Bilirubin-protein interactions monitored by difference spectroscopy.

Difference spectroscopy is used to monitor bilirubin-protein interactions, to assess the residual binding capacity of proteins for bilirubin. A change in the difference spectra monitored at 482 nm is directly proportional to bound bilirubin up to a molar ratio of bilirubin to albumin of approximately 1; increasing bilirubin beyond the 1:1 molar ratio does not further change the difference spectra. After excess free bilirubin is added, the change in the difference spectrum is proportional to the residual binding capacity of the serum for bilirubin. The risk of kernicterus among neonates may be assessed by monitoring the residual bilirubin binding capacity of serum. This report summarizes our research effort leading to an assay method which requires only 40 microliter of serum and can be completed in less than 10 min.