URINE . 519 



the filtrate until the heavy metals are completely precipitated. Filter again. Pass 

 a current of air through the filtrate to remove all hydrogen sulphide (test with lead 

 acetate paper). Neutralize this final nitrate with calcium carbonate and remove 

 the carbon dioxide with a current of air. 



The neutral nitrate is then treated with an amount of allantoin reagent in excess 

 of that required to precipitate the allantoin as indicated by a preliminary test. 

 (The allantoin reagent is a solution containing 5 per cent mercuric acetate and 20 

 per cent of sodium acetate. The reagent when used must be clear.) 



Allow to stand for half an hour (not too long) and then filter. A measured 

 amount of the filtrate is taken and treated with about 10 c.c. of iron-ammonium alum 

 and the red solution decolorized with dilute sulphuric acid. (The solution is not 

 completely decolorized but retains a faint greenish tint.) Any calcium sulphate 

 precipitate formed at this point may be disregarded. Titrate with N/io ammonium 

 thiocyanate solution to a yellow color, which increases in intensity on the addition 

 of 1-2 drops more of solution. The titration should not be carried out in artifi- 

 cial light and some practice is required to get the exact end point. The thiocya- 

 nate solution should be standardized occasionally against standard silver nitrate 

 solution. 



Calculation. One c.c. of N/io NEUSCN solution corresponds to 0.00436 gram 

 of allantoin. The limit of error of the method is about 5 mg. for the daily output 

 of allantoin. In the calculation of course all losses through filtration, etc., must 

 be considered. 



For the considerable modifications required in carrying out the determination 

 on human urine with its very low content of allantoin see the section by Wiechowski 

 in Neubauer-Huppert. 1 



Interpretation. Allantoin may be found in very small amounts in human urine 

 (5-15 mg. per day), appearing to be mainly, though not entirely, exogenous in origin. 

 It forms, however, the principal end-product of the purine metabolism of practically 

 all mammals other than man and the anthropoid apes. Thus over 90 per cent of 

 the purine-allantoin nitrogen excretion of the dog, the cow, and the pig occurs in 

 this form. In these animals its origin is from exogenous and endogenous purines, 

 and its excretion is influenced by much the same factors as is that of uric acid in 

 man. 



2. Determination by Difference. Method of Plimmer and Skelton. 2 Allan- 

 toin is transformed into urea and determined as such in the acid-magnesium chloride 

 method of Folin for urea in urine (page 495). Urease, however, has no effect upon 

 allantoin. Therefore, determine the urea + allantoin of the urine according to Fo- 

 lin's procedure (see Urea), and also determine the true urea according to the 

 urease method (see Urea). The difference between the results thus obtained rep- 

 resents allantoin. If sugar is present it must be removed before applying Folin' s 

 procedure. 



Hippuric Acid 



i. Method of Folin and Flanders. 3 Principle. The hippuric acid 

 is hydrolyzed to benzole acid in alkaline solution and then the solution 

 is boiled with strong nitric acid to remove pigments and emulsifying 



1 Wiechowski: Neubauer-Huppert: Analyse des Harns, Wiesbaden, 1913, p. 1076. 



2 Plimmer and Skelton: Bioch. /., 8, 70 and 641, 1914. 



3 Folin and Flanders: Jour. Biol. Chem , n, 257, 1912. 



