Effect of divalent cations on the surface-pressure-area (-A) profiles of human meibomian lipids

Purpose: Previously we have shown that the major proteins found in tears are able to penetrate a bovine Meibomian lipid film. This implies that the outer layer of tear film is not a lipid blanket, but also includes proteins and these are likely to contribute to this layer’s role in reducing surface tension and evaporation. The composition of bovine Meibomian lipids differs from human Meibomian lipids, so we have now investigated whether the surface tension properties and protein penetration of human Meibomian lipids differ from bovine Meibomian lipids. We also investigated the effects of divalent cations on the surface tension properties of human Meibomian lipids. Methods: Human Meibomian lipids were collected by extrusion from the eyelids. These lipids dissolved in chloroform were applied to the surface of an artificial tear solution (AT) on a dual barrier Langmuir trough and the dynamic surface pressure was measured using a Wilhelmy plate. Temperature was maintained at 20°C or 37°C. The effects of different sized divalent cations on the surface pressure-area (Π-A) profile were measured. Barium, calcium, or magnesium ions at various concentrations (0.2mM-1mM) were included in the subphase. Protein penetration of the Meibomian lipid layer was also measured by applying proteins into the subphase. Results: Π-A profiles of human Meibomian lipids showed continuous increase in pressure with almost no hysteresis at but showed pronounced hysteresis at 20°C. The maximum pressure obtained was ~20mN/m when the estimated surface area per molecule was 0.5Å2 indicating multiple layers had formed. Even at these high surface pressures, proteins normally found in the tear film (lysozyme, and lactoferrin) were able to penetrate the lipid layer after they were added into the subphase. Lysozyme penetrated more readily than lactoferrin and both penetrated much more quickly at 37°C. Only subtle differences could be seen between in the pressure area profiles for the different cations in the subphase. Particularly notable was the area per molecule was reduced compared with no divalent cations in the subphase. Conclusions: Π-A profiles of human Meibomian lipids differ from those of bovine Meibomian lipids at 20°C but were similar at 37°C. Proteins found in tears are able to penetrate human Meibomian lipids even at pressures where multiple lipid layers must have formed. This supports the idea that the outer layer of the normal tear film comprises proteins as well as lipids. Divalent cations have subtle effects on the lipid layer which might be an important consideration in excipients used in eye-drop formulations.