Abstract
Purpose: :
Lipocalin (TL) is a major tear protein, and it binds lipids. It is equivocal as to how it interacts with the Meibomian lipid layer. The purpose of this study was to investigate how it interacts with human and bovine Meibomian lipid films and how the presence of lysozyme (LZ) or lactoferrin (LF) might affect this interaction.
Methods: :
Human TL was purified by size exclusion and ion exchange chromatography under non-denaturing conditions. The surface activity of TL by itself was tested on a Langmuir trough. Its adsorption and interaction with Meibomian lipids was examined by injecting TL (35µL of 1mg/mL), TL-LZ, or TL-LF mixtures into a buffered aqueous subphase (80mL) and observing their penetration into human or bovine Meibomian lipid films held at a pressure of 10mN/m spread on a buffered subphase in a Langmuir trough. Interaction was monitored by changes in pressure and fluorescence microscopy. Experiments were conducted at 20°C and 37°C. Pressure-area isocycles were also carried out.
Results: :
Purified TL resulted in a single 17kDa band on a SDS-PAGE gel. TL by itself was less surface active than LZ or LF. TL much more readily adsorbed to a human Meibomian lipid film than a bovine Meibomian lipid film. However, its adsorption was relatively slow compared with LZ or LF. Adsorption was much faster and resulted in a much higher equilibrium pressure at 37°C compared with 20°C. Adsorption of TL to a human Meibomian lipid layer caused it to gel (fibrous appearance). Such gelling was not seen after adsorption to a bovine Meibomian lipid layer. TL-LZ and TL-LF mixtures adsorbed more quickly than TL alone, and interacted quite differently with the Meibomian lipid films. Again the lipid layer became very stable after penetration, even at 37°C, but there were distinct protein rich regions that corresponded with identical lipid rich regions.
Conclusions: :
TL interacts differently with bovine and human Meibomian lipids. By itself, TL adsorption into a Meibomian lipid layer is very slow and unlikely to be physiologically significant. However, mixed with LZ or LF, in similar ratios to that encountered in the tears, adsorption is much more rapid. This indicates that the behaviour of TL in tears is affected by the presence of other tear proteins.
Keywords: cornea: tears/tear film/dry eye • lipids