Heterogeneity of bile pigment conjugates as revealed by chromatography of their anthranilate azopigments

Abstract

1. Azopigments derived from conjugated bile pigments by coupling with the diazonium salt of ethyl anthranilate are analysed conveniently by quantitative t.l.c. or by column chromatography on CM-cellulose. 2. By chromatographic studies combined with a series of chemical tests six groups of azopigments were demonstrable in preparations from bile and from icteric urine of man. Azobilirubin and its β-d-monoglucuronide have hitherto been considered to be the only major derivatives that can be obtained from human bile pigments. In the present work, other azopigments accounted for 30–40% of the total azopigment material, and the amounts of these showed considerable variation among biological fluids. 3. The divergence of the present results from earlier work is probably related to the use of milder diazotization conditions and of chromatographic techniques with a high resolving power. 4. The thin-layer chromatographic systems developed allow rapid and quantitative analysis of azopigments derived from bile pigments.

The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile

“…The structures of the first two azopigment conjugates have been well established by chemical and enzymic analysis, combined g.l.c. and mass spectrometry, and nuclearmagnetic-resonance spectrometry (Heirwegh et al, 1970;Fevery et al, 1971;Compernolle et al, 1971;Gordon et al, 1974Gordon et al, , 1976, but there has been some controversy about the nature of pentose-containing conjugates (Kuenzle, 1970). Because of this and for the other reasons outlined above, we have additionally characterized the structure of azopigment a2.…”

“…In this reaction the tetrapyrrole is split into two dipyrrolic molecules and then coupled with a diazonium salt (Heirwegh et al, 1973). Although this procedure is valid, and has been used to elucidate the chemical nature of the half-molecules (Heirwegh et al, 1970;Compernolle et al, 1971;Fevery et al, 1971;Gordon et al, 1974Gordon et al, , 1976, it does not permit one to determine if bilirubin is secreted as mixed, di-, or mono-conjugates.…”

“…The compounds formed include azopigment ao or dipyrrolic azo-A bilirubin, azopigment 3 or azobilirubin monoglucuronide (Heirwegh et al, 1970;Gordon et al, 1976), azopigment a3 or azobilirubin monoglucoside (Heirwegh et al, 1970;Gordon et al, 1974), and, in the pigments from dog gall-bladder bile, azopigment a2. The latter has previously been identified as a pentose-containing azopigment, azobilirubin monoxyloside Compernolle et al, 1971), by mass-spectrometric and n.m.r.…”

Changes in the Composition of Bilirubin‐IX Isomers During Human Prenatal Development

“…Bilirubin-IXP and bilirubin-1x6 are extremely hydrophilic because polar propionyl, carbonyl and imino groups are exposed on the molecular surface and therefore they cannot form intramolecular hydrogen bonds. It has been confirmed that IXg and 1x6 isomers are excreted from the liver into the bile without conjugation (Heirwegh et al, 1970;Yamaguchi et al, 1979b).…”

“…However, small amounts of other bilirubin isomers as well as several similar unidentified pigments have been found under both physiological and pathological conditions in vivo (Heirwegh et al, 1970;Gordon et al, 1977;.…”