Identification of purine nucleoside phosphorylase deficiency in dried blood spots by a non-radiochemical assay using reversed-phase high-performance liquid chromatography.
Purine nucleoside phosphorylase (PNP) deficiency results in severe T cell dysfunction and hypouricemia. An assay to measure PNP activity in dried blood spots was developed using reversed-phase HPLC. The assay was linear with reaction times between 5 and 12.5 minutes, and protein concentrations ranging from 0.4 to 1.8 mg/ml. The intra-assay CV and the inter-assay CV for the complete assay was <3.6%. The PNP activity in a control blood spot, stored at 4 degrees C, remained stable for at least one year. In a patient suffering from a PNP deficiency, the residual PNP activity was only 0.3% compared to that observed in controls (1431 +/- 238 nmol/mg/h, n = 114). The PNP activity (483 +/- 35 nmol/mg/h, n = 3) in heterozygotes for the c.614A > C mutation (p.E205A) in the PNP gene was 34% compared to controls. Thus, the analysis of the PNP activity in blood spots can readily detect patients with a PNP deficiency.
van Kuilenburg
A B P
AB
Academic Medical Center, Emma Children's Hospital, Laboratory Genetic Metabolic Diseases, Amsterdam, the Netherlands. a.b.vankuilenburg@amc.uva.nl
Zoetekouw
L
L
Meijer
J
J
Kuijpers
T W
TW
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D011683:Purine-Nucleoside Phosphorylase / Q000097:blood* / Q000172:deficiency*
D011683:Purine-Nucleoside Phosphorylase