Adaptation of the Bloor Oxidation Procedure to the Determination of Small Quantities of Cholesterol as Digitonid.

Abstract

Micro methods for the estimation of cholesterol have hitherto been based almost entirely upon the color reaction with acetic anhydrid and concentrated sulphuric acid. The presence of interfering color producing substances in blood and tissue extracts frequently makes it highly problematical whether or not this reaction gives a true picture of the cholesterol concentration. The gravimetric estimation as the digitonid, proposed by Windaus 1 is impracticable where the amounts to be weighed are as small as those available in the average blood analysis.

We have, therefore, attempted to adapt the digitonin precipitation to micro determination by oxidation of the digitonid according to the method used by Bloor 2 for fatty acids and phospholipins; i. e., with silver-chromate sulfuric acid (Nicloux reagent) and normal K₂Cr₂O₇. Since this oxidation is not specific, the largest problem involved has been the separation of the digitonid from the excess digitonin used in precipitation, and from contaminating substances of a fatty nature. Digitonin is a saponin. Consequently, the surface tension of its water solutions is such as to make separation of the digitonid by centrifugalization impossible. Filtration of the digitonid from water solutions of digitonin has proved quantitative. However, the physical nature of the digitonid precipitate is such as to make quantitative removal of very small amounts from the filter and sides of the flask impracticable. We have, therefore, devised, with the assistance of Mr. D. J. Kooyman, a small Gooch filter tube made from a piece of Pyrex glass tubing 45 mm. long and 18 mm. in diameter, constricted somewhat at one end and fitted with a perforated porcelain disk (cut from the bottom of an ordinary Gooch crucible). This, after the filtration and washing is completed, may be placed bodily inside the flask used for the oxidation in such a way that the oxidizing solution may be made to come in contact with every part of the tube and the asbestos mat. The asbestos used for the filter must be prepared by long continued (72 to 120 hrs.) heating at 120-130° C. with bichromate-sulphuric acid which is frequently renewed. The asbestos is washed free of bichromate and suspended in distilled water in the usual way. This filter differs from that used by A. von Szent Györgyi 3 in that it makes possible complete oxidation of the digitonid by allowing control of time and temperature.