Bilirubin Binding in the Plasma of Newborns: Critical Evaluation of a Fluorescence Quenching Method and Comparison to the Peroxidase Method

Abstract

SummaryBilirubin binding affinities and capacities and apparent unbound ("free") bilirubin levels were determined in serum samples from 47 high-risk newborns, in 22 samples of cord serum, and in serum samples from 15 Greek children with marked hyperbilirubinemia, by both fluorescence quenching and peroxidase methods. The free fatty acid:albumin molar ratio was also determined for serum samples from high-risk newborns. In vitro and in vivo measurements suggest that free fatty acids are rarely present at levels that produce significant displacement of bilirubin, which is in agreement with previous studies. The two bilirubin binding assays showed only fair correlation with sizable discrepancies for many specimens. Technical difficulties inherent in the fluorescence quenching method and possible sources of error are discussed. Our observations suggest that routine application of these two assays as the primary criterion for therapeutic intervention (e.g., exchange transfusion) is premature.

Molecular Basis of Indomethacin-Human Serum Albumin Interaction

“…Figure 7B shows a Scatchard plot of the interaction. The value of the association constant for binding of the ®rst molecule of bilirubin to albumin (5Á71 6 10 7 mol À1 ) was found to be in close agreement with previous reports (Berde et al 1984;Minchiotti et al 1993). Upon addition of higher concentrations of indomethacin (i.e.…”

“…Binding of ligands to receptor proteins often produces a decrease in protein¯uorescence. This quenching phenomenon has previously been used by several workers to study bilirubin±albumin (Levine 1977;Berde et al 1984;Mir et al 1992;Minchiotti et al 1993;Tayyab & Trivedi 1995) and other ligand±protein interactions (Eftink & Ghiron 1981;Gonzalez-Jimenez et al 1995). In the present study we used the same technique to study the interaction of indomethacin with HSA.…”

Effect of storage and freezing on unbound bilirubin measurement

“…Emerging evidence suggests that the fraction of TSB not bound to albumin, referred to as the unbound bilirubin concentration (B f ), may be a better predictor of bilirubin-induced neurotoxicity than TSB. [1][2][3] A semi-automated, Food and Drug Administration approved method based on the peroxidase method has been most widely used to measure B f [4][5][6] but is not routinely available in clinical laboratories. [7] Current bilirubin research is aimed at evaluating the usefulness of B f .…”

Neonatal Bilirubin Toxicity

“…Medium chain fatty acids (12 to 14 carbons in length) may actually enhance bilirubin binding. More recent evidence suggests that the first 2 molecules of fatty acid bind to sites some distance from the primary bilirubin site, and that the fatty acids may alter bilirubin binding by causing significant conformational changes in albumin which affect the distant bilirubin sites (Berde et al 1979(Berde et al , 1984. The mechanism mediating this effect is controversial.…”