HPLC法测定人血红细胞巯基嘌呤甲基转移酶的活性

Determination of thiopurine methyltransferase activity in human red blood cells by HPLC

目的：建立测定人血红细胞中巯基嘌呤甲基转移酶（thiopurine methyltransferase，TPMT）活性的HPLC方法。方法：以6-硫鸟嘌呤（6-TG）为底物，S-腺苷-L-甲硫氨酸为甲基供体，删催化6-TG生成甲基化产物6-甲基硫鸟嘌呤，100℃加热终止反应并沉淀蛋白后，加60％高氯酸再次沉淀蛋白，离心后进样20止检测。色谱柱：Shim-pack CLC-ODS（6mm×150mm，5μm）；流动相：甲醇-水-三乙胺（24：76：0．4），磷酸调pH至4.8；流速：1．0mL/min；检测波长：313nm。结果：该方法在100-1700μg／mL浓度范围内线性良好，r=0．9997；高、中、低浓度样本的日内、日间RSD为2．28％～6．48％；回收率为100．56％～118．71％（n=5）。结论：所建立的方法稳定可靠、精密度好，适合临床大规模推广应用，

Aim：To develop a convenient HPLC for the determination of thiopurine methyltransferase （TPMT） activity in red blood cells. Methods： Based on the TPMT-catalyzed effection,6-thioguanine （6-TG） was converted to 6-methylthioguanine （6-MTG） with S-adenosyl-L-methionine as methyl donor.The incubate was heated to 100 ℃ to stop the reaction and 60% HClO4 was added for the precipitation of protein. 6-MTG was quantitated by RP-HPLC on CLC-ODS colunm （6 mm × 150 mm, 5 μm）. The mobile phase consisted of methanol-water-triethylamine （24：76：0.4,adjusted to pH 4.8 with phosphoric acid） and was run at a flow rate of 1.0 mL/min. The elution was monitored at the wavelength of 313 nm. Results： The linear concentration range was 100- 1 700 ng/mL, with correlation coefficient of 0.999 7. The RSDs of the spiked samples were below 6.48 % for inter-and intra-run validation. The recoveries at low, middle and high concentrations of the spiked samples ranged between 100.56 % and 118.71% （ n = 5 ）. Conclusion： The develeloped method is sensitive, accurate and specific. It is suitable for the determination of the TPMT activity in red blood cells in clinical chemistry.