| Project/Area Number |
08670487
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Legal medicine
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| Research Institution | Tottori University |
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Principal Investigator |
IKEBUCHI Jun Department of Legal Medicine, Faculty of Medicine, Tottori University, Research Associate, 医学部, 助手 (30150361)
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| Co-Investigator(Kenkyū-buntansha) |
IRIZAWA Yoshito Department of Legal Medicine, Faculty of Medicene, Tottori University, Professor, 医学部, 教授 (90112226)
YUASA Isao Department of Legal Medicine, Faculty of Medicene, Tottori University, Assistant, 医学部, 講師 (00093633)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | drug metabolizing enzyme / genetic polymorphism / genotype / phenotype / individual difference / interethnic difference / toxicogenetics / toxicology / チトクロームP-450遺伝子 / N-アセチル転移酵素遺伝子 / 遺伝的多型性 |
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| Research Abstract |
N-acetyltansferase (NAT) in human liver catalyzes N-acetylation of various arylamine-containing drugs and xenobiotics. Human NAT isoenzymes are encoded at two loci, and the NAT2 locus is polymorphic.
Acentylation polymorphism has been studied by several methods with a test drug, such as isoniazid, and ethnic variation in N-acetylation capacity has been reported.
In this study, we prepared the DNA samples from the blood of unrelated healthy Japanese, and developed a rapid and simple genotyping method using a polymerase chain reaction (PCR) based restriction fragment length polymorphism. NAT2 genotypes were determined based on the fact that four alleles in the NAT2 gene differe at single base substitutions which modify restriction enzyme cleavage sites. The sequence containing the polympophic sites was amplified, and the cleavage of the PCR product by the restriction enzymes was detected by polyacrylamide gel electrophoresis. Furthermore, NAT2 phenotypes were determined by measuring the molar ratio of caffeine urinary metabolites, 5-acetylamino-6-formylamino-3-methyluracil (AFMU) /1-methylxanthine (1X), taken at 4-5hr after caffeine ingestion.
Consequently, the genotypes observed were NAT2^<**>1/^<**>1, NAT2^<**>1/^<**>2, NAT2^<**>1/^<**>3, NAT2^<**>1/^<**>4 and NAT2^<**>3/^<**>4. In vivo metabolic ratio (AFMU/1X) of wild-type was 1.55, whereas hetero-type was 0.87, and homo-mutated type 0.12. The genotype of NAT2 correlated with the phenotype.
These results proved that genotyping assays of drug matabolizing enzymes would play more important role in assessing the severity and predicting the outcome of poisoning for forensic and clinical toxicology.
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