Bilirubin conjugates identification

In liver, bilirubin undergoes enzymic conjugation and is secreted in bile in the form of water-soluble derivatives. A large body of literature279 exists on the separation and identification of bilirubin conjugates appearing in normal and pathologic bile, icteric sera, and urine. 

Extrahepatic biliary atresia may involve all or part of the extrahepatic bifiary tree. The gallbladder is usually absent. Involvement of the hepatic or common duct leads to the characteristic syndrome of severe cholestatic jaundice. It occurs in 1 in 10,000 births, with females more commonly affected than males. Jaundice and pruritus usually appear in the first week. Stools are pale and the urine is tea colored. Jaundice is deep, but the aminotransferases are only mildly elevated. If jaundice persists beyond 14 days of age, a direct or conjugated bilirubin measurement must be performed to exclude biliary atresia. If it is elevated, the urine should be tested for bile and the stool color inspected if the color is not green or yellow, biliary atresia is likely. Early identification of this condition is essential if these infants are to benefit fi"om the operation of portoenterostomy, which should be performed no later than 60 days after birth. If portoenterostomy is not successful, liver transplantation is the treatment of choice. Children rarely live beyond 3 years unless the lesion is surgically correctable. 

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. 

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