Measurement of bilirubin-protein conjugates in serum and application to human and rat sera.

Evidence suggests that the esterified bilirubins in serum of patients with conjugated hyperbilirubinemia can react with serum protein, predominantly albumin, producing bilirubin-protein conjugates. To specifically measure this pigment fraction, we now have developed an assay that requires neither sample preincubation nor special equipment and is based on (1) selective removal of the bilirubins that are reversibly bound to serum protein by using organic solvent extraction, and (2) subsequent measurement of the bilirubin-protein conjugates in the denatured protein pellet by a diazo method. The accuracy of the bilirubin-protein conjugate assay was verified by determining the individual recoveries of the various pigment fractions known to occur in patient serum samples. Near-complete removal (approximately 97%) of unconjugated bilirubin and its various monoesterified and diesterified bilirubins from the serum proteins was demonstrated, with quantitative recovery of total serum protein (greater than 99%) and of bilirubin-protein conjugates (greater than or equal to 95%; tested with purified bilirubin-albumin conjugate). The assay has a demonstrated linearity for bilirubin-protein conjugate concentrations between 3 mumol/L and 170 mumol/L, is precise (total and within-day coefficient of variation, assessed over a 12-month period, less than or equal to 5%), and is essentially free of interference by hemoglobin (for hemoglobin concentrations up to 5.0 gm/L). A fair linear correlation (r = 0.975) was found between the bilirubin-protein conjugate assay results and the values for diazo-positive pigment in serum that is not accounted for by unconjugated bilirubin and its esters. Bilirubin-protein conjugates were found only in sera of patients and rats with conjugated hyperbilirubinemia, not in those with unconjugated hyperbilirubinemia.