URINE 495 



Close the test-tube by means of a rubber stopper carrying an empty narrow 

 "calcium chloride tube" (1.5 cm. by 25 cm., without bulb) as a condenser. Sus- 

 pend the test-tube and condenser above a micro-burner (see page 487) by means 

 of a burette clamp or some similar device in such a way that they may be easily 

 raised or lowered. Heat gently, using a bottomless beaker or some similar 

 device as a wind shield if needed. The acetate will soon dissolve (two minutes) 

 and the mixture begin to boil. At this point the indicator begins to melt showing 

 that the desired temperature (i53-i6oC.) has been reached. Continue the 

 boiling in a gentle, even manner for ten minutes at the end of which time the 

 decomposition of the urea is complete. Remove the apparatus from the flame 

 and dilute the contents with 5 c.c. of water. 1 Add an excess of alkali (2 c.c. of a 

 saturated solution of sodium hydroxide or potassium carbonate) and remove 

 the liberated ammonia by means of a strong air current (see page 487). The 

 ammonia may be caught in a 100 c.c. volumetric flask which contains about 35 

 c.c. of ammonia-free water and 2 c.c. of N/io acid. With a stiong air current 

 this process requires only about ten minutes. Determine the ammonia colori- 

 metrically against i mg. of nitrogen in the form of ammonium sulphate. For 

 the colorimetric procedure see the total nitrogen determination page 485. 



4. Method of Folin and Denis. 2 Principle. Sugar interferes with the de- 

 composition of urea. This was formerly believed to be due to the formation of 

 nitrogenous "melanins," 3 but is more probably due to the formation of definite, 

 stable ureids. 4 This difficulty may be overcome by proper dilution of the urine 

 thus preventing the formation of the ureids. Because of this great dilution titration 

 procedures are inapplicable, and the colorimetric procedure is applied. 



Procedure. Dilute i c.c. of the urine with 20 to 100 volumes of ammonia-free 

 water and decompose i c.c. of this dilute urine with potassium acetate and acetic 

 acid as described under the method of Folin and Pettibone on page 494. 



By means of an air current remove the ammonia to a second test-tube which 

 contains about 2 c.c. of water and 0.5 c.c. of N/io hydrochloric acid. Add to the 

 contents of this tube about 2 c.c. of water and 3 c.c. of the diluted (i : 5) Nessler- 

 Winkler solution (page 490). Wash this colored solution into a 10 c.c. volumetric 

 flask and dilute it to the mark with ammonia-free water. Transfer the entire vol- 

 ume to a dry cylinder of a Duboscq colorimeter and determine the depth of color 

 against a standard containing i mg. of nitrogen per 100 c.c. of solution. For the 

 detailed colorimetric procedure see the method for total nitrogen, page 494. 



5. Method of Folin. 5 Principle. The urea is decomposed by heating with 

 magnesium chloride and hydrochloric acid. The ammonia is distilled off and de- 

 termined by titration. This method was one of the first accurate methods for the 

 determination of urea. Allantoin is also included in the results from this method 

 but occurs only in minute quantities in human urine. The method is not applicable 

 to urines containing sugar as ureides are formed. For such urines the method of 

 Folin and Denis (above) or the urease method using aspiration (page 491) may 

 be employed. The earlier Mb'rner-Sjoqvist method 6 has been combined with that 



1 This water should be added by means of a pipette through the calcium chloride tube so 

 as to rinse the sides of the tube and the bottom of the rubber stopper from any possible traces 

 of ammonium acetate. Not more than 5 c.c. of water should be used for this purpose. 



2 Folin and Denis: Jour. Biol. Chem., u, 520, 1912. 

 3 Morner: Skand. Arch. Physiol., 14, 319. 



4 Folin: Am. Jour. Physiol., 13, 46, 1905. 

 6 Folin: Am. Jour. Physiol., 13, 46, 1905. 

 8 Morner: Skand. Arch. Physiol., 14, 297, 1903. See also Fourth Edition of this book. 



