Amperometric Determination of Total Cholesterol in Serum, with Use of Immobilized Cholesterol Ester Hydrolase and Cholesterol Oxidase

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Abstract
We describe an electrochemical method for simple, rapid, and economical assay of total serum cholesterol with use of immobilized cholesterol esterase (EC 3.1.1.13) and cholesterol oxidase (EC 1.1.3.6). A rotating porous cell was specially designed to hold the immobilized enzymes firmly and to allow the reaction mixture to pass through the en¬zyme layer easily, thus catalyzing the enzymatic trans¬formation quickly. Hydrogen peroxide resulting from the catalytic reactions was measured amperometrically at +0.60 V vs. a standard calomel electrode. The calibration curve for total serum cholesterol was linear from 0 to 5.00 g/liter. The method is specific, precise, and inexpensive. Our results correlate well with those obtained by the method of Abell et al. [Stand. Methods Clin. Chem. 2, 26 (1958)] , the correlation coefficient being 0.992. Ascorbic acid or bilirubin in concentrations up to 100 mg/liter do not interfere. The immobilized enzymes are stable, and the same immobilized-enzyme stirrer can be used for at least 200 accurate, reproducible assays.

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