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

Abstract

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.