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. 1. 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. 2. The bilirubin-IX isomers were structurally characterized (a) at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and (b) by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. 3. The absorption spectrum of bilirubin-IX alpha differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin-IXbeta allows easy differentiation from bilirubin-IXdelta. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXalpha- and non-alpha isomers. 4. The bilirubin-IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro-scale by (a) t.l.c. with respect to reference compounds and (b) subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. 5. 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.

Bile Pigment Composition and Bilirubin Esterification in the Developing Chick

“…The following reference con~pounds were prepared as reported previously: bilirubin C-8 monomethyl ester, bilirubin C-12 monomethyl ester, bilirubin dimethyl ester, and xanthobilirubic acid monomethyl ester (18); bilirubin-IXP, 1x7, and -1X6 in their unesterified form, monomethyl ester form, and dimethyl ester form (19); bilirubin-IIIa and -XIIIa (20); and the dimethyl esters of biliverdin-IXa, -IXP, -IXy, and 1x6 (18). The monomethyl ester and unesterified forms of the latter four verdin isomers were prepared by partial saponification (18), and were isolated by thin-layer chromatography using chloroform/methanol/water (40:9:1, vollvol) as solvent system.…”

“…Azopyrromethenes prepared by trcatment of dog gallbladder bile with diazotized 0-ethyl anthranilate were employed as reference material for structural analysis of bilirubins in the avian body fluids (19).…”

Hepatic jaundice - a review

“…[1] [2] It is commonly associated with itchiness. [3] The feces may be pale and the urine dark. [4] Jaundice in babies occurs in over half in the first week st is not a problem.…”

“…[7] The itchiness may be helped by draining the gallbladder or ursodeoxycholic acid. [3] The word jaundice is from the French jaunisse, meaning "yellow…”

“…[7] The itchiness may be he draining the gallbladder or ursodeoxycholic acid. [3] The word jaundice is from the French jaunisse, meaning "yellow disease". [11] Types On the basis of causes Jaundice can be classified into three types.…”