282 PRACTICAL PHYSIOLOGY 



liberated ammonia less the amount neutralised by the preformed 

 ammonia in the urine (which must be determined by separate experi- 

 ment) multiplied by 0-003 gives in grammes the amount of urea in the 

 urine used. For ammonia determination, see p. 286. 



A smaller apparatus with tubes of about 100 c.c. capacity, and using 

 fiftieth normal acid, has been found useful for estimating small amounts 

 of urea. The quantities for this apparatus are 5 c.c. of urea-containing 

 fluid (content not more than 10 mgms. urea) ; 1 gm. soy bean meal ; 

 and | c.c. saturated sodium carbonate solution. Aeration with the 

 small apparatus is complete in twenty minutes. 



Since the time required for complete liberation of the ammonia 

 depends on the velocity of the air-current and the amount of soy bean 

 meal added, it is well to determine that all conditions are correct by 

 doing one estimation on a solution containing a known amount of urea. 

 The soy bean meal liberates a certain small amount of ammonia itself, 

 so that, for accurate work, it is necessary to estimate this by doing a 

 blank experiment using water in place of urea solution. 



Folin Method. Sometimes it is convenient and useful to use an acid 

 hydrolysis method, such as that introduced by Folin, where the urine 

 is heated to 150-160 C. in the presence of an acid. 



Weigh 20 gms. of crystallised magnesium chloride into an Erlenmeyer 

 flask (250-300 c.c. capacity), then add 5 c.c. urine, 6 c.c. concentrated 

 HC1, a small piece of hard paraffin wax or a few c.c. of liquid paraffin and 

 finally a drop or two of an aqueous solution ( 1 per cent. ) of alizarin red. 

 A special safety tube (a U-shaped condenser) is then inserted in the 

 mouth of the flask and then the mixture is boiled, best on a hot plate, 

 until distinct " bumping " commences. The heat is then lowered 

 until each drop as it falls back (about three times a minute) into the 

 flask causes a slight bump. Heating is continued for at least two hours. 

 If the indicator shows any appearance of a red colour during the 

 hydrolysis a few drops of the acid distillate in the condenser must be 

 returned to the flask by tilting the condenser. When the hydrolysis is 

 complete the contents of the Erlenmeyer are transferred to a distilling 

 flask, as in the Kjeldahl method, and after rendering just alkaline with 

 caustic soda the ammonia is distilled off. Deduct the value for ammonia 

 which must also be determined (see p. 286). 



N 

 1 c.c. J-Q- H 2 SO 4 = -003 gm. urea = -0014 gm. urea nitrogen. 



This method serves excellently for the indirect estimation of allantoin 

 as both urea and allantoin if present are hydrolysed under the above 

 conditions, whereas by the urease method urea alone is attacked. 

 Therefore if the amount of nitrogen obtained by the urease method is 

 deducted from the yield by the acid hydrolysis method the difference 

 may be assumed to be due to allantoin. 



Estimation of Uric Acid. Hopkins- Folin Method. Reagent neces- 

 sary. (1) Uranium solution; 500 gms. ammonium sulphate, 5 gms. 

 uranium acetate and 60 c.c. 10 per cent, acetic acid in 650 c.c. water. 



(2) 2Q potassium permanganate. (3) 10 per cent, ammonium sulphate 



solution. 



100 c.c. urine is measured into a tall, narrow cylinder and 25 c.c. of the 

 uranium solution is added in order to precipitate mucoid substances. 

 The mixture is allowed to stand without stirring for about half an hour. 



