Preventing Kernicterus: Serum Albumin-Bilirubin Binding

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DESCRIPTION (provided by applicant): Unconjugated bilirubin (UCB) is the normal, but toxic end product of red blood cell catabolism and circulates in the plasma. However, because of its very low aqueous solubility of <70 nM, it is transported primarily by human serum albumin (HSA). UCB is conjugated in the liver into a water-soluble compound, conjugated bilirubin, for excretion into the bile. In near term and full term infants the enzyme responsible for conjugating bilirubin is not yet fully functional and UCB levels can exceed 175 ?M, though never exceeding the binding capacity of HSA, with more UCB being presumably pushed into the lower affinity binding sites. Since the circulating HSA now contains a high amount of bound UCB, UCB can more easily diffuses through plasma membranes, allowing UCB to accumulate in tissues, causing jaundice. Similarly, these high circulating levels of TSB (>428 ?M) also diffuse across the blood brain barrier where it causes severe neurological deficits in untreated infants. This disease is Kernicterus, or bilirubin-induced encephalopathy, and causes athetoid cerebral palsy, deafness, upgaze abnormalities, and hypoplasia of baby teeth enamel. Since this devastating disease is preventable and reversible with the proper treatment, the American Academy of Pediatrics has developed treatment guidelines. In order to have healthy birth outcomes and to prevent these children from becoming dependent on the healthcare system, it is imperative to efficiently and quickly identify those infants in need of treatment. For this, measures of the total serum bilirubin in the body are performed to determine the potential for bilirubin toxicity. However, this measurement has been deemed a poor determinant of bilirubin toxicity. In order to rectify this, a more complete understanding of the mechanisms of transport of bilirubin throughout the body is needed. We propose to determine the primary and secondary binding sites on HSA for UCB. The exact binding site(s) of UCB have evaded years of research, and these sites and their relationships to fatty acid (FA) and drug binding sires needs to be known at the molecular level. We will use novel applications of 1D and 2D Nuclear Magnetic Resonance (NMR) Spectroscopy, to study aqueous complexes of UCB with HSA and with/without FAs at their physiological concentrations. For direct studies of binding and identification of the primary bilirubin binding site, we will use 13C-labeled bilirubin synthetic analogs together with our methods established for identifying FA sites, namely drug displacement techniques and HSA mutants. Additionally, for clinical applications, it is critical to identify the lower affinity sites and determine in a site specific manner how FA and drug binding forces UCB into the lower affinity sites. These additional studies will utilize unlabeled bilirubin and displacement by 13C-labeled FA. Not only does this proposal identify the primary and secondary bilirubin binding sites on HSA, but will also study how drug binding affects bilirubin binding, for example, predicting whether binding of therapeutic drugs may have an unintended effect of displacing bilirubin into lower affinity sites, and thus increasing the chances for accumulation of UCB in the tissues and brain.