Grant D M, Mörike K, Eichelbaum M, Meyer U A
Department of Pharmacology, University of Basel, Switzerland.
J Clin Invest. 1990 Mar;85(3):968-72. doi: 10.1172/JCI114527.
The biochemical basis underlying the genetic polymorphism of drug N-acetylation was investigated using a combination of in vivo and in vitro assays for arylamine N-acetyltransferase (NAT) activity and content in human liver. The acetylator phenotype of 26 surgical patients was determined using caffeine as an innocuous probe drug by measurement of the 5-acetyl-amino-6-formylamino-3-methyluracil to 1-methylxanthine molar ratio in urine. Liver wedge biopsies from these patients and livers from 24 organ donors were then used for measurement of N-acetyltransferase activity with the substrate sulfamethazine and for quantitation of immunoreactive N-acetyl-transferase protein. In vivo (caffeine metabolites in urine) and in vitro (sulfamethazine acetylation) measures of N-acetyl-transferase activity correlated very highly (r = 0.98). Moreover, in all subjects tested, slow acetylation both in vivo and in vitro was associated with a decrease in the quantity of immunodetectable N-acetyltransferase protein in liver cytosol relative to that seen in cytosols from rapid acetylator livers. Two kinetically distinct enzyme activities, designated NAT-1 and NAT-2, were partially purified from low- and high-activity livers and their relationship to acetylator status was determined. Low acetylation capacity was related to decreases in the liver content of both of these immunologically related proteins. The results demonstrate that genetically defective arylamine N-acetylation is due to a parallel decrease in the quantity of two structurally and functionally similar acetylating enzymes.
采用体内和体外相结合的方法,对人肝脏中芳胺N-乙酰基转移酶(NAT)活性和含量进行检测,以研究药物N-乙酰化遗传多态性的生化基础。通过测定尿中5-乙酰氨基-6-甲酰氨基-3-甲基尿嘧啶与1-甲基黄嘌呤的摩尔比,以咖啡因作为无害探针药物,确定了26名外科手术患者的乙酰化表型。然后,将这些患者的肝楔形活检组织以及24名器官捐献者的肝脏用于测定底物磺胺二甲嘧啶的N-乙酰基转移酶活性,并对免疫反应性N-乙酰基转移酶蛋白进行定量分析。N-乙酰基转移酶活性的体内(尿中咖啡因代谢产物)和体外(磺胺二甲嘧啶乙酰化)测定结果高度相关(r = 0.98)。此外,在所有测试对象中,体内和体外的慢乙酰化均与肝细胞溶质中免疫可检测的N-乙酰基转移酶蛋白数量相对于快乙酰化肝脏细胞溶质中的减少有关。从低活性和高活性肝脏中部分纯化出两种动力学特性不同的酶活性,分别命名为NAT-1和NAT-2,并确定了它们与乙酰化状态的关系。低乙酰化能力与这两种免疫相关蛋白在肝脏中的含量降低有关。结果表明,遗传性芳胺N-乙酰化缺陷是由于两种结构和功能相似的乙酰化酶数量同时减少所致。