Disruption and destabilization of meibomian lipid films caused by increasing amounts of ceramides and cholesterol.

PURPOSE: We evaluated quantitatively direct effects of ceramide (Cer) and free cholesterol (FC) on meibomian lipid films (MLF) using a Langmuir trough (LT) and a Brewster angle microscope (BAM). METHODS: Meibum was obtained from healthy volunteers. A series of mixtures of meibum with Cer or FC (mixed MLF) taken in different ratios were tested. Standard rheologic parameters, such as elasticity and hysteresis of MLF, were computed. BAM was used to study the morphology of MLF. RESULTS: Pure MLF were capable of withstanding multiple compression/expansion cycles with little hysteresis observed (1.9 J/G meibum). The films made of either pure CER or pure FC were clearly collapsible, and had much higher rigidity and hysteresis than pure meibum. Adding progressively higher amounts of CER or FC to meibum had a strong impact on the rigidity, stability, and morphology of the mixed MLF: their hysteresis increased many fold compared to pure meibum. A concomitant increase in the rigidity and collapsibility of the mixed MLF was observed. CONCLUSIONS: Cer and FC changed the surface properties of mixed MLF in a way that implied their destabilization and/or disruption. One of the mechanisms that might lead to these effects is strong aggregation of meibum lipids with FC or Cer that leads to the formation of smaller particles of meibum surrounded by a thinner layer of FC or Cer. As Cer and FC can be elevated in meibum and the tear film because of certain pathologic processes, or can be of exogenous nature, our results can explain (partially) a less stable tear film in those subjects.

Evaluation and quantitation of intact wax esters of human meibum by gas-liquid chromatography-ion trap mass spectrometry.

PURPOSE: Wax esters (WE) of human meibum are one of the largest group of meibomian lipids. Their complete characterization on the level of individual intact lipid species has not been completed yet. We obtained detailed structural information on previously uncharacterized meibomian WE. METHODS: Intact WE were separated and analyzed by means of high-temperature capillary gas-liquid chromatography (GLC) in combination with low voltage (30 eV) electron ionization ion trap mass spectrometry (ITMS). 3D (mass-to-charge ratio [m/z] versus lipid sample weight versus signal intensity) calibration plots were used for quantitation of WE. RESULTS: We demonstrated that GLC-ITMS was suitable for analyzing unpooled/underivatized WE collected from 14 individual donors. More than 100 of saturated and unsaturated WE (SWE and UWE, respectively) were detected. On average, UWE represented about 82% of the total WE pool. About 90% of UWE were based on oleic acid, while less than 10% were based on palmitoleic acid. The amounts of poly-UWE were <3% of their mono-UWA counterparts. SWE were based primarily on C(16)-C(18) fatty acids (FA) in overall molar ratios of 22:65:13. A pool of C(16:0)-FA was comprised of a 20:80 (mol/mol) mixture of straight chain and iso-branched isomers, while the corresponding ratio for C(18:0)-FA was 43:57. Interestingly, C(17:0)-FA was almost exclusively branched, with anteiso- and iso-isomers found in a ratio of 93:7. CONCLUSIONS: GLC-ITMS can be used successfully to analyze more than 100 individual species of meibomian WE, which were shown to comprise 41 ± 8% (wt/wt) of meibum, which made them the largest group of lipids in meibum.

Effects of free fatty acids on meibomian lipid films.

The purpose of this study was to evaluate the impact of free fatty acids (FFA), namely oleic (OA) and linoleic (LA) ones, on meibomian lipid films (MLF) using a Langmuir trough (LT) and a Brewster angle microscope (BAM). Human meibum was collected from healthy volunteers. A Tris-buffered saline (TBS, pH 7.4) was used as the control aqueous subphase for LT experiments. Then, varying amounts of OA and LA were dissolved in TBS to make FFA-containing subphases. Predetermined amounts of meibum were loaded onto the surface of the (TBS/±FFA) subphases to form MLF. Then, surface pressure-area (π/A) isotherms of MLF were recorded. Standard rheological parameters such as rigidity, elasticity, and hysteresis, were computed. In a separate experiment, OA and LA were pre-mixed with meibum at different weight ratios prior their spreading onto the control TBS subphase, and the (π/A) isotherms of the resulting mixed films of meibum and FFA were studied and analyzed in the same fashion as described above. When studied at the normal corneal temperature of 34 °C with the (TBS/-FFA) subphase, meibum formed stable films. When (TBS/+FFA) subphase was used, both FFA quickly disrupted the MLF, acting in a similar fashion. BAM revealed that the most dramatic changes in the structure of MLF occurred in the range of OA concentrations between 5 and 15 µM. However, this effect was apparent even with 2.5 µM OA. When OA was pre-mixed with meibum, but was absent from the subphase, it caused gradual concentration-dependent changes in the (π/A) isotherms, but the MLF did not disappear from the surface. Thus, tested FFA showed a remarkable ability to disrupt, and/or prevent the formation of, human MLF, which could contribute to the onset of those forms of dry eye disease that are associated with enhanced activity of lipolytic enzymes, such as chronic blepharitis.

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.

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.