Kröplin T, Weyer N, Gutsche S, Iven H
Department of Pharmacology, Lübeck Medical University, Germany.
Eur J Clin Pharmacol. 1998 May;54(3):265-71. doi: 10.1007/s002280050457.
To develop a non-radioactive assay to measure thiopurine S-methyltransferase (TPMT) activity. The assay was used to study the distribution of TPMT activity in a healthy German population.
The assay is based on the conversion of 6-thioguanine (6-TG) to 6-methylthioguanine (6-MTG) using non-radiolabelled S-adenosyl-L-methionine (SAM) as the methyl donor. At the end of the incubation period (60 min) 6-MTG is extracted into chloroform/2-propanol and quantitated by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection at Ex 315 nm and Em 390 nm.
The method is rapid, sensitive and reproducible, with an interassay CV of 6.7% (quality control sample with TPMT activity of 43 nmol 6-MTG x g(-1) Hb x h(-1)) and thus suitable for routine monitoring of TPMT activity. The TPMT activity of 219 healthy German blood donors showed the known trimodal distribution with a range from 1.3 to 68.3 nmol 6-MTG x g(-1) Hb x h(-1) with a median value of 38.8 nmol 6-MTG x g(-1) Hb x h(-1). When the cut-off value for intermediate to high activity was set at 23.5 nmol 6-MTG x g(-1) Hb x h(-1), 14.1% belonged to the group with intermediate and 83.6% to the group with high TPMT activity. Five individuals had a very low TPMT activity of <2 nmol 6-MTG x g(-1) Hb x h(-1). Genetic analysis revealed that these persons were found either homozygote for the variant allele *3A (n = 3) or they were compound heterozygotes for the variant alleles *3A/*3C (n = 2). With these alleles for low TPMT activity they would run an increased risk of myelosuppression in case of treatment with standard doses of thiopurine drugs.
开发一种非放射性检测方法来测定硫嘌呤甲基转移酶（TPMT）活性。该检测方法用于研究TPMT活性在健康德国人群中的分布情况。
该检测方法基于以非放射性标记的S-腺苷-L-甲硫氨酸（SAM）作为甲基供体，将6-硫鸟嘌呤（6-TG）转化为6-甲硫鸟嘌呤（6-MTG）。在孵育期结束时（60分钟），将6-MTG萃取到氯仿/2-丙醇中，并通过反相高效液相色谱（HPLC）在激发波长315nm和发射波长390nm处进行荧光检测来定量。
该方法快速、灵敏且可重复，批间变异系数为6.7%（TPMT活性为43nmol 6-MTG×g⁻¹Hb×h⁻¹的质量控制样品），因此适用于TPMT活性的常规监测。219名健康德国献血者的TPMT活性呈现出已知的三峰分布，范围为1.3至68.3nmol 6-MTG×g⁻¹Hb×h⁻¹，中位数为38.8nmol 6-MTG×g⁻¹Hb×h⁻¹。当中等至高活性的临界值设定为23.5nmol 6-MTG×g⁻¹Hb×h⁻¹时，14.1%属于中等活性组，83.6%属于高TPMT活性组。有5个人的TPMT活性非常低，<2nmol 6-MTG×g⁻¹Hb×h⁻¹。基因分析显示，这些人要么是变异等位基因3A的纯合子（n = 3），要么是变异等位基因3A/*3C的复合杂合子（n = 2）。携带这些低TPMT活性等位基因的人在使用标准剂量硫嘌呤类药物治疗时，骨髓抑制风险会增加。
