CHOLESTEROL IN BLOOD OR BLOOD SERUM 155 



to room temperature, filled to the mark with alcohol-ether, mixed, 

 and filtered. The filtered liquid if placed in a tightly stoppered 

 bottle in the dark will keep unchanged for a considerable time so 

 that if it is not convenient to complete the determination at once, 

 the sample may be carried to the above stage and left till a more 

 suitable time. 



By running the blood slowly into the large quantity of alcohol- 

 ether, as above, the protein material is precipitated in finely divided 

 form and under these conditions the short heating combined with 

 the great excess of solvent is adequate for complete extraction of 

 serum or plasma. The extraction, while not quite so complete 

 in the case of whole blood, is believed to be better (because of 

 the higher values obtained) than that obtained by any other 

 method in use at the present time. 



The determination of fat (see page 151) may be made upon 

 the same sample of blood plasma or serum. 



Determination. 10 c.c. of the alcohol-ether extract are meas- 

 ured into a small flat-bottomed beaker and evaporated just to 

 dryness on a water bath or electric stove. Any heating after 

 dryness is reached produces a brownish color which passes into 

 the choloroform and renders the subsequent determination diffi- 

 cult or impossible. The cholesterol is extracted l from the dry 

 residue by boiling out three or four times with successive small 

 portions of chloroform and decanting into a 10 c.c. glass-stoppered, 

 graduated cylinder after cooling (5 c.c. or less) are then made up 

 to 5 c.c. The solution should be colorless but not necessarily 

 clear, since the slight turbidity clears up on adding the reagents. 



5 c.c. of a standard cholesterol solution in chloroform 2 (con- 

 taining 0.5 mg. of cholesterol) are measured into a similar 10 c.c. 

 cylinder. 



To each of the solutions are added 2 c.c. of acetic anhydride 

 and 0.1 c.c. of concentrated sulfuric acid, the solutions mixed by 

 inverting several times, then set away in the dark for fifteen 



1 In order to get an adequate extraction with the small amounts of chloro- 

 form used, an excess (3 or 4 c.c.) should be added each time and the mixture 

 allowed to boil down to half its volume or less, before decanting. 



2 It is convenient to make the cholesterol standard in two strengths: 

 (a) the stock solution containing 0.2 gm. of cholesterol (Kahlbaum) in 200 c.c. 

 chloroform; and (b) the standard solution for use, made by diluting 10 c.c. 

 of the above to 100 c.c. with chloroform. 5 c.c. of this latter solution will 

 contain 0.5 mg. 



