CHOLESTEROL 157 



about one minute). Draw off the chloroform layer into a flask 

 or beaker and add 5 to 10 gms. of anhydrous sodium sulphate, 

 bring to boiling on a water-bath or hot plate, filter into a 100 c.c. 

 volumetric flask and dilute to the mark with chloroform. (The 

 washing and treatment with anhydrous sodium sulphate and heat 

 are very important as they bring about the removal of substances 

 which if present render the subsequent comparison of colors very 

 difficult if not quite impossible.) Transfer 10 c.c. of this extract 

 to a small glass-stoppered bottle of about 20 c.c. capacity, add 

 4 c.c. of pure acetic anhydride, 0.2 c.c. of concentrated sulphuric 

 acid and shake. (If the acetic anhydride has become brownish 

 or yellowish satisfactory results are impossible, due to the develop- 

 ment of a brownish-green color. When such discoloration has 

 occurred the acetic anhydride should be redistilled before using. 

 The anhydride should distill at about 136 C. However, the distil- 

 late between 134 and 140 C. may be used.) Place in a water- 

 bath or incubator at 35 to 38 C. and allow to stand fifteen 

 minutes in the dark. A blue-green color is developed. At the 

 same time a series of standards are prepared and treated in the 

 same manner. The color of the unknown is compared, in a colorim- 

 eter, with that of the most similar of the standards. The average 

 of several closely agreeing readings should be taken. Such read- 

 ings should be made within a period of twenty to thirty minutes. 

 Four standards are kept, these being prepared by dissolving in 

 100 c.c. portions of chloroform; (1) 2 mg., (2) 4 mg., (3) 6 mg., 

 (4) 8 mg., respectively of pure cholesterol. (It is convenient to 

 make up a stock solution of pure cholesterol containing 100 mg. 

 in 100 c.c. of pure chloroform, diluting this as required to make 

 up the four standards suggested above.) In preparing the stand- 

 ards for comparison 10 c.c. portions of each of the above standard 

 solutions are taken, placed in small glass-stoppered bottles and 

 treated with acetic anhydride and sulphuric acid as was the 

 unknown. The standards then represent, with the above quanti- 

 ties and dilutions, cholesterol concentrations of: 100 mg., 200 mg., 

 300 mg., and 400 mg., respectively, in 100 c.c. of the original 

 blood or serum sample used. The 4 mg. standard solution is usually 

 satisfactory, but for blood of very low or high cholesterol content 

 the other standards should be employed. 

 Calculation. 



Standard reading X Cholesterol Equivalent of standard , 



% ss - . TT . - where x 



Unknown reading 



