Mass-spectrometric structure elucidation of dog bile azopigments as the acyl glycosides of glucopyranose and xylopyranose

Abstract

1. The structures of the alpha(2)- and alpha(3)-azopigments, prepared by diazotization of dog bile with ethyl anthranilate, were shown by mass spectrometry and g.l.c. to correspond to azobilirubin beta-d-xylopyranoside and azobilirubin beta-d-glucopyranoside respectively. 2. Both azopigments consist of a mixture of two methyl vinyl isomers having structures (IIIa) and (IIIb) for the alpha(2)-azopigment and structures (IVa) and (IVb) for the alpha(3)-azopigment. Separation of methyl vinyl isomers was obtained by t.l.c. or column chromatography performed on the acetylated azopigments. Hydrolysis of the less polar acetates derived from components (IIIa) and (IVa) gave rise to the azopigment (Ia), whereas hydrolysis of the more polar acetates derived from components (IIIb) and (IVb) gave rise to the azopigment acid (Ib). The positions of methyl and vinyl substituents in compounds (Ia) and (Ib) were assigned on the basis of their n.m.r. spectra. 3. Molecular ions in the mass spectra of the trimethylsilyl and acetyl derivatives of the azopigments indicated the presence of a pentose and a hexose conjugating sugar. 4. The ester functions linking the sugars to the propionic acid side chain of azobilirubin were demonstrated by ammonolysis and identification of the amide of azobilirubin as the aglycone derivative. 5. The sugar moieties were shown to occur as xylopyranose (alpha(2)) and glucopyranose (alpha(3)), bound at C-1, by application of a sequence of reactions performed on a micro-scale. The sugar hydroxyl groups were acetylated and the 1-acyl aglycone removed selectively by treatment with hydrogen bromide in acetic acid. Hydrolysis of the 1-bromo sugar acetates followed by acetylation afforded the alpha- and beta-xylopyranose tetra-acetates and alpha- and beta-glucopyranose penta-acetates, identified by a combination of g.l.c. and mass spectrometry. 6. The validity of this degradation scheme was confirmed (a) by g.l.c.-mass spectrometry identification of the alpha- and beta-1-propionyl derivatives of glucopyranose tetra-acetate, obtained from the alpha(3)-azopigment after final reaction with propionic anhydride; (b) by subjecting the acetates of alphabeta-glucopyranose, alphabeta-xylofuranose and alphabeta-glucofuranose to the same sequence of reactions.

Unconjugated Bilirubin and an Increased Proportion of Bilirubin Monoconjugates in the Bile of Patients with Gilbert's Syndrome and Crigler-Najjar Disease

“…In dog bile, they have been identified as the f3-D-xylopyranoside and the f8-D-glucopyranoside of azodipyrrole, respectively (31,33). The same conclusion has been reached for human bile (Fevery, unpublished work).…”

“…Before the assays, bile samples from the normal controls and adult patients were diluted 21 to 51-fold with distilled water. To obtain sufficient sensitivity in the assay, bile of patients with Crigler-Najjar disease was diluted only 3 to 11-fold, except in the child S (101-fold (19,(32)(33)(34). The ratio of the a,,-EA (pH 2.7) over total azopigment was calculated, thus permitting the determination of the relative amounts of mono-and diconjugated bilirubin-IXa (see below, Validity of methods).…”

Bilirubin conjugates in bile of man and rat in the normal state and in liver disease

“…The disaccharidic conjugating structures described by Kuenzle (8) may perhaps be sought among these fractions. The absence of phosphomolybdate-positive material in purified preparations of azopigments-P and -Y, and evidence obtained previously (4)(5)(6)(7), indicate that these azopigments are not artifacts in the sense that they would represent molecular complexes with other components. It therefore appears that they represent chemical entities which can be studied in relation to physiological and pathological states.…”

“…Cholestasis is known to influence markedly the cholesterol and bile salt compositions because of decreased secretion and increased hepatic synthesis. The presence of (3-and -y-azopigments in the chromatograxms could therefore be due to interaction with components from bile although this is unlikely in the light of previous work (4)(5)(6)(7)(8). As a further test of the previous conclusion that the (3- (26) purified preparations of (3-and y-azopigments were shown to be free of bile salts.…”

“…Dog bile contained relatively large amounts of P-D-xylopyranoside and P-Dglucopyranoside of bilirubin (5,6) whereas the P-D-glucuronoside dominated in bile of rat (4,7). A much more complex composition (4) with existence of disaccharidic conjugating groups (8) was demonstrated for human T-tube bile.…”