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


Roshan B. Colah, Department of Haematogenetics, National Institute of Immunohaematology, Indian Council of Medical Research, 13th Floor, New Multistoried Building, K.E.M Hospital Campus, Parel, Mumbai 400012, India. Tel.: +9122 24138518; Fax: +9122 24138521; E-mail: colahrb@gmail.com


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.