Photometric determination of total cholesterol in plasma of serum by a modified Liebermann-Burchard reaction.

The wide-spread use of photoelectric calorimeters in the field of quantitative clinical chemistry has brought with it the realization that many of the visual calorimetric methods in use are grossly inaccurate and that their use may be justified only on the basis of an arbitrary range of normal values for the given procedure. This is particularly true in the case of total cholesterol values which have a reported normal range from about 100 to 400 mg. per cent. The principal sources of the inaccuracies for the cholesterol methods employing the Liebermann-Burchard reaction are well known and have been recently discussed in some detail in a paper by Sperry and Brand (1). They mill be mentioned here only because in the procedure presented by the authors the experimental studies of the LiebermannBurchard reaction were run under conditions somewhat different from those of most other investigators~ (24). Instead of the usual organic solvents, e.g. chloroform, alcohol-ether, alcohol-acetone, etc., used for extracting cholesterol and cholesterol esters from biological fluids, the authors have followed the lead of Drekter (5) who used acetic anhydride as the medium for the simultaneous precipitation of the proteins and the extraction of the total cholesterol present in these fluids. Because of the extreme rapidity of development of the maximal color density, pure acetic anhydride was unsuited for the photometric determination of total cholesterol. It was found experimentally that a mixture of acetic anhydride and 1,4-dioxane (3:2 by volume) not only would extract cholesterol and cholesterol esters from biological fluids with the simultaneous precipitation of proteins, but in addition would reduce the rate of development of maximal color density to a point at which the Liebermann-Burchard reaction might be used to obtain accurate photometric readings. The method presented here is unlike any other found in the literature in that no attempt is made to remove the water from the biological fluid;