The colorimetric estimation of total cholesterol in whole blood, serum, plasma and other biological material

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Abstract
The method is based on the results obtained from a critical study of the various factors influencing the accuracy of the colorimetric method for the estimation of cholesterol by means of the Liebermann-Burchard reaction. The yellow color occurring in ether-alc. extracts prepared from blood or tissues, can be completely removed, without loss of cholesterol, by means of finely divided silica gel. For accurate color development, the cholesterol must be in the free state; the chloroform used must be completely water free; the acetic anhydride-conc. H2SO4 (20:1) mixture (18[degree]) should be prepared not more than 10 mins. before use; and the reaction should take place in the dark at 18[degree] for exactly 60 mins. Saponification of the cholesterol esters is best accompanied by heating at 100[degree] for 90 mins. with a 2:1 mixture of 10% aq. KOH and amyl alcohol. The paper is illustrated by 2 charts showing the rates of color development for free cholesterol at 18, 25, and 35[degree]C under standard conditions and comparative rates of color development for cholesterol and its esters at 18[degree].

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