Photometric Determination of Total and Free Cholesterol and the Cholesterol Ester Ratio of Serum by a Modified Liebermann-Burchard Reaction

Abstract

A previously published method (Jour. Biol. Chem. 164 657. 1946) for total cholesterol was extended to the detn. of free cholesterol and the cholesterol ester ratio of serum. Use of acetic anhydride-dioxane extracts of serum permits the employment of higher temps. and greater concn. of sulfuric acid than is usually employed in the Liebermann-Burchard reaction and eliminates differences in the reaction rates between cholesterol and various cholesterol esters. This was shown to be true for known mixtures of free cholesterol and the acetate, laurate, palmitate, stearate, oleate and digitonide. The method, as applied to human sera, permits the accurate determination of total cholesterol (without saponification) and of the free cholesterol as the digitonide. The cholesterol ester ratio is calculated by difference and for 25 normal sera was found to vary from 73.8 to 78.8% (avg. 76.9%) with a standard deviation of + 1.5%.