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Three new very long chain polyunsaturated fatty acids (VLC PUFA) belonging to the omega-3 family have been identified in meibum samples collected by Schirmer strips. These VLC PUFA, namely FA (32:3), FA (34:3) and FA (36:3), were detected in O-acyl-ω-hydroxy fatty acids using a molecular network approach, and as free fatty acids. Identification was supported by retention time prediction model, exact mass determination and isotopic patterns. Double bond location was determined using cross metathesis reaction associated to tandem mass spectrometry. In meibum, synthesis of these VLC PUFA is likely to be mediated by elongation of very long chain fatty acids 4 enzyme. The biological role of these newly VLC PUFA and their occurrence in other tissues and biological fluids remains to be elucidated., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Purpose: To investigate the alteration in lipid composition of meibum, objective clinical signs, and subjective symptoms associated with aging and meibomian gland (MG) dysfunction (MGD)., Methods: In 10 MGD patients [4 males/6 females, mean age: 65.6 ± 7.9 years (range: 50-79 years)] and 24 healthy volunteer subjects [young subjects: 6 males/6 females, mean age: 25.7 ± 3.8 years (range: 20-35 years), elderly subjects: 6 males/6 females, mean age: 58.4 ± 7.5 years (range: 50-79 years)], three objective clinical signs were evaluated: MG orifice obstruction, meibum score, and tear film lipid layer interference pattern. Subjective symptoms were analyzed via a 15-item questionnaire. After careful collection of meibum samples, comprehensive lipid analysis was performed via liquid chromatography-mass spectrometry. Data was analyzed via JMP® ver. 13 (SAS Institute, Inc., Cary, NC) statistical analysis software., Results: In the MGD patients and elderly subjects, there was a significant decrease in non-polar lipids such as cholesterol esters (ChEs), while a significant increase in polar lipids [cholesterol (Ch), (O-acyl)-ω-hydroxy fatty acid (OAHFA), and free fatty acid (FA)] in total lipids (Tukey-Kramer test: p < 0.05). Triglyceride was significantly increased only in MGD patients (p < 0.05). Symptom scores representative of vision quality (i.e., blurred vision/haziness) were significantly negatively-correlated with the ratio of the non-polar lipid ChE, while significantly positively correlated with the polar lipids Ch, OAHFA, and FA (Spearman's rank correlation coefficient: p < 0.05)., Conclusions: Our findings revealed that both MGD and aging affect the composition ratio of major meibum lipids, resulting in the appearance of subjective symptoms., (Copyright © 2021. Published by Elsevier Inc.)

Purpose: Sphingolipids (SPL) play a role in cell signaling, inflammation, and apoptosis. The purpose of this study was to examine meibum and tear SPL composition in individuals with poor versus good meibum quality., Methods: Individuals were grouped by meibum quality (n = 25 with poor quality, case group and n = 25 with good quality, control group). Meibum and tears were analyzed with liquid chromatography-mass spectrometry (LC-MS) to quantify SPL classes. Semiquantitative and relative composition (mole percent) of SPL and major classes, Ceramide (Cer), Hexosyl-Ceramide (Hex-Cer), Sphingomyelin (SM), Sphingosine (Sph), and sphingosine 1-phosphate (S1P) were compared between groups., Results: Demographic characteristics were similar between the two groups. Overall, individuals with poor meibum quality had more SPL pmole in meibum and tears than controls. Relative composition analysis revealed that individuals with poor meibum quality had SPL composed of less Cer, Hex-Cer, and Sph and more SM compared to individuals with good quality meibum. This pattern was not reproduced in tears as individuals with poor meibum quality had SPL composed of a similar amount of Cer, but more Hex-Cer, Sph and SM compared to controls. In meibum, SPL pmole and relative composition most strongly correlated with MG metrics while in tears, SPL pmole and relative composition most strongly correlated with tear production. SPL in both compartments, specifically Cer pmole in meibum and S1P% in tears, correlated with DE symptoms., Conclusion: SPL composition differs in meibum and tears in patients with poor vs good meibum quality. These findings may be translated into therapeutic targets for disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: This study evaluated the presence and roles of cholesteryl esters (CEs) and wax esters (WEs) from human tear film and meibum in meibomian gland dysfunction (MGD)., Methods: Out of 195 enrolled subjects, 164 and 179 subjects provided tear and meibum samples, respectively. Subjects were classified into normal, asymptomatic MGD, MGD, and mixed (MGD & aqueous deficient). The precorneal tear film (PCTF) thinning rate (evaporation) was measured using optical coherence tomography. Lipids extracted from tear and meibum samples were infused into a SCIEX 5600 TripleTOF mass spectrometer. CE and WE intensities quantified with Analyst 1.7 TF and LipidView 1.3 were compared across disease groups in MetaboAnalyst 5.0 and correlated with PCTF thinning rates., Results: The numbers of unique CEs and WEs identified in the samples were 125 and 86, respectively. Unsupervised Principal Component (PC) analysis and supervised Partial Least Square Discriminant analysis exhibited little separation among groups for both CEs and WEs in tears and meibum. Spearman's correlation analyses showed no association between either the first or second PC scores with PCTF thinning rates., Conclusion: The abundances of human PCTF and meibum-derived CEs and WEs were independent of MGD disease status and PCTF thinning (evaporation). CEs and WEs alterations do not contribute to alterations in tear film dynamics in MGD, such as has been demonstrated by the (O-acyl) ω-hydroxy fatty acids (OAHFAs)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recent clinical findings suggest that mucomimetic polymers (MMP) can alter not only the texture of the aqueous tear but also the spreading and structure of the tear film (TF) lipid layer, thereby allowing for their synchronized performance in vivo. Thus, we aimed to evaluate in vitro (i) the capability of pharmaceutically applicable MMP to ensure the formation of post-evaporative ferning patterns (a characteristic feature of the "healthy" tear colloid) and (ii) the MMP interactions with human meibum films accessed in the course of blink-like deformations via Langmuir surface balance and Brewster angle microscopy (BAM). Four MMP were used- hyaluronic acid (HA), cross-linked hyaluronic acid (CHA), carboxymethyl cellulose (CMC) and gellan gum (GG)- at the concentrations of 0.0001%, 0.001%, 0.01%, 0.05% and 0.1%. Significant differences were observed in the MMP fern formation capability: CHA (≥0.001%) > HA (≥0.01%) = CMC (≥0.01%) > GG (≥0.05%). All MMP affected the spreading of meibum, with BAM micrographs revealing thickening of the films. CHA was particularly efficient, showing concentration-dependent enhancement of tear ferning and of meibomian layer structure, surfactant properties and viscoelasticity. Thus, endogenous and exogenous MMP may play key roles for the concerted action of the TF layers at the ocular surface, revealing novel routes for TF-oriented therapeutic applications.

Sexually mature male deer are known to rub-urinate, a process where urine is deposited on the tarsal gland. The resulting mixture of compounds from urine and secretions from the tarsal gland are used to signal sex, age, maturation status, and other information at close distance. We examined the difference in metabolites of tarsal gland extracts from male and female whitetail deer, Odocoileus virginianus, harvested during the mating season. Using NMR spectroscopy and high-pressure liquid chromatography linked to high resolution mass spectrometry (HPLC/HR-MS) we identified a homologous series of four male-specific compounds. The compounds are novel glycine conjugates of 10-hydroxy-6,9-oxido fatty acids, which we term cervidins A-D. Cervidins were deemed to possess the absolute configuration 6S,9R,10R through comparison of their spectroscopic data with those of known compounds. In addition, cholesterol 3-sulfate and 3-(3-hydroxyphenyl)-propanoic acid were found to be present in the extracts. Our results clearly demonstrate the diversity of potential semiochemicals contained in the mammalian integument.

The precorneal tear film keeps the eye surface moist and helps to maintain normal eye function. The outermost lipid layer of the tear film, which attenuates tear film evaporation, contains meibum secreted from the meibomian gland. Most meibum lipids are neutral, including wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs), along with some polar lipids including free fatty acids (FFAs), O-acyl-ω-hydroxy fatty acids (OAHFAs), and trace phospholipids. Detection of neutral lipids by mass spectrometry (MS) is challenging due to interference from impurities, particularly when working with minute-volume meibum samples. Here, we describe procedures for sample preparation and MS analysis of these elusive meibum lipids that can be used to examine dry eye disease mechanisms. Because the method described here minimizes impurity peaks for lipids generally, neutral and otherwise, it may be applied to high-sensitivity analysis of other biological samples.

Meibum is a lipid secretion that is produced by holocrine Meibomian glands (MGs). MGs are a specialized type of sebaceous glands that are embedded in the human eyelids. Chemically, meibum and sebum are different. A detailed characterization of lipidome and transcriptome of MG is required to deconvolute a complex and poorly characterized array of biosynthetic reactions (termed meibogenesis ) that lead to formation of meibum. Changes in the composition and quality of meibum have been linked to various ocular disorders, some of which are more prevalent in males, while others in females. To establish the role of gender in meibogenesis in humans, we characterized MG transcriptomes and lipidomes of females and males, and identified signature genes of meibogenesis in both genders. Specimens of MG tissues were subjected to mRNA microarray analyses. Chemical composition of meibum samples was assessed chromatographically and mass spectrometrically. Both targeted and untargeted approaches were used. About 290 signature genes of meibogenesis were identified. The analyses of their expression patterns demonstrated no major differences between the genders. Lipid profiling of major classes of meibomian lipids, such as wax esters, cholesteryl esters, free cholesterol, ( O )-acylated omega-hydroxy fatty acids (OAHFA), cholesteryl esters of OAHFA, and triacylglycerols, also demonstrated only minor (and random) differences in these lipids. The results of transcriptomic analyses correlated well with lipidomic data. Taken together, our data imply that in males and females, meibogenesis proceeds in a similar fashion, yielding secretions with similar, highly conserved, compositions. This finding is important for designing novel, gender-independent diagnostic and therapeutic approaches to various MG-related diseases and pathological conditions.

Purpose: The purpose of this work was to optimize methodology to analyze the human tear film lipids by using untargeted, direct infusion electrospray ionization-mass spectrometry (ESI-MS) to establish the analytical approach for a large-scale clinical translational study of tear film lipids in ocular surface disease, particularly associated with the O-acyl-omega hydroxy fatty acids (OAHFAs)., Methods: Meibum and tear samples were collected from both eyes of five normal subjects without ocular disease using two different microcapillary collection tubes, glass and polytetrafluoroethylene (PTFE). An untargeted lipidomics approach was used to analyze the lipids in human tear and meibum samples using direct infusion ESI-MS in positive and negative ion modes. Direct and indirect quantification methods were evaluated., Results: The amount of OAHFAs measured in tears using these techniques was approximately 0.7-0.8% of the total lipids. More phospholipids, including phosphatidylcholine and sphingomyelin, were detected in the tear samples associated with glass microcapillaries compared to PTFE., Conclusions: Reliable assessment of lipids in small volumes of tear film is possible using high resolution, untargeted ESI-MS in positive and negative ion modes. Using this technique, the concentration of OAHFAs can be quantified, as can the presence of other polar lipids., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose/aim: To assess the effectiveness of polytetrafluoroethylene (PTFE) tubes in the collection of human tears and meibum., Materials and Methods: This was a prospective study that enrolled 10 healthy human subjects. Both the tear film and meibum were sampled using PTFE tubes in the right eye of all subjects. In the left eyes, either 5-µL or 1-µL glass microcapillary tubes were used to collect tears, and 0.5-µL glass microcapillary tubes were used to collect meibum. The lipids from the samples were extracted and analyzed using mass spectrometry (SCIEX TripleTOF 5600, Framingham, MA, USA). The absolute peak intensities of the omega-acyl hydroxy fatty acids (OAHFA), cholesterol esters (CE), and wax esters (WE) obtained for both methods were summed and compared between collection methods., Results: A total of 10 subjects completed the study (five female, mean age: 35.7 ± 7.9 years). Using the mass spectrometer output, the median (first quartile, third quartile) summed intensity units of OAHFA, CE, and WE collected associated with tears using PTFE were 516 (125, 1315), 7946 (2571, 19,915), and 38,892 (139,630, 174,082), all of which were significantly higher (all p ≤ 0.04) than those collected from glass microcapillaries (91 (41, 408), 2463 (1389, 6042), and 11,109 (7465, 37,371), respectively). The median summed intensity units of OAHFA, CE, and WE associated with meibum (1958 (1417, 3502), 11,726 (8434, 87,691), and 84,771 (52,657, 206,050), respectively) using PTFE were not significantly different (all p ≥ 0.13) than those associated with glass microcapillaries (1502 (699, 4407), 10,781 (3287, 38,205), and 77,381 (26,590, 178,213), respectively)., Conclusions: PTFE tubes, which are thought to be lipophilic, were associated with more measurable lipids from the tear film than glass microcapillaries. There was no difference between collection methods in lipid profiles when used with meibum.

The ( O -acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids., (Copyright © 2018 Hancock et al.)

Elevated levels of acyl chain saturation of meibomian lipids are associated with vastly different effects: from enhanced tear film (TF) stability in infants to shortened TF breakup time in meibomian gland disease patients. Thus it is important to study the effect of saturation on the surface properties of human meibum (MGS). Therefore, MGS films (1, 2, 3, 4, 5, 10, 25, 50, 67, and 100% saturation) were spread at the air/water interface of a Langmuir surface balance. The layers' capability to reorganize during dynamic area changes was accessed via the surface pressure (π)-area (A) compression isotherms and step/relaxation dilatational rheology studies. Film structure was monitored with Brewster angle microscopy. The raise in the % (at ≥10%) of saturation resulted in the formation of stiffer, thicker, and more elastic films at π ≥ 12 mN/m with the effects being proportional to the saturation level. At the same time, at low (≤10 mN/m) π the raise in saturation resulted in altered spreading and heterogeneous structure of MGS layers. The strong impact of saturation on MGS surface properties correlates with our recent spectroscopy study, which demonstrated that saturation induced increase of MGS acyl chain order, phase transition temperature, and cooperativity.

Purpose/aims: Dry eye is a common multi-factorial disease characterized by tearfilm dysfunction. Diagnosis of dry eye requires documentation of tear status with non-invasive tear break up times (NIBUT), tear meniscal height (TMH), and tearfilm lipid layer. These parameters objectively assess tear stability and indirectly, the functional impact of meibomian gland dysfunction. While it is obvious that portable instrumentation gives broader access than fixed instrumentation, its value would be limited if it is unreliable and its precision and accuracy substantially worse than fixed instruments., Materials/methods: Because of the lack of systematic reviews concerning the Tearscope, we performed a scoping review of the literature to determine 1. the ranges of the tear parameters as measured by the device, 2. relationship between these measurements and other clinical variables, 3. Information about intra or inter-observer agreement, and comparison with another technique, where available., Results: We found 22 reports related to NIBUT, 18 reports concerning tearfilm lipid morphology (LM) based on classification of interference patterns, and 8 reports on TMH. While publication of these reports indicates an acceptance of the reliability of portable instruments, we interrogated these studies further to evaluate the results obtained from portable devices and found that there was no equivalent measurements in fixed devices (LM) or measurements differ considerably from fixed devices (NIBUT). In the case of TMH, portable measurements were similar to those by traditional slit-lamp microscopy. There were relatively few studies on TMH measured using handheld devices, but it was found to be correlated to NIBUT but not the Schirmer test values., Conclusions: Imaging algorithms in portable tearfilm instruments should be further standardized to facilitate wider adoption and evaluation. In the next few years, new emerging technologies will employ a strategy of non-invasive tear testing that is portable and relatively inexpensive, and these will enhance adoption of such tools in the clinic.

The function of the meibomian gland in the upper and lower eyelids is critical to maintaining homeostasis at the ocular surface. Highly specialized meibocytes within the gland must differentiate and accumulate intracellular lipid droplets that are released into the tear film following rupture of the cell membrane. Proteases and their inhibitors have been recognized as key players in remodeling extracellular matrices and promoting the normal integrity of glandular tissue. They modulate a wide range of biological processes, such as cell proliferation and differentiation, and can contribute to disease when aberrantly expressed. Deciphering the role of proteolytic activity in the meibomian gland offers an opportunity to gain a more comprehensive and fundamental understanding of the developmental, physiological, and pathological processes associated with this gland., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The surface properties of meibomian secretion (MGS), the major constituent of the tear film (TF) lipid layer, are of key importance for TF stability. The interfacial properties of canine, cMGS, and feline, fMGS, meibum films were studied using a Langmuir surface balance. These species were selected because they have blinking frequency and TF stability similar to those of humans. The sample's performance during dynamic area changes was evaluated by surface pressure (π)-area (A) isocycles and the layer structure was monitored with Brewster angle microscopy. The films' dilatational rheology was probed via the stress-relaxation technique. The animal MGS showed similar behavior both between each other and with human MGS (studied previously). They form reversible, non-collapsible, multilayer thick films. The relaxations of canine, feline, and human MGS films were well described by double exponential decay reflecting the presence of two processes: (1) fast elastic process, with characteristic time τ < 10 s and (2) slow viscous process, with τ > 100 s-emphasizing the meibum layers viscoelasticity. The temperature decrease from 35 to 25 °C resulted in decreased thickness and lateral expansion of all MGS layers accompanied with increase of the π/A hysteresis and of the elastic process contribution to π relaxation transients. Thus, MGS films of mammals with similar blinking frequency and TF stability have similar surface properties and stress relaxations unaltered by the interspecies MGS compositional variations. Such knowledge may impact the selection of animal mimics of human MGS and on a better understanding of lipid classes' impact on meibum functionality.

Tear film lipid layer is a complex lipid mixture forming the outermost interface between eye and environment. Its key characteristics, such as surface tension and structural stability, are governed by the presence of polar lipids. The origin of these lipids and exact composition of the mixture are still elusive. We focus on two minor polar lipid components of the tear film lipid later: sphingomyelin and ceramide. By employing coarse grain molecular dynamics in silico simulations accompanied by Langmuir balance experiments we provide molecular-level insight into behavior of these two lipids in a tear film lipid layer model. Sphingomyelin headgroups are significantly exposed at the water-lipids boundary while ceramide molecules are incorporated between other lipids frequently interacting with nonpolar lipids. Even though these two lipids increase surface tension of the film, their molecular-level behavior suggests that they have a stabilizing effect on the tear film lipid layer., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Wax esters (WE) are one of the predominant lipid types in human meibomian gland secretions (meibum) and account for 40-50 % of total meibum lipids. Recently, we managed to quantify 51 isomeric groups of intact WE in normal human meibum samples by direct infusion electrospray ionization mass spectrometry (ESI-MS), with each WE peak in the MS spectrum corresponding to one isomeric group (Chen et al, Invest Ophthalmol Vis Sci 54(8):5730-53, 2013). However, the information of the isomeric composition in each group was not obtained. In this study, tandem mass spectrometry (MS/MS) was applied to quantify relative amounts of these isomers using the intensities of the corresponding diagnostic ions after appropriate correction and normalization. This data was combined with the previous obtained mole fraction of each isomeric group to total WE in human meibum to determine the corresponding percentage of each isomer. A total of 23 of the most abundant WE peaks of different molecular weights (corresponding to 85.3 % of the total amount of WE) in human meibum were studied and resulted in quantification of 92 WE species. The quantitative information of composition of WE in human meibum will help better understand their role in the tear film.

Purpose: The inhibition of the rate of evaporation (R evap ) by surface lipids is relevant to reservoirs and dry eye. Our aim was to test the idea that lipid surface films inhibit R evap ., Methods: R evap were determined gravimetrically. Hydrocarbon chain conformation and structure were measured using a Raman microscope. Six 1-hydroxyl hydrocarbons (11-24 carbons in length) and human meibum were studied. Reflex tears were obtained from a 62-year-old male., Results: The Raman scattering intensity of the lipid film deviated by about 7 % for hydroxyl lipids and varied by 21 % for meibum films across the entire film at a resolution of 5 μm 2 . All of the surface lipids were ordered. R evap of the shorter chain hydroxyl lipids were slightly (7%) but significantly lower compared with the longer chain hydroxyl lipids. R evap of both groups was essentially similar to that of buffer. A hydroxyl lipid film did not influence R evap over an estimated average thickness range of 0.69 to >6.9 μm. R evap of human tears and buffer with and without human meibum (34.4 μm thick) was not significantly different. R evap of human tears was not significantly different from buffer., Conclusions: Human meibum and hydroxyl lipids, regardless of their fluidity, chain length, or thickness did not inhibit R evap of buffer or tears even though they completely covered the surface. It is unlikely that hydroxyl lipids can be used to inhibit R evap of reservoirs. Our data do not support the widely accepted (yet unconfirmed) idea that the tear film lipid layer inhibits R evap of tears., Competing Interests: None of the authors had a conflict of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Sebum may contribute to the composition of the tear film lipid layer naturally or as a contaminant artifact from collection. The aims of this study were to determine: if sebum changes the rheology of meibum surface films; if the resonance near 5.2 ppm in the 1H-NMR spectra of sebum is due to squalene (SQ); and if sebum or SQ, a major component of sebum, interacts with human meibum., Methods: Human meibum was collected from the lid margin with a platinum spatula. Human sebum was collected using lipid absorbent tape. Langmuir trough technology was used to measure the rheology of surface films. Infrared spectroscopy was used to measure lipid conformation and phase transitions. We used 1H-NMR to measure composition and confirm the primary structure of SQ., Results: The NMR resonance near 5.2 ppm in the spectra of human sebum was from SQ which composed 28 mole percent of sebum. Both sebum and SQ lowered the lipid order of meibum. Sebum expanded meibum films at lower concentrations and condensed meibum films at higher concentrations. Sebum caused meibum to be more stable at higher pressures (greater maximum surface pressure)., Conclusions: Physiological levels of sebum would be expected to expand or fluidize meibum making it spread better and be more surface active (qualities beneficial for tear film stability). Sebum would also be expected to stabilize the tear film lipid layer, which may allow it to withstand the high shear pressure of a blink.

Purpose: An experimental platform to replicate the human tear film on a contact lens is presented. The influence of interfacial viscoelasticity in stabilizing in vitro model tear films against breakup and dewetting is investigated using this instrument., Methods: Model tear films consisting of bovine meibomian lipids (meibum) spread on either PBS or artificial tear solution (ATS) are created. The interfacial shear rheology of these films is measured as a function of temperature. The dewetting dynamics of these films is then investigated using the Interfacial Dewetting and Drainage Optical Platform (i-DDrOP) on top of silicone hydrogel (SiHy) contact lenses at 23 and 35°C. The film breakup times are evaluated using two parameters: onset of film breakup, Tonset for thick films (∼100 μm), and tear breakup times, TBU for thin films (∼1 μm). Thin film thinning rates as a result of evaporation are also calculated., Results: The ATS/meibum films have the largest surface rheology and correspondingly show the largest Tonset times at both 23 and 35°C. The parameter TBU is also significantly larger for ATS/meibum (TBU ∼ 40 seconds) compared with that of ATS and PBS/meibum films (TBU ∼ 30 seconds) at room temperature. However, at 35°C, all three model tear films exhibit similar TBU ∼ 17 seconds and average rate of thinning of -4 μm/minute., Conclusions: Tear film stability is influenced by both surface rheology and evaporation. The in vitro tear breakup times and thinning rates of model tear films at 35°C are in good agreement with in vivo measurements previously reported, highlighting the utility of the i-DDrOP for in vitro tear film breakup research.

Purpose: Our aim was to quantitate day-to-day changes in the tear and meibum lipid profile of individual subjects in a pilot study of healthy humans., Methods: Matched tear and meibum samples were obtained from four subjects on three consecutive days. Quantitative lipid profiles of human basal tears and meibum were compared using multivariate analysis by principal components., Results: Substantial differences in the lipid profile between subjects were observed, while lipid profiles were steady across the three consecutive days of sampling. Multivariate principal component analysis demonstrated that lysophosphatidylcholine was the largest variant lipid class between subjects in tears, while wax esters comprised the most variation between subjects in meibum secretions., Conclusion: Interday variability is shown to be much smaller than interpatient variability, suggesting that tears and meibum subjects both have unique profiles in humans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To evaluate the effectiveness and safety of a single thermodynamic treatment system (LipiFlow) for meibomian gland dysfunction (MGD)., Methods: Retrospective series case study. Forty-eight subjects with meibomian gland dysfunction were analyzed before and after 12 minutes LipiFlow system treatment. All subjects were examined before, 4 weeks and 12 weeks after this treatment. Subjective symptoms, lipid layer thickness (LLT), expressible meibomian gland, tear break-up time, meibomian gland assessment and ocular surface staining were measured. Statistical analysis was performed using SPSS 11.5 software. Analysis included those independent sample two-tailed t-tests for comparison of the mean change from baseline to 4 weeks and baseline to 12 weeks after Lipiflow treatment. Statistically significant difference was based on α=0.05 (P<0.05)., Results: A total of 48 cases completed the 12-week follow-up. MGD patients with LipiFlow treatment had a significant reduction in the Ocular Surface Disease Index (OSDI) scores with 45.36±19.34 before treatment, 23.82±11.94 at 4 weeks (t=2.009, P=0.035) and 25.66±14.12 at 12 weeks (t=1.976 P=0.038). LipiFlow resulted in a higher number of expressible glands from 2.91±1.13 (baseline) to 6.27±2.37 (4 weeks, t=3.505, P<0.001) and 5.15±2.08 (12 weeks, t=2.004, P= 0.027) and change of secretion quality from 6.18±2.48 (baseline) to 13.55±3.46 (4 weeks,t=2.698,P=0.005) and 12.67±3.41 (12 weeks,t=2.403,P=0.009). In addition, a single thermodynamic treatment increase the LLT from (42.13±9.67)nm (baseline) to (59.02±16.39)nm (4 weeks, t=2.971, P=0.002) and (54.65±12.52)nm (12 weeks, t=2.021, P=0.021). The rate of partial blink was relieved from 0.37±0.30 (baseline) to 0.15±0.14 (4 weeks, t=3.428, P=0.035) and 0.12±0.13 (12 weeks, t=1.986, P=0.026). BUT was increased from (4.73± 2.34)s (baseline) to (9.32 ± 2.18)s (4 weeks, t=3.385, P<0.001) and (9.91 ± 3.01)s (12 weeks, t=3.253, P< 0.001). There were no unanticipated or serious device-related adverse events reported. Compared with baseline, there was no statistically significant difference in BCVA, corneal staining and intraocular pressure (P=0.141, 0.376, 0.421)., Conclusion: The LipiFlow system was a safety and effectiveness treatment of MGD in the 12-week study.

This review critically evaluates a broad range of literature in order to show the relationship between meibum, tear lipids and the tear film lipid layer (TFLL). The relationship of meibum composition to dry eye syndrome is briefly discussed. The review also explores the interactions between aqueous and the TFLL by examining the correlations between meibomian lipids and lipids extracted from whole tears, and by considering protein adsorption to the TFLL from the aqueous. Although it is clear to the authors that a normal tear film resists evaporation, an emerging idea from the literature is that the main purpose of the TFLL is to allow the spread of the tear film and to prevent its collapse onto the ocular surface, rather than to be an evaporative blanket. Current models on the possible structure of the TFLL are also examined., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A series of different types of wax esters (represented by RCOOR') were systematically studied by using electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (MS/MS) along with pseudo MS(3) (in-source dissociation combined with MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer. The tandem mass spectra patterns resulting from dissociation of ammonium/proton adducts of these wax esters were influenced by the wax ester type and the collision energy applied. The product ions [RCOOH2](+), [RCO](+) and [RCO-H2O](+) that have been reported previously were detected; however, different primary product ions were demonstrated for the three wax ester types including: (1) [RCOOH2](+) for saturated wax esters, (2) [RCOOH2](+), [RCO](+) and [RCO-H2O](+) for unsaturated wax esters containing only one double bond in the fatty acid moiety or with one additional double bond in the fatty alcohol moiety, and (3) [RCOOH2](+) and [RCO](+) for unsaturated wax esters containing a double bond in the fatty alcohol moiety alone. Other fragments included [R'](+) and several series of product ions for all types of wax esters. Interestingly, unusual product ions were detected, such as neutral molecule (including water, methanol and ammonia) adducts of [RCOOH2](+) ions for all types of wax esters and [R'-2H](+) ions for unsaturated fatty acyl-containing wax esters. The patterns of tandem mass spectra for different types of wax esters will inform future identification and quantification approaches of wax esters in biological samples as supported by a preliminary study of quantification of isomeric wax esters in human meibomian gland secretions.

In our previously study, we reported lower tear volume in with an n-3 fatty acid deficient mice and that the docosahexaenoic acid and total n-3 fatty acid levels in these mice are significantly reduced in the meibomian gland, which secretes an oily tear product. Furthermore, we noted very long chain fatty acids (≥25 carbons) in the meibomian gland. To verify the detailed mechanism of the low tear volume in the n-3 fatty acid-deficient mice, we identified the very long chain fatty acids in the meibomian gland, measured the fatty acid composition in the tear product. Very long chain fatty acids were found to exist as monoesters. In particular, very long chain fatty acids with 25-29 carbons existed for the most part as iso or anteiso branched-chain fatty acids. n-3 fatty acid deficiency was decreased the amount of meibum secretion from meibomian gland without change of fatty acid composition. These results suggest that the n-3 fatty acid deficiency causes the enhancement of evaporation of tear film by reducing oily tear secretion along with the decrease of meibomian gland function., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The ocular tear film is a complex structure composed of a number of elements. While all of these components serve valuable functional and structural roles, the external lipid layer has been a focus because it is known to play a critical role in dry eye. Traditionally, meibomian gland phospholipids have been considered to be the vital amphiphilic molecules needed to create an interphase between the outer nonpolar lipid layer and inner aqueous layers, yet recent work has called this theory into question. The purpose of this review is to clarify the current understanding of the origins, identity, and significance of polar tear lipids. Studies indicate that both phospholipids and ω-hydroxy fatty acids likely play a critical role in tear film stability. Studies also indicate that polar lipids likely originate from multiple sources and that they are integrally involved in ocular surface disease. Additional studies are needed to fully understand the origins and significance of polar tear lipids, because to date only correlational evidence has described their hypothesized origins and functions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background/aims: It has been suggested that eye makeup could interact with human meibum causing a decrease in the stability of the tear film. The aim of this pilot study was to measure makeup-human meibum interactions in vitro., Methods: Human meibum-makeup interactions were quantified by measuring order-to-disorder lipid phase transitions using infrared spectroscopy., Results: Makeup products exhibited lipid phase transition temperatures that were much higher than those for meibum. One product increased the lipid phase transition temperature by 4.2°C when combined with human meibum causing a large increase (from 30 to 49%) in the order of the meibum-lipid hydrocarbon chains and significantly decreased the minimum frequency, enthalpy and entropy of the phase transition of human meibum. Another eyeliner caused no significant (p < 0.05) change in the phase transition parameters of human meibum., Conclusion: Infrared spectroscopy may be used to measure interactions between human meibum and makeup. One makeup product increased the lipid order (viscosity) which could have adverse effects on tear film stability. Modern cosmetics are highly regulated and relatively safe to use; however, it could be beneficial to design makeup products that do not interact with meibum, especially since women have a higher prevalence of dry eye symptoms., (© 2015 S. Karger AG, Basel.)

We report a rapid liquid chromatography/quadrupole Orbitrap Fourier transform mass spectrometry (LC-FTMS) method for identifying free fatty acids (FAs) and (O-acyl)-ω-hydroxyFAs (OAHFAs) in human meibum without derivatization. Meibum is a lipid-rich secretion and an important component of the tear film lipid layer. FAs are commonly detected by gas chromatography (GC) or GC/MS after methyl ester derivatization. We developed high-throughput lipid profiling using LC-FTMS and lipid identification software, Lipid Search, without derivatization and applied the method to human meibum. Chromatographic separation was performed on a C18 column. We selected negative electrospray ionization [M-H](-), and applied high-resolution full scan mode to FA analysis and data-dependent MS(2) mode to OAHFA analysis. High-resolution Orbitrap MS proved to be an excellent tool for the rapid analysis of lipids from meibum, and 100 FA and 61 OAHFA molecular species were detected. The analysis times were 12 and 16.5min, respectively. Very long chain FAs (up to C37) and OAHFAs (up to C56) were also detected. The results clearly showed that retention time correlates with the number of double bonds and carbon chains. This LC/MS method can be applied to the identification of FAs and OAHFAs in human meibum., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-ω-hydroxyfatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model.

Purpose: To quantify the molecular lipid composition of patient-matched tear and meibum samples and compare tear and meibum lipid molecular profiles., Methods: Lipids were extracted from tears and meibum by bi-phasic methods using 10:3 tert-butyl methyl ether:methanol, washed with aqueous ammonium acetate, and analyzed by chip-based nanoelectrospray ionization tandem mass spectrometry. Targeted precursor ion and neutral loss scans identified individual molecular lipids and quantification was obtained by comparison to internal standards in each lipid class., Results: Two hundred and thirty-six lipid species were identified and quantified from nine lipid classes comprised of cholesterol esters, wax esters, (O-acyl)-ω-hydroxy fatty acids, triacylglycerols, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine. With the exception of phospholipids, lipid molecular profiles were strikingly similar between tears and meibum., Conclusions: Comparisons between tears and meibum indicate that meibum is likely to supply the majority of lipids in the tear film lipid layer. However, the observed higher mole ratio of phospholipid in tears shows that analysis of meibum alone does not provide a complete understanding of the tear film lipid composition.

Tear film stability decreases with age however the cause(s) of the instability are speculative. Perhaps the more saturated meibum from infants may contribute to tear film stability. The meibum lipid phase transition temperature and lipid hydrocarbon chain order at physiological temperature (33 °C) decrease with increasing age. It is reasonable that stronger lipid-lipid interactions could stabilize the tear film since these interactions must be broken for tear break up to occur. In this study, meibum from a pool of adult donors was saturated catalytically. The influence of saturation on meibum hydrocarbon chain order was determined by infrared spectroscopy. Meibum is in an anhydrous state in the meibomian glands and on the surface of the eyelid. The influence of saturation on the surface properties of meibum was determined using Langmuir trough technology. Saturation of native human meibum did not change the minimum or maximum values of hydrocarbon chain order so at temperatures far above or below the phase transition of human meibum, saturation does not play a role in ordering or disordering the lipid hydrocarbon chains. Saturation did increase the phase transition temperature in human meibum by over 20 °C, a relatively high amount. Surface pressure-area studies showing the late take off and higher maximum surface pressure of saturated meibum compared to native meibum suggest that the saturated meibum film is quite molecularly ordered (stiff molecular arrangement) and elastic (molecules are able to rearrange during compression and expansion) compared with native meibum films which are more fluid agreeing with the infrared spectroscopic results of this study. In saturated meibum, the formation of compacted ordered islands of lipids above the surfactant layer would be expected to decrease the rate of evaporation compared to fluid and more loosely packed native meibum. Higher surface pressure observed with films of saturated meibum compared to native meibum suggests greater film stability especially under the high shear stress of a blink., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

(O-acyl) ω-hydroxy fatty acids (OAHFAs) are a recently found group of polar lipids in meibum. Since these lipids can potentially serve as a surfactant in the tear film lipid layer, the surface properties of a molecule of this lipid class was investigated and compared with a structurally related wax ester and a fatty acid. (O-oleyl) ω-hydroxy palmitic acid was synthesized and used as the model OAHFA. It was spread either alone or mixed with human meibum on an artificial tear buffer in a Langmuir trough, and pressure-area isocycle profiles were recorded at different temperatures and compared with those of palmityl oleate and oleic acid. These measurements were accompanied by fluorescence microscopy of meibum mixed films during pressure-area isocycles. The pressure area curves indicated that pure films of the model OAHFA are as surface active as oleic acid films, cover a much larger surface area than either palmityl oleate or oleic acid and show a distinct biphasic pressure-area isocycle profile. The OAHFAs appeared to remain on the aqueous surface and show only a minor re-arrangement into multi-layered structures during repetitive pressure area isocycles. All these properties can be explained by OAHFAs binding weakly to the aqueous surface via an ester group and strongly via a carboxyl group. By contrast, the pressure area profiles of palmityl oleate films indicate that they form multi-layers and oleic acid presumably forms micelles and desorbs into the subphase. When mixed with meibum, similar features as for pure films were observed. In addition, meibum-OAHFA films appeared very homogeneous; a feature not seen with other mixtures. In conclusion these data support the notion that the tested OAHFA is a very potent surfactant which is important in spreading and stabilising meibomian lipid films., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this investigation was to better understand lipid composition in human meibum., Methods: Intact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation., Results: The absolute amount (μmol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 ± 4% and 40 ± 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 ± 2% and 25 ± 1% for CEs and 14 ± 1% and 86 ± 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 α,ω Type II DEs, and 21 ω Type I-St DEs., Conclusions: Major neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive.

Purpose: We examined in vitro the evaporation-retarding effect of wax esters (WEs). The WEs resembled closely the most abundant WE species in meibum., Methods: A custom-built system was used to measure the evaporation rates through WE layers applied to the air-water interface at 35°C and, as a reference, at 30°C and 41°C. Additionally, the melting points of the WEs were determined. The organization and stability of the WE layers were assessed using Brewster angle microscopy (BAM) and Langmuir film experiments, respectively., Results: Four of 19 WEs retarded evaporation at 35°C: behenyl palmitoleate (BP), behenyl oleate (BO), behenyl linoleate (BLN), and behenyl linolenate (BLNN) decreased evaporation by 20% to 40%. BP was the most effective evaporation retardant. At 30°C the most effective retardants were BLN and BLNN decreasing evaporation by ~50%, whereas BP and BO decreased evaporation by only 5% to 10%. At 41°C, each lipid decreased evaporation by only 2% to 4%. The evaporation-retardant WEs all melted within 2°C of physiological temperature. BAM images showed that the evaporation-retardant WE layers spread somewhat uniformly and possibly exhibited areas of condensed lipid. The isotherms suggested that WE layers were surface pressure tolerant but unstable under compression-relaxation cycles., Conclusions: The evaporation-retarding effect is dependent on the physicochemical properties of the WEs at given temperature, and therefore, the effect most likely arises from a certain phase of the WE layer. However, WEs as such are poor surfactants and need to be accompanied by polar lipids to form stable lipid layers.

Recent NMR studies suggest that unsaturation may contribute to tear film instability in adults and loss of cholesteryl esters and squalene could reduce tear film stability in adults with meibomian gland dysfunction. The proton resonances were tentatively assigned in those studies. In this current investigation, meibum from seven infants and children, one adult and a pool of adult meibum have been analyzed using an NMR spectrometer with greater sensitivity and spectral resolution. The goals of this work are to confirm/correct the previous assignments and to determine possible age-related changes in composition. The initial resonance assignments were confirmed using heteronuclear single quantum correlation spectroscopy. Because there were no significant interferences in the spectral region corresponding to the resonances for cholesteryl and wax esters, the areas of these resonances were used to calculate their molar ratios. We calculated a wax ester:cholesteryl ester molar ratio of 1:0.57 ± 0.05 for all our meibum samples and there were no age-related differences. At lower film thicknesses, the rate of evaporation measured in vitro was lower for wax esters mixed with a long chain cholesteryl ester compared to wax esters alone. However, the film thicknesses tested were non-physiological. Longer chain cholesteryl esters increase the interactions between hydrocarbon chains. Hydrocarbon chains were more saturated in meibum from infants and children compared to adults. Unsaturation may contribute to tear film instability in adults. Loss of cholesteryl ester and squalene could destabilize tear film in adults with meibomian gland dysfunction., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human meibomian gland secretions (meibum) are the major lipid component of the human preocular tear film. The predominant lipid classes found in meibum include waxes (WE), cholesteryl esters (CE), and varying amounts of cholesterol (Chl). The classical sulfo-phospho-vanillin assay (SPVA), adapted for a microplate reader, was used to quantitate lipids in meibum. To account for varying reactivities of different lipids in SPVA, a model meibomian lipid mixture (MMx) that approximated the WE/CE/Chl composition of meibum was developed and used to quantitate meibomian lipids. The overall SPV responses of MMx and meibum were found to be close, with similar intermediate and final reaction products for both. Saturated WE that had not been expected to be reactive were found to be SPV-positive. A reaction mechanism for these compounds in SPVA which involves the formation of alkenyl ethers is proposed and discussed. Tested proteins were non-reactive in SPVA. Thus, by comparing the results of gravimetric analyses of meibum samples with the results of a properly calibrated SPVA, it was estimated that the SPV-reactive lipid content of dry meibum in tested samples was about 78 % (w/w). The SPV method can also be adopted for analyzing other types of complex lipids secretions, such as sebum, as well as whole lipid extracts from other lipid-enriched organs and tissues, if proper standards are chosen.

Meibomian gland secretions (or meibum) are produced by holocrine meibomian glands and are secreted in melted form onto the ocular surface of humans and animals to form a protective tear film lipid layer (TFLL). Its protective effect strongly depends on the composition and, hence, thermotropic behavior of meibum. The goal of our study was to quantitatively evaluate the melting characteristics of human meibum and model lipid mixtures using differential scanning microcalorimetry. Standard calorimetric parameters, e.g. changes in calorimetric enthalpy, transition temperatures Tm, cooperativity of melting, etc. were assessed. We found that thermotropic behavior of meibum resembled that of relatively simple mixtures of unsaturated wax esters, but showed a lower change in calorimetric enthalpy, which can be indicative of a looser packing of lipids in meibum compared with pure standards and their simple mixtures. The cooperativity of melting of meibomian lipids was comparable to that of an equimolar mixture of four oleic-acid based wax esters. We demonstrated that the phase transitions in meibum start at about 10-15°C and end at 35-36°C, with Tm being about 30°C. The highly asymmetrical shape of the thermotropic peak of meibum is important for the physiology and biophysics of TFLL., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Purpose: We explore the unique rheological and structural properties of human and bovine meibomian lipids to provide insight into the physical behavior of the human tear-film lipid layer (TFLL)., Methods: Bulk rheological properties of pooled meibomian lipids were measured by a commercial stress-controlled rheometer; a home-built interfacial stress rheometer (ISR) probed the interfacial viscoelasticity of spread layers of meibomian lipids. Small- and wide-angle x-ray scattering detected the presence and melting of dispersed crystal structures. Microscope examination under cross polarizers provided confirmation of ordered crystals. A differential scanning calorimeter (DSC) analyzed phase transitions in bulk samples of bovine meibum., Results: Bulk and interfacial rheology measurements show that meibum is extremely viscous and highly elastic. It is also a non-Newtonian, shear-thinning fluid. Small- and wide-angle x-ray diffraction (SAXS and WAXS), as well as differential scanning calorimetry (DSC) and polarizing microscopy, confirm the presence of suspended lamellar-crystal structures at physiologic temperature., Conclusions: We studied meibum architecture and its relation to bulk and interfacial rheology. Bovine and human meibomian lipids exhibit similar physical properties. From all structural probes utilized, we find a melt transition near eye temperature at which lamellar crystals liquefy. Our proposed structure for the tear-film lipid layer at physiologic temperature is a highly viscoelastic, shear-thinning liquid suspension consisting of lipid lamellar-crystallite particulates immersed in a continuous liquid phase with no long-range order. When spread over on-eye tear, the TFLL is a duplex film that exhibits bulk liquid properties and two separate interfaces, air/lipid and water/lipid, with aqueous protein and surfactantlike lipids adsorbed at the water/lipid surface.

Purpose: In vitro studies indicate that surface tension and surface viscosity of the tear film lipid layer (TFLL) are governed by interactions between meibomian lipids and proteins from the aqueous layer. The role of minor tear proteins with strong lipophilic properties or those correlated with pathological states is still unknown. The discovery of lung surfactant proteins (SPs) in tears and keratin in normal and abnormal meibomian gland excretions warrants investigation into their effects on the surface activity of meibomian lipid films., Methods: Commercial keratin and bovine lung SPs were used in vitro to assess the surface pressure of meibomian lipid films using a Langmuir trough., Results: The pressure-area profiles of meibomian lipid films seeded with SPs (2.5 μL; 0.1 μg) demonstrated hybrid characteristics between meibomian lipid films alone and SPs alone but reached much higher maximum surface pressures (approximately 30 vs. 24 mN/m). Microscopically, the appearance of meibomian lipid films was not altered by SPs. Maximum surface pressure of meibomian films premixed with keratin was much higher than meibum alone. The pressure-area isocycles appeared more like those of meibomian lipids with a low concentration of protein and more like pure keratin films at a high concentration., Conclusions: The data strongly indicate that SPs and keratin likely interact with the TFLL. SPs are likely to act as strong surfactants and to reduce the surface tension of the lipid layer. Excess concentrations of keratin as identified in patients with meibomian gland dysfunction could disrupt the normal structure of the meibomian lipid film.

Purpose: Meibomian lipid films have very complex physical properties that enable them to be compressed and expanded without collapsing. These properties can be attributed to the self assembly of the individual components, mainly wax and cholesteryl esters (WE and CE). Here, the surface pressure properties of WEs and CEs films have been compared to evaluate their contributions to meibomian lipid films., Methods: Films of different WEs and CEs were spread on a Langmuir trough and their surface pressure area profiles were compared with a particular emphasis on the effects caused by the degree of saturation of the alkyl/alkene chains., Results: Fully saturated WEs and CEs formed unstable films that collapsed upon compression. Very unsaturated waxes and CEs tended to have two distinct phases, one that reflects interaction with the aqueous subphase, while the second appeared to be with the multilayered bulk of the lipid film. With aging of the films, the WEs tended to move off the surface into the bulk. When meibomian lipid films were seeded with large amounts of WEs, only minor changes could be seen unless the WE was very unsaturated., Conclusions: These data are consistent with meibomian lipid films having a surfactant layer with a complex bulk layer external to this. It is speculated that the bulk layer contains thermotropic smectic chiral liquid crystals of CEs that are interacting with the WEs. This structure would tend to prevent collapse of the meibomian lipids onto the ocular surface and be very tolerant of lipophilic contaminants.

Purpose: Secretions that are produced by meibomian glands (also known as meibum) are a major source of lipids for the ocular surface of humans and animals alike. Many animal species have been evaluated for their meibomian lipidomes. However, there have been a very small number of studies in which the animals were compared with humans side by side. Therefore, the purpose of this study was to compare meibum collected from humans and three typical laboratory animals, canines, mice, and rabbits, for their meibomian lipid composition in order to determine which animal species most resembles humans., Methods: High pressure liquid chromatography (HPLC) and gas-liquid chromatography (GLC) in combination with mass spectrometry were used to evaluate lipidomes of all tested species., Results: Among three tested animal species, mice were found to be the closest match to humans in terms of their meibomian lipidomes, while canines were the second closest species. The lipids of these three species were close to each other structurally and, for most lipid classes, quantitatively. The rabbit meibomian lipidome, on the other hand, was vastly different from lipidomes of all other tested species. Interestingly, a previously described class of lipids, acylated omega-hydroxy fatty acids (OAHFA), was found to be present in every tested species as the major amphiphilic component of meibum., Conclusions: Our side by side comparison of the rabbit and the human meibum demonstrated their vast differences. Thus, the rabbit seems to be a poor animal model of the human tear film, at least when studying its biochemistry and biophysics.

Meibum is the primary component of the tear film lipid layer. Thought to play a role in tear film stabilization, understanding the physical properties of meibum and how they change with disease will be valuable in identifying dry eye treatment targets. Grazing incidence X-ray diffraction and X-ray reflectivity were applied to meibum films at an air-water interface to identify molecular organization. At room temperature, interfacial meibum films formed two coexisting scattering phases with rectangular lattices and next-nearest neighbor tilts, similar to the Ov phase previously identified in fatty acids. The intensity of the diffraction peaks increased with compression, although the lattice spacing and molecular tilt angle remained constant. Reflectivity measurements at surface pressures of 18 mN/m and above revealed multilayers with d-spacings of 50 Å, suggesting that vertical organization rather than lateral was predominantly affected by meibum-film compression.

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.

Protein-meibum and terpenoids-meibum lipid interactions could be important in the etiology of meibomian gland dysfunction (MGD) and dry eye symptoms. In the current model studies, attenuated total reflectance (ATR) infrared (IR) spectroscopy was used to determine if the terpenoid β-carotene and the major proteins in tears and meibum affect the hydrocarbon chain conformation and carbonyl environment of wax, an abundant component of meibum. The main finding of these studies is that mucin binding to wax disordered slightly the conformation of the hydrocarbon chains of wax and caused the wax carbonyls to become hydrogen bonded or experience a more hydrophilic environment. Lysozyme and lactoglobulin, two proteins shown to bind to monolayers of meibum, did not have such an effect. Keratin and β-carotene did not affect the fluidity (viscosity) or environment of the carbonyl moieties of wax. Based on these results, tetraterpenoids are not likely to influence the structure of meibum in the meibomian glands. In addition, these findings suggest that it is unlikely that keratin blocks meibomian glands by causing the meibum to become more viscous. Among the tear fluid proteins studied, mucin is the most likely to influence the conformation and carbonyl environment of meibum at the tear film surface., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: Human tear film stability decreases with increasing age. In this study, the changes in meibum composition were measured in search of markers of tear film instability., Methods: (1)H NMR nuclear magnetic resonance (NMR) spectra of 43 normal donors aged 1 to 88 years were acquired., Results: Compared with meibum from adolescents and adults, meibum from infants and children contains less CH(3) and C═C groups and an increased aldehyde-to-lipid hydroperoxide ratio., Conclusions: It is reasonable that tear film stability is higher in infants than in adults. Their meibum contains less CH(3) and C═C groups and higher levels of protein, and as a result, the lipid is more ordered because of the tighter and stronger lipid-lipid interactions. For water to evaporate, it must first pass through the tight lipid-lipid barrier. For tears to break up, lipid-lipid interactions must be broken. It is reasonable that because the lipid-lipid interactions are stronger in infants' and children's tears compared with those of adolescents and adults, the tear film in the younger groups is more stable and provides a better barrier to evaporation than does the tear film of adults. Lipid saturation could be the critical feature in meibum that stabilizes tears in infants.

Human Meibomian gland secretions (MGS) are a complex mixture of diverse lipids that are produced by Meibomian glands that are located in the upper and the lower eyelids. During blinking, MGS are excreted onto the ocular surface, spread and mix with aqueous tears that are produced by lachrymal glands, and form an outermost part of an ocular structure called "the tear film" (TF). The main physiological role of TF is to protect delicate ocular structures (such as cornea and conjunctiva) from desiccating. Lipids that are produced by Meibomian glands are believed to "seal" the aqueous portion of TF by creating a hydrophobic barrier and, thus, retard evaporation of water from the ocular surface, which enhances the protective properties of TF. As lipids of MGS are interacting with underlying aqueous sublayer of TF, the chemical composition of MGS is critical for maintaining the overall stability of TF. There is a consensus that a small, but important part of Meibomian lipids, namely polar, or amphiphilic lipids, is of especial importance as it forms an intermediate layer between the aqueous layer of TF and its upper (and much thicker) lipid layer formed mostly of very nonpolar lipids, such as wax esters and cholesteryl esters. The purpose of this review is to summarize the current knowledge on the lipidomics of human MGS, including the discussions of the most effective modern analytical techniques, chemical composition of MGS, biophysical properties of Meibomian lipid films, and their relevance for the physiology of TF. Previously published results obtained in numerous laboratories, as well as novel data generated in the author's laboratory, are discussed. It is concluded that despite a substantial progress in the area of Meibomian glands lipidomics, there are large areas of uncertainty that need to be addressed in future experiments., (Copyright © 2011 Elsevier Ltd. All rights reserved.)