Covalently protein-bound bilirubin conjugates in cholestatic disease of dogs

We investigated the occurrence of covalently protein-bound bilirubins in the plasma of dogs with hyperbilirubinemia attributable to hepatobiliary diseases or Coombs test-positive hemolysis. The bilirubins in plasma were measured with the conventional Van den Bergh reaction, by treatment with diazotized p-iodoaniline, and by high-performance liquid chromatography of bilirubin and its methylesters after alkaline methanolysis. All but one dog had covalently protein-bound bilirubin conjugates. The concentration and the fraction of total bilirubins varied in all diseases investigated, but they tended to be low in primary hemolysis. The "biliprotein" complex accounted for 2 to 94% of total plasma bilirubins. Because biliprotein usually is not cleared by the liver, but has a half-life comparable with that of albumin, it prevents the evaluation of the actual state of the underlying disease. Measurement of the total bilirubin concentration exclusively with the Van den Bergh reaction, therefore, is clinically useless. Other methods should be introduced for routine bilirubin assays, permitting the measurement of noncovalently bound pigment as a meaningful estimate of the course of the disease.