高效液相色谱法测定人红细胞中巯基嘌呤甲基转移酶的活性

Determination of human red blood cell thiopurine methyltransferase activity by high-performance liquid chromatographic method

目的:建立一种改良的高效液相色谱法测定人红细胞中巯基嘌呤甲基转移酶(thiopurinemethyltransferase,TPMT)的活性。方法:采用反相高效液相色谱法直接测定酶促反应的产物6-甲基巯基嘌呤的浓度,从而计算红细胞中TPMT的活性。以6-巯基嘌呤为底物,S-腺苷-L-甲硫氨酸作为甲基供体,红细胞裂解液在一定的条件下进行孵化,然后经乙酸乙酯萃取处理后,用HPLC分析。色谱柱为hypersilBDSC18,流动相为水-甲醇-三乙胺(磷酸调至pH3.5)(76.8∶23∶0.2),紫外检测波长为290nm。结果:6-甲基巯基嘌呤在10～200μg/L范围内呈良好的线性关系,相关系数r=0.9994,最低检测限为3μg/L(S/N=3),平均回收率为86%～94%。我们运用该法测定了273例健康中国成年人红细胞中TPMT的活性,其平均活性为(11.96±3.27)nmol/h·mlPackedRBC,男性和女性的平均TPMT活性分别为(12.08±3.38)nmol/h·mlPackedRBC和(11.73±3.07)nmol/h·mlPackedRBC,两者无显著性差异。结论:本分析方法具有灵敏准确,重复性好,安全可靠,易于推广等特点,能满足巯基嘌呤类药物药代动力学研究和临床用药监测的要求。

Objective：To investigate thiopurine methyltransferase（TPMT） activity distribution in Chinese Hail population. Methods：The RP- HPLC method is based on the dirct quantitation of 6 -methly -mercaptopurine（6- MMP） produced from 6 -mercaptopurine by crude erythrocyte lysates and non - radioactive S - adenosyl - L - methionine is the methly donor. The 6 - MMP is extracted into ethyl acetate and quantitated by HPLC. The analysis involved a C18 column as stationary phase and water -methanol -triethylamine（76. 8：23： 0. 2） ,adjusted to pH 3. 5 with phosphoric acid ,as mobile phase. The flow rate was 1.0 ml/min,and UV detection wavelength was at 290 nm. Results：The limit of detection was 3 μg/L The calibration curve was linear between 10 -200 pg/L with a correlation coefficient of 0. 9994. The recoveries of 6 - MMP samples were 86% -94%. The mean value of TPMT activity in 273 healthy Chinese people were （11.96±3. 27）mnol/h · ml Packed RBC ,male（ 12. 08±3. 38）nmol/h·ml Packed RBC, and female（ 11.73±3. 07 ）nmol/h · ml Packed RBC. Conclusion：The method is economic, simple and rapid. It can be applied to pharmacokinetic study of TPMT and 6 -MP.