Abstract
Since the Liebermann-Burchard reaction was adopted by Grigaut (1) for the quantitative determination of serum total cholesterol, many modified procedures of the original method have been made. The various procedures have differed in extraction method and in the reagents used in the color development. The separation of cholesterol from its esters with digitonin has had limited application because of the time and labor required in the involved and delicate technique. The errors which are encountered in the Liebermann-Burchard color reaction of different cholesterol and cholesterol ester methods have been described by Sperry (2). Because of the different methods of extraction in use, the wide range of accepted normal values for total cholesterol, the difficulty of performing cholesterol ester determinations, and the instability of color produced in the Liebermann-Burchard reaction, an investigation of the possible improvement in the method of determination of cholesterol and its ester was undertaken. The method reported has been satisfactorily used over the past 2 years for the determination of several hundred serum cholesterols and cholesterol esters.
Highlights
Maximum color density of chloroform extracts of total cholesterol and cholesterol ester of serum was reached in the same time intervals at 030” as those shown in Fig. 2 for cholesterol
Single and average duplicate determinations for both sexes were within the range of 159 to 261 mg. of total cholesterol, and 116 to 203 mg. of cholesterol ester per 100 ml. of serum
Cholesterol and cholesterol ester agreed to within 5.0 per cent or less. These values are in close agreement with those of Sobel and Mayer (i’), who found normal serum total cholesterol to range between 160 and 267 and cholesterol ester between 118 and 202 mg. per 100 ml. of serum
Summary
Mix cautiously 4 parts of cold acetic anhydride and 1 part of cold sulfuric acid (O-5”) in a stoppered. Published with the permission of the Chief Medical Director, Department of Medicine and Surgery, Veterans Administration, who assumes no responsibility for the opinions expressed or conclusions drawn by the authors Of ash-free cholesterol (m.p. 149”) in chloroform and dilute to 100 ml. With redistilled chloroform in the same manner as the stock standard and store in the refrigerator. 1, 2, 3, 4, 5 ml., of working cholesterol standard to photometer cuvett’es and dilute each to 5 ml. Of ice-cold fresh acetic anhydride-sulfuric acid reagent, mix, and read the color as directed in “Procedure.” Add 2 ml. of ice-cold fresh acetic anhydride-sulfuric acid reagent, mix, and read the color as directed in “Procedure.”
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