Identification of new Omega-3 very long chain poly-unsaturated fatty acids in meibomian gland secretions.

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.

Meibum sphingolipid composition is altered in individuals with meibomian gland dysfunction-a side by side comparison of Meibum and Tear Sphingolipids.

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.

Alteration in meibum lipid composition and subjective symptoms due to aging and meibomian gland dysfunction.

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.

Human meibum and tear film derived cholesteryl and wax esters in meibomian gland dysfunction and tear film structure.

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).

Mass Spectrometric Analysis of Meibomian Gland Lipids.

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.

Interactions of Meibum and Tears with Mucomimetic Polymers: A Hint towards the Interplay between the Layers of the Tear Film.

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.

Cervidins A-D: Novel Glycine Conjugated Fatty Acids from the Tarsal Gland of Male Whitetail Deer, Odocoileus virginianus.

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.

Comparative Transcriptomic and Lipidomic Analyses of Human Male and Female Meibomian Glands Reveal Common Signature Genes of Meibogenesis.

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.

Untargeted lipidomic analysis of human tears: A new approach for quantification of O-acyl-omega hydroxy fatty acids.

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.

Comparison of Collection Methods for the Measure of Human Meibum and Tear Film-Derived Lipids Using Mass Spectrometry.

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.

Effects of Lipid Saturation on the Surface Properties of Human Meibum Films.

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.

Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids.

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.

Review of Literature on Measurements of Non-invasive Break Up Times, Lipid Morphology and Tear Meniscal Height Using Commercially Available Hand-held Instruments.

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.

Proteolytic activity in the meibomian gland: Implications to health and disease.

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.

Surface properties and exponential stress relaxations of mammalian meibum films.

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.

Behavior of sphingomyelin and ceramide in a tear film lipid layer model.

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.

Compositional Analysis of Wax Esters in Human Meibomian Gland Secretions by Direct Infusion Electrospray Ionization Mass Spectrometry.

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.

Evaporation and Hydrocarbon Chain Conformation of Surface Lipid Films.

PURPOSE: The inhibition of the rate of evaporation (Revap) by surface lipids is relevant to reservoirs and dry eye. Our aim was to test the idea that lipid surface films inhibit Revap. METHODS: Revap 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 µm2. All of the surface lipids were ordered. Revap of the shorter chain hydroxyl lipids were slightly (7%) but significantly lower compared with the longer chain hydroxyl lipids. Revap of both groups was essentially similar to that of buffer. A hydroxyl lipid film did not influence Revap over an estimated average thickness range of 0.69 to >6.9 µm. Revap of human tears and buffer with and without human meibum (34.4 µm thick) was not significantly different. Revap 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 Revap of buffer or tears even though they completely covered the surface. It is unlikely that hydroxyl lipids can be used to inhibit Revap of reservoirs. Our data do not support the widely accepted (yet unconfirmed) idea that the tear film lipid layer inhibits Revap of tears.

Sebum/Meibum Surface Film Interactions and Phase Transitional Differences.

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.