Time-dependent degenerative transformations in the lipidome of chalazia.

The aim of this prospective study was to conduct histopathologic and lipidomic analyses of chalazia, in order to evaluate time-dependent changes in the lesion. Samples of surgically excised chalazia were collected over a period of 12 months from 10 patients (mean age 41 years; range, 23-58) with clinically diagnosed chalazia, who underwent scheduled surgery. The ages of chalazia varied from 2 to 28 weeks. To confirm the clinical diagnoses, the morphology of collected tissue samples was evaluated histologically after hematoxylin and eosin staining. The lipids from individual chalazia were analyzed by high-performance liquid chromatography-mass spectrometry and compared with authentic lipid standards and with the lipids of meibum collected from normal controls. We observed gradual, lesion age-dependent transformation of the lipidome of chalazia from an almost normal meibum-like composition to a very different kind of lipidome. A rapid initial increase in the free cholesterol content was followed by a gradual replacement of extremely long chain meibomian-type lipids with a mixture of shorter-chain cholesteryl esters of the C14-C18 family, triacylglycerols, ceramides, phospholipids and sphingomyelins. In addition, a rapid disappearance of wax esters and cholesteryl esters of (1-O)-acyl-omega-hydroxy fatty acids from the lipidome of aging chalazia was observed. Our results are indicative of dramatic, time-dependent changes in the lesion that may involve cholesterol as a trigger and/or a marker of subsequent degeneration of the meibomian lipidome. We hypothesize that early inhibition of these transformations may be useful in reversing the course of the disease.

Biophysical and morphological evaluation of human normal and dry eye meibum using hot stage polarized light microscopy.

PURPOSE: To study melting characteristics and the morphology of human and mouse meibum. METHODS: Hot stage cross-polarized light microscopy (HSPM) and immunohistochemical approaches were used. RESULTS: Isolated human meibum, and meibum of mice (either isolated or within the meibomian ducts of mice), were found to be in liquid-crystal state at physiological temperatures. Melting of both types of meibum started at approximately 10°C and was completed at approximately 40°C. Melting curves of isolated meibum and meibum inside the meibomian ducts were multiphasic with at least two or three clearly defined phase transition temperatures, typically at approximately 12 ± 2°C (minor transition), 21 ± 3°C, and 32 ± 3°C, regardless the source of meibum. Melting was highly cooperative in nature. Samples of abnormal human meibum collected from dry eye patients with meibomian gland dysfunction often showed an increased presence of nonlipid, nonmelting, nonbirefringent, chloroform-insoluble inclusions of a protein nature. The inclusions were positively stained for cytokeratins. The presence of these inclusions was semiquantitatively characterized using a newly proposed 0 to 4 scale. In the presence of large amounts of these inclusions, melting characteristics of meibum and its structural integrity were altered. CONCLUSIONS: HSPM is an effective tool that is suitable for biophysical and morphological evaluation of meibum. Morphological properties and melting characteristics of human meibum were found to be similar to those of mice. Abnormal meibum of many dry eye patients contained large quantities of nonlipid, protein-like inclusions, which were routinely absent in meibum of normal controls.

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.

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 C(18:n) (n = 1-4) unsaturated FAs ranking in the following order of abundance: C(18:1)>C(18:2)>C(18:3)>C(18:4). The major fatty alcohols (FAls) found in WE were of saturated nature and varied from C(18:0) to C(28:0). The three most abundant species were C(18:1)-FA esters of C(24:0), C(25:0), and C(26:0)-FAl. Typically, a major compound based on C(18:1)-FA and a saturated FAl was accompanied by a few related compounds based on a C(18:2), C(18:3), and C(18:4)-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)-omega-hydroxy-FAs (OAHFAs) were observed. The main detected OAHFAs were based on very long-chain omega-hydroxy-FA (C(30:1), C(32:1), and C(34:1)) acylated through their omega-hydroxyls by a C(18:1)-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.

Effects of daily and overnight wear of a novel hyper oxygen-transmissible soft contact lens on bacterial binding and corneal epithelium: a 13-month clinical trial.

OBJECTIVE: To test prospectively a new biologic rationale for an advanced hyper oxygen-transmissible lens (HOTL) providing prospects for safer daily (DW) or extended (EW) contact lens wear. DESIGN: Prospective, randomized, double-masked, single-center, 13-month clinical trial. PARTICIPANTS: One hundred sixty-eight patients completed the DW study (1 month): control lens (n = 70); HOTL (n = 98). One hundred thirty-six patients finished 1 year of EW: controls (n = 56), HOTL (n = 25, 6 nights; n = 55, 30 nights). TESTING: Irrigation chamber to collect corneal surface cells, confocal microscopy, tear collection at baseline, 2, and 4, weeks of DW, and 24 hours, 1, 3, 6, 9, and 12 months of EW. MAIN OUTCOME MEASURES: (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness (CET); (3) superficial cell area (SCA); (4) epithelial surface cell exfoliation (DESQ); and (5) tear lactate dehydrogenase (LDH). RESULTS: Daily wear with control lens increased PA binding from 5.90 +/- 2.60 to 7.81 +/- 3.04 bacteria per cell (P < 0.01); HOTL wear increased PA binding significantly less (5.31 +/- 1.87-5.98 +/- 2.26; P < 0.01). Daily wear produced no significant changes in CET or SCA. Significant decreases in DESQ were seen with both lenses with no significant intergroup differences. Tear LDH increased significantly in DW with HOTL wear versus control (P = 0.0017), but not after 1 month of subsequent EW (P = 0.533). One to 3 months of EW with control lens showed significantly higher PA binding than HOTL wear (P < 0.01); binding adaptively decreased thereafter, returning to baseline at 9 to 12 months. Lens EW produced significantly enlarged SCA, thinning of CET (except 6-night HOTL wear), and decreased DESQ (P < 0.01). Some adaptive recovery was seen with CET and DESQ, but not SCA; importantly, the data indicated no significant difference between 6- or 30-night EW for all outcomes. CONCLUSIONS: Hyper oxygen-transmissible lens wear (DW or EW) produced significantly decreased PA binding compared with control lens wear, with no significant difference in wearing schedule (6 nights vs. 30 nights); additionally, there was a remarkable and unexpected adaptive recovery in the first 6 months of all soft lens wear, with a return to baseline PA binding levels and partial recovery for the other outcomes except SCA at 1 year. These results suggest that HOTL use should result in a decrease in the incidence of and risk(s) for lens-related microbial keratitis and that further epidemiologic studies should consider time in adapted EW in future risk and incidence analyses.