The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile.
Bilirubin and its conjugates were extracted from either dog gall-bladder bile or bile-containing human duodenal juice into chloroform containing 10mm-tetraheptylammonium chloride. The intact bilirubin tetrapyrroles were then separated by t.l.c. Structural elucidation was made after coupling of the individual pigments with diazonium salts. Four azopigments were detected: azopigment alpha(o) or dipyrrolic azobilirubin; azopigment delta or dipyrrolic azobilirubin monoglucuronide; azopigment alpha(3) or dipyrrolic azobilirubin monoglucoside; and, from dog gall-bladder bile, azopigment alpha(2). The last conjugate required further verification of its structure. After methanolysis, it was shown by combined g.l.c.-mass spectrometry to contain xylose in a 1:1 molar ratio with the azopigments of bilirubin. Human bile contained 86% bilirubin diglucuronide, 7% bilirubin monoglucuronide monoglucoside diester, 4% bilirubin monoglucuronide and 3% bilirubin. Dog gall-bladder bile had a considerably different composition; it contained 47% bilirubin diglucuronide, 40% bilirubin monoglucuronide monoglucoside diester, 8% bilirubin monoglucuronide, 4% bilirubin diglucoside, 1-2% bilirubin and traces of conjugates containing xylose. The total bilirubin content and proportions of the conjugates did not change in bile that was frozen and stored at -20 degrees C under N(2), whereas in the chloroform/tetraheptylammonium chloride extract, similarly stored, total pigment was slowly lost and the diglucuronide conjugate converted into the monoglucuronide.
D000818:Animals; D001391:Azo Compounds / Q000032:analysis; D001646:Bile / Q000032:analysis*; D001663:Bilirubin / Q000302:isolation & purification*; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D004285:Dogs; D004386:Duodenum; D005704:Gallbladder; D006801:Humans; D007419:Intestinal Secretions / Q000032:analysis*; D013058:Mass Spectrometry; D011758:Pyrroles / Q000302:isolation & purification*; D013997:Time Factors
D001391:Azo Compounds; D011758:Pyrroles; D001663:Bilirubin