Application of the Schoenheimer-sperry Method to the Determination of Cho- Lesterol and Cholesterol Esters in Tissues*

In the course of studies planned on cholesterol metabolism, it was felt necessary to develop a microtechnique for cholesterol that would be suitable for analysis in tissues. It has been pointed out that a direct Liebermann-Burchard reaction method is dependent upon too many variables, some of which cannot be regulated, to be accurate for calorimetric work. There is also the possibility of other chromogenic substances interfering with the color reading. Because of these factors, the basic method of Schoenheimer and Sperry (1) was chosen. It provides for a finer separation of cholesterol from other chromogenic substances by means of a digitonide precipitation and an indirect Liebermann-Burchard reaction for color development, the variables of which can be regulated. Colorimetry was carried out with the use of a calorimeter equipped with microcells and a light filter. The filter is necessary because digitonin by this method produces a slight color which is avoided by reading between 610 and 630 rnp, the maximum absorption for digitonin. The method of extraction is that of a simple reflux, completeness of which is absolutely essential for accurate results.