URINE 543 



flask, and wash the dried residue with hot water. 1 Add 10 c.c. of concentrated 

 sulphuric acid to the washed residue, heat on the water-bath for five to ten 

 minutes, dilute with 100 c.c. of water, and titrate the blue solution with a very 

 dilute solution of potassium permanganate. 2 The end point is indicated by the 

 dissipation of all the blue color from the solution and the formation of a pale 

 yellow color. 



Beautiful plates of indigo blue sometimes appear in the chloroform extract 

 of urines containing abundant indican. In urines preserved by thymol the 

 determination of indican is interfered with unless great care is taken in washing 

 the chloroform extract with dilute alkali. Care should be taken, therefore, to 

 make the indican determination upon fresh urine, before the addition of the 

 preservative. 



Plasencia 3 has suggested a method which is shorter than Ellinger's and ac- 

 cording to its sponsor, just as accurate. 



Calculation. One cubic centimeter of the diluted permanganate solution 

 is equivalent to about 0.15 mg. of indigo. Ellinger claims that one -sixth of the 

 amount determined must be added to the value obtained by titration in order to 

 secure accurate data. This correction should always be made. 



Interpretation. From 4-20 mg. of indican are excreted per day by 

 normal men. In normal individuals the variations are dependent 

 mainly upon the diet. A meat diet increases the indican excretion, 

 while a milk or carbohydrate-rich diet decreases it. Pathologically 

 the greatest increases are found in disorders involving increased 

 putrefaction and stagnation of intestinal contents. Bacterial de- 

 composition of body protein as in gangrene, putrid pus formation, etc., 

 gives rise to increases. 



Phenols 



Colorimetric Method of Folin and Denis. 4 Principle. This method 

 is based upon the fact that phenols yield with a solution of phospho- 

 tungstic-phosphomolybdic acid and alkali a deep blue color the depth 

 of which is proportional to the amount of such substances present. 

 Traces of protein, which may be present in the urine, and uric acid 

 give a blue color with the reagent and are removed by precipitation 

 with an ammoniacal silver solution and colloidal iron as a preliminary 

 to the determination of the phenols. 



Procedure. Removal of Interfering Substances. Place 10 c.c. of ordinary 

 urine, or 20 c.c. of a dilute urine in a 50 c.c. volumetric flask. To this add an 



1 The washing should be continued until the wash water is no longer colored. Ordi- 

 narily two or three washings are sufficient. If a separation of indigo particles takes place 

 during the washing process, the wash water should be filtered, the indigo extracted with 

 chloroform, and the usual method applied from this point. 



2 A "stock solution" of potassium permanganate containing 3 grams per liter should be 

 prepared, and when needed for titration purposes a suitable volume of this solution should 

 be diluted with 40 volumes of water. The potassium permanganate solution may be 

 standardized with pure indigo. 



3 Plasencia: Revista de Medicina y Cirugia., 17, i, 1912. 



4 Folin and Denis: /. Biol. Chem., 22, 305, 1915. 



