Unpacking the human tear film

Associate Professor Igor Butovich from the Department of Ophthalmology, University of Texas’ Soutwestern Medical Center in Dallas, delivered a Visiting Fellowship presentation at the University of New South Wales’ School of Optometry and Vision Science (SOVS), in late 2018.Butovich has spent the best part of 40 years researching the human tear film. Much of the minutiae we now know about it is a direct result of what he, his colleagues, and graduate students have discovered over that time.However, his research interests are far broader that just the tear film, and include areas such as lipidomics, enzymology, and bioanalytical chistry. The sophistication of the latter powers much of his and his team’s research output.His lab’s bioanalytical analysis endeavours target ocular aspects of biochistry, biophysics, and physiology. Experimentally, his lab uses High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), mass spectrometry (in combination with HPLC if relevant), Nuclear Magnetic Resonance (NMR) spectroscopy, and Ultraviolet (UV) spectrometry.His current focus is on causes of, and appropriate treatments for, Dry Eye (DE). To assist with the laboratory side of that pursuit, he is researching the most appropriate animal model of DE and the human tear film.Almost paradoxically, although Butovich and his colleagues’ work is well referenced in the Tear Film & Ocular Surface Society (TFOS) DEWS 2 report (2017), he was not a mber of any DEWS 2 sub-committee. However, he was a mber of the Research sub-committee of the DEWS 1 report (2007).The Meibomian GlandButovich opened his presentation by describing the Meibomian Glands (MGs) collectively as a lipid-making machine that is unique to mammals. They have been 25 to 40 glands in the upper eyelid, and 20 to 30 in the lower eyelid.The MGs are holocrine glands first described in 1666 by German scholar Heinrich Meibom. Infrared photography is the preferred method of imaging th. The tears thselves are a less obvious entity, because the tear film is not of uniform thickness and has a varying structure.{{image2-a:r-w:250}}Using HPLC, GC, and mass spectrometry, Butovich has shown that meibum – the lipid-rich secretory product of the MGs – includes sterols, squalene, fatty acids, alcohols, saturated and unsaturated wax esters and short-chain cholesterol esters. Those secretions are part of a defence mechanism that protects the ocular surface from hazardous environmental factors and desiccation.Using mass spectrometry’s high-resolution spectra and Negative and Positive Ion Modes (NIM/PIM), the MG’s lipids have been analysed. Butovich cautioned researchers against using direct infusion or ‘shotgun-type’ experiments, as they can result in fragmentation of lipids that may be misinterpreted as novel entities.In his lab, only intact lipids are used, 95% of which are non-polar and 5% amphiphilic (having hydrophilic and hydrophobic components).He gave the major classes of meibum lipids as:

• Sterols (mainly cholesterol)
• Cholesterol esters (straight and branched)
• Cholesterol esters of (O-Acyl)-omega-Hydroxy Fatty Acids (OAHFA)
• Diacylated OAHFA (2 acyl groups added)
• OAHFA
• Triacylglycerols
• Free Fatty Acids (FFAs)
• Wax esters (straight chains)
• Wax esters (branched chains)

To monitor the reliability and repeatability of the intricate laboratory analyses undertaken, six repetitive injects taken from the same sample are compared when overlaid.Butovich stated that the results were ‘ultra’ reproducible, as long as the apparatus worked. Tear samples taken from different animals proved to be almost identical with only minor differences detected. No difference was found between the sexes as far as meibum lipids and wax esters were concerned.The most significant difference found involved cholesterol esters, but those sex differences proved to be totally random. A slight increase in the shorter chain cholesterol esters was noted in fales. The most variable lipid in the sexes proved to be triacylglycerol fractions.In summary, he observed that there were few differences between the meibum of individual humans and the meibum of humans and mice were almost the same.Other observations he made included:

• The relatively high level of wax esters, cholesterol esters, and cholesterol esters of OAHFA
• The mouse had highly enriched palmitoleic acid (C16:1)
• The low degree of unsaturated fatty acid chains
• Iso and anteiso-branching fatty acids
• A high level of odd-numbered fatty acids

According to Butovich, the factors that make meibum unique include: the ratio of identified classes of lipids, the extre length of its components, extensive hydroxylation of fatty acids and alcohols, iso and anteiso-branching of Meibomian lipids such as waxes, and the presence of unique complex lipids with several ester bonds.Butovich and colleagues call the network of enzymatic reactions responsible for the biosynthesis of meibum ‘meibogenesis’.They postulate that the factors responsible for the process of meibogenesis and the uniqueness of meibum lipids might be: the activity of a known enzyme, the activity of a yet unknown enzyme, or both those possibilities acting together.Butovich stated categorically that meibogenesis and lipidgenesis were not the same process. Iso-branching indicates that the branch point of fatty acids is at their penultimate carbon atom in a carbon chain, whereas ante-branching indicates their branch point is the ante-penultimate, meaning two carbon atoms from the chain’s end.Interestingly, meibum lipids are unlike the lipids from other body locations (humans and mice) with sebum from sebaceous glands being the closest ‘relative’.It comes as little surprise that Butovich’s lab now deals almost exclusively with research into Meibomian Gland Dysfunction (MGD), including its role in DE disease. As there are still many unknowns, there rains an outstanding clinical need for efficacious treatments, and the lab’s MG-secretion research rains directly relevant.{{image3-a:c-w:1015}}