The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile

Abstract

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.

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

“…The phospholipids and bile pigments were precipitated from bile by using acetone saturated with MgCI2, and the bile pigments were then solubilized in water and extracted into chloroform containing 10mM-tetraheptylammonium chloride (Gordon et al, 1976). Approx.…”

“…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.…”

“…To this end we have devised a simple reproducible method for the isolation of the native bilirubin tetrapyrroles from bile-containing human duodenal juice and dog gall-bladder bile. This procedure was first used in the separation of bilirubin diglucuronide (Gordon et al, 1976). The method has now been improved and the other tetrapyrroles present in dog and human bile have been identified.…”

“…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.…”

“…Molar equivalence between the conjugating moiety and azopigment was not documented in this case. Since problems in azopigment identification tend to arise when this equivalence is neglected (Gordon et al, 1976), it seemed to us appropriate to confirm the nature of azopigment a2. These results are also presented here.…”