300 PHYSIOLOGICAL CHEMISTRY 



the characteristic dense acid fumes begin to fill the test tube. 

 This is usually accomplished -in from 3 to 7 minutes. When the 

 fumes are unmistakable, cut down the size of the flame so that 

 the contents of the tube are just visibly boiling, and close the 

 mouth of the test tube with a watch glass or a very small Erlen- 

 meyer flask. Continue the heating very gently for 2 minutes 

 from the time the fumes began to be unmistakable, even if the 

 solution has become clear and colorless at the end of 20 to 40 

 seconds. If the oxidations are not visibly finished at the end 

 of 2 minutes the heating must be continued until the solution is 

 nearly colorless. Such cases are very rare; the oxidation is 

 almost invariably finished within the first minute. Allow the 

 contents to cool for 70 to 90 seconds and then add 15 to 25 c.c. 

 of water. Cool further, approximately to room temperature, 

 and add water to the 35 c.c. mark. Add, preferably with a 

 pipette, 15 c.c. of Nessler solution. Insert a clean rubber stopper 

 and mix. If the solution is turbid, centrifuge a portion before 

 making the color comparison with the standard. The standard 

 most commonly required is 0.3 mg. of N (in the form of am- 

 monium sulfate) in a 100 c.c. flask. Add to it 2 c.c. of the 

 sulfurie-phosphoric acid mixture, about 50 c.c. of water, and 

 30 c.c. of Nessler solution. Fill to the mark and mix. The 

 unknown and the standard should be Nesslerized at approxi- 

 mately the same time. If the standard is set at 20 mm. for the 

 color comparison, 20 divided by the reading and multiplied by 

 30 gives the nonprotein nitrogen in mg. per 100 c.c. of blood. 



Determination of Urea by Urease Decomposition and Distil- 

 lation. (Folin and Wu. Jour. Biol. Chem., xxxviii, 94, 1919). 

 Transfer 5 c.c. of the tungstic acid blood filtrate to a clean 

 and dry Pyrex ignition tube (capacity about 75 c.c.). The 

 graduated Pyrex tubes recommended for the nonprotein nitro- 

 gen determination should never be used for urea determina- 

 tions, because they have contained Nessler solutions and Nessler 

 solutions leave behind films of mercury compounds which de- 

 stroy the urease. If those tubes must be used, they should first 

 be washed with nitric acid to remove the mercury films. Add 



