Jadwiga C. Wojtowicz

Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum

Human meibum was targetly analyzed for the presence of intact wax esters (WEs) and related compounds by means of reverse-phase HPLC in combination with ion trap mass spectrometry. The major detected WEs were based on C18:n (n = 1–4) unsaturated FAs ranking in the following order of abundance: C18:1>C18:2>C18:3>C18:4. The major fatty alcohols (FAls) found in WE were of saturated nature and varied from C18:0 to C28:0. The three most abundant species were C18:1-FA esters of C24:0, C25:0, and C26:0-FAl. Typically, a major compound based on C18:1-FA and a saturated FAl was accompanied by a few related compounds based on a C18:2, C18:3, and C18:4-FA. Contrary to previous reports, no epoxy-WEs or epoxy-FAs were detected in fresh and 1-year-old meibum samples. More than 20 (O-acyl)-ω-hydroxy-FAs (OAHFAs) were observed. The main detected OAHFAs were based on very long-chain ω-hydroxy-FA (C30:1, C32:1, and C34:1) acylated through their ω-hydroxyls by a C18:1-FA. Due to their amphiphilic anionogenic nature, OAHFAs may be responsible for stabilization of the tear film lipid layer by creating an interface between the vast pool of strictly nonpolar lipids of meibum (WEs, cholesteryl esters, etc.) and the aqueous subphase beneath it, a role previously attributed to phospholipids.

Pilot, Prospective, Randomized, Double-masked, Placebo-controlled Clinical Trial of an Omega-3 Supplement for Dry Eye

Purpose: To investigate the potential effect of dietary supplementation with omega-3 fatty acid on lipid composition of meibum, aqueous tear evaporation, and tear volume in patients with dry eye. Methods: In a pilot, prospective, randomized, double-masked study, patients with dry eye received a daily dose of fish oil, containing 450 mg of eicosapentaenoic acid, 300 mg of docosahexaenoic acid, and 1000 mg of flaxseed oil (TheraTears Nutrition; Advanced Vision Research, Woburn, MA) for 90 days. There were 2 patient visits: baseline and final. At these visits, patients completed the ocular surface disease index to score subjective symptoms, and slit-lamp examinations, breakup time, corneal staining, Schirmer type I, fluorophotometry, evaporometry, and collection of meibomian gland secretion samples for lipid composition analysis were performed. Results: A total of 36 patients with dry eye completed the study. At the end of the study, 70% of the patients became asymptomatic, whereas for the placebo group, 7% of the symptomatic patients became asymptomatic. Schirmer testing and fluorophotometry suggested that the omega-3 supplement increased tear secretion. The lipid composition of the samples collected from the omega-3 group was found to be very similar to that from the placebo group. No trends between groups were seen for other objective parameters. Conclusions: Dietary supplementation with omega-3 fatty acids in dry eye showed no significant effect in meibum lipid composition or aqueous tear evaporation rate. On the other hand, the average tear production and tear volume was increased in the omega-3 group as indicated by both Schirmer testing and fluorophotometry.

Meibomian Lipid Films and the Impact of Temperature

PURPOSE There is evidence that, in cold conditions, the temperature of human eyelids and of the ocular surface drops well below normal physiological levels. This may have a detrimental impact on the stability and functionality of the human tear film and the tear film lipid layer. The goal of this project was to quantitatively examine the possible impact of temperature on the latter. METHODS Meibum samples were collected by using a soft-squeezing technique and were studied in a Langmuir trough. The obtained surface pressure and area isotherms were analyzed to determine the biophysical parameters of thin meibomian lipid film (MLF): the lift-off area, collapse pressure, two-dimensional elasticity, and hysteresis and their dependence on temperature. RESULTS MLF was found to be highly susceptible to changes in temperature. At temperatures below the physiological level, the MLF became stiff and shrank considerably. The shrinkage left a large portion of the air-water interface uncovered with lipid molecules. The effect was shown to be reversible. On reheating, the lipids melted and respread to restore the original film. There was a fundamental difference observed between three-dimensional melting of dry meibum in bulk and the two-dimensional melting in MLF at the air-water interface. Bulk meibum melted in a narrower temperature range and showed a much higher cooperativity of melting. CONCLUSIONS Temperature critically influences MLF. Low temperature leads to stiffening of the film, which loses its ability to form continuous layers at the air-water interface. These effects were shown be of a cooperative nature, manifesting in relatively narrow concentration and temperature ranges.

Changes in the Evaporation Rate of Tear Film After Digital Expression of Meibomian Glands in Patients With and Without Dry Eye

Purpose: To evaluate the effect of excess meibum on tear evaporation rate in patients with and without dry eye. Methods: Eleven healthy subjects and 16 patients with dry eye were tested. The dry eye group was divided into 2 subgroups: classic keratoconjunctivitis sicca (KCS) with clear and easily expressed meibum and KCS with meibomian gland dysfunction (MGD) with turbid secretions and difficult-to-express meibum. Evaporative measurements were performed at baseline and after digital expression of meibomian glands at 12, 24, 36, and 48 minutes. Two ranges of relative humidity were used, 25% to 35% and 35% to 45%. The data were expressed as microliters per square centimeter per minute. Results: An increase in the evaporation rate of the tear film was noted for all measurements at both relative humidities in the classic KCS and KCS with MGD groups compared with healthy subjects (P < 0.05). The average evaporation rates at relative humidities of 25% to 35% and 35% to 45% were 0.056 ± 0.016 and 0.040 ± 0.008 for the classic KCS group; 0.055 ± 0.026 and 0.037 ± 0.019 for the KCS with MGD group and 0.033 ± 0.012 and 0.023 ± 0.008 for the healthy group. Also, a decrease in the evaporation rate was observed in the healthy and KCS with MGD groups between baseline and the first measurement after digital expression for both relative humidities (P < 0.05). The classic KCS group did not show any changes after expression. Conclusions: Classic KCS and KCS with MGD groups showed an increase in tear evaporation rates compared with the healthy group. Aqueous tear evaporation diminished in the healthy and KCS with MGD groups after expression of meibomian glands. However, this effect was transient and negligible after the second measurement.

Historical brief on composition of human meibum lipids.

Meibomian glands (MG) secrete an oily substance, meibum, that spreads across the ocular surface and mixes with secretions produced by other ocular structures to create a thin film. The protective efficacy of the tear film is believed to be related to the chemical composition of the lipid layer. We reviewed the literature describing the composition of human MG secretions and have provided an overview on methods of collecting meibum samples, methods of lipid analyses, and the results obtained in previous studies. The usefulness and quality of the data obtained about meibum depend on proper sampling and the analytical techniques used. Historically, several methods have been developed, which have yielded contradictory data regarding meibum sample collection and analytical techniques. Based on review of the literature, the major lipids present in meibum are of nonpolar origin: waxes, sterols, and sterol esters, followed by triacylglycerides and fatty acids. The amphiphilic lipids, diacylglycerides, were reported in fewer studies, and monoacylglycerides were reported in only two. Information on the composition of the polar lipids is more controversial. Meibum phospholipids were found in small amounts (16% or less) in some studies, but not in others. Thus, meibum is a complex mixture of lipid molecules. Historical analytical inconsistencies may be partly explained by limitations of past analytical procedures and by the consideration that the tear film lipid layer may have contributing sources other than meibum.

Differential scanning calorimetric evaluation of human meibomian gland secretions and model lipid mixtures: transition temperatures and cooperativity of melting.

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.

Assessment and impact of the time of day on aqueous tear evaporation in normal subjects.

Objectives To determine the impact of the time of day on aqueous tear (AT) evaporation in normal subjects on two consecutive days. Methods In a controlled laboratory setting, morning and afternoon AT evaporation was tested in 19 normal subjects, at the same time of day on two consecutive days. Evaporometry was used at two ranges of relative humidity (RH) 25% to 35% and 35% to 45%. Results Mean AT evaporation rates were 0.069 ± 0.024 for 25% to 35% RH and 0.049 ± 0.018 for 35% to 45% (P = 0.001). There were significant differences for both RH between time of day (P < 0.05) on day 1, but not observed on day 2. Variation between days showed no difference for RH during the afternoon, but there was a difference during the morning (P = 0.042). Conclusions The data are remarkably consistent between study days, but there is more fluctuation during the morning than in the afternoon. Therefore, to further standardize AT evaporation study protocol, we recommend perform evaporometry measurements during the afternoon rather than the morning, because our results showed less variability during the afternoon test between days.

Effect of systane and optive on aqueous tear evaporation in patients with dry eye disease.

Objective: To compare the effect on aqueous tear (AT) evaporation rate of Systane and Optive at 30 min postinstillation in patients with dry eye. Methods: In a crossover study of 20 patients with keratoconjunctivitis sicca, the evaporation rate of AT was measured. Evaporometry was used at two relative humidity (RH) ranges of 25% to 35% and 35% to 45%. The measurements were made at baseline (before the instillation of the study agent) and at 30 min after the instillation of 40 μL of either Systane or Optive per randomization assignment per visit with a 1-week interval between visits. Results: No significant effects on AT evaporation rates at both RHs were found between study agents. Conclusions: In our study, neither Systane nor Optive has a significant impact on AT evaporation at 30 min postinstillation in patients with dry eye.

Dissecting lipid metabolism in meibomian glands of humans and mice: An integrative study reveals a network of metabolic reactions not duplicated in other tissues.

Lipids comprise the bulk of the meibomian gland secretion (meibum) which is produced by meibocytes. Complex arrays of lipogenic reactions in meibomian glands, which we collectively call meibogenesis, have not been explored on a molecular level yet. Our goals were to elucidate the possible biosynthetic pathways that underlie the generation of meibum, reveal similarities in, and differences between, lipid metabolism in meibomian glands and other organs and tissues, and integrate meibomian gland studies into the field of general metabolomics. Specifically, we have conducted detailed analyses of human and mouse specimens using genomic, immunohistochemical, and lipidomic approaches. Among equally highly expressed genes found in meibomian glands of both species were those related to fatty acid elongation, branching, desaturation, esterification, reduction of fatty acids to alcohols, and cholesterol biosynthesis. Importantly, corresponding lipid products were detected in meibum of both species using lipidomic approaches. For the first time, a cohesive, unifying biosynthetic scheme that connects genomic, lipidomic, and immunohistochemical observations is outlined and discussed.

Corneal thickness association with ocular and corneal high-order aberrations.

Purpose: To investigate the association among central corneal thickness, peripheral corneal thickness (PCT), and wavefront aberrations in the anterior cornea, posterior cornea and the whole eye in myopia. Methods: Sixty-four eyes of 64 myopic subjects were evaluated with a Pentacam rotating Scheimpflug camera (Oculus, Inc., WA) corneal topographer for: (1) wavefront aberrations from the anterior and posterior corneal surface, (2) corneal thickness (central and peripheral), and (3) with a wavefront aberration-supported cornea ablation wavefront analyzer (Wavescan, Visx, Inc., Santa Clara, CA) for wavefront aberrations generated in the whole eye. Relationships between the wavefront aberrations and corneal thickness were analyzed. Results: The mean age of subjects was 34.75 ± 10.08 years. The central corneal thickness was 550.5 ± 28.459 &mgr;m. The mean peripheral thickness was 629.9 ± 32.1 &mgr;m. Central and PCTs were not significantly correlated with corneal or ocular high-order aberrations. Intraocular pressure was significantly correlated with ocular trefoil (r = −0.307, P=0.001). Conclusions: Central and PCT were not significantly associated with either anterior or posterior corneal Zernike aberrations; in addition, no association with the whole ocular wavefront aberrations was found.