Evidence for Phospholipids on the Surface of Human Tears.

Purpose: The structure of tears has been theoretically considered three tiers with lipids at the air interface, aqueous and proteins in the subphase, and anchored mucins on the corneal epithelial surface. While many lipid and protein species have been identified in tears by mass spectrometry, the localization of the major components within the tear film structure remains speculative. The most controversial components are phospholipids. Although surface active, phospholipids have been presumed to be bound entirely to protein in the aqueous portion of tears or reside at the aqueous-lipid interface. Herein, the possibility that phospholipids are adsorbed at the air-surface interface of tears is interrogated. Methods: Polarization-modulated Fourier transform infrared reflective absorption spectroscopy (PM-IRRAS) was used to study the presence of phosphate signals at the tear surface. In order to constrain the depth of signal detection to the surface, an extreme grazing angle of incident radiation was employed. Nulling ellipsometry was used to confirm the presence of monolayers and surface thicknesses when surface active reagents were added to solutions. Results: Surface selection of PM-IRRAS was demonstrated by suppression of water and phosphate signals in buffers with monolayers of oleic acid. Phosphate signals were shown to reflect relative concentrations. Absorption peaks attributable to phospholipids were detected by PM-IRRAS on the human tear film surface and were augmented by the addition of phospholipid. Conclusions: The data provide strong evidence that phospholipids are present at the surface of tears.