高效液相色谱荧光检测法测定鸟氨酸脱羧酶(ODC)活性

HPLC determination of ornithine decarboxylase (ODC) activity with fluorescence detection

目的:利用改进后的HPLC分析方法,评价鸟氨酸脱羧酶(ODC)的活性。比较前列腺肿瘤患者和正常人血液中ODC的活性。方法:以TSKgelODS-80TM凝胶色谱柱(150 mm×4.6 mm,5μm)为分析柱,甲醇-水(8∶2)保持3 min,从4 min到14min,甲醇比率由80%梯度增至100%梯度洗脱,流速为0.8 mL.min-1,激发波长为340 nm,发射波长为515 nm,柱温为50℃,进样量10μL。按内标法定量。结果:前列腺肿瘤患者血液和组织中ODC活性约为正常人血液和组织中ODC活性的2～3倍。在实验条件下,ODC酶促反应符合一般酶促反应动力学规律,Km=87.0481μmol.L-1,Vmax=3.4532 pmol.min-1.L-1。结论:方法准确可靠,可用于人血中鸟氨酸脱羧酶活性的测定。

Obiective： A fluorescent assay for the investigation of Ornithine decarboxylase （ODC） activity in plasma by HPLC has been developed. Method： This methods used TSKgel ODS-80^TM C18（150 mm 4.6 mm, 5 μm） with methanol- distilled water 8：2 balance columniation 3 min later, 4-14 min methanol grads from 80 % to 100 % as the mobile phase, 0.8 mL·min^-1 as flow rate, 340 nm as excitation, 515 nm as emission, 50 ℃ as temperature, 10 μL as the injection volume. Content uniformity was expressed by internal standard method. Results： The results showed that the ODC activity in prostate cancer patients was 2-3 times higher than those of normal controls. At the experiment conditions, it is found that ODC catalyzes to putrescine corresponding to regularity of general anzyme catalyzes reaction, the Km value and Vmax value were 87.0481 μmol·L^-1 and 3.4532 pmol·min^-1·L^-1. Conclusion： The present HPLC method was more sensitive, accurate and rapid for assessment of ODC activity.