Year

2013

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

The tear film is a complex mixture of proteins, mucins and lipids that forms a layer over the anterior surface of the eye and is vital in ocular health. The outer portion of the tear film contains a thin lipid layer, which prevents the excess evaporation of the aqueous phase and aids in the stability of the tear film. The tear film lipid layer contains both polar and non-polar lipids, with the polar lipids thought to act as a surfactant that spread the non-polar lipids across the aqueous surface to produce a stable tear film. Despite the importance of tear film lipids, their complexity and the small volume of samples collected (of which lipids only comprise a small proportion) has made identification of the complete tear lipidome difficult. The use of animal models or analysis of meibomian gland secretions (meibum) – believed to be the major source of tear lipids – have been utilised in an attempt to overcome these difficulties, however recent studies suggest their profiles may not be a true representation of human tear film lipids. The identification of the polar lipidome in tears and meibum has been particularly controversial. While earlier studies reported that phospholipids played an important role in this, more recent studies have failed to detect any significant concentrations of phospholipid. It is this uncertainty that highlights the need for a more detailed analysis into the lipid profile, particularly for polar lipids.

Changes in the tear film have been implicated in contact lens spoliation and intolerance, with tear film components rapidly depositing onto contact lenses following insertion. Research into the deposition of proteins onto contact lenses has been extensive, with contact lens material shown to be a factor in protein deposition.1 Investigations into the deposition of lipids have been more limited, and many studies using in vitro experiments or the measurement of total lipid only. Furthermore, there has been little work into the effect of contact lens wear on tear lipids, which may have implications for contact lens intolerance and contact lens related dry eye. The aim of this thesis was therefore to provide a detailed analysis of phospholipids in human basal tears, meibum and deposited onto contact lenses using electrospray ionisation tandem mass spectrometry.

This thesis established optimised methods for the extraction and analysis of phospholipids from very small volumes of tears (~5 μL). This was achieved using a biphasic extraction followed by nano-electrospray ionisation tandem mass spectrometry. Utilising these methods, the unequivocal presence of phospholipids in human basal tears could be determined. The identification of individual molecules in sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipid classes was established and their concentration was determined. These same phospholipid classes were identified in human meibum samples, however comparison of the phospholipid profiles between tears and meibum showed significant differences, suggesting that meibum may not be the sole source of phospholipids in tears.

The analysis of in vivo deposition of tear film lipids onto contact lenses confirmed that tear phospholipids deposit onto contact lenses. The concentration of both polar and non-polar lipids deposited was significantly affected by contact lens material, with a differential efficacy of lens care solutions also observed.

Tear phospholipid levels were affected by contact lens wear, with both short (i.e., 30 min) and long term (30 days) wear showing significant differences in phospholipid concentration. While there were no differences between contact lens materials, significant differences were observed with the combination of lens and time, but were not consistent across all phospholipids. This is likely due to the large variability of phospholipid concentrations across samples. The methods optimised within this thesis would allow for the analysis of the more abundant lipids, particularly cholesterol esters and the newly characterised (O-acyl)-ω- hydroxy fatty acids, which may show more definitive changes with contact lens wear. This could provide valuable information for research into contact lens biocompatibility, as well as the changes in tear lipids associated with contact lens intolerance and dry eye disease.

The concentration of total phospholipid in tears was measured at 17 ± 2 pmol/mg tear. At these low concentrations, it is unlikely that phospholipids are solely responsible for spreading of the non-polar lipids across the aqueous phase. If phospholipids do play a role in spreading of the tear film lipid layer then it is likely in cooperation with more abundant surfactants such as (O-acyl)-ω-hydroxy fatty acids, which have garnered much attention recently. The work presented in this thesis has confirmed that (O-acyl)-ω-hydroxy fatty acids are not only present in meibum, but also in human tears and deposited onto contact lenses in vivo. Furthermore, the comprehensive structural characterisation undertaken shows that their properties make them ideal candidates to act as a surfactant within the tear film. This could have major implications for tear film stability, dry eye disease and contact lens intolerance.

This thesis answers the question over the presence of phospholipids in human tears, confirming their presence but showing that they are not observed in concentrations high enough to be the sole surfactant provider in the tear film. While phospholipids are present in meibum, their profile is different to that of tears, suggesting that they may not be the sole provider of tear phospholipids. These tear phospholipids are shown to deposit onto contact lenses, with contact lens wear affecting the concentration of phospholipids in tears.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.