Effects of keratin and lung surfactant proteins on the surface activity of meibomian lipids.

PURPOSE: In vitro studies indicate that surface tension and surface viscosity of the tear film lipid layer (TFLL) are governed by interactions between meibomian lipids and proteins from the aqueous layer. The role of minor tear proteins with strong lipophilic properties or those correlated with pathological states is still unknown. The discovery of lung surfactant proteins (SPs) in tears and keratin in normal and abnormal meibomian gland excretions warrants investigation into their effects on the surface activity of meibomian lipid films. METHODS: Commercial keratin and bovine lung SPs were used in vitro to assess the surface pressure of meibomian lipid films using a Langmuir trough. RESULTS: The pressure-area profiles of meibomian lipid films seeded with SPs (2.5 µL; 0.1 µg) demonstrated hybrid characteristics between meibomian lipid films alone and SPs alone but reached much higher maximum surface pressures (approximately 30 vs. 24 mN/m). Microscopically, the appearance of meibomian lipid films was not altered by SPs. Maximum surface pressure of meibomian films premixed with keratin was much higher than meibum alone. The pressure-area isocycles appeared more like those of meibomian lipids with a low concentration of protein and more like pure keratin films at a high concentration. CONCLUSIONS: The data strongly indicate that SPs and keratin likely interact with the TFLL. SPs are likely to act as strong surfactants and to reduce the surface tension of the lipid layer. Excess concentrations of keratin as identified in patients with meibomian gland dysfunction could disrupt the normal structure of the meibomian lipid film.

Interfacial dilatational viscoelasticity of human meibomian lipid films.

PURPOSE/AIM: The meibomian lipid layer is able to withstand the enormous stresses and deformations that occur during blinking due to the combination of its elastic and viscous properties. The purpose of this study was to measure the dilatational viscoelasticity of in vitro meibomian lipid films and compare how these properties differ between room temperature and physiological temperatures. Viscoelasticity was also compared with meibomian lipid films seeded with cholesterol or ß-carotene (the levels of these lipid species change in disease states). MATERIALS AND METHODS: Dilatational viscoelasticity (E) was measured using an oscillating pendant drop method. Measurements were carried out on spread films at the air-water interface as a function of frequency (0.1256-12.56 rad/s) at various temperatures between 18-43 °C. RESULTS: Generally, E gradually decreased as the overall temperature was increased. At both 37 and 20 °C, films demonstrated that the elastic modulus (E') was more dominant than the viscous modulus (E″), indicating films were more solid-like than fluid-like, regardless of temperature. E' and E″ were also dependant on frequency, indicating some molecular rearrangements of the lipid molecules as films were compressed and expanded. Films seeded with cholesterol or ß-carotene showed a modest increase in the moduli. CONCLUSIONS: These results are consistent with previous findings which have predicted and indicated that the meibomian lipid layer is a viscoelastic film at the air-liquid interface. These properties are integral to how the tear film lipid layer is able to maintain its structure, and hence integrity of the ocular surface.

Adsorption of human tear lipocalin to human meibomian lipid films.

PURPOSE: Tear lipocalin (Tlc) is a major lipid binding protein in tears and is thought to have an important role in stabilizing the Meibomian lipid layer by transferring lipids to it from the aqueous layer or ocular surface, or by adsorbing to it directly. These possible roles have been investigated in vitro using human Tlc. METHODS: Tlc was purified from human tears by size exclusion chromatography followed by ion exchange chromatography. Three additional samples of the Tlc were prepared by lipidation, delipidation, and relipidation. The lipids extracted from the purified Tlc were analyzed by HPLC-MS followed by fragmentation. Adsorption of these different forms of Tlc to a human Meibomian lipid film spread on the surface of an artificial tear buffer in a Langmuir trough were observed by recording changes in the pressure with time (Pi-T profile) and monitoring the appearance of the film microscopically. These results were compared with similar experiments using a bovine Meibomian lipid film. RESULTS: The results indicated that Tlc binds slowly to a human Meibomian lipid film compared with lysozyme or lactoferrin, even at 37 degrees C. The adsorption of Tlc to a human Meibomian lipid film was very different from its adsorption to a bovine Meibomian lipid film, indicating the nature of the lipids in the film is critical to the adsorption process. Similarly, the different forms of Tlc had quite distinct adsorption patterns, as indicated both by changes in Pi-T profiles and the microscopic appearance of the films. CONCLUSIONS: It was concluded that human Tlc was capable of adsorbing to and penetrating into a Meibomian lipid layer, but this process is very complex and depends on both the types of lipids bound to Tlc and the lipid complement comprising the Meibomian lipid film.