Relationships of bilirubin binding parameters

The apparent unbound bilirubin concentration by the "peroxidase" method (U) and the total unconjugated bilirubin in blood (T), albumin-bound bilirubin (B), and reserve bilirubin binding capacity (R) by the bilirubin hematofluorometer were measured in 164 specimens from 98 neonates and in a series of artifactual specimens, made by adding bilirubin to the blood of a single adult donor. Linear correlations between U and (B/R) were found for both the prepared specimens (r = 0.99) and the clinical specimens (r = 0.87), with the slopes of the regression lines being close to the reciprocal of the albumin-bilirubin binding constant, a prediction of the mass action law. An excellent linear correlation was observed for the prepared specimens (r = 0.96) between U and (T--B), the concentration of bilirubin bound to low-affinity secondary sites ("loosely bound bilirubin"). A simple model for low-affinity binding of bilirubin in blood predicts this simple relation. A significant linear correlation between U and (T--B) was found for the clinical specimens, although the correlation was less good (r = 0.72), as one would expect. The demonstrated simple linear relationships between U, (B/R), and (T--B) support the hypothesis that both the hematofluorometer and peroxidase methods provide valid measurements of bilirubin binding status.