Kernicterus, which is caused by the delivery of excessive amounts of bilirubin to the brain, is a devastating neurological problem in neonates. The planned experiments are organized to define how bilirubin is carried in the plasma, delivered to the membranes of the central nervous system, flip-flop to the cytosolic surface of the membranous hemileaflet and then is made available for presumed intracellular toxicity. To this end, three specific aims are proposed. The first focuses on the distribution of bilirubin in plasma and, in particular, HDL and its associated apolipoprotein D (apo D). Preliminary data indicate that HDL is the major non-albumin carrier of bilirubin and that apo D has a very high affinity for this organic anion. Therefore, the studies are designed to demonstrate which of the components of HDL are the most important bilirubin binders and how this might be affected by triacylglycerol, cholesterol, and cholesteryl esters. Reconstitution experiments will enable this determination. In the second specific aim, the kinetics of bilirubin delivery to defined membranes and the determination of transmembrane flip-flop will be studied. Unilamellar liposomes and native cellular membranes will be utilized. In additional studies, cell culture systems will be used. To this end, a series of studies using stop flow fluorometry, 13C-nmr, and fluorescence parallax studies will be performed at various pH, membrane lipid composition, and bile salts. Because bilirubin appears to bind at the surface of phospholipids, its effect on the binding of HDL uptake by isolated liver cells and HepG2 cells will be studied as well. In the third specific aim, the question of apo D expression will be addressed. It is thought that apo D expression in the brain binds bilirubin and perhaps protects the CNS from bilirubin toxicity. Therefore, experiments will be performed to examine the ontogenic expression of apo D in the rat. Furthermore, vitamin A has been shown to enhance the production of apo B and therefore its effect will also be studied. Furthermore, advantage will be taken of homozygous Gun rats which either do or do not develop kernicterus although they have the same serum bilirubin levels. In these rats, variations in apo D expression will be studied.
| Zucker, S D; Goessling, W; Bootle, E J et al. (2001) Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching. J Lipid Res 42:1377-88 |
