A Novel Approach to Measure Flip-flop of Very Long Chain Saturated Fatty Acids (VLCFA) in Model Membranes

VLCFA (>18 carbons) are normally present in very low proportions compared to other FA in humans, and their elevation accompanies debilitating and sometimes lethal neurological disorders. To understand whether VLCFA are causative agents or biomarkers requires detailed knowledge of their transport and metabolism, studies of which are often impeded by their extremely low aqueous solubility. Here we focus on how rapidly VLCFA can move across a protein-free phospholipid bilayer. To overcome the solubility limitation, we prepared aqueous complexes of VLCFA with methyl-β-cyclodextrin (CD), and tested whether VLCFA dissociate rapidly enough to study the kinetics of their transport in membranes. This VLCFA/CD complex was used for delivering VLCFA to small and large unilamellar vesicles (phosphatidylcholine) containing fluorescent probes, pyranine and fluorescein-phosphatidylethanolamine (FPE). Pyranine, a water-soluble pH probe, was trapped inside vesicles, and FPE, a membrane surface potential probe, was added to the outer monolayer of the vesicles. Using spectrofluorometry, FPE detected rapid binding of VLCFA (half-time <4 sec), which established that desorption from the CD carrier was at least this rapid. Pyranine detected the transbilayer movement (flip-flop) of VLCFA by a rapid decrease in pH (t1/2 <4 sec). Together these experiments demonstrate rapid binding and flip-flop of VLCFA with 20 to 26 carbons across the bilayer. The magnitude of the intensity changes were dependent on the concentration of VLCFA added. The kinetics of flip-flop was independent of temperature, media viscosity and lipid composition of phospholipid bilayer. Our results suggest that putative proteins such as Adrenoluekodystropy protein (ALDP) are not required for the flip-flop of VLCFA between membrane leaflets and that other mechanisms for the physiological role(s) of ALDP in neuropathophysiology require investigation.