536 PHYSIOLOGICAL CHEMISTRY 



times. A greater dilution is not desirable. The urine is treated with i per cent of 

 sulphuric acid and about 3 c.c. of acetic acid for each day's volume. Test a small 

 quantity of the urine to determine the amount of 50 per cent phosphotungstic acid 

 required to completely precipitate it. The bulk of the urine is then treated on this 

 basis with sufficient solid phosphotungstic acid to precipitate it completely. Stir 

 to dissolve the acid and allow to stand for several hours. Filter with suction, first 

 lining the filter with infusorial earth by rubbing up a little of the substance with some 

 of the urine mixture and filtering with suction. To some ordinary lead oxide in a 

 mortar add a small amount of the filtrate and stir until it becomes warm, then add 

 the rest of the filtrate and stir, adding more oxide if necessary until the solution 

 reacts alkaline due to the formation of basic lead acetate. 



Filter again. The filtrate should give no precipitate with basic lead acetate. 

 A measured volume of the filtrate is then treated with measured volumes of acetic 

 acid and silver nitrate solution to completely precipitate any chlorides present. 

 Filter again preferably through infusorial earth. Pass hydrogen sulphide through 

 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 filtrate with calcium carbonate and remove 

 the carbon dioxide with a current of air. 



The neutral filtrate 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 thiocyanate 

 solution should be standardized occasionally against standard silver nitrate 

 solution. 



Calculation. One c.c. of N/io NH^SCN 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. 



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



