294 PRACTICAL PHYSIOLOGY 



As mentioned above, be ta-oxy butyric acid may be estimated separ- 

 ately by previous removal of the aceto-acetic acid and acetone. Take 

 25 c.c. of the nitrate from the copper lime precipitation in an open 

 flask, add 100 c.c. water and 2 c.c. 17N. sulphuric. Boil for ten 

 minutes over the free flame. Cool, measure, return to flask, make up 

 volume to 127 c.c., add 8 c.c. of the sulphuric acid and 35 c.c. of the 

 mercuric sulphate. Connect with reflux condenser and then proceed 

 as before. 



1 gm. precipitate = 2-64 gms. beta-oxybutyric per 100 c.c. as 

 acetone. Beta-oxybutyric acid = acetone value x 1-793. 



Van Slyke states that there should be a preliminary test of the 

 materials, using distilled water in place of urine. No precipitate of 

 any kind should be formed. 



A good rough approximation to the amount of aceto-acetic acid and 

 acetone present may be obtained by using a modified ammonia estima- 

 tion apparatus. Put 510 c.c. urine in a large boiling test tube fitted 

 with appropriate rubber stopper, add 2-3 drops 5 per cent, sulphuric 

 acid, connect with an absorption tube or flask containing 510 c.c. 

 Scott- Wilson cyanide reagent. 1 Place tube containing the urine in a 

 waterbath at 70-75 and] start suction pump so as to run a moderately 

 rapid air stream through the urine. If acetone is present, even if only 

 in traces, the reagent becomes turbid. The degree of turbidity may be 

 compared with that given by a standard solution of acetone containing 

 0-5 mg. in a riephelometer or Duboscq colorimeter. (See Marriott, 

 J. Biol Chem., 16 and 18; Folin and Denis, J. Biol Ch&m., 18.) 



Quantitative Estimation of Lactic Acid (Ryffel). Lactic acid can be 

 shown to be excreted in minute amount, 0-004 gm. per hour during 

 the day and about half this at night by a perfectly normal man on 

 an ordinary diet. If the man is made to perform strenuous work 

 there may be a very considerable rise in the output, so much indeed 

 that it may be determined roughly by the thiophene test. Render 

 the urine alkaline with sodium carbonate, evaporate, extract with 

 alcohol, evaporate alcohol, dissolve residue in a little water, strongly 

 acidify with phosphoric acid and then thoroughly extract with ether. 

 Ether is shaken up with dilute sodium carbonate solution, the alkaline 

 solution decolorised by boiling with charcoal, filtered and evaporated 

 to dryness. Residue dissolved in 5 c.c. cone. H 2 SO 4 and the thiophene 

 test applied (see p. 230). 



The distillation method devised by Ryffel gives good results even 

 when only small amounts are to be dealt with. This method depends 

 on the fact that lactic acid, when heated above 140 C. with sulphuric 

 acid, yields acetaldehyde quantitatively according to the following 

 equation : 



CH 3 . CHOH . COOH = CH 3 . CHO + H . COOH. 



40 c.c. of the liquid, which must be free from sugar and nearly free 

 from protein, are placed in a 500 c.c. Jena distillation flask. 45 c.c. 



1 Reagent. Dissolve 10 gms. mercuric cyanide in 600 c.c. water and add 

 to this solution 180 gms. pure caustic soda also dissolved in 600 c.c. water. 

 Pour in a slow stream, stirring vigorously, into the mixture 400 c.c. of water 

 containing 2-9 gms. silver nitrate. If properly made the silver dissolves 

 completely, giving a clear solution. Usually there is slight turbidity ; set aside 

 for three to four days to settle ; decant off clear supernatant liquid. When 

 reagent stands a new sediment gradually forms so that solution deteriorates 

 slowly. Will keep good several months. 



