A new simple approach for the determination of pyrimidine 5'-nucleotidase activity in human erythrocytes using an ELISA reader.

ABSTRACT

INTRODUCTION: 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 METHODS: 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 RESULTS: 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 CONCLUSION: 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.