Bilirubin conjugates isolation

As yet only a limited number of bilirubin conjugates can be synthesized in vitro. Mono- and diglucuronosides of bilirubin predominate in rat bile (F6, Hll, 03, S6). In accordance with these observations, isolated liver preparations and liver slices from rat (S6), and rat hepatoma cells (R9)... 

Icterogenin is a triterpenic acid, isolated from Lippia rehmanii, which causes jaundice in sheep (R12). This compound reduces excretion of bilirubin, conjugated bilirubin, BSP, and phylloerythrin in rabbits (H7) and, more specifically, the maximal transport rate of BSP and bilirubin in man (A16). On histological examination, livers from animals that have received icterogenin show changes in the number and location of lyso-... 

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. 

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. 

Bunamiodyl (B19, S26) and iodopanoic acid (S17) cause an elevation of BSP retention. These compounds, and the related iodinated aryl compound iodipamide, competitively inhibit the enzyme S-aryl glutathione transferase (B38). However, these compounds also affect the excretion of bilirubin (B21, B33), which is excreted as a glucuronide, of indocyanine green (H4), which is not conjugated by the liver, and of conjugated bilirubin (B21). Bunamiodyl and iodipamide reduce the uptake of BSP by liver slices (B6, B7) and by the isolated perfused liver (B19). 

Finally it must be stressed that a sulfate of bilirubin does not occur naturally, despite the fact that such a compound has been repeatedly inferred to represent the alkali-stable fraction. Experiments performed with a synthetic sulfate compound (Kuenzle, 1970b,c) have shown that diis or a similar derivative would not have escaped detection with the method employed to isolate the alkali-labile conjugates. 

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