The formation of mixed phospholipid: bile salt micelles in the bile and their secretion into and absorption from the intestinal lumen are essential to the excretion of cholesterol, the digestion and absorption of fats, and the formation of chylomicrons. The long- term objective of this research is to understand how these highly water-insoluble phospholipids are rapidly transferred between the different hydrophobic environments during their movement from liver hepatocytes to intestinal enterocytes. This proposal focuses on three steps in the overall process: 1) transfer between micelles; 2) transfer from micelles to the enterocyte brush-border membrane; and 3) transfer across the brush-border membrane. Fluorescent (NBD)-labeled phospholipids and lysophospholipids will be used to monitor the rapid transfer involved in each of the above cases. The spectral properties of the NBD fluorophore (sensitivity of quantum yield to the polarity of the environment, self-quenching at high concentrations, and quenching by Co++) will be used to detect the transfer of NBD-labeled phospholipids from one location to another. Using these techniques, a fluorometer equipped with a stopped-flow rapid mixing device can detect the fast phospholipid transfer that occurs between micelles and membranes, and across membranes. Kinetic modeling will be used to determine the mechanism or mechanisms involved in each of the above processes.
