Enzymatic rate method for measuring cholesterol in serum

An enzymatic rate assay is described for measuring cholesterol in serum. Cholesterol is analyzed by mixing 5 mul of sample with a reagent consisting of cholesterol esterase, cholesterol oxidase, catalase, acetylacetone, methanol, and hydroxypolyethoxydodecane in a ammonium phosphate buffer at pH 7.0. The rate of increase in absorbance of the dihydrolutidine product is measured at 37 degrees C and 405 nm. The change in absorbance between 4 and 10 min is used to calculate the cholesterol concentrations by using simultaneously determined free cholesterol standards. The change is linearly related to cholesterol concentration up to 4 g/liter. Samples containing bilirubin up to 200 mg/liter, uric acid up to 200 mg/liter, and hemoglobin up to 1 g/liter, or certain drugs (clofibrate, phenobarbital, nicotinic acid, salicylate, Ketochol, and Ovral) gave no interference. Ascorbic acid added to serum caused a positive interference. Lipemic samples gave values that were slightly lower than did the method of Abell et al., used for comparison. Our kinetic assay, compared with the method of Abell et al., the enzymatic assay used with Abbott's Bichromatic Analyzer, and the Technicon SMA 12/60 enzymatic procedure gave correlation coefficients of 0.992, 0.985, and 0.986, respectively.