Separation of Photoproducts of Bilirubin by Thin-Layer Chromatography, and their Diazo Coupling Reactions

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We have described the diazo coupling reactions of the photoproducts of bilirubin separated by thin-layer chromatography using silicagel FM258 plate-Wako (Wako Pure Chemical Industries, Ltd.) and a mixture of chloroform: methanol: acetic acid (97: 2: 1, v/v) as a developing solvent. In the bilirubin solution irradiated with a fluorescent lamp (visible light), eleven substaces including biliverdin were detected. The ten substances separated other than biliverdin were classified into three groups, with absorption maxima of about 450 nm (six substances), 420 nm (three substances) and 388 nm (one substance). All these photoproducts reacted with the diazonium salt of sulfanilic acid to form a purple product with absorption maximum of about 550 nm. However, upon oxidation with a nitrous acid solution, the six photoproducts with absorption maxima of about 450 nm each yielded a green product with absorption maxima at about 700 and 370 nm, and the four photoproducts with absorption maxima of about 420 or 388 nm yielded a colorless substance with an absorption peak of about 360 nm. In addition, it was confirmed that bilirubin IIIα and XIIIα were also produced from bilirubin IXα by irradiation with a fluorescent lamp.
Japan Society of Clinical Chemistryの論文