554 PHYSIOLOGICAL CHEMISTRY 



ing, is determined without the /3-hydroxybutyric acid exactly as the total 

 acetone bodies, except that (i) no dichromate is added to oxidize the 0-hydroxy- 

 butyric acid and (2} the boiling must continue for not less than 30 nor more than 

 45 minutes. Boiling for more than 45 minutes splits off a little acetone from 

 /3-hydroxybutyric acid even in the absence of chromic acid. 1 



Determination of /3-hydroxybutyric Acid. The /3-hydroxybutyric acid alone 

 is determined exactly as total acetone bodies except that the preformed 

 acetone and that from the acetoacetic acid are first boiled off. To do this the 

 25 c.c. of urine filtrate plus 100 c.c. of water are treated with 2 c.c. of the 50 per 

 cent sulphuric acid and boiled in the open flask for 10 minutes. The volume of 

 solution left hi the flask is measured in a cylinder. The solution is returned to 

 the flask, and the cylinder washed with enough water to replace that boiled off 

 and restore the volume of the solution to 127 c.c. Then 8 c.c. of the 50 per 

 cent sulphuric acid and 35 c.c. of mercuric sulphate are added. The flask is 

 connected under the condenser and the determination is continued as 

 described for total acetone bodies. 



Titration of the Precipitate hi the Above Methods. Instead of weighing the 

 precipitate, one may wash the contents of the Gooch, including the asbestos, 

 into a small beaker with as little water as possible, and add 15 c.c. of 

 normal HC1. The mixture is then heated, and the precipitate quickly dissolves. 

 In case an alundum crucible is used, it is set into the beaker of acid until the 

 precipitate dissolves, and then washed with suction, the washings being added 

 to the beaker. In place of using either a Gooch or alundum crucible one may, 

 when titration is employed, wash the precipitate without suction on a small 

 quantitative filter paper, which is transferred with the precipitate to the beaker 

 and broken up with a rod in 15 c.c. of normal HC1. 



In order to obtain a good end-point hi the subsequent titration it is necessary 

 to reduce the acidity of the solution. For this purpose it has been found that the 

 addition of excess sodium acetate is the most satisfactory means. Six to 7 c.c. 

 of 3 M acetate are added to the cooled solution of redissolved precipitate. Then 

 the 0.2 M KI is run hi rapidly from a burette with constant stirring. If more than 

 a small amount of mercury is present, a red precipitate of HgI 2 at once forms, and 

 redissolves as soon as 2 or 3 c.c. of KI hi excess of the amount required to form 

 the soluble K<>Hgl4 have been added. If only a few mg. of mercury are present, 

 the excess of KI may be added before the HgI 2 has had tune to precipitate so 

 that the titrated solution remains clear. In this case not less than 5 c.c. of the 

 0.2 M KI are added, as it has been found that the final titration is not satisfac- 

 tory if less is present. The excess of KI is titrated back by adding 0.05 M 

 HgCl 2 from another burette until a permanent red precipitate forms. Since 



1 Blank Determination of Precipitate from Substances in Urine Other than the Acetone Bodies, 

 The 25 c.c aliquot of urine filtrate is treated with sulphuric acid and water and boiled 

 10 minutes to drive off acetone. The residue is made up to 175 c.c. with the same amounts 

 of mercuric sulphate and sulphuric acid used in the above determinations, but without 

 chromate, and is boiled under the reflux for 45 minutes. Longer boiling splits off some 

 acetone from /3-hydroxybutyric acid, and must therefore be avoided. The weight of 

 precipitate obtained may be subtracted from that obtained in the above determination. 

 The blank is so smaU that it appears to be relatively significant only when compared 

 with the small amounts of acetone bodies found in normal or nearly normal urines. In 

 routine analyses of diabetic urines it is not determined. 



Tests of Reagents. When the complete total acetone bodies determination, including 

 the > 'preliminary copper sulphate treatment, is performed on a sample of distilled water 

 instead of urine no precipitate whatever should be obtained. This test must not be 

 omitted. 



