconitine and its analogues in forensic and clinical toxicology.

Time-of-flight (TOF)-mass spectrometry (MS) is not a new technique. In 1980s, TOF-MS was used as an analyzer for matrix-assisted laser desorption ionization (MALDI) this combination much contributed to the proteome research. An orthogonal acceleration technique enabled TOF-MS to be an analyzer for the electrospray (ESI) interface a quadrupole/orthogonal acceleration time-of-flight tandem mass spectrometer (QqTOF-MS/MS) was developed in 1990s. De Leenheer's group developed some methods for identification of drugs of abuse, e.g. cocaine, amphetamines and opiates by QqTOF-MS/MS. However, this system is quite expensive and not easy to be handled a single TOF-MS system thus can be an alternative technique for actual analyses of drugs and poisons. So far, the single TOF-MS has been applied mainly in environmental toxicology it will be extensively applied in the field of forensic toxicology, because TOF-MS has higher mass resolution power and full spectral sensitivity. On the contrary, the dynamic range of TOF-MS is sometimes inferior to those of other MS or MS/MS systems however, in an AccuTOF® mass spectrometer, developed by JEOL, its dynamic range has been much improved. Therefore, this system seems promising for both identification and quantitation of drugs of abuse and poisons. Our data have shown that the AccuTOF® MS system is very useful in analyzing aconitine alkaloids in human serum samples. ©2001 Japanese Association of Forensic Toxicology. All rights reserved.