Effect of serum dilution on apparent unbound bilirubin concentration as measured by the peroxidase method

I studied the effects of serum dilution and oxidation of albumin-bound bilirubin on the accuracy of results by the peroxidase method for assessing bilirubin-albumin binding. The apparent concentration of unbound bilirubin decreases with dilution of bilirubin-enriched serum or defatted-albumin solutions, the effect being more marked with serum. The decrease in apparent unbound bilirubin does not appear to be due to slow oxidation of albumin-bound bilirubin. Instead, the bilirubin-albumin complex has a much lower apparent dissociation rate constant (K-1) in serum (0.0033 [SD 0.0002] s-1) than in solutions of defatted-albumin (0.017 [SD 0.006] s-1), causing the dissociation of the complex to be rate-limiting when serum is analyzed at the currently recommended 40-fold dilution and peroxidase concentration (0.11 mu mol/L). In addition, dilution appears to enhance bilirubin binding by serum but not by defatted-albumin solutions. Decreasing the serum dilution and peroxidase concentration may significantly improve the accuracy of the peroxidase test. The actual correlation between clinical bilirubin toxicity and specific unbound bilirubin concentrations, however, remains to be determined.