摘要
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.
出处
《中国药师》
CAS
2013年第2期196-199,共4页
China Pharmacist