The Meibomian puzzle: combining pieces together

Abstract

The purpose of this review was to summarize the available information on lipidomic analysis of human meibum and tear film, and critically evaluate the pertinent past and present analytical procedures and results obtained in various laboratories. Human meibum was shown to be a very complex mixture of lipids of various classes. For decades, their exact structures have remained elusive. Because of the limitations of the then-current techniques, most of the complex lipids that constitute meibum could not be analyzed as whole molecules and required prior hydrolysis and/or transesterification of the entire lipid pool. These procedures effectively made it very difficult, and often impossible, to reconstruct the complete structures of the original intact compounds, which prompted us to call this The Meibomian Puzzle. Modern techniques such as high-performance liquid chromatography in combination with mass spectrometry help in solving this puzzle by allowing a researcher to detect and analyze intact molecules of complex lipid compounds, even if present in extremely low concentrations. This current de-facto standard procedure in lipidomic analysis of natural lipids and their mixtures is compared with other experimental techniques such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, gas chromatography, and thin layer chromatography, among the others. The results obtained by older techniques, and their limitations and deficiencies are discussed. It appears that some of the earlier findings did not withstand a scrupulous re-evaluation and need to be modified and/or corrected. The most intriguing development is the virtual absence in meibum of typical phospholipids - an important group of amphiphilic compounds whose role in the human tear film was thought to be to stabilize the entire tear film structure. Instead, another group of previously unidentified compounds, very long chain (O-acyl)-omega-hydroxy fatty acids, appears to be a stabilizing factor which might be related to tear film stability and deterioration. Thus, these compounds may become an important target in biochemistry and (patho)physiology of ocular surface and dry eye research.

Figure 1

Human meibum collected using the eyelid squeezing method and a spatula. Note meibum solidification at room temperature

Figure 2

Partial observation APCI-MS spectra of human meibum recorded in the positive (upper panel) and the negative (lower panel) ion modes in an NP HPLC experiment. The HPLC-MS experiments were performed as described earlier(Butovich et al., 2007a).

Scheme 1

Structures of representative simple and complex lipids relevant to lipidomic analysis of meibum, tear film, and aqueous tears. Hydrolyzable bonds are marked with dashed lines. Complex lipids are color-coded to show their continuous non-hydrolizable fragments.

Scheme 2

Structures of representative polar and amphiphilic lipids relevant to lipidomic analysis of meibum, tear film, and aqueous tears. Ionizable polar groups are shown in red; nonionizable polar groups – in blue; nonpolar groups – in black.

Scheme 3

Comparison chart of common chromatographic methods of meibum lipid analyses.

Scheme 4

Comparison chart of common spectroscopic methods of meibum lipid analyses

Scheme 5

Comparison chart of stand-alone mass spectrometric methods in lipid analysis

Scheme 6

Proposed structure of the tear film (on the left) and the amphiphilic lipid sublayer formed of very long chain (O-acyl)-ω-hydroxy fatty acids and diacyl glycerols (on the right). *Di- and tri-esters as defined by Nicolaides et al (Nicolaides and Santos, 1985). The locations of double bonds in the structures of the very long chain ω hydroxy fatty acids are tentative (Butovich et al., 2009b).