高效液相色谱法测定小儿急性淋巴细胞白血病红细胞中硫嘌呤甲基转移酶活性的研究

HPLC analysis of TPMT activity in children with acute lymphocytic leukemia

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 mereaptopurine，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；中溶液（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。

Objective To establish an HPLC method to measure thiopurine S-methyhransferase（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. Resets Good tinearity 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. 093 μg/L （ S/N = 3 ）. Conclusion This method is simple and rapid With low detection limit, and suitable for MT pharmacokinetics and clinical drag monitoring assay.