Abstract
Cholesterol esters react more rapidly than free cholesterol with sulfuric acid and acetic anhydride to form the characteristic green color of the Liebermann-Burchard reaction. This difference, first observed for cholesterol palmitate 1 has been found to hold for the acetate and oleate ester as well. This difference in the reactivity of the free and combined cholesterol influences the results obtained by the usual colorimetric methods, and it is certain that in several of the widely used procedures true color equivalence is not attained.
By developing the Liebermann-Burchard reaction at 0–2°C, only the esters will develop color while the free cholesterol remains practically colorless, thus allowing the determination of cholesterol esters in the presence of free cholesterol. Then by completing the reaction at 38°C, total cholesterol can be determined in the same solution. This procedure, which obviates the use of digitonin, is carried out as follows:
Alcohol-ether extracts prepared according to Bloor, 2 containing the equivalent of 1 cc. of blood serum, are evaporated to dryness at a temperature of 60–70° C. The dry residue is extracted with anhydrous chloroform∗ which is filtered through cotton into a glass-stoppered 10 cc. cylinder. Two standard solutions, one containing cholesteryl oleate equivalent to 1.6 mg. cholesterol† in 10 cc. chloroform, the other cholesterol in the same amount and volume, are measured into similar cylinders. Both standard and unknown solutions are cooled in a refrigerator for 10 minutes. Acetic anhydride and sulfuric acid are mixed in the proportion of 1 cc. of acetic anhydride to 0.025 cc. of concentrated sulfuric acid, then cooled to 0–2°C. One cc. of the cold mixture is added to the cooled standard and unknown solutions.
Get full access to this article
View all access options for this article.