Analysis of Comparison of Human Meibomian Lipid Films and Mixtures with Cholesteryl Esters In Vitro Films using High Resolution Color Microscopy

Abstract

PURPOSE. The lipid layer of the tears has been studied in vivo using high resolution color microscopy (HRCM). The purpose of these experiments was to gain further insight into the structure of the lipid layer by applying HRCM to in vitro meibomian lipid films.METHODS. Films of human meibomian lipids, cholesteryl nervonate, cholesteryl palmitate, or their mixtures, were spread on a Langmuir trough. Changes to the films were monitored using HRCM as the films were compressed to different surface pressures. The penetration of albumin into a meibomian lipid film also was studied.RESULTS. Small amounts of meibomian lipids at low pressures formed very thin films estimated to be 5.2 nm thick. Compression caused spots to appear in the films. At higher concentrations, micro lenses were a feature of the film. Cholesteryl nervonate formed a multilayered oil slick that did not change with surface pressure. Cholesteryl palmitate formed a stiff film that collapsed at high compression. Mixtures of cholesteryl nervonate and meibomian lipids showed that they mixed to increase surface pressures above that of the individual components. HRCM also allowed albumin to be seen penetrating the meibomian lipid film.CONCLUSIONS. HRCM combined with in vitro surface pressure measurements using a Langmuir trough is useful for modeling meibomian lipid films. The films often resemble the appearance of the lipid layer of in vivo films. The data indicate that the lipid layer might be modeled best as a duplex film containing an array of liquid crystals. (Invest Ophthalmol Vis Sci. 2012; 53:4710-4719) DOI:10.1167/iovs.12-10022 T he seminal work of Holly and his co-workers 1-6 signalled the beginnings of trying to understand the physical properties of the tear film in relationship to its chemical components. At that time, the lipid layer was perceived as just a blanket preventing evaporation, 7 but Holly began to investigate the role of meibomian lipids in spreading of the tear film across the ocular surface and stabilising the film. This led to a model involving lipids and then a mucin lipid mixture spread from the lid margins across the ocular surface upon eye opening. 2,3 Since this time, to understand further the structure of the lipid layer, a great deal of effort has been placed on determining the composition of meibomian lipids from humans and animals. [8][9][10][11][12][13][14][15][16][17][18][19] The analysis from several groups now suggests a composition of mainly cholesteryl and wax esters (~80%), triglycerides (~3%), and omega hydroxy acyl fatty acids (~5%). [8][9][10][11][12][13][14][15][16][17][18][19] Although the major portion being wax and cholesteryl esters is consistent across the literature, the ratios vary considerably from 13-68% for wax esters and 8-67% for cholesteryl esters. The latest analyses indicate that cholesteryl esters increase with age 18 and wax esters in human meibum estimates are around 40%.19 From their own analysis of meibomian lipid composition, McCulley and Shine proposed a model that described the major lipi...

The real reason for having a meibomian lipid layer covering the outer surface of the tear film – A review

“…Nevertheless, a feature of their models was the formation of clusters of hydrophobic lipids into lenses under high surface pressures. These resemble the spots seen using high resolution lipid layer microscopy of the TFLL in vivo (King- Smith et al, 2011) and of meibomian lipid films in vitro (Millar and King-Smith, 2012). The formation of such lenses may be critical for allowing the spread of the lipid layer following a blink.…”

Four Characteristics and a Model of an Effective Tear Film Lipid Layer (TFLL)

“…3 Meibum is little altered by compression-expansion cycles; thus, for any surface area, surface pressure during expansion is not much different from the surface pressure during compression. 3 More importantly, the surface pressure functions for repeated compression-expansion cycles are similar to that for the first cycle, 3-4,26 indicating that meibum is “respreadable.”…”

“…Additionally, monolayers having high evaporation resistance (characteristic 1), such as saturated fatty acids and their esters, 2 tend to show poor respreadability (characteristic 2). 3,4 Thus, lipid layer composition and structure must involve compromise and trade-off between apparently conflicting characteristics.…”

“…3 In addition to the changes in surface pressure, the appearance of the lipid layer at the end of the first compression-expansion cycle may be quite different from the original appearance. 4 If an artificial meibum substitute were composed of lipids that collapsed irreversibly, then the substitute would fail to provide a barrier to evaporation.…”

“…As an example, cholesteryl nervonate, derived from the mono-unsaturated nervonic acid, has much more repeatable compression-expansion cycles than cholesteryl palmitate, derived from the saturated palmitic acid. 4 …”