311 EFFECTS OF SERUM DILUTION AND OXIDATION OF ALBUMIN-BOUND BILIRUBIN ON THE DETERMINATION OF THE UNBOUND BILIRUBIN CONCENTRATION BY THE PEROXIDASE METHOD

Abstract

The peroxidase test, although a sensitive and precise method for determining the unbound bilirubin concentration in serum from jaundiced neonates, has been reported to be inaccurate due to errors from sample dilution as well as oxidation of albumin-bound bilirubin. These potential errors were investigated in serum and defatted albumin solutions. A decrease in the peroxidase-determined apparent unbound bilirubin concentration (AUBC) with increasing serum dilution was found in bilirubin-enriched umbilical cord sera as well as sera from jaundiced infants, but was less marked in bilirubin-enriched defatted albumin solutions. The dilutional decrease in AUBC did not appear to be due to slow oxidation of albumin-bound bilirubin. Instead, the bilirubin-albumin complex was found to have a much lower dissociation rate constant in serum (K-1 ± SD = 3.3 ± 0.2 × 10-3 sec-1) than in defatted albumin solutions (K-1 ± SD = 1.7 ± 0.6 × 10-2 sec-1), causing the dissociation of the complex to be rate-limiting when serum is analyzed at the currently recommended dilution (1:40) and peroxidase concentration (5.0 μg/ml). In addition, there appears to be a dilutional enhancement of bilirubin binding by serum, but not by defatted albumin. Decreasing the serum dilution and peroxidase concentration should significantly improve the accuracy of the peroxidase test. Further studies are needed to clarify the effects of dilution on serum binding of bilirubin.