BILE 2Oy 



consists principally of bilirubin in combination with calcium; biliverdin 

 is sometimes found in small amount. The cholesterol calculus is the one 

 found most frequently in man. These may be formed almost entirely 

 of cholesterol, in which event the color of the calculus is very light, or 

 they may contain more or less pigment and inorganic matter mixed 

 with the cholesterol, which tends to give us calculi of various colors. 

 For discussion of cholesterol see page 355. 



EXPERIMENTS ON BILE 



1. Reaction. Test the reaction of fresh ox bile to litmus, phenolphthalein and 

 Congo red. 



2. Nucleprotein. Acidify a small amount of bile with dilute acetic acid. A 

 precipitate of nucleoprotein forms. Bile acids will also precipitate here under 

 proper conditions of acidity. 



3. Inorganic Constituents. Test for chlorides, sulphates, and phosphates 

 (see page 59). 



4. Tests for Bile Pigments. Practically all of these tests for bile 

 pigments are based on the oxidation of the pigment, by a variety of 

 reagents, with the formation of a series of colored derivatives, e.g., 

 biliverdin (green), bilicyanin (blue), choletelin (yellow). 



(a) Gmelin's Test. To about 5 c.c. of concentrated nitric acid in a test-tube 

 add 2-3 c.c. of diluted bile carefully so that the two fluids do not mix. At the 

 point of contact note the various colored rings, green, blue, violet, red and reddish 

 yellow. Repeat this test with different dilutions of bile and observe its delicacy. 



(b) Rosenbach's Modification of Gmelin's Test. Filter 5 c.c. of diluted bile 

 through a small filter paper. Introduce a drop of concentrated nitric acid into 

 the cone of the paper and note the succession of colors as given in Gmelin's test. 



(a) Nakayama's Reaction. To 5 c.c. of diluted bile in a test-tube add an equal 

 volume of a 10 per cent solution of barium chloride, centrifugate the mixture, pour 

 off the supernatant fluid, and heat the precipitate with 2 c.c. of Nakayama's reagent. 1 

 In the presence of bile pigments the solution assumes a blue or green color. 



(d) Huppert's Reaction. Thoroughly shake equal volumes of undiluted bile 

 and milk of lime in a test-tube . The pigments unite with the calcium and are pre- 

 cipitated. Filter off the precipitate, wash it with water, and transfer to a small 

 beaker. Add alcohol acidified slightly with hydrochloric acid and warm upon a 

 water-bath until the solution becomes colored an emerald green. 



In examining urine for bile pigments, according to Steensma, this procedure 

 may give negative results even in the presence of the pigments, owing to the fact 

 that the acid-alcohol is not a sufficiently strong oxidizing agent. He therefore 

 suggests the addition of a drop of a 0.5 per cent solution of sodium nitrite to the 

 acid-alcohol mixture before warming on the water-bath. Try this modification also. 



(e) Hammarsten's Reaction. To about 5 c.c. of Hammarsten's reagent 2 in a 



1 Prepared by combining 99 c.c. of alcohol and i c.c. of fuming hydrochloric acid con- 

 taining 4 grams of ferric chloride per liter. 



2 Hammarsten's reagent is made by mixing i volume of 25 per cent nitric acid and 19 

 volumes of 25 per cent hydrochloric acid and then adding i volume of this acid mixture 

 to 4 volumes of 95 per cent alcohol. 



