E. Uchiyama

Lipids of human meibum: mass-spectrometric analysis and structural elucidation

The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MSn). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MSn detection of the analytes (HPLC API MSn). The major precursor ions were isolated and subjected to further sequential fragmentation in MSn experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M − H2O + H]+) and oleic acid (OA; m/z 283; [M + H]+) were found. The other major compounds of the general molecular formula CnH2n-2O2 were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas CnH2n-4O2 and CnH2nO2. Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MSn analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components.

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.

Liquid Chromatography–Mass Spectrometric Analysis of Lipids Present in Human Meibomian Gland Secretions

The purpose of the study was to qualitatively characterize the major lipid species present in human meibomian gland secretions (MGS) by means of high-performance liquid chromatography with atmospheric pressure ionization mass spectrometric detection of the analytes (NP HPLC-MS). Two different NP HPLC-MS methods have been developed to analyze lipid species that were expected to be present in MGS. The first method was optimized for the analysis of relatively nonpolar lipids [wax esters (WE), di- and triacyl glycerols (DAG and TAG), cholesterol (Chl) and its esters (Chl-E), and ceramides (Cer)], while the second method was designed to separate and detect phospholipids. The major lipid species in MGS were found to be WE, Chl-E, and TAG. A minor amount of free Chl (less then 0.5% of the Chl-E fraction) was detected in MGS. No appreciable amounts of DAG and Cer were found in MGS. The second NP HPLC-MS method, capable of analyzing model mixtures of authentic phospholipids (e.g. phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin) in submicrogram/mL concentrations, showed little or no presence of these species in the MGS samples. These observations suggest that MGS are a major source of the nonpolar lipids of the WE and Chl-E families for the tear film lipid layer (TFLL), but not of the previously reported phospholipid components of the TFLL.

Increased evaporative rates in laboratory testing conditions simulating airplane cabin relative humidity: an important factor for dry eye syndrome.

Purpose. To quantitatively explore the relationship between low relative humidity conditions, as experienced in airplane cabins during flight, and increases in aqueous tear evaporation as a potential explanation for increased dry eye symptoms noted by people when in low humidity environmental conditions. Methods. Prospective experimental laboratory study. Evaporative rates under two different ranges of increasing relative humidity, from 20% to 25% (similar to the relative humidity in airplane cabins or arid regions) and from 40% to 45% (similar to the relative humidity in nonarid regions), were obtained from 18 patients with dry eye and 11 healthy subjects. Results. Statistically significant differences were found within all groups: patients with dry eye (divided into keratoconjunctivitis sicca [P=0.001] and meibomian gland dysfunction [P=0.007]) and healthy subjects (P≤0.001). The mean increase in evaporative rate across all study subjects was 99.72% (P≤0.001) when the eyes were exposed to the lower humidity condition. Conclusions. These studies quantitatively show the negative impact of environmental low relative humidity conditions, including those associated with commercial airplane travel, on aqueous tear evaporation dynamics. The increased evaporative rate is similar in healthy subjects and patients with dry eye. These findings provide useful data for the development and evaluation of treatment paradigms for any person who notes dry eye symptoms in low relative humidity environments.

Impact on ocular surface evaporation of an artificial tear solution containing hydroxypropyl guar.

Objective. To determine whether any acute effects on evaporative parameters are produced when using a solution containing Hydroxypropyl (HP) (Systane) versus normal saline solution in the eyes of patients with Keratoconjunctivis Sicca at 30 and 60 minutes postinstillation. Methods. Randomized double-blinded placebo-control 2-period cross-over clinical trial. Twelve patients with a clinical diagnosis of Keratoconjunctivis Sicca were enrolled in this study. Aqueous tear evaporation was measured at baseline, i.e., before the application of drops on the eye, and at 30 and 60 minutes after instillation of one 40 &mgr;L drop of either the HP-Guar containing drop or normal saline on two separate days. Statistical analysis included descriptive data analysis and paired t test. Results. Hydroxypropyl-Guar resulted in a decrease in aqueous tear evaporation at 30 minutes postinstillation under 25% to 35% relative humidity (RH) (13.2% reduction, P=0.044) and 35% to 45% RH (10% reduction, P=0.028) conditions. The effect of HP-Guar at 60 minutes postinstillation also decreased aqueous tear evaporation but to a lesser degree. Normal saline solution produced no statistically significant increases and decreases of evaporation. Conclusions. Aqueous tear evaporation contributes significantly to aqueous tear loss and is humidity dependent. An HP-guar containing solution decreased aqueous tear evaporation 30 and 60 minutes after application. The use of topical medication with known antievaporative effect may be beneficial in dry eye therapy. This effect may also be achieved in normal eyes or sub-clinical dry eyes when in low RH environments.

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.

Pattern of vital staining and its correlation with aqueous tear deficiency and meibomian gland dropout.

Purpose. To assess the relationship between the degree and anatomic location of ocular surface vital staining and the degree of aqueous tear deficiency in patients with dry eye, as measured by the Schirmer test, and the presence of meibomian gland dropout. Methods. Twenty-two patients with dry eye (nine men and 13 women; mean age, 60 years) were prospectively enrolled in the study. Eleven subjects with no ocular disease were considered control subjects. Lissamine green vital staining, Schirmer test, and meibomian gland dropout were evaluated. Results. Patients were stratified based on their vital staining pattern (presence or absence of corneal staining). A progressive decrease in the Schirmer test result was found when the vital staining score increased (r = –0.655; P<0.0001). Patients with dry eye showed an increase in meibomian gland dropout when compared to control subjects (P≤0.001). Conclusions. The presence of corneal vital staining correlates with a more severe dry eye as shown by the Schirmer test. Patients with dry eye have an increased meibomian gland dropout. The degree and pattern of vital staining correlate with the severity of dry eye.