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Urinary & serum oxalate determination by oxalate oxidase immobilized on to affixed arylamine glass beads
Reprint requests: Prof. C.S. Pundir, Department of Biochemistry & Genetics, Dean Faculty of Life Sciences, M.D. University, Rohtak 124 001, India. e-mail: pundircs@rediffmail.com
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Received: ,
Abstract
Background & objectives:
Although the measurement of oxalate in urine and serum by Amaranthus leaf oxalate oxidase immobilized on free arylamine glass beads is highly sensitive and specific, the handling of glass beads is tedious and cumbersome. The present study was undertaken to overcome this problem.
Methods:
Partially purified Amaranthus spinosus leaf oxalate oxidase was immobilized through diazotization onto arylamine glass beads affixed on the surface of a plastic strip by a non reactive fixative and employed for oxalate determination in urine and serum samples collected from healthy individuals and urinary stone formers.
Results:
The immobilized enzyme retained 56 per cent of its initial activity with a conjugation yield of 40 mg/g support. The strip bound enzyme showed maximum activity at pH 3.5 when incubated at 40°C for 15 min. The minimum detection limit of the method was 0.01μM/l in the urine and 2.5 mM/l in the serum. The analytical recovery of added oxalate was 97.7+1.2 per cent in urine and 92.0+2.4 per cent in serum. Within and between assay coefficient of variation (CV) were 4.6 and 5.2 per cent in urine and 7.4 and 5.8 per cent in serum respectively. A good correlation for oxalate in urine (r1= 0.99) and in serum (r2= 0.92) was obtained between Sigma kit method and the present method. The strip could be reused 150 times over a period of 2 months, when stored at 4°C in reaction buffer.
Interpretation & conclusions:
Immobilization of Amaranthus leaf oxalate oxidase on to affixed glass beads provided enormous ease in its reuse for determination of oxalate in urinary and serum samples.
