ANALYSIS OF TISSUES 603 



I. CHOLESTEROL. 



The most accurate method of estimating cholesterol in tissues is that 

 adopted by Gardner and his co-workers, the method in the case of blood hav- 

 ing been described by Gardner and Fraser. 1 This procedure is for the estima- 

 tion of cholesterol and cholesterol esters. The blood is weighed and mixed 

 with plaster of Paris ; the dry mass is finely powdered and extracted for 

 several days (or weeks) in a Soxhlet extractor with ether. 



Gravimetrically. 



(a) Cholesterol. 



The ethereal extract is evaporated to dryness, the residue is weighed and 

 taken up with 95 per cent, alcohol. Excess of digitonin dissolved in 95 per 

 cent, alcohol is added and the mixture, after standing some time, is evapor- 

 ated to dryness in a vacuum desiccator. The precipitate of the insoluble 

 digitonin cholesteride 



is washed by decantation with ether until the washings give no residue on 

 evaporation. The washings are filtered through a Gooch crucible, or better a 

 tared filter paper, which has been similarly treated. The excess of digitonin 

 is dissolved with distilled water, the washing being continued until the water 

 leaves no residue on evaporation. The precipitate is dried at 1 1 o, cooled 

 and weighed. 



(b) Cholesterol Ester. 



The ethereal washings are saponified with excess of sodium ethoxide, the 

 ethereal solution is filtered from soap and the soap washed with ether. The 

 ethereal solution and washings are freed from soap, alkali and alcohol by 

 washing with water, dried with calcium chloride and evaporated to dryness. 

 The dry residue is dissolved in 95 per cent, alcohol and treated as under (a). 



(c) Total Cholesterol. Cholesterol and Cholesterol Ester. 



It is preferable to estimate the total cholesterol in the tissues and to deduct 

 the amount of cholesterol so as to obtain the amount of cholesterol as ester. 

 A second portion of material is required. It is dried and extracted with ether 

 as described (a), the ethereal solution is saponified with sodium ethoxide and 

 filtered from soap, which is washed with ether, the ethereal solution and 

 washings are freed from alkali, etc., with water, dried, evaporated and the residue 

 dissolved in alcohol and precipitated with digitonin as under (a). 



In these experiments the whole of the blood is used and it is divided into 

 two portions as nearly equal in weight as possible. 



Total cholesterol is about ;o8 per cent. 

 Cholesterol ester is about '04 ,, 



Colorimetrically. 



Grigaut has described a method for estimating cholesterol in small 

 quantities of blood or serum and tissues ; the cholesterol is extracted with 

 alcohol and ether, Liebermann's reaction is performed and the colour com- 

 pared with the colour produced in the same way with a known amount of 

 cholesterol. 



It is carried out as follows : 



2 c.c. of serum are placed in a specially made separating funnel of a 

 total volume of about 50 c.c. and with a narrow portion to contain about 15 c.c., 

 points showing the level of 15 c.c. and 30 c.c. being marked ; 60 per cent. 

 alcohol containing -5 per cent, of sodium hydroxide is added as far as the 



1 Proc. Royal Soc., 1910, B, 82, 560. 



