【摘要】 目的探讨急性淋巴细胞白血病患儿红细胞中的硫嘌呤甲基转移酶(TPMT)活性的高效液相色谱(HPLC)测定法。方法采用反相HPLC直接测定酶促反应的产物浓度,从而计算红细胞中TPMT的活性。以S-腺苷-L-甲硫氨酸(S-adenosyl-L-methionine,SAM)作为甲基供给体,6-硫鸟嘌呤(6-thioguanine,6-TG)作为酶反应底物,TPMT催化6-TG生成2-氨基-6-甲基巯基嘌呤(2-amino-6-methyl mercaptopurine,6-MTG),采用高氯酸溶液终止反应及沉淀蛋白,分离上清液进行色谱分析。色谱柱为AichromBond-1 C18柱(5μm,4.6mm×150mm);等梯度洗脱,流动相为乙腈:0.01mol/L磷酸钾缓冲溶液(用盐酸调pH=2.74)(6∶94,V/V),流速为1.0mL/min;柱温为35℃;进样量为50μL。荧光检测器检测,激发波长为310nm,发射波长为390nm。结果6-MTG在0~250μg/L范围内呈良好的线性关系,相关系数r=0.9999,最低检出限为0.093μg/L(S/N=3),回收率为81.4~106.3%。结论本方法操作简便、分析速度快、检出限低、重现性好、易于推广,能满足巯基嘌呤类药物代谢动力学研究和临床用药监测常规分析。更多还原

【Abstract】 Objective To establish an HPLC method to measure thiopurine S-methyltransferase(TPMT) activity of children with acute lymphocytic leukemia(ALL).Methods The assay was based on 6-thioguanine(6-TG) which changed to 2-amino-6-methyl mercaptopurine(6-MTG) in enzymatic reaction using S-adenosyl-L-methionine(SAM) as the methyl donor and catalyzed by TPMT.The reaction was terminated and educed protein by perchloric acid.Then analyze the supernatant with chromatography.The product concentration was directly assayed by RP-HPLC with a chromatographic apparatus AichromBond-1 C18 reverse-phase column(4.6mm×150mm,5μm) and at the temperature of 35℃.The mobile phase was acetonitrile-0.01 mol/L phosphate buffer(6:94,v/v) with a flow rate of 1.0 ml/min.The sample introduction was 50μL.The excitation wavelength was set at 310 nm and emission wavelength was 390 nm.Results Good linearity was shown on 6-MTG concentration range of 0~250 μg/L.The methodology recovery was 81.4~106.3% and the lowest detection limit was 0.093μg/L(S/N=3).Conclusion This method is simple and rapid with low detection limit,and suitable for MT pharmacokinetics and clinical drug monitoring assay.更多还原