Abstract: We have reported a simple method of determination of plasma oxalate using a Cl- and NO3- 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, H2O2 generated from plasma oxalate by immobilized oxalate oxidase is measured colorimetrically at 520 fim 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 <11.08, 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 NO3- but also provides the reuse of glass beads and thus reduces the cost of analysis for routine.

Abstract: Porcine liver uricase immobilized on alkylamine glass (pore diameter 55 nm) through glutaraldehyde coupling has been found useful in the determination of uric acid in urine. The minimum detection limit was 12.0 µg/0.1 mL sample and the recovery of added uric acid was 91%. The coefficient of variation (CV) were <6.0% and <2.0% for within and between batch assay respectively. A good correlation (r=0.93) was found between urate values obtained by a standard commercial method and the present method.

Abstract: Commercial cholesterol esterase from bovine pancreas and cholesterol oxidase from Brevibacterium recombinant type have been immobilized individually and co-immobilized on to arylamine glass beads (pore diameter, 55 nm) through diazotization. A method for discrete analysis of total cholesterol in serum was developed employing individually immobilized cholesterol esterase (0.36 mg/50 mg of glass beads) and cholesterol oxidase (0.41 mg/50 mg of glass beads) or co-immobilized cholesterol esterase and cholesterol oxidase (0.56 mg/100 mg of glass beads). Peroxidase from horseradish immobilized on to arylamine glass (0.9 mg/50 mg of glass beads) was common in both the cases. 4-Aminophenazone (0.25 mg/1.5 mL of reaction mixture) and phenol (0.5 mg/1.5 mL of reaction mixture) were used to form dye. In the method, cholesterol ester is hydrolyzed by cholesterol esterase to free fatty acid and cholesterol, which is oxidized by cholesterol oxidase to cholestenone and H2O2. H2O2 is determined enzymatically with horseradish peroxidase by additive coupling of 4-aminophenazone with phenol, and the resulting quinioneimine dye is measured at 520 nm, (epsilon = 4.0 x 10^-4). The lower detection limit of the method was 42.8 mg/l for individually immobilized enzymes and 21.4 mg/l for co-immobilized enzymes. Within-day and between-day coefficients of variation were < 1.5% and < 4.0% respectively for individually immobilized enzymes and < 1.0% and < 2.5% respectively for co-immobilized enzymes. A good correlation (r = 0.99) was found between total serum cholesterol obtained by the present method and a commercial enzo-kit method employing free enzymes. The individually immobilized/co-immobilized enzymes did not show much loss of activity after their 300 uses, when stored at 4°C in distilled water. The co-immobilized enzymes showed better efficiency in terms of sensitivity, linearity and precision compared with individually immobilized enzymes.