Studies on bilirubin isomers Part 1. Separation and characterization of bilirubin isomers

The commercial bilirubin obtained from Daiichi Pure Chemical Co. Ltd. was separated into three fractions according their movability from the front (upper, middle and lower bands) by McDonagh's thin layer chromatography. Each fraction was examined by mass spectrometry, nuclear magnetic resonance (N.M.R.) spectra and photochemical methods. The following results were obtained. 1) Mass spectra specific to bilirubin, the parent ion at m/e 584 and the fragment ions at m/e 299 & 286 were obtained from all these fractions. 2) Signals of methyl groups in bilirubin molecule on N.M.R. spectra of these fractions were observed as follows. Upper band: at 2.03 and 2.16 p.p.m. of C-13 and C-17, middle band: at 1.92 and 2.00 p.p.m. of C-2 and C-7, 2.03 and 2.16 p.p.m. of C-13 and C-17, and lower band: at 1.91 and 2.00 p.p.m. of C-2 and C-7, respectively. From these observations it was concluded the bands coincide with bilirubin Ⅲα, Ⅸα and XⅢα respectively. 3) Absorption maxima of bilirubin Ⅲα, Ⅸα and XⅢα in chloroform were found at 455~458nm, 453~455nm and 449~453nm respectively. Absorption maxima of their diazotized sulfanylate solution (pH 1.3) were 548~556nm, 540~548nm and 533~540nm respectively. Absorption maxima of their diazotized ethylanthranilate solution (pH 1.4) were 524~527nm, 527~530nm and 530~533nm respectively. After acidification with hydrochloric acid to pH 0.5, absorption maxima of their diazotized sulfanylate solution were moved to 577~584nm, 570~577nm and 563~570nm. 4) E(max). of the isomers in chloroform were calculated to be 64,900 for bilirubin Ⅲα, 62,900 for bilirubin Ⅸα and 53,700 for bilirubin XⅢα respectively.