Purpose:The degree of unsaturation in hydrocarbon chains (HCunsat) is likely to play an important role in determining the stability, order, and fluidity of lipids in the tear film lipid layer. The HCunsat of meibum lipids was investigated in relationship with age, sex, dry eye, Sjögren's syndrome, Parkinson's disease, and treatment for allogeneic hematopoietic stem cell transplantation. Methods:Meibum was expressed and HCunsat was measured using 1H-NMR resonances at 5.33 and 2.00 ppm. HCunsat levels were normalized to the level of wax esters, cholesteryl esters, and hydrocarbons. Results:HCunsat levels, normalized to lipid esters, were found to be 20% to 40% lower in individuals aged >50 years compared with those <50 years (P < 0.05) and showed a linear age-dependent decrease with age (P < 0.001). No substantial changes were observed with sex and race, except for a 24% reduction in a small Hispanic group (P = 0.0078) compared with Caucasians. A cohort with dry eye had a ∼13% lower level of HCunsat, whereas cohorts susceptible to dry eye such as those with Sjögren's syndrome, Parkinson's, and graft-versus-host disease had a ∼35% lower HCunsat levels compared with a cohort without dry eye (P < 0.05). Experimental deviations were minimal (<4%). Conclusions:Meibum HCunsat decreases with age and is lower with dry eye, Sjögren's syndrome, and Parkinson's disease cohorts compared with a cohort without dry eye. This decrease in HCunsat is expected to contribute to lipid aggregation, decreased lipid spreading, and meibomian gland outflow. ¹H-NMR was effective for quantifying major meibum lipids.

The tear film lipid layer (TFLL) is an outermost component of the tear film. The TFLL plays a critical role in maintaining ocular hydration, protecting the corneal surface, and preserving visual clarity. By stabilizing the tear film and minimizing evaporative water loss, the TFLL helps to prevent tear film breakup. Disruptions in the structural organization or chemical composition of the TFLL can compromise its function and cause dry eye disease (DED). Molecular-level mechanisms governing the TFLL behavior remain poorly understood. This study presents large-scale, atomistic molecular dynamics (MD) simulations of TFLL incorporating physiological lipid compositions. Model accuracy was validated by comparison to previously reported experimental properties. The model exhibited reasonable densities and lipid ordering, as previously observed in spectroscopic studies. Shear thinning behavior was observed, consistent with known viscoelastic characteristics. Lateral diffusion coefficients were in reasonable agreement with experimental data. The resulting atomistic representation is a reproducible, high-resolution molecular model that will enable future investigations into TFLL properties. The model structure provides mechanistic insights into tear film structuring and establishes a foundation for future computational work that can inform the development of more effective, long-lasting therapies for DED.

Purpose:Meibum compositional, structural, and functional relationships have been inferred indirectly. The current study measured the relationships among cholesteryl esters (CEs), lipid hydrocarbon chain unsaturation (HCunsat), meibum protein, and meibum structure, directly on the same samples as correlation does not necessitate cause. Methods:Exactly 367 meibum samples were collected from a diverse group of donors, categorized by race, sex, and age including cohorts with individuals with (with CDED) and without (Cn) dry eye disease (DED) where "C" refers to cohort and "n" refers to normal. Meibum composition was measured using 1H-NMR. Meibum phase transition characteristics were measure using infrared spectroscopy. Results:A decrease in HCunsat and CEs with DED results in stiffer, more tightly packed, and less mobile lipids. Meibum proteins have an opposite effect on CDED lipids than CE and HCunsat, moderating structural changes due to compositional variation. Lipid hydrocarbon chain order and phase transition temperature were higher in DED than in healthy eyes, regardless of age, but both decreased with age in DED, indicating reduced order in older donors. In meibum without protein, DED samples showed greater stiffness and higher phase transition temperatures, even at similar unsaturation levels, whereas protein presence minimized these differences. Conclusions:A decrease in HCunsat and CEs with DED would likely inhibit the flow of meibum from the meibomian glands and cause a discontinuous patchy tear film lipid layer, resulting in deteriorated spreading, decreased surface elasticity, and an unstable tear film. Therapies to increase HCunsat and CEs could be successful in treating DED.

Purpose:To visualize the behavior of perfluorohexyloctane (PFHO), an eye drop to treat dry eye disease (DED), on the surface of saline in vitro and on the human ocular surface using infrared emissivity. Methods:Emissivity videos were used to measure the spreading and disappearance rates of PFHO on saline (with and without mucin for spreading rate) and layered over a 125 nm film of meibum on the surface of saline using a TearView camera. Ocular surface emissivity was videoed in a volunteer without DED before and after instillation of 1 drop of PFHO. Videos were exported and converted to still photographs, and grayscale levels measured. Results:PFHO formed a layer over saline that spread at a mean (SD) rate of 0.89 (0.5) cm2/s and disappeared at 0.0760 (0.0055) mu L/min, consistent with reported rates of evaporation for PFHO. Mucin in the subphase did not alter spreading rate (P > 0.2). In vitro, a single drop of PFHO spread over the top of a 125-nm thick film of meibum remaining for 3.3 hours. In the volunteer, an increase in emissivity was detected on the ocular surface for >= 5 hours. Conclusions:PFHO quickly spread to form a layer over the surface of saline or meibum in vitro and was detected on the ocular surface in vivo for >= 5 hours after topical administration. This supports findings that PFHO forms a long-lasting barrier to evaporation at the air-liquid interface of the tear film and thus reduces signs and symptoms of DED.

OBJECTIVE:Semifluorinated alkanes (SFAs) are amphiphilic molecules with a fluorinated carbon segment (F) and a hydrocarbon segment (H). Perfluorohexyloctane ophthalmic solution (F6H8; MIEBO®) is an anti-evaporative agent for dry eye that forms a long-lasting layer on the surface of the tear film. This in vitro study evaluated the impact of SFA chain length on intermolecular interactions, evaporation rate (Revap), and inhibition of Revap of saline to compare the anti-evaporative properties of various SFAs. METHODS:The SFAs F4H2, F4H5/CsA (cyclosporine ophthalmic solution 0.1 %; Vevye®), F6H7, F6H8, and F6H10 were evaluated. The Revap of 1 mL of each SFA and saline alone or saline with 11 or 100 μL of the SFA layered over top were evaluated gravimetrically at 35 °C. Total sample weight was recorded every 10 min over 100 min, and Revap was obtained from the slope of the best-fit line obtained by least squares linear regression analysis. The relative order (fluidity) of the SFAs was evaluated using Fourier transform infrared spectroscopy. RESULTS:The mean (SEM) inherent Revap (mg/min) was 102 (25), 6.0 (0.6), 0.58 (0.20), 0.15 (0.017), and 0.12 (0.09) for F4H2, F4H5/CsA, F6H7, F6H8, and F6H10, respectively (n = 3-9). The Revap of saline with a 100 μL F6H7, F6H8, and F6H10 layer was inhibited by 40 %, 80 %, and 66 %, respectively (p < 0.001 vs saline), and unchanged by F4H2 and F4H5/CsA. Only F6H8 had a significant inhibitory effect on Revap of saline when applying an 11 μL drop (p < 0.0001 vs saline; n = 10-25). The SFA-SFA intermolecular interactions were greater with longer H and F chain lengths. CONCLUSIONS:SFA chain length and volatility correlated with inhibition of the Revap of saline with the longest chain length SFAs F6H8 and F6H10 having the maximal effect. These data support the suitability of F6H8 as a treatment for evaporative dry eye disease.

Purpose: Changes in meibum composition and quantity in meibomian gland dysfunction (MGD) result in tear film instability and dry eye. This exploratory study aimed to identify changes in (O-acyl)-ω-hydroxy fatty acid (OAHFA) and hydrocarbon chain (HC) unsaturation levels in meibum related to the presence and severity of MGD. Methods: Meibum samples were collected from 3 cohorts of adults with no MGD, mild-to-moderate MGD, and severe MGD in a noninterventional clinical trial (NCT01979887). OAHFAs, cholesterol esters (CE), HC unsaturation, and HC length in the meibum samples were quantified with 1H-nuclear magnetic resonance spectroscopy using 2 methods of normalization. Results: Meibum samples from 62 subjects were analyzed: 21 non-MGD, 21 mild-to-moderate MGD, and 20 severe MGD. Meibum OAHFA and CE levels and HC unsaturation were reduced with increasing severity of MGD, with most pairwise comparisons significant (P < 0.05, t-tests), following the order non-MGD > mild-to-moderate MGD > severe MGD. Regardless of the resonances used for normalization, each pairwise comparison of OAHFA, CE, and HC unsaturation levels in MGD (combined severities) versus non-MGD samples was significant (P < 0.01, t-test). Analysis using various normalization equations showed reductions of 20%-22% for OAHFAs, 51%-57% for CE, and 36%-66% for HC unsaturation in MGD (combined severities) compared with non-MGD. HC length was not altered in MGD (combined severities) compared with non-MGD samples (t-test). Conclusions: Meibum OAHFA, CE, and HC unsaturation levels were reduced in MGD and were lowest in the severe MGD cohort. These findings may contribute to the understanding of the pathophysiology of MGD.

Objective: One-hundred percent perfluorohexyloctane (PFHO) is a water-free, preservative-free eye drop approved by the Food and Drug Administration in the United States for the treatment of dry eye disease. PFHO has shown relief of dry eye signs and symptoms in clinical trials and has potent antievaporative action in vitro. The objective of this study was to measure the level of oxygen in PFHO.Methods: T1 relaxation times (time taken for proton spins to translate from a random alignment to an alignment with the main magnetic field) for fluorine-19 in perfluorohexyloctane were measured using fluorine-19 nuclear magnetic resonance spectroscopy. The level of oxygen was interpolated from published data.Results: The hydrogen-1 and fluorine-19 nuclear magnetic resonance spectra of PFHO were well resolved and the resonance assignments and intensities were as expected. The T1 values calculated for the CF3 group resonance in the current study was 0.901 seconds and 1.12 seconds at 25 & DEG;C and 37 & DEG;C, respectively. The T1 values for the CF2 group resonances increased by 17% to 24% with an increase in temperature from 25 & DEG;C to 37 & DEG;C. The mean (SD) partial pressure of oxygen in PFHO was calculated to be 257 (36) mm Hg and 270 (38) mm Hg at 25 & DEG;C and 37 & DEG;C, respectively.Conclusions: The current study confirms that PFHO contains a significant amount of oxygen, more so than that calculated for tears in equilibrium with air. Once instilled on the eye, PFHO is not expected to be a barrier to the oxygen necessary for a healthy cornea and may in fact deliver nonreactive oxygen to the cornea to facilitate healing in patients with dry eye disease.& COPY; 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Objective: Perfluorohexyloctane (PFHO) MIEBOTM, formerly (NOV03) is a single component, water-free eye drop approved by the Food and Drug Administration in the United States for the treatment of dry eye disease. We evaluated the in vitro inhibitory effect of PFHO on the evaporation rate (R-evap) of saline. Methods: Evaporation rates were measured gravimetrically at 25 degrees C or 35 degrees C. The evaporation rate (R evap) of phosphate-buffered saline (PBS) was measured following the application of 11-20 0 mu L PFHO or 10 0 mu L artificial tears (Soothe XP [Bausch + Lomb, Bridgewater, New Jersey], Systane Balance [Alcon, Fort Worth, Texas], and Systane Ultra [Alcon]). The effect of PFHO on the R evap of PBS was further evaluated following the addition of 50 mg/mL mucin to PBS and compared with that of meibum lipid collected from a 68 year-old White volunteer. Results: At 25 degrees C the mean (SEM) R evap of PBS alone or PFHO alone was 4.06 (0.06) and 0.137 (0.004) mu m/min, respectively. Layering 100 mu L PFHO over PBS inhibited the R evap of PBS by 81% (P < 0.0001), whereas artificial tears had no effect. The presence of mucin attenuated the inhibition of the R evap of PBS by PFHO by 17% (P < 0.00 01). At 35 degrees C, the R evap of PBS was inhibited by 88% when layering 100 mu L PFHO over PBS and 28% when applying a single 11 mu L drop of PFHO (P value < 0.0001 for both). Meibum lipid inhibited the R evap of PBS by 8% at this temperature, whereas the combination of a drop of PFHO plus meibum inhibited the R evap of PBS by 34%. Conclusions: PFHO significantly inhibited the R evap of saline in this in vitro model. The data support the idea that PHFO may form an antievaporative layer on the tear film surface and may be a functional substitute for the native tear-film lipid layer in patients with dry eye disease. (c) 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/

Purpose:Information on the relationship between meibum lipid composition and severity of meibomian gland dysfunction (MGD) is limited. The purpose of this study was to analyze the molecular components of meibum collected from individuals with no MGD, mild-to-moderate MGD, and severe MGD.Methods:Adults with and without MGD were enrolled in a prospective, multicenter, exploratory clinical trial (ClinicalTrials.gov Identifier: NCT01979887). Molar ratios of cholesteryl ester to wax ester (RCE/WE) and aldehyde to wax ester (Rald/WE) in meibum samples were measured with 1H-NMR spectroscopy. Results were evaluated for participants grouped by MGD disease status and severity (non-MGD, mild-to-moderate MGD, and severe MGD), as defined by maximum meibum quality scores, Schirmer test results, and Subject Ocular Symptom Questionnaire responses.Results:Sixty-nine meibum samples from 69 individuals were included in the analysis: 24 non-MGD, 24 mild-to-moderate MGD, and 21 severe MGD. Mean RCE/WE was 0.29 in non-MGD, 0.14 in mild-to-moderate MGD (P = 0.038 vs. non-MGD, 51% lower), and 0.07 in severe MGD (P = 0.16 vs. mild-to-moderate MGD, 52% lower; P = 0.002 vs. non-MGD, 76% lower). Mean Rald/WE was 0.00022 in non-MGD, 0.00083 in mild-to-moderate MGD (P = 0.07 vs. non-MGD, 277% higher), and 0.0024 in severe MGD (P = 0.003 vs. mild-to-moderate MGD, 190% higher; P < 0.001 vs. non-MGD, 992% higher).Conclusions:RCE/WE was lowest and Rald/WE was highest in the severe MGD cohort, suggesting that these meibum constituent molar ratios may result from the pathophysiology associated with MGD and can impact ocular surface lipid and tear film homeostasis. These findings may potentially help identify targets for MGD treatment.

In a recent study (Ewurum et al., 2021), wax (WE) and sterol esters (CE) from human meibum secretions (MGS) were separated and reconstituted with controlled WE/CE ratios (0%, 20%, 30%, 40%, 50%, 75% and 100% CE weight fractions). It was found that the alterations in the CE content of WE/CE mixtures modified the hydrocarbon chain conformation and packing of the mixture. A major question that emerges is whether the spectroscopic packing parameters determined for bulk meibum translate to a change in the performance of meibomian layers at the air/water interface, as it is the surface film functionality that is crucial for the performance of MGS at the ocular surface. The study of human meibum films with Langmuir surface balance was performed to access the surface properties at blink-like deformations of the film area. Surface pressure (π)-area (A) isocycles and stress relaxations were used to assess the layer's reorganization during area cycling and dilatational elasticity, respectively. The morphology of the films was monitored by Brewster angle microscopy. It was found that the increased order and chain melting temperature of the bulk samples correlated with a raise in the maximum surface pressure attained at minimal surface area and in the transient dilatational modulus of the meibomian layers. Such correlations may allow for development of an improved understanding between the bulk and surface properties of human meibum and of other natural and synthetic tear lipid films.

Patients with Sjӧgren's syndrome (SS) have dry eye associated with meibomian gland dysfunction (MGD). The meibum from donors with dry eye due to MGD but without SS (MMGD) presents with lower levels of cholesteryl ester, less straight chains, and more ordered hydrocarbon chains compared with meibum from donors without MGD (Mn). The aim of the current study was to compare the composition and hydrocarbon chain conformation of meibum from donors with Sjögren's syndrome (Mss) to Mn and MMGD. Meibum was expressed from patients with SS using an ILUX instrument (Alcon Inc., Fort Worth TX). All of the nine meibum donors with SS were female. Meibum composition was characterized using 1H-NMR and meibum hydrocarbon chain conformation was measured using fourier transform infrared spectroscopy. Meibum from every donor with SS measured contained a significantly (P < 0.01) higher cholesteryl ester/wax ester ratio and more straight chains compared with donors without SS or dry eye. None of the nine phase transitional parameters were significantly different, P > 0.05, for Mss compared with Mn. Nor was the CH3/CH2 band height ratio used to estimate the number of hydrocarbon CH3 and CH2 moieties different, P = 0.22, for Mss compared with Mn. In conclusion, the compositional differences between Mss compared with Mn did not result in differences in any of the nine meibum lipid phase transitional parameters measured. The compositional differences observed between Mss and Mn could be markers for or contribute to SS as the differences could lead to tear film lipid packing differences other than conformational differences.

Lens and tear film lipids are as unique as the systems they reside in. The major lipid of the human lens is dihydrosphingomylein, found in quantity only in the lens. The lens contains a cholesterol to phospholipid molar ratio as high as 10:1, more than anywhere else in the body. Lens lipids contribute to maintaining lens clarity, and alterations in lens lipid composition due to age are likely to contribute to cataract. Lens lipid composition reflects adaptations to the unique characteristics of the lens: no turnover of lens lipids or proteins; the lowest amount of oxygen of any tissue; and contains almost no intracellular organelles. The tear film lipid layer (TFLL) is also unique. The TFLL is a thin (100 nm) layer of lipid on the surface of tears covering the cornea that contributes to tear film stability. The major lipids of the TFLL are wax esters and cholesterol esters that are not found in the lens. The hydrocarbon chains associated with the esters are longer than those found anywhere else in the body (as long as 32 carbons), and many are branched. Changes in the composition and structure of the 30,000 different moieties of TFLL contribute to the instability of tears. The focus of the current review is how spectroscopy has been used to elucidate the relationships between lipid composition, conformational order and function, and the etiology of cataract and dry eye.

PURPOSE:Patients with Parkinson's disease (PD) exhibit unstable tear films. Tear film lipid composition and structure are related to tear film stability and dry eye and tear lipids have not been characterized in people with PD. The aim of this study is to characterize Meibum tear lipids in donors with PD using 1H-NMR and infrared spectroscopy.METHODS:Three cohorts were compared: meibum from donors with PD (Mp) n = 10, meibum from donors with PD and dry eye (Mpd) n = 3, meibum from donors without PD (Mn) n = 29.RESULTS:There were no significant differences, P > 0.05, in hydrocarbon branching for Mp compared with Mn. Mn contained twice as much cholesteryl esters compared with Mp, P < 0.0001. The cooperativity of the phase transition was significantly 37% lower for Mp compared with Mn, P < 0.0001. Mpd was much more ordered (stiffer) with compared with Mp and Mn, P < 0.0001.CONCLUSION:Changes in meibum lipid composition and structure could be a marker for and/or contribute to increase the susceptibility of dry eye in patients with PD. A less cooperative phase transition for Mp compared with Mn indicates that Mp was more heterogeneous and/or contained more contaminants than Mn. The data support the idea that more ordered lipid contributes to dry eye.