Bilirubin conjugates phenylazo derivatives

The overall procedure employed by the author (Kuende, 1970a) to isolate the phenylazo derivatives of the bile bilirubin is shown diagrammati-cally in Fig. 3. Human hepatic bile served as the source of bilirubin conjugates. A total of 15.3 liters of bile was processed in this investigation. The bile was sampled postoperatively from cholecystectomized patients through a T tube. Bile specimens from several patients were pooled, and were kept frozen until processed further. The isolation was carried out in three steps. Gare was taken to apply the mildest of conditions at all stages. 

The alkali-lability of azo pigment B4 was then exploited to obtain both the conjugating and the pigment moiety on a preparative scale. The pigment moiety could easily be identified as the phenylazo derivative of bilirubin by spectroscopic methods (see Section IV,B,l,a). In contrast, the identification of the conjugating moiety was more exacting. 

Several bilirubin conjugates have been isolated from human hepatic bile as the phenylazo derivatives. The major bilirubin conjugates are excreted, not as was formerly thought in the form of glucuronides, but as the acyl glycosides of three aldobiouronic acids, one hexurono l-hexuronic acid, and one pseudoaldobiouronic acid. The conjugation of human metabolites with acidic disaccharides of the above types has not been reported previously. 

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