INDICAN TESTS. 731 



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 l ). 



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- 

 acid 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 2 ), 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 3 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- 

 glucuronic acid (SCHMIEDEBERG) . Such an acid has been found in the 

 urine of animals after the administration of the sodium-salt of o-nitro- 

 phenylpropiolic acid (G. HOPPE-SEYLER). PORCHER and HERVIEUX 4 

 have obtained indoxyl sulphuric acid in dogs and asses under similar 

 conditions. 



Free indigo, and in fact indirubin as well as indigotin, occur in rare cases in 

 the undecomposed urine. GROBER and WANG have recently observed such cases. 

 According to STEENSMA 5 traces of free indol occur always in the urine. 



1 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. 

 Centralbl., 8, 54: 



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

 kowski, ibid., 42. 



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



4 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 14; G. Hoppe-Seyler, ' Zeitschr. f. 

 physiol. Chem., 7 and 8; Porcher and Hervieux, Journ. de Physiol., 7. 



5 Grober, Munch, med. Wochenschr., 1904; Wang, Salkowski's Festschrift, 1904; 

 Steensma, Maly's Jahresb., 40, 314. 



