title: A new simple approach for the determination of pyrimidine 5'-nucleotidase activity in human erythrocytes using an ELISA reader.
abstract: BACKGROUND
  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
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Haematogenetics, National Institute of Immunohaematology (Indian Council of Medical Research), Parel, Mumbai, India.
authors.forename: P
authors.initials: P
authors.lastname: Warang
authors.forename: P
authors.initials: P
authors.lastname: Kedar
authors.forename: K
authors.initials: K
authors.lastname: Ghosh
authors.forename: R
authors.initials: R
authors.lastname: Colah
mesh_terms: D015720:5'-Nucleotidase / Q000097:blood* / Q000172:deficiency; D000745:Anemia, Hemolytic, Congenital / Q000201:enzymology / Q000473:pathology; D017487:Ankyrins / Q000172:deficiency; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology*; D006801:Humans; D041781:Jaundice, Obstructive / Q000201:enzymology / Q000473:pathology; D013103:Spherocytosis, Hereditary
chemical_list: D017487:Ankyrins; D015720:5'-Nucleotidase