Meibum lipid compositional changes with meibomian gland dysfunction reflect changes in hydrocarbon chain conformation and lipid-lipid interaction strength. Spectroscopic techniques are useful in studying the lipid-lipid interactions and conformation of lipid from individual patients. (ClinicalTrials.gov number, NCT00803452.).

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Phospholipids (PLs) are important sources of lipid second messengers that participate in cell signaling pathways. Consequently, their analysis in biological tissues has received increased attention. Current approaches for PL analysis include an extraction step and subsequent identification of the main PL classes by either 31P NMR spectroscopy or chromatographic separation followed by mass spectrometric detection. Previous in vitro studies revealed regional changes in the PL composition of mammalian lenses at different growth stages. In this report, we demonstrate the feasibility of direct in situ analysis of two relevant PL classes, phosphatidylcholines (PCs) and sphingomyelins (SMs), in slices of fresh or fixed (2.5% formaldehyde) mammalian lenses. The chosen matrix was p-nitroaniline, as it generated superior sensitivity when compared to 2,5-dihydroxybenzoic acid, the compound most commonly used for in vitro analysis of PLs by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Regional differences in the relative amounts of PCs and SMs were in agreement with trends demonstrated by previous in vitro studies. Fresh and fixed tissue of the same lens gave comparable relative levels of PCs and SMs. In situ analysis of PLs by MALDI-TOF MS offers a rapid and sensitive tool for the mapping of PLs in biological tissues.

Purpose-Meibomian gland dysfunction (MGD) is a common clinical problem that is often associated with evaporative dry eye disease. Alterations of the lipids of the meibomian glands have been identified in several studies of MGD. This prospective, observational, open label clinical trial documents the improvement in both clinical signs and symptoms of disease as well as spectroscopic behavior of the meibomian gland lipids after therapy with topical azithromycin ophthalmic solution.Methods-Subjects with symptomatic MGD were recruited. Signs of MGD were evaluated with a slit lamp. Symptoms of MGD were measured by the response of subjects to a questionnaire. Meibum lipid (ML) lipid-lipid interaction strength, conformation and phase transition parameters were measured using Fourier transform infrared spectroscopy (FTIR).Results-In subjects with clinical evidence of MGD changes in ordering of the lipids and resultant alteration of phase transition temperature were identified. Topical therapy with azithromycin relieved signs and symptoms and restored the lipid properties of the meibomian gland secretion towards normal.Conclusions-Improvement in phase transition temperature of the meibomian gland lipid with the determined percent trans rotomer composition of the lipid strongly suggests that the ordering of the lipid molecules is altered in the disease state (MGD) and that azithromycin can improve that abnormal condition toward normal in a manner that correlates with clinical response to therapy.

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Sphingomyelin (SM) is the most prevalent sphingolipid in the majority of mammalian membranes. Proton and 31P nuclear magnetic resonance spectral data were acquired to establish the nature of intra- and intermolecular H-bonds in the monomeric and aggregated forms of SM and to assess possible differences between this lipid and dihydrosphingomyelin (DHSM), which lacks the double bond between carbons 4 and 5 of the sphingoid base. The spectral trends suggest the formation of an intramolecular H-bond between the OH group of the sphingosine moiety and the phosphate ester oxygen of the head group. The narrower linewidth and the downfield shift of the resonance corresponding to OH proton in SM suggest that this H-bond is stronger in SM than in DHSM. The NH group appears to be involved predominantly in intramolecular H-bonding in the monomer. As the concentration of SM increases and the molecules come in closer proximity, these intramolecular bonds are partially disrupted and the NH group becomes involved in lipid-water interactions. The difference between the SM and DHSM appears to be not in the nature of these interactions but rather in the degree to which these intermolecular interactions prevail. As SM molecules cannot come as close together as DHSM molecules can, both the NH and OH moieties remain, on average, more intramolecularly bonded as compared to DHSM.

Purpose Meibomian gland dysfunction (MGD) is a common clinical problem that is often associated with evaporative dry eye disease. Alterations of the lipids of the meibomian glands have been identified in several studies of MGD. This prospective, observational, open label clinical trial documents the improvement in both clinical signs and symptoms of disease as well as spectroscopic characteristics of the meibomian gland lipids after therapy with topical azithromycin ophthalmic solution and oral doxycycline treatment. Methods Subjects with symptomatic MGD were recruited. Signs of MGD were evaluated with a slit lamp. Symptoms of MGD were measured by the response of subjects to a questionnaire. Meibum lipid-lipid interaction strength, conformation and phase transition parameters, and meibum protein content were measured using Fourier transform infrared spectroscopy (FTIR) and principal component analysis (PCA). Terpenoids, short chain CH3 moieties, lipid oxidation, wax, cholesterylesters and glycerides were measured with a proton nuclear magnetic resonance (1H-NMR) spectrometer. Results Topical therapy with azithromycin and oral therapy with doxycycline relieved signs and symptoms and restored the lipid properties of the meibomian gland secretion towards normal. Compared to 4 weeks of azithromycin treatment reported in our previous study, oral doxycycline treatment was slightly less effective in improving foreign body sensation and the signs of plugging and secretion. In subjects with clinical evidence of MGD, changes in ordering of the lipids and phase transition temperature were brought closer to normal with azithromycin treatment than doxycycline treatment. Treatment with doxycycline but not azithromycin restored the FTIR PCA scores and relative area of the 1H-NMR resonance at 1.26 ppm. Both doxycycline and azithromycin treatment restored the levels of the relative areas of the 1H-NMR resonances at 5.2 and 7.9 ppm to normal levels. The level of meibum protein and meibum lipid oxidation were not influenced by azithromycin or doxycycline treatment. Conclusions The mechanism of action of doxycycline may be different than that of azithromycin in therapy of MGD. It is notable that when carotenoids in meibum are low, as in MGD, the tear film is unstable and patients have the signs and symptoms of dry eye. When carotenoids are restored with azithromycin and doxycycline treatment, tear film stability is restored and patients no longer have the signs and symptoms of dry eye.

The changes in the amount of lipids with age and cataract support the idea that glycerolipids are selectively oxidized over lipids with fewer double bonds, such as sphingolipids. As a result of the elevation of sphingolipid levels with species, age, and cataract, lipid hydrocarbon chain order, or stiffness, increases. Increased membrane stiffness may increase light-scattering, reduce calcium pump activity, alter protein-lipid interactions, and perhaps slow fiber cell elongation.