Penetration of tear proteins into a meibomian lipid layer

Abstract: : Purpose: It is generally believed that a lipid layer forms the outer layer of the pre–ocular tear film and helps maintain tear film stability by lowering its surface tension (increasing surface pressure). Proteins of the aqueous layer of the tear film (which lies beneath the lipid layer) may also contribute to reducing surface tension by adsorbing to the lipid layer or penetrating the lipid layer. The purpose of this study was to compare the penetration of lysozyme, a tear protein, into films of meibomian lipids and phospholipids held at different surface pressures to determine if it were likely that lysozyme is part of the surface layer of the tear film. Methods:A lipid film (meibomian lipids, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), or phosphatidylethanolamine (PE)) was spread onto the surface of a buffered aqueous subphase held at 20ºC or 37ºC in a Langmuir trough. The film was compressed to predetermined pressures and lysozyme was injected into the subphase. The area of the film was held constant and the rate of change in surface pressure was monitored to determine penetration of lysozyme into the surface film. Their behaviour at the surface was examined using epifluorescence microscopy of films doped with a small amount of fluorescently tagged lipid and fluorescently tagged protein. Results:Lysozyme, at concentrations 100 times less than normally occur in tears, readily penetrated a meibomian lipid layer at all pressures tested (max. = 20mN/m). It also penetrated PG, PS, and PE lipid layers, but at a pressure of 20mN/m or above it was excluded. It was unable to penetrate a PC layer, even at low pressure (10mN/m) or at 37ºC. Epifluorescence showed that the protein had penetrated to the surface and the pattern of mixing depended upon the lipid at the surface. Lysozyme was dispersed between the meibomian lipids and there were areas of high lipid or high protein concentrations. For PS, PG, and PE a striped pattern was observed with lysozyme forming a mostly complementary pattern with the lipids i.e. they did not mix. Even though lysozyme did not penetrate the PC layer, epifluorescence showed that it stabilized this layer which then retained its normal patterning of liquid condensed and liquid expanded areas. Conclusions: Lysozyme is likely to be present at the surface of the tear film where it contributes to an increase in surface pressure and helps to stabilize the meibomian lipids in this layer.