HYDROPHOBIC INTERACTION OF LYSOPHOSPHOLIPIDS AND BILE-SALTS AT SUBMICELLAR CONCENTRATIONS

A series of environment-sensitive, fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)monoacylphosphatidylethanolamine (N-NBD-lysoPE) probes of differing acyl chain length (C12 - C18) was used to demonstrate the hydrophobic interaction between lysophospholipids and two different bile salts at concentrations below their respective critical micelle concentrations (cmc's). Formation of submicellar aggregates in the presence of bile salt - phospholipid mixed micelles could facilitate lipid absorption in the intestine. To ensure the use of submicellar lysolipid concentrations in the experiments, the cmc of each fluorescent lysolipid probe was determined by concentration-dependent self-quenching. The cmc values obtained for the various N-NBD-lysoPE probes were as follows (μM): monolauroyl, ≥ 40; monomyristoyl, 4; monopalmitoyl, 0.3; monostearoyl, 0.04. Probe concentrations well below their respective cmc's were used in all experiments. The fluorescence of a solution of each lysolipid probe was monitored as the concentration of bile salt was gradually increased. The increase in fluorescence was taken as a measure of the ability of the bile salt molecules to complex with the probe molecule, thereby increasing the fluorescent yield of the lysolipid probe molecule. Determination of the cmc of the bile salts in the presence of the lysolipid probe was made in parallel with the fluorescence measurement by monitoring the increase in light scattering of the solution. Both bile salts were shown to induce maximal increases in fluorescence of the N-NBD-lysoPE derivatives at concentrations of bile salt well below their respective cmc values, indicating the existence of submicellar lysolipid-bile salt aggregates. Examination of the data within the framework of the Hill equation model for cooperativity indicated that between two and five molecules were a minimum estimate for the number of bile salts required to give maximal fluorescence. The affinity of the bile salt - lysolipid interaction was found to increase with the hydrophobicity of both the bile salts (TDC > cholate) and the lysolipid (C18 > C16 > C14 > C12).