324 PHYSIOLOGICAL CHEMISTRY. 



pigments produced in one day is not over 200 milligrams prob- 

 ably ; the physiological meaning of the formation is not known. 

 The iron of the original hematin is largely retained by the 

 substance of the liver cells. 



Preparation of Bilirubin. The pigment cannot be easily obtained from 

 bile because of the small amount present, but may be obtained from the 

 pathological concretions known as gall-stones, which will be described 

 later. Powder several grams of these stones from cattle very fine and 

 exhaust thoroughly with ether, then repeatedly with boiling water to take 

 out cholesterol and bile acids. In the residue the bilirubin exists as an 

 insoluble calcium compound; this is decomposed by the addition of a little 

 dilute hydrochloric acid, after which- what is left is washed thoroughly 

 with hot water, and then 'with alcohol to leave the pigment in a still better 

 condition for extraction. Finally extract with chloroform in which the 

 substance is relatively soluble. On evaporating the chloroform crude 

 bilirubin is secured, which after washing with alcohol may be recrystallized 

 from hot chloroform or from dimethylaniline, in which it dissolves in 

 the proportion of about I to 100 cold or i to 30 hot. By several crystal- 

 lizations it is possible to obtain a product pure enough to employ as a 

 standard for spectroscopic measurements. 



By exposing an alkaline solution to the air or by treating with a little 

 acid and sodium peroxide, bilirubin is converted into biliverdin. The lat- 

 ter free substance is not soluble in water, chloroform or ether. 



The Bile Pigment Tests. Some of these are extremely delicate and 

 have long been used for the recognition of bile, especially in urine. For 

 the tests to be given the bilirubin alkali in very dilute solution may be 

 used, or a diluted bile. 



GMELIN'S TEST. In a test-tube take a few cubic centimeters of nitric 

 acid containing some nitrous acid. Over this pour carefully the weak bile 

 solution to be tested. At the junction point colored rings appear which 

 result from the formation of oxidation products of the bilirubin. The 

 colors appear in this order from above down: green, blue, violet, red and 

 yellowish. Of these the green is the most characteristic; the other shades 

 represent more advanced stages in the oxidation. For success in the test 

 the bile solution must not be too strong, and the amount of nitrous acid 

 in the nitric acid must be small. 



HAMMARSTEN'S TEST. Use as reagent a mixture of strong nitric acid 

 and strong hydrochloric acid in the proportion of about I to 50 by vol- 

 ume. This mixture must stand some time before use, or until it becomes 

 yellow. It keeps a long time. For the practical test mix i cubic centi- 

 meter of the acid with 4 cubic centimeters of alcohol and add a drop or 

 two of the bilirubin solution to be tested. A permanent green color 

 appears, but if stronger oxidation is secured by adding more of the acid 

 mixture the colors change as in the Gmelin test. The reaction can be 

 well applied to urine. 



