BLOOD ANALYSIS 285 



and run in from a burette a standard solution of potassium sulphocyanate (approxi- 

 mately o.i per cent) 1 until a very faint pinkish -brown color is obtained throughout 

 the solution. The end point which consists in the faintest trace of color, can be 

 detected only when the titration is performed on a pure white surface. A con- 

 trol beaker with i drop excess of sulphocyanate should be on hand for compari- 

 son. A whole cubic centimeter of sulphocyanate may be run in after the end 

 point is reached without very greatly darkening the shade. 



Calculation. By this procedure acetone preformed and from diacetic acid 

 are determined together. The amount of acetone and diacetic acid combined, 

 in terms of acetone, may then be calculated by multiplying the number of cubic 

 centimeters of the KSCN solution used by the equivalent of i c.c. of this solution 

 in acetone as determined by standardization. 1 To obtain the amount of acetone 

 and diacetic acid in 100 c.c. of blood the result must of course be multiplied by 10. 



(b) Determination of 0-Hydroxybutyric Acid. The residue in the Kjeldahl 

 flask from the above determination is used in the determination of /3-hydroxy- 

 butyric acid. While still hot, precipitate it with about 8 c.c. of 10 per cent sodium 

 carbonate, boil a moment, filter on a Biichner funnel and wash with hot water. 

 To the clear filtrate add 15 c.c. of basic lead acetate (U.S.P.) and 10 c.c. of strong 

 ammonia and make to definite volume (150 c.c.) with water. Allow the precipi- 

 tate to settle and then filter off on a dry, folded filter. Take an aliquot of the 

 clear filtrate (about 125 c.c.) and boil it to expel the greater part of the ammonia. 

 Cool and add dilute sulphuric acid to precipitate the excess of lead as sulphate 

 and filter. Add 10 c.c. of 50 per cent sulphuric acid and transfer the whole to a 

 Kjeldahl flask provided with a dropping funnel. The contents of the flask are 

 distilled and a solution of potassium bichromate is run in from the dropping funnel 

 at such a rate that the liquid always retains some yellow color and the volume 

 remains at about 100 c.c. It is rarely necessary to add more than about o.i gram 

 of bichromate and an excess is to be avoided. Slow distillation is continued for 

 two hours and about 100 c.c. of distillate is collected. The tip of the delivery tube 

 must always remain under the surface of the water in the receiving flask. Add 

 20 c.c. of hydrogen peroxide, make slightly alkaline with NaOH and distil again. 

 Catch the distillate in small Erlenmeyer flasks containing an excess of the Scott- 

 Wilson acetone reagent (at least 30 c.c. for each milligram of acetone) and de- 

 termine the acetone according to the procedure outlined in the preceding method 

 for acetone and diacetic acid. Calculate the /3-hydroxybutyric acid in terms of 

 acetone and express as milligrams of acetone per 100 c.c. of blood. 



9. Cholesterol. Method of Autenrieth and Funk. 2 Principle 

 The blood or serum is boiled with strong alkali to saponify the fats. 

 The alkaline solution is extracted with chloroform. The chloroform 

 is dried and clarified by means of anhydrous sodium sulphate and 

 nitration and then treated with sulphuric acid and acetic anhydride, 

 and the characteristic color reaction of the Liebermann-Burchard test 



1 The sulphocyanate solution should be standardized against pure acetone treated 

 in the same manner as the final distillate above. According to Scott- Wilson it may also 

 be standardized against a pure solution of mercuric nitrate and the equivalent of i c.c. of 

 the KSCN solution in milligrams of Hg determined. According to this author i mg. of Hg 

 is equivalent to 0.058 mg. of acetone. 



2 Autenrieth and Funk: Munch, med. Woch., 60, 1243, 1913. A slight modification 

 has been suggested by Bloor (Jour. Biol. Chem., 23, 317, 1915). 



