HPLC法间接测定人和猪红细胞硫嘌呤甲基转移酶活性及其差异分析

HPLC Determination and Diversity Analysis of Thiopurine Methyltransferase Activity in Human and Pig

目的建立测定红细胞内6-甲基-巯基嘌呤（6-MMP）浓度的HPLC法，换算出红细胞和肝组织中硫嘌呤甲基转移酶（TPMT）活性，探讨猪与人肝脏TPMT酶活性的差异。方法使用岛津2010C系列高效液相色谱仪，Symmetry C18（150mm×3．9mm，5μm）色谱柱。样品用乙酸乙酯萃取后进样，0．1％的乙酸：甲醇为流动相梯度洗脱，总流速1．0mL／min，280nm波长检测，内标法定量。结果标准曲线在6．25-100nmol／mL范围内有良好线性，方法回收率为98．08%～100．05％，萃取回收率为88．1％～92．4％，批内RSD为1．0％～1．5％，批间RSD为1．0%～4．4％。测定显示人TPMT活性为（17．45±3．62）U，家猪TPMT活性为（7．65±1．35）U，版纳猪TPMT活性为（8．73±1．55）U。结论本方法适用于临床和科研中测定人和猪红细胞和肝组织TPMT活性。实验结果提示猪对嘌呤类药物的代谢能力低于人类。

Objective To develop an HPLC method for the determination of 6-methylmercaptopurine concentration and then numerate the thiopurine methyltransferase （TPMT） activity in erythrocyte via formula and analysis of the TPMT activity difference between human and pig. Methods The chromatographic apparatus SHIMADZU GC-2010C was used. The stationary phase was a Symmetry Cis reverse-phase column（150 mm × 3.9 mm, 5 μm）. The mobile phase was 0. 1% acetic acid-methanol and the flow rate 1.0 mL/min. The samples were extracted by ethyl acetate and injected automatically. They were measured at UV 280 nm, 2-amino-6-methyl- mercaptopurine was used as the internal standard. Results The retention times for 6-MP, AMMP and 6-MMP were 4. 4 min, 7.7 min and 9.3 min respectively. The calibration curves were linear over the range of 6. 25-100 nmol/mL, the methodology recovery was 98. 08%-100. 05%. The extraction recovery of 6-MMP was 88. 1%- 92.4%. The within-group RSD 1.0%-1.5% and inter-group RSD 1.0%-4. 4%. The levels of TPMT activity of human, pig and Banna Minipig Inbred Line （BMI） were （17.45±3.62） U, （7.65±1.35） U and （8. 73±1.55） U respectively. Conclusion The method is suitable for determination of TPMT activity in erythrocyte of different patients and species in that of clinical medicine and scientific research. TPMT activity of pig and BMI is obviously lower than human＇s.