平田 ゆかり

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.

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 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.

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.

The Aconitum species (Ranunculaceae) are widely distributed in northern Asia and North America. Their roots are popularly used in herbal medicines in China and Japan. Many cases of accidental, suicidal and homicidal intoxication with this plant have been reported; some of these were fatal because the toxicity of Aconitum is very high. It is thus important to detect and quantify Aconitum alkaloids in body fluids, with high sensitivity. We have developed a simple and sensitive method for measuring four kinds of Aconitum alkaloids (aconitine, hypaconitine, jesaconitine and mesaconitine) by LC/electrospray (ESI)-time-of-flight (TOF)-MS. For all of them, only molecular ions were observed at an orifice voltage of 75 V; at 135 V, base peaks corresponding to [M - 60 + H](+) ions were observed. These four compounds and methyllycaconitine (internal standard) in human plasma samples were purified by solid-phase extraction. The four extracted compounds were completely separated in mass chromatograms; the calibration curves showed good linearity in the range 10-300 ng/ml, and the detection limits were estimated to be 0.2-0.5 ng/ml. Using our method, we also determined the amounts of these compounds in tuber samples. The present method is applicable in clinical and forensic toxicology. Copyright (c) 2006 John Wiley & Sons, Ltd.