Bilirubinoid-Azo色素に関する研究 第3編 黄疸尿より分離した直接bilirubinに於けるdiazo反応について

According to the method described by Komuta, jaundiced urine was salted out with ammonium sulfate, and the bilirubin, collected on a filter paper, was extracted in methanol. After the addition of the double dose of chloroform to the methanol solution, the dark-reddish precipitation was obtained, that was the ester-form bilirubin. On the other hand, its supernatant was dried in vacuo and then dissolved it in water, followed by the addition of chloroform to that solution. The dark-brown flocculate appeared at borderline of the both solution, that was considered to be an unknown bilirubinoid forming the intermediate zone. A spectrophotometrical examination was performed on the diazo reaction's process of the ester-form bilirubin and the unknown bilirubinoid. The following results were obtained; After the addition of the diazo reagent to the methanol solution of ester-form bilirubin, a color with reddish-violet ton was appeared rapidly, while a maximum on the absorption curve of the azopigments appeared after a little while, and its absorption maximum was shown at 530 to 540 mμ. A blue-violet color appeared macroscopically after the addition of HCl to the ester-form bilirubin-azopigment, its absorption maximum moved to 560 mμ, while no change in its absorption curve was seen after leaving this acid azopigment in the room temperature for 48 hours. In adding the diazo reagent to the methanol solution of the bilirubinoid forming the intermediate zone, its absorption curve also moved as like as that in the ester-form bilirubin. On the other hand, the absorption maximum of its azopigment showed at 550 mμ, and there was no appearance of a color with the blue ton by the addition of HCl to this azopigment. Moreover, its absorption maximum moved only a little toward 560 mμ, namely to the side of the long wavelength, and this acid azopigment showed no particular change on its absorption curve after leaving it in the room temperature for 72 hours.