Simple non-radiochemical HPLC-linked method for screening for purine metabolism disorders using dried blood spot

Background: Pathologies associated with rare inherited disorders affecting purine metabolic pathways range from renal failure to neurological dysfunction and immunodeficiency. The disorders are usually diagnosed by measuring enzyme activities in hemolysates. A non-radiochemical HPLC-linked method is described for simultaneous determination of the activities of hypoxanthine-guanine phosphoribosyltransferase (HPRT: E.2.4.2.8.), adenine phosphoribosyltransferase (APRT: E.2.4.2.7.), adenosine deaminase (ADA: E.3.5.4.4.) and purine nucleoside phosphorylase (PNP: E.2.4.2.1.) in dried blood spots. Method: 7-mm-diameter blood spots stored at 4 °C or room temperature were transferred to an Eppendorf tube and eluted with 500-μl 0.1 mol/l Tris–HCl buffer, pH 7.4. The eluate was added to substrate solutions and incubated at 37 °C. Reaction products were analysed by HPLC. Results and conclusions: The enzyme activities tested in spot eluates were similar to those in erythrocyte lysates from the same subjects. None of the enzymatic activities tested were significantly affected by different storage temperatures. The main advantages of the proposed method are small blood volume required, easy sample collection and transfer, and accurate results. The method is therefore suitable for screening inborn errors of purine metabolism even in newborns.