DETECTION AND ESTIMATION OF UROBILIN. 747 



and if the urine contains urobilin, it is first allowed to undergo alkaline 

 fermentation, when the urobilin is converted into urobilinogen. The 

 urine is acidified with tartaric acid and extracted with ether. The 

 foreign pigments are precipitated from the ethereal solution by petroleum 

 ether; the ether solution is washed with water, evaporated, and the residue 

 allowed to stand with water for several hours at 38 C., when the urobilino- 

 gen is transformed into urobilin. The urobilin can now be precipitated 

 with ammonium sulphate, and the dried precipitate extracted with absolute 

 alcohol. This urobilin has about three times as much extinction ability 

 as HALT'S urobilin (hydrobilirubin) . Other methods of preparation 

 have been suggested. 



The urobilinogen is prepared by shaking the urine, directly after 

 adding sodium bicarbonate, with chloroform (FISCHER and MEYER- 

 BETZ). In regard to details we must refer to the original publication. 



The detection of urobilin can sometimes be done directly on the urine. 

 Otherwise the urine is shaken with ether, amyl alcohol or chloroform and 

 these solutions tested. According to ScHLESiNGER 1 the urine can also be 

 precipitated by an equal volume of a saturated solution of zinc acetate in 

 alcohol and the filtrate directly tested for the fluorescence and absorption. 

 GRIMBERT 2 has suggested a method for the separate testing for urobilin and 

 urobilinogen by using the chloroform, after shaking the urine therewith. 

 For the detection of urobilin we always make use of the color of the acid 

 or alkaline solutions, the absorption spectrum and the beautiful fluorescence 

 of the ammoniacal solution containing zinc chloride. For the detection 

 of urobilinogen we make use of ERHLICH'S reagent, and the property of 

 the colorless solution of being changed into urobilin in the air and light. 



In the quantitative estimation of urobilin we proceed as follows, 

 according to G. HOPPE-SEYLER : 3 100 cc. of the urine are acidified with 

 sulphuric acid and saturated with ammonium sulphate. The precipitate 

 is collected on a filter after some time, washed with a saturated solu- 

 tion of ammonium sulphate, and repeatedly extracted with equal parts 

 of alcohol and chloroform after pressing. The filtered solution is treated 

 with water in a separatory funnel until the chloroform separates well 

 and becomes clear. The chloroform solution is evaporated on the water- 

 bath in a weighed beaker, the residue dried at 100 C., and then extracted 

 with ether. The ethereal extract is filtered, the residue on the filter dis- 

 solved in alcohol, and transferred to the beaker and evaporated, then 

 dried and weighed. According to this method G. HOPPE-^EYLER found 

 0.08-0.14 gram of urobilin in one day's urine of a healthy person, or an 

 average of 0.123 gram. 



The urobilin can also be determined according to the method sug- 

 gested by CHARNAS for its preparation and urobilin can also be deter- 

 mined spectroscopically by the method suggested by SAiLLET. 4 Further 

 details will be found in the original publications and in larger handbooks. 



The quantitative estimation of urobilinogen can be accomplished 

 spectroscopically by means of EHRLICH'S reagent, as suggested by CHARNAS. 



1 Deutsch. med. Wochenschr., 1903. 



* Compt. rend. soc. biol., 70. 



Virchow's Arch., 124. 



4 Charnas, 1. c.; Saillet, 1. c.; see also Tsuschija, Zeitschr. f. exp. Path. u. Ther., 7. 



