Synthesis and separation by thin layer chromatography of bilirubin IX isomers. Their identification as tetrapyrroles and dipyrrolic ethyl anthranilate azo derivatives

Abstract

Procedures for the synthesis, separation and determination of structure of the bilirubin IX isomers are described. The four biliverdin IX isomers were prepared by oxidative cleavage of haemin and were separated as their dimethyl esters. The individual esters were reduced with NaBH4, and the bilirubin esters obtained were subjected to alkaline hydrolysis yielding the corresponding bilirubin IX isomers. The bilirubin IX isomers were structurally characterized at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. The absorption spectrum of bilirubin IXα differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin IXβ allows easy differentiation from bilirubin IXδ. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXα and non α isomers. The bilirubin IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro scale by t.l.c. with respect to reference compounds and subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. Pronounced differences in polarity, i.e., solvent distribution, between the bilirubin IX isomers indicate that a re evaluation of conclusions reached previously with regard to the presence in, or absence from, biological fluids of some isomers and their relative amounts is needed.