Individual Difference in Drug Metabolish and Disposition : Toxicological Significance of Genotypes and Phenotypes of Human Polymorphic Arylamine N-acetyltransferase

人肝脏中的n -乙酰转移酶（NAT）可催化各种芳胺类药物和外源药物的n -乙酰化。人类NAT同工酶在两个位点编码，而NAT2位点是多态的。用异烟肼等试验药物研究了n -乙酰化多态性，并报道了n -乙酰化能力的种族差异。本研究从无血缘关系的健康日本人血液中提取DNA样本，建立了一种基于限制性内切片段长度多态性的快速、简单的基因分型方法。根据NAT2基因的4个等位基因在修饰限制性内切酶切割位点的单碱基替换上的差异，确定了NAT2基因型。扩增含有多亲性位点的序列，用聚丙烯酰胺凝胶电泳检测限制性内切酶对PCR产物的切割作用。此外，通过测量咖啡因尿液代谢物5-乙酰氨基-6-甲酰基氨基-3-甲基尿嘧啶(AFMU) /1-甲基黄嘌呤（1X）的摩尔比，测定咖啡因摄入后4-5hr的NAT2表型。因此,观察到的基因型是NAT2 ^ < * * > 1 / ^ < * * > 1, NAT2 ^ < * * > 1 / ^ < * * > 2, NAT2 ^ < * * > 1 / ^ < * * > 3, NAT2 ^ < * * > 1 / ^ < * * > 4和NAT2 ^ < * * > 3 / ^ < * * > 4。野生型的体内代谢比（AFMU/1X）为1.55，异型为0.87，同源突变型为0.12。NAT2基因型与表型相关。这些结果表明，药物代谢酶基因分型分析在法医和临床毒理学评估中毒严重程度和预测结果方面具有更重要的作用。

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.