694 URINE. 



latter suggested by PORCHER and HERVIEUX 1 ). With OBERMAYER'S 

 reagent the test is performed as follows : 



The acid urine (if alkaline it must be acidified with acetic acid) (ELLIN- 

 GER) is precipitated with basic lead acetate, 1 cc. for every 10 cc. of 

 the urine. 20 cc. of the filtrate are treated in a test-tube with an equal 

 volume of pure concentrated hydrochloric acid (specific gravity 1.19) 

 which contains 2-4 grams ferric chloride to the liter, and 2-3 cc. chloro- 

 form are added and the mixture immediately thoroughly shaken. The 

 chloroform is thereby colored more or less blue, depending upon the 

 amount of indican. Besides indigo blue we may also have indigo red 

 produced, whose formation has been explained in various ways. The 

 quantity of indigo red becomes greater the more slowly the oxidation 

 takes place, and especially when the decomposition takes place in the 

 warmth (see the works of ROSIN, BOUMA, WANG, MAILLARD, ELLINGER 

 and HERVIEUX 2 ). 



According to ELLINGER the source of the indigo-red formation may be the 

 isatin that is produced by the superoxidation of the indoxyl by the action of 

 the reagent, and this isatin forms indigo red with the indoxyl in the hydrochloric- 

 aeid solution. MAILLARD, on the contrary, is of the view that the blue substance 

 which is taken up by the chloroform from the urine mixed with hydrochloric 

 acid is not indigotin (indigo-blue), but another substance, called by him hemi- 

 indigotin, which in alkaline solution polymerizes immediately into indigotin, 

 while in acid reaction it is converted into indirubin (indigo red). * 



The chloroform solution of indigo obtained in the indican test may be 

 used in the quantitative colorimetric determination by comparison with 

 a solution of indigo in chloroform of known strength (KRAUSS and ADRIAN) . 

 WANG and others convert the indigo into indigo-sulphonic acid by con- 

 centrated sulphuric acid and titrate with potassium permanganate. 

 There is still doubt as to the surest and most trustworthy method for 

 the determination of indican, and especially as to the question how the 

 indigo residue is to be washed (see WANG, BOUMA, ELLINGER, and SAL- 

 KOWSKI 3 ), and for this reason we shall refer only to the works cited above. 



Because of the difficulty arising from the production of indirubin 

 in addition to indigotin, BOUMA has recommended the conversion of 

 all the indoxyl into indirubin by boiling the urine with hydrochloric 

 acid containing isatin. The indirubin can be taken up by chloroform 

 and determined by titration with potassium permanganate and sul- 

 phuric acid after purification of the chloroform residue. OERUM 4 has 

 also worked out a colorimetric method of estimation based upon BOUMA'S 

 method. 



Indol seems also to pass into the urine as a glucuronic acid, indoxyl- 



1 Jaffe", Pfliiger's Arch., 3; Obermayer, Wien. klin. Wochenschr., 1890; Salkowski, 

 Zeitschr. f. physiol. Chem., 57; Porcher and Hervieux, Zeitschr. f. physiol. Chem., 39. 



2 Rosin, Virchow's Arch., 123; Bouma, Zeitschr. f. physiol. Chem., 27, 30, 32, 

 39; Wang, ibid., 25, 27, 28; Ellinger, ibid., 38 and 41; Maillard, Bull. soc. chim., Paris 

 (3), 29, and Compt. rend., 136; also L'indoxyle urinaire et les couleurs qui en derivent, 

 Paris, 1903, and Zeitschr. f. physiol. Chem., 41; Hervieux, see Bioch. Centralb., 8, 54. 



3 Krauss, Zeitschr. f. physiol. Chem., 18; Adrian, ibid., 19; Wang, ibid., 25; Sal- 

 kowski, ibid., 42. 



4 Bouma, Zeitschr. f. physiol. Chem., 32; Oerum, ibid., 45. 



