Department of Haematogenetics, National Institute of Immunohaematology (Indian Council of Medical Research), Parel, Mumbai, India.
Int J Lab Hematol. 2012 Jun;34(3):232-6. doi: 10.1111/j.1751-553X.2011.01381.x. Epub 2011 Nov 14.
Pyrimidine 5' nucleotidase type I (P5'N-1) deficiency is the most frequent abnormality of cell nucleotide metabolism causing hereditary non spherocytic hemolytic anemia (HNSHA). The aim of this study was to develop a simple method of determination of P5'N-1 activity in human erythrocytes using an ELISA reader
Determination of P5'N-1 activity is based on the liberation of inorganic phosphorus (Pi) after incubation with uridine monophosphate/cytidine monophosphate. Inorganic phosphorus (Pi), a product of the enzymatic reaction is directly quantitated from its ultraviolet absorbance. Purine/Pyrimidine nucleotides ratio (OD 260: OD 280) was also measured
P5'N-1 deficient patients showed reduction in P5'N-1 activity (Mean ± SD; 4.06 ± 0.66 using an ELISA reader & 6.25 ± 1.37 using a spectrophotometer) as compared to the normal control group (ELISA reader: 13.24 ± 3.42 & Spectrophotometer: 18.25 ± 3.20). Heterozygotes showed intermediate activity (ELISA reader: 6.06 ± 0.48 & Spectrophotometer: 8.06 ± 1.28), however they would have been missed on screening using the Purine/Pyrimidine nucleotides ratio
Determination of P5'N-1 activity by using an ELISA reader is a new, simple, less time consuming and reliable method. It also avoids the use of radioactive material or HPLC which is a significant advantage.
嘧啶 5'核苷酸酶 1 型（P5'N-1）缺乏症是导致遗传性非球形细胞溶血性贫血（HNSHA）的最常见细胞核苷酸代谢异常。本研究旨在开发一种使用 ELISA 读取器测定人红细胞 P5'N-1 活性的简单方法。
P5'N-1 活性的测定基于与尿苷单磷酸/胞苷单磷酸孵育后无机磷（Pi）的释放。酶促反应的产物无机磷（Pi）可直接通过其紫外吸光度进行定量。嘌呤/嘧啶核苷酸比值（OD260：OD280）也进行了测量。
与正常对照组相比（ELISA 读取器：13.24 ± 3.42；分光光度计：18.25 ± 3.20），P5'N-1 缺乏症患者的 P5'N-1 活性降低（平均±SD；ELISA 读取器：4.06 ± 0.66；分光光度计：6.25 ± 1.37）。杂合子表现出中间活性（ELISA 读取器：6.06 ± 0.48；分光光度计：8.06 ± 1.28），但在使用嘌呤/嘧啶核苷酸比值进行筛查时会被遗漏。
使用 ELISA 读取器测定 P5'N-1 活性是一种新的、简单、耗时更少且可靠的方法。它还避免了使用放射性物质或 HPLC，这是一个显著的优势。
