A Revision of the Schoenheimer-Sperry Method for Cholesterol Determination

The original Schoenheimer-Sperry method for cholesterol determination (1) has been modified at several points.It is the purpose of this report to describe the revised procedure with particular emphasis on a recent investigation of the technique used for the precipitation and washing of cholesterol digitonide. EXPERIMENTALExcept for changes in the precipitation and washing technique, the modified Schoenheimer-Sperry method was used in the form described below for all determinations of total and free cholesterol.The blood serums studied were obtained from healthy human subjects (2); 1 or 2 ml.were extracted in a total volume of 25 or 50 ml., respectively.Aliquot portions of the extracts were precipitated by four different procedures.In Procedure A, a 0.4 per cent solution of digitonin in water was used with no other changes in the method as described below.This is the procedure which has been employed in our laboratory for some time.The solution was about 2 months old at the time the investigation was started, and about 9 months old at the end.In Procedure B, a dilute alcoholic digitonin solution, prepared as described by Sobel and Mayer (3), was used.This is the modified method, adopted as a result of this study, and described below.Procedure C was the same as Procedure B, except that precipitation was continued for only 3 hours at a temperature of 37".In Procedure D, free cholesterol was precipitated with the dilute alcoholic digitonin solution for 3 hours at 37", and stirring rods and the acetoneether washing were omitted.This is the procedure recommended by Sobel and Mayer (3); it was applied exactly as they described it except that 2 ml. of extract and 1 ml. of digitonin solution were employed instead of the larger volumes used by them.All determinations were carried out in duplicate. ResultsComparison of Aqueous and Dilute Alcoholic Dlgitonin Solutions-Procedures A and B were applied to twenty-five samples of serum.In one 97