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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。
Objective To investigate the thiopurine methyltransferase (TPMT) activity in erythrocytes of children with acute lymphoblastic leukemia by high performance liquid chromatography (HPLC). Methods Reverse phase HPLC was used to directly determine the product concentration of enzymatic reaction to calculate the activity of TPMT in erythrocytes. Using S-adenosyl-L-methionine (SAM) as a methyl donor and 6-thioguanine (6-TG) as enzyme reaction substrates, TPMT The 6-TG was catalyzed to generate 2-amino-6-methyl mercaptopurine (6-MTG). The perchloric acid solution was used to terminate the reaction and precipitate the protein. The supernatant was separated and chromatographed. The column was AichromBond-1 C18 column (5μm, 4.6mm × 150mm). The mobile phase consisted of acetonitrile: 0.01mol / L potassium phosphate buffer solution (adjusted to pH = 2.74 with hydrochloric acid) V) at a flow rate of 1.0 mL / min. The column temperature was 35 ° C. The injection volume was 50 μL. Fluorescence detector detection, excitation wavelength of 310nm, emission wavelength of 390nm. Results The 6-MTG showed a good linearity in the range of 0-250 μg / L with a correlation coefficient of 0.9999 and a minimum detectable limit of 0.093 μg / L (S / N = 3). The recoveries ranged from 81.4% to 106.3%. Conclusion The method is simple, rapid, low detection limit, good reproducibility, easy to popularize, and can meet the needs of thiopurines pharmacokinetics and routine monitoring of clinical drug use.