One-minute electrochemical enzymic assay for cholesterol in biological materials

In this rapid and specific micro-scale electrochemical enzymic assay for cholesterol and cholesterol esters, 10 microL of standard or sample is injected directly into a heated (50 degrees C) thermostated, oxystated cuvet containing pH 7.25 buffer, cholesterol oxidase (EC 1.1.3.6), and cholesterol esterase (EC 3.1.1.13). The cholesterol esters are hydrolyzed by the esterase, and the cholesterol is simultaneously oxidized by the oxidase. The hydrogen peroxide produced from oxidation of the unesterified cholesterol is measured by a polarographic anode covered with an acetate/polycarbonate membrane. The membrane allows hydrogen peroxide to diffuse to the platinum anode, where it is oxidized, but prevents the diffusion of ascorbic acid, uric acid, and bilirubin to the electroactive surface. Turbidity does not interfere. The correlation (r) between results by our method and the Abell-Kendall method for 105 samples of serum was 0.9994 and for 105 samples of plasma was 0.9997. Our method is convenient for the analysis of high-density lipoprotein cholesterol in plasma and serum supernates and in many kinds of tissue homogenates. Its limitations are also described.