210 PHYSIOLOGICAL CHEMISTRY 



calculi, cholesterol-calcium-pigment calculi, calcium- pigment calculi, and 

 calculi made up almost entirely of inorganic material. This last class 

 of calculus is formed principally of the carbonate and phosphate of 

 calcium and is rarely found in man although quite common to cattle. 

 The calcium-pigment calculus is also found in cattle, but is more 

 common to man than the inorganic calculus. This calcium-pigment 

 calculus ordinarily consists principally of bilirubin in combination with 

 calcium; biliverdin is sometimes found in small amount. The choles- 

 terol 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 tend to give us 

 calculi of various colors. 



For discussion of cholesterol see page 3 73 . 



EXPERIMENTS ON BILE 



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

 Congo red. 



2. Nucleoprotein. 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 58). 



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. 



(c) Huppert-Cole Test. 1 Boil about 15 c.c. of the fluid in a test tube. 

 Add two drops of a saturated solution of magnesium sulphate, then add a 10 

 per cent solution of barium chloride, drop by drop, boiling between each addi- 

 tion. Continue to add the barium chloride until no further precipitate is ob- 

 tained. Allow the tube to stand for a minute. Pour off the supernatant fluid 

 as cleanly as possible or use a centrifuge. To the precipitate add 3 to 5 c.c. 

 of 97 per cent alcohol, two drops of strong sulphuric acid, and two drops of a 

 5 per cent aqueous solution of potassium chlorate. Boil for half a minute and 



dole's "Practical Physiological Chemistry," 6th edition, p. 268, 1920. 



