HPLC法测定人红细胞中次黄嘌呤鸟嘌呤磷酸核糖转移酶活性

Activity Determination of Hypoxanthine Guanine Phosphoribosyl Transferase in Human Red Cells by HPLC

目的:建立一种检测次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)活性的高效液相色谱法,并检测患者红细胞中HGPRT活性。方法:在5-磷酸核糖-1-焦磷酸(PRPP)条件下,HGPRT催化次黄嘌呤生成次黄嘌呤核苷酸(IMP),高氯酸沉淀蛋白后进行高效液相色谱法分析。采用Hypersil ODS C_(18)(150 mm×4.6 mm,5μm)柱;流动相为磷酸氢盐缓冲液-乙腈(99.6:0.4),流速1 ml·min^(-1),在262 nm处检测IMP。应用新建立的高效液相色谱法测定68例服用过硫唑嘌呤的肾移植患者红细胞中HGPRT活性。结果:在该条件下,测得的线性范围是20～600μmol·L^(-1),检测限是20μmol·L^(-1),其日间和日内精密度均小于5%,方法回收率和提取回收率分别在100.81%～101.91%和99.05%～101.40%范围内。65例患者红细胞HGPRT活性范围为44.59～123.86 U,平均(87.83±21.55)U。结论:该法操作简便、灵敏度高、重复性好,适用于临床常规检测。

Objective ： To develop an assay method for the activity determination of hypoxanthine guanine phosphoribosyl transfer- ase（HGPRT） in human red cells by HPLC. Method： In the presence of 5-phosphorylribosel pyrophosphate （PRPP）, hypoxanthine was changed into hypoxanthine nucleotide （IMP） under the catalysis of HGPRT. The sample was deposited by perchloric acid and then analyzed by HPLC. A Hypersil ODS Cl8 （150 mm × 4.6 mm,5 μm） column was used. The mobile phase was potassium phosphate buffer-acetonitrile （99.6： 0.4） and the flow rate was 1 ml min-l. The detection wavelength of IMP was 262 nm. The HGPRT activity in the red cells of 68 kidney transplantation patients treated by azathioprine was detected. Result： The standard curve was linear within the range of 20-600μmol L-l , and LLOQ was 20μmol L-1. The intra- and inter-day preeisions were both less than 5% , and the method recovery and the extraction recovery was 100.81% -101.91% and 99.05 % -101.40% , respectively. The HGPRT activity range of 65 patients was 44.59-123.86 U with the average value of （ 87.83 ± 21.55 ） U. Conclusion ： The method is simple, sensitive and repeatable, which is applicable in the HGPRT activity detection in the clinical practice.