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

Summary: Bilirubin 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.