Oxygen-Carrying Capacity of Perfluorohexyloctane, a Novel Eye Drop for Dry Eye Disease.

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 °C and 37 °C, respectively. The T1 values for the CF2 group resonances increased by 17% to 24% with an increase in temperature from 25 °C to 37 °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 °C and 37 °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.

In Vitro Inhibition of Evaporation with Perfluorohexyloctane, an Eye Drop for Dry Eye Disease.

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 (Revap) of saline. Methods: Evaporation rates were measured gravimetrically at 25°C or 35°C. The evaporation rate (Revap) of phosphate-buffered saline (PBS) was measured following the application of 11-200 µL PFHO or 100 µL 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 Revap 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°C the mean (SEM) Revap of PBS alone or PFHO alone was 4.06 (0.06) and 0.137 (0.004) µm/min, respectively. Layering 100 µL PFHO over PBS inhibited the Revap of PBS by 81% (P < 0.0001), whereas artificial tears had no effect. The presence of mucin attenuated the inhibition of the Revap of PBS by PFHO by 17% (P < 0.0001). At 35°C, the Revap of PBS was inhibited by 88% when layering 100 µL PFHO over PBS and 28% when applying a single 11 µL drop of PFHO (P value < 0.0001 for both). Meibum lipid inhibited the Revap of PBS by 8% at this temperature, whereas the combination of a drop of PFHO plus meibum inhibited the Revap of PBS by 34%. Conclusions: PFHO significantly inhibited the Revap 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.

Lipid conformational order and the etiology of cataract and dry eye.

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.

A spectroscopic approach to measuring meibum lipid composition and conformation in donors with SjÓ§gren's syndrome.

Patients with SjÓ§gren'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.

In-vitro and ex-situ regional mass spectral analysis of phospholipids and glucose in the vitreous humor from diabetic and non-diabetic human donors.

The causes of vitreous humor (VH) liquefaction remain unclear. Diabetes accelerates this process and other ocular diseases. The weakening of the blood-retina barrier observed with diabetes could enhance the rate of transfer of relatively small molecules such as glucose (Glu) and phospholipids (PLs) from the retina to the VH. Glucose and PLs have been detected previously in VH but their regional distributions are not known. The mapping of Glu and PLs in VHs from subjects with and without diabetes could reveal the roles of these molecules in VH liquefaction. Diabetic and non-diabetic human eyes were acquired from the Kentucky Lions Eye Bank and frozen immediately. Each VH was removed and halved along the sagittal plane. One half was stamped on a matrix assisted laser desorption ionization (MALDI) plate. Either p-Nitroanaline (26 mg/mL MeOH:CHCl3) or 2,5-dihydroxybenzoic acid (20 mg/mL H2O:acetonitrile) was used as matrix. Glu and PLs were extracted from the remaining sections and analyzed. Data were acquired using a MALDI-mass spectrometer. The levels of Glu and PLs were significantly greater in VH from diabetics (VHd) compared with VH from non-diabetics (VHnd). VHds showed the highest relative levels of PLs in the posterior VH, followed by the anterior and central regions. Throughout the entire VH, the most abundant PLs were phosphatidylcholines followed by sphingomyelins. For Glu, the relative intensities were ~3 times higher in the posterior region of VHd (12 ± 1.3) compared with VHnd (6.5 ± 0.7) VHs. Regional studies showed that relative to the posterior VHd, the Glu levels were lower in the anterior (8.1 ± 1.0) and central (6.7 ± 0.8) regions. For the VHnds, the values for the central and anterior regions were 5.9 ± 1.2 and 4.7 ± 0.9, respectively. PLs and Glu are most abundant in the posterior region relative to the central and anterior zones of VHs. This trend was observed in VHd and VHnd, but PLs and Glu levels were significantly higher in VHds. These results support the possibility that higher levels of Glu and PLs accelerate VH liquefaction in diabetic patients. As liquefaction begins in the posterior region, the higher abundance of PLs and Glu in this zone also suggests that they may play a role in liquefaction. The specific molecular interactions affected by Glu and PLs in the collagen/hyaluronan/water network need to be examined.

Structural Differences in Meibum From Teenagers Without and With Dry Eye and Allogeneic Hematologic Stem Cell Transplantations.

Although dry eye occurs mostly in adults, dry eye may be induced in teens receiving allogeneic hematologic stem cell transplantations (AHSCT). Changes in meibum composition and structure has been associated with dry eye. The structure of meibum from teens with dye eye or teens with dry eye and AHSCT has not been studied, so in this study, we compared the structure of meibum from teens receiving AHSCT that had a dry eye with meibum from teens without AHSCT and without dry eye symptoms.

Lipid Saturation and the Rheology of Human Tear Lipids.

Elevated levels of acyl chain saturation of meibomian lipids are associated with enhanced tear film (TF) stability in infants to shortened TF breakup time with meibomian gland dysfunction. Thus, the effect of saturation on the surface properties of human TF lipids (TFLs) using a Langmuir surface balance and Brewster angle microscopy was studied. Lipid phase transitions were measured using infrared spectroscopy. The raise in the % of saturation resulted in thicker, and more elastic films at π = 12 mN/m, with the effects being proportional to the saturation level. At the same time, at lower (≤10 mN/m) π, the raise in saturation resulted in an altered spreading and modified structure of TFL layers. The strong impact of saturation on TFL surface properties correlated with a saturation induced increase of the TFL acyl chain order, phase transition temperature, and lipid-lipid interactions. The native TFL order and πmax were significantly greater, compared with native meibum collected from the same individual. Aggregation of lipids on the tear surface due to saturation was not as significant as it was for meibum. Although the surface pressure/area isotherms for TFL were similar for meibum, differences in rheology and phase transition parameters warrant the study of both.

The optical properties of rat, porcine and human lenses in organ culture treated with dexamethasone.

Organ culturing of lenses is useful for testing the direct effect of moieties such as cations, glucose, oxygen, antioxidants or drugs on the lens. Lens opacity changes in organ cultured lenses have never been measured quantitatively. Our aims were twofold: to establish the time course of lens opacity changes in organ cultured rat, porcine and human lenses and to test the utility of our system to measure opacity changes in lenses treated with dexamethasone. Human, porcine and rat lenses were incubated with or without 5 µM dexamethasone in minimum essential medium for 13 days. Lens absorbance and light scattering were measured using a spectrometer. Lens opacity was graded from lens photographs. Sixteen human lenses were obtained from 10 donors. Light scattering accounted for all of the absorbance of the human lens above 550 nm, 72% of the absorbance at 500 nm and 50% of the absorbance at 450 nm. Similarly, for the porcine lens, light scattering accounted for all of the absorbance above 400 nm and 40% of the absorbance at 350 nm. For the rat lens, scattering accounted for all of the absorbance above 325 nm, and 37% of the absorbance at 300 nm. In the rat lens spectrum, the broad absorbance peak near 600 nm was unique to the rat lens. After day five in organ culture, the level of light scattering, absorbance and optical grade of human lenses were higher in dexamethasone treated lenses compared with those without dexamethasone. Similar differences were observed for rat lenses after one day in organ culture. The opacity of porcine lenses did not change with dexamethasone treatment. In conclusion, this study showed that dexamethasone-induced opacity in human and rat lenses could be measured which is important to future studies to test the positive or negative efficacy of moieties that cause or ameliorate cataract.

Insights into Tear Film Stability from Babies and Young Adults: A Study of Human Meibum Lipid Conformation and Rheology.

Babies have the most stable tears and people with dry eye have the least stable tears. Meibum may contribute to tear film stability, so in this study, the hydrocarbon chain conformation and rheology of meibum from babies was studied for the first time. Infrared spectroscopy was used to measure lipid phase transitions. Rheology was measured using Langmuir film technology. Meibum from 25 donors 1 to 13 years old was compared with meibum from 18 donors 13 to 25 years old. The phase transition temperature and lipid order (stiffness) increased with increasing age from 1 to 25 years. The increase in meibum lipid order from 1 to 25 years of age may contribute to the instability of the tear film with age and contribute to films with a higher reciprocal compressibility modulus that are not as compressible and not as viscoelastic. Changes in the lipid phase transition parameters of meibum lipid with dry eye are an exacerbation of the changes observed with age. The lower reciprocal compressibility moduli of meibum films from children and babies compared with meibum from adults reiterates higher stability in their films which spread better, resist deformation, and facilitates their ability to be quickly restored after blinking.

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.

Whales, lifespan, phospholipids, and cataracts.

This study addresses the question: why do rats get cataracts at 2 years, dogs at 8 years, and whales do not develop cataracts for 200 years? Whale lens lipid phase transitions were compared with the phase transitions of other species that were recalculated. The major phospholipids of the whale lens were sphingolipids, mostly dihydrosphingomyelins with an average molar cholesterol/phospholipid ratio of 10. There was a linear correlation between the percentage of lens sphingolipid and lens lipid hydrocarbon chain order until about 60% sphingolipid. The percentage of lens sphingolipid correlated with the lens lipid phase transition temperature. The lifespan of the bowhead whale was the longest of the species measured and the percentage of whale lens sphingolipid fit well in the correlation between the percentage of lens sphingolipid and lifespan for many species. In conclusion, bowhead whale lens membranes have a high sphingolipid content that confers resistance to oxidation, allowing these lenses to stay clear relatively longer than many other species. The strong correlation between sphingolipid and lifespan may form a basis for future studies, which are needed because correlations do not infer cause. One could hope that if human lenses could be made to have a lipid composition similar to whales, like the bowhead, humans would not develop age-related cataracts for over 100 years.

Regional distribution of phospholipids in porcine vitreous humor.

This project explores the regional phospholipid distribution in porcine vitreous humor, retina, and lens. Matrix-assisted laser desorption mass spectrometry has been used previously to image lipids, proteins, and other metabolites in retinas and lenses. However, the regional composition of phospholipids in vitreous humors is not known. To address this issue, we have applied this mass spectral method to explore the regional phospholipid distribution in porcine vitreous humor both ex-situ and in-vitro. To establish the possible source(s) of phospholipids in the vitreous humor, compositional studies of the lens and retina were also pursued. Due to the overall low levels of phospholipids in vitreous humor, it was necessary to optimize the experimental approaches for ex-situ and in-vitro studies. The sensitivity observed in the spectra of methanol extracts from the lens and retina was higher than that for methanol:chloroform extracts, but the compositional trends were the same. A fourfold improvement in sensitivity was observed in the analysis of vitreous humor extracts obtained with the Bligh and Dyer protocol relative to the other two extraction methods. For ex-situ studies, the 'stamp method' with para-nitroaniline as the matrix was chosen. Throughout the vitreous humor, phosphatidylcholines were the most abundant phospholipids. In-vitro results showed higher relative levels of phospholipids compared to the 'stamp' method. However, more details in the regional phospholipid distribution were provided by the ex-situ approach. Both in-vitro and ex-situ results indicated higher levels of phospholipids in the posterior vitreous region, followed by the anterior and central regions. The posterior region contained more unsaturated species whereas more saturated phospholipids were detected in the anterior region. The observed trends suggest that the phospholipids detected in the posterior vitreous humor migrate from the retina and associated vasculature while those present in the anterior regions are likely to derive from the lens. Not all species found in the lens were observed in the vitreous humor. For example, whereas cholesterol was present in lens extracts, it was not detected in the vitreous humor. Overall, the higher relative abundance of unsaturated species in the posterior vitreous humor and also present in the retina suggests that these species may be able to disrupt the water-collagen-hyaluronan network and contribute to vitreous liquefaction.

Human Meibum Age, Lipid-Lipid Interactions and Lipid Saturation in Meibum from Infants.

Tear stability decreases with increasing age and the same signs of instability are exacerbated with dry eye. Meibum lipid compositional changes with age provide insights into the biomolecules responsible for tear film instability. Meibum was collected from 69 normal donors ranging in age from 0.6 to 68 years of age. Infrared spectroscopy was used to measure meibum lipid phase transition parameters. Nuclear magnetic resonance spectroscopy was used to measure lipid saturation. Increasing human meibum lipid hydrocarbon chain unsaturation with age was related to a decrease in hydrocarbon chain order, cooperativity, and in the phase transition temperature. The change in these parameters was most dramatic between 1 and 20 years of age. Meibum was catalytically saturated to determine the effect of saturation on meibum lipid phase transition parameters. Hydrocarbon chain saturation was directly related to lipid order, phase transition temperature, cooperativity, changes in enthalpy and entropy, and could account for the changes in the lipid phase transition parameters observed with age. Unsaturation could contribute to decreased tear film stability with age.

Pilot study of the influence of eyeliner cosmetics on the molecular structure of human meibum.

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.

Surface Properties of Squalene/Meibum Films and NMR Confirmation of Squalene in Tears.

Squalene (SQ) possesses a wide range of pharmacological activities (antioxidant, drug carrier, detoxifier, hydrating, emollient) that can be of benefit to the ocular surface. It can come in contact with human meibum (hMGS; the most abundant component of the tear film lipid layer) as an endogenous tear lipid or from exogenous sources as eyelid sebum or pharmaceuticals. The aims of this study were to determine (i) if SQ is in tear lipids and (ii) its influence on the surface properties of hMGS films. Heteronuclear single quantum correlation NMR confirmed 7 mol % SQ in Schirmer's strips extracts. The properties of SQ/hMGS pseudo-binary films at the air/water interface were studied with Langmuir surface balance, stress-relaxation dilatational rheology and Brewster angle microscopy. SQ does not possess surfactant properties. When mixed with hMGS squalene (i) localized over the layers' thinner regions and (ii) did not affect the film pressure at high compression. Therefore, tear SQ is unlikely to instigate dry eye, and SQ can be used as a safe and "inert" ingredient in formulations to protect against dry eye. The layering of SQ over the thinner film regions in addition to its pharmacological properties could contribute to the protection of the ocular surface.

13C and 1H NMR ester region resonance assignments and the composition of human infant and child meibum.

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.

Lipid order, saturation and surface property relationships: a study of human meibum saturation.

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.

Relationship Between Human Meibum Lipid Composition and the Severity of Meibomian Gland Dysfunction: A Spectroscopic Analysis.

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.

Wax-tear and meibum protein, wax-ß-carotene interactions in vitro using infrared spectroscopy.

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 ß-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 ß-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.

Correlations between bulk and surface properties of meibomian lipids with alteration of wax-to-sterol esters content.

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.