Determination of total cholesterol in serum by cholesterol esterase and cholesterol oxidase immobilized and co-immobilized on to arylamine glass

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 hydrolysed by cholesterol esterase to free fatty acid and cholesterol, which is oxidized by cholesterol oxidase to cholestenone and H2O2. H2O2 is determined enzymically with horseradish peroxidase by additive coupling of 4-aminophenazone with phenol, and the resulting quinoneimine dye is measured at 520 nm, (e = 4.0×10^-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.