Determination of Urinary Oxalate with Arylamine Glass-Bound Sorghum Oxalate Oxidase and Horseradish Peroxidase

A.K Bhargava,C.S Pundir,M Thakur

doi:10.3126/ijasbt.v4i3.15768

Abstract

We describe an enzymic colourimetric method for determination of oxalate level in urine using arylamine glass-bound sorghum leaf oxalate oxidase and horseradish peroxidase. The method is based on quantification of H2O2 generated from oxidation of urinary oxalate by immobilized oxalate oxidase, by a colour reaction consisting of 4-aminophnazone, phenol and immobilized peroxidase as chromogen. Minimum detection limit of the method was 0.05 mmol/l. Analytical recovery of added oxalate in urine was 96.8± 3.0% (mean ±S.D.). Within and between day coefficient of variation (CV) for urinary oxalate in urine were < 3.5% and <6.46 % respectively. The urinary oxalate values in apparently healthy and urinary stone formers as measured by the present method were correlated with those by modified Sigma Kit method (r= 0.929). The method has the advantages that it provides ca 200 times reuse of oxalate oxidase and peroxidase and free from interferences by Cl- and NO3- normally found in urine. Int J Appl Sci Biotechnol, Vol 4(3): 346-351

Highlights

Determination of oxalate in urine is essential for the diagnosis and medical management of various forms of hyperoxaluria, leading to urinary stone disease, malabsorption, steatorrhoea, illeal disease and ethylene glycol poisoning
We have reported the immobilization of sorghum oxalate oxidase and horseradish peroxidase onto arylamine glass beads and studied their properties (Pundir et al; 1999a; Pundir et al;1999b )
Immobilization of Oxalate Oxidase Oxalate oxidase purified from sorghum leaves(2.0mg/ml 0.02M sodium phosphate buffer, pH 7.0) was immobilized onto arylamine glass beads through diazotization according to reference (Pundir et al.,1999a).One hundred mg arylamine glass beads were taken in a 10ml conical flask kept on ice bath

Summary

Introduction

Determination of oxalate in urine is essential for the diagnosis and medical management of various forms of hyperoxaluria, leading to urinary stone disease, malabsorption, steatorrhoea, illeal disease and ethylene glycol poisoning. Immobilization of enzyme(s) onto insoluble support allows its reuse and reduce the cost of the method. We have developed an enzymatic colorimetric method for determination of urinary oxalate using alkylamine glass bound sorghum leaf oxalate oxidase and horseradish peroxidase (Thakur and Pundir,1999). Immobilization of an enzyme on arylamine glass through diazotization has no such problem, as it enables the inter-deposition of a spacer arm between two reduced steric interactions and yields higher enzyme to carrier conjugation ratio than by other coupling procedures (Foster, 1980). We have reported the immobilization of sorghum oxalate oxidase and horseradish peroxidase onto arylamine glass beads and studied their properties (Pundir et al; 1999a; Pundir et al;1999b ). The present report describes the method of urinary oxalate determination employing arylamine glass bound sorghum leaf oxalate oxidase and horseradish peroxidase and its evaluation

Material and Methods

Results and Discussion

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