Factors affecting the binding of bilirubin to serum albumins: validation and application of the peroxidase method.

The unbound "free" bilirubin concentration (Bf), not the total bilirubin concentration, is the critical determinant of cellular uptake and toxicity of bilirubin. We compared Bf measured by a modified peroxidase method with published data obtained with ultrafiltration and examined conditions that affect the affinity (KF) of human (HSA) and bovine (BSA) serum albumin for bilirubin. The peroxidase and ultrafiltration methods yielded similar KF values that decreased with increasing HSA concentration and the presence of 50 mM chloride. When related to ionic strength, inhibition of BSA-bilirubin binding by chloride, bromide, and sulfate were similar, whereas phosphate buffer had a smaller effect. KF was lower at 37 degrees C than at 25 degrees C for HSA but not for BSA. KF for BSA was similar at pH 7.4 and 8.0. BSA and FCS had similar binding properties. The close agreement of Bf and KF values determined by the peroxidase method with published results obtained by ultrafiltration validates both methods and supports the use of the peroxidase method as a practical technique for measuring Bf under steady state conditions in minimally diluted serum or culture medium.