Studies on Urobilinogen Part 1. Spectrophotometric studies on the coloured substances of urobilinogen by the Ehrlich's aldehyde reaction

The spectrophotometric studies were made on the coloured solutions prepared by the addition of the Ehrlich's aldehyde reagent into the urobilinogen solution (stercobilinogen) prepared from urine and stool, the mesobilirubinogen solution prepared from crystalline bilirubin by a reduction with natrium amalgam, and their salt and mesobilinogen dimethylester etc. And the results are as follows. 1. Both the urobilinogen solution in which stercobilinogen is mainly included and the mesobilinogen solution coloured with the Ehrlich's aldehyde reagent, display the absorption maxima at 560 mμ and 490 mμ at the pH 4.2. 2. When the hydrogen ion density of the above coloured solution becomes below 4.0, the absorption maxima display at 558 mμ and 490 mμ or 492 mμ. Both of these absorption maxima are not moved by the change of pH. 3. In proportion as the pH of the coloured solution becomes high more than 42, the absorption maximum at 560 mμ shifts slightly to the short wave length and displays at 556 mμ or 554 mμ, but the absorption maximum at 490 mμ shifts to the long wave length in a relatively prominent degree and displays at 506 mμ or 510 mμ. As the pH of the coloured solution becomes showing a alkaline reaction, the absorption maximum at 560 mμ disappears and the absorption maximum at 490 mμ shows shifting to the opposite direction, the short wave length. 4. On the use of methanol, as a solvent, instead of water, both of them display the absorption maxima at 550 mμ and 490 mμ at the pH 5.4. And no differences are observed, on the change of the pH, between the coloured substances of stercobilinogen and mesobilinogen with the Ehrlich's aldehyde reagent. 5. As for the absorption maximum of mesobilinogen is observed shifting to the long wave length for 1 or 3 mμ more than that of stercobilinogen under the acid-and weak acid condition of pH. 6. The coloured substances of the sodium-, potassium- and ammonium salts of urobilinogen with the Ehrlich's aldehyde reagent display the same absorption maxima by the change of the pH value, but the absorption maximum shifts to the short wave length for 2 mμ on the occasion of preparing the salts by adding several times of the computation dosis of caustic soda, caustic potash and ammonia more than on the occasion of preparing the salts by adding the computation dosis of them or the absorption maximum of the coloured substance of stercobilinogen itself with the Ehrlich's aldehyde reagent at the pH 1.4-1.0, and it displays at 556 mμ. 7. When the Ehrlich's aldehyde reaction is obtained immediately after the process being moved into caustic soda from the petroleum ether stratum on the preparation of stercobilinogen from stool, the absorption maximum displays only at 556 mμ at the pH 1.6 without showing the absorption maximum around 490 mμ. Therefore, it is thought that the absorption maximum at 420 mμ is not coexisted with the absorption maximum around 560 mμ when the oxidation is prevented on the preparation of urobilinogen or during it's process, and that the absorption maximum of the coloured substance with the Ehrlich's aldehyde reaction displays only one around 560 mμ and the absorption maximum around 490 mμ belongs to that of the urobilin oxygenated from urobilinogen. 8. The coloured substance of mesobilinogen dimethylester with the Ehrlich's aldehyde reagent displays the absorption maxima at 550 mμ and 488 mμ at the pH 3.8. 9. The attitude of mesobilinogen dimethylester to various solvents is markedly different from that of bilirubin dimethylester, and mesobilinogen dimethylester is more unstable to an oxidation than mesobilinogen.