Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase

Abstract

We have reported a simple method of determination of plasma oxalate using a Cl − and NO 3 − insensitive oxalate oxidase purified from grain sorghum leaf and commercially available peroxidase from horseradish [Pundir et al., Ind. J. Biochem. Biophys., 35 (1998) 120–122]. The present report describes the immobilization of both the enzymes onto alkylamine glass, their kinetic properties and application for discrete analysis of plasma oxalate. In the analytic method, H 2O 2 generated from plasma oxalate by immobilized oxalate oxidase is measured colorimetrically at 520 nm by oxidative coupling with 4-aminophenazone, and phenol catalyzed by immobilized peroxidase. The minimum detection limit of the method is 2.5 μmol/l. Analytic recovery of added oxalate in plasma was 89.5±4.1% (mean±S.D.). The within and between day CV for plasma oxalate measurement were <9.37 and <11.0%, respectively. The normal range of plasma oxalate as measured by the present method was 3.6 to 5.7 μmol/l. The method is not only free from interference by plasma Cl − and NO 3 − but also provides the reuse of glass beads and thus reduces the cost of analysis for routine.