316 PRACTICAL PHYSIOLOGY 



divided by the reading of the unknown and multiplied by 30 gives the 

 non-protein nitrogen in nigs, in 100 c.c. blood. 



Determination of Urea. Put 5 c.c. of the tungstic blood filtrate in 

 a clean hard glass tube (200 x 25 mm.), add 2 drops of a phosphate 

 mixture (60 gms. monosodium phosphate and 179 gms. crystallised 

 disodium phosphate dissolved in warm water and diluted to 1 litre) 

 and 1 c.c. of an active urease solution. Immerse the test tube in warm 

 water (40 55 C.) for five minutes. In another test tube with a 25 c.c. 

 mark place 2 c.c. of 0-05 N hydrochloric acid. After the addition to the 

 urease tube of a dry pebble, a drop or two of paraffin and 2 c.c. saturated 

 borax solution connect the two tubes by means of glass tubing, carrying 

 two rubber corks and of such a length that the tubing on the receiver 

 side dips beneath the surface of the acid. Having inserted both corks 

 boil at a moderately fast, and as uniform as possible, rate for four minutes. 

 Then slip off receiver, leaving connecting tube still inside, but free from 

 acid solution and continue boiling for another minute. Rinse the end 

 of the delivery tube with a little water, cool the distillate under the tap 

 and dilute to about 20 c.c. Prepare standard solution of ammonium 

 sulphate by diluting 3 c.c. of the standard in a 100 c.c. flask to about 

 75 c.c. Nesslerise by adding to standard flask 10 c.c. Nessler solution 

 and to the test tube 2-5 c.c., dilute both to appropriate volume (i.e. 

 100 c.c. and 25 c.c. respectively) and do colorimetric reading. 



Calculation. Set standard at 20 m. Divide 20 by reading of un- 

 known and multiply by 15. Result is urea nitrogen in mg. in 100 c.c. 

 blood. 



If an autoclave is available and there are many ureas to be done it is 

 much more convenient to hydrolyse 5 c.c. of the filtrate in a hard glass 

 test tube with 1 c.c. normal HC1 at 150 for ten minutes. (Cover mouth 

 of test tube with tinfoil. ) In the subsequent distillation of the ammonia 

 on account of the acid present 2 c.c. of 10 per cent, sodium carbonate 

 must be used in place of borax. 



(If preferred the 0-05 N HC1 used for receiving the ammonia distilled 

 over may be titrated with 0-05 N alkali instead of using the Nessler 

 method. The same statement applies to the estimation of non-protein 

 nitrogen if the ammonia is distilled off and received in 0-05 N acid.) 



Determination of Preformed Creatinine. Pipette 10 c.c. of blood 

 filtrate into one small flask and into another similar flask 5 c.c. of 

 standard creatinine solution (v. i. ) and 15 c.c. water. Add to the blood 

 filtrate 5 c.c. and to the standard creatinine solution 10 c.c. of freshly 

 prepared alkaline picrate solution (25 c.c. saturated solution of pure 

 picric acid mixed with 5 c.c. 10 per cent, sodium hydroxide). Allow 

 both to stand eight to ten minutes then do colour comparison in the 

 ordinary way. This comparison should be completed within fifteen 

 minutes after the addition of the picrate. Also it is advisable to have, 

 if possible, several standard creatinine solutions available in case of 

 meeting with very abnormal blood creatinines. If necessary, however, 

 the unknown may be further diluted by the addition of dilute alkaline 

 picrate solution (1 part solution: 2 parts water). 



The standard (blood) creatinine solution. Add from a known solution 

 of pure creatinine (pure creatinine may be prepared by the method of 

 Benedict, J. Biol. Chem., 1914, 18, 183) an amount containing 6 mg. of 

 creatinine and 10 c.c. of normal hydrochloric acid to water contained in a 

 litre flask, mix and dilute to mark. Solution may be kept as stock 

 after addition of a few drops of toluol. 5 c.c. of this solution contain 



