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.

Ultra-thin donor tissue preparation for endothelial keratoplasty with a double-pass microkeratome.

PURPOSE: To quantify and describe practically a novel technique for donor tissue preparation in Descemet stripping endothelial keratoplasty to approach the superior visual outcomes of Descemet membrane endothelial keratoplasty. DESIGN: Experimental laboratory investigation. SETTING: Institutional. STUDY POPULATION: Eleven human donor corneas. INTERVENTION: Double-pass of microkeratome over donor corneas-first with a thicker cutting depth and subsequently with a thinner cutting depth. MAIN OUTCOME MEASURES: Donor tissue profiles and residual bed thicknesses. RESULTS: After the first pass of the microkeratome, the average cut thickness using the 250-µm cutting head was 342.5 ± 14.8 µm (range, 332 to 353 µm), that using the 300-µm head was 343.8 ± 39.2 µm (range, 315 to 411 µm), and that with the 350-µm head was 467.7 ± 50.1 µm (range, 419 to 519 µm). We used the 200-µm cutting head only once with a cut depth of 210 µm. For the second pass, when using the 110-µm head, the cutting depth averaged to 167.8 ± 28.8 µm (range, 133 to 203 µm). The 130-µm cutting head yielded a cut depth of 199.7 ± 24.4 µm (range, 180 to 227 µm). Two corneas were perforated during the second pass. The average final thickness of the residual bed was 121 ± 32.2 µm (range, 52 to 160 µm). CONCLUSIONS: Double-pass harvest for ultra-thin Descemet stripping automated endothelial keratoplasty could improve optical outcomes by obtaining donor Descemet stripping automated endothelial keratoplasty tissue with thinner residual beds. Further studies are needed with larger sample sizes to establish algorithms for appropriate cutting head thickness in each pass. Potential additional endothelial cell loss with the second pass of the microkeratome also should be evaluated.

Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction.

PURPOSE: Instability of the tear film with rapid tear break-up time is a common feature of aqueous-deficient and evaporative dry eye diseases, suggesting that there may be a shared structural abnormality of the tear film that is responsible for the instability. It may be that a change in the normal meibum lipid composition and conformation causes this abnormality. Principle component analyses of infrared spectra of human meibum indicate that human meibum collected from normal donors (Mn) is less ordered than meibum from donors with meibomian gland dysfunction (Md). In this study the conformation of Md was quantified to test this finding. METHODS: Changes in lipid conformation with temperature were measured by infrared spectroscopy. There were two phases to our study. In phase 1, the phase transitions of human samples, Mn and Md, were measured. In phase 2, the phase transitions of model lipid standards composed of different waxes and cholesterol esters were measured. RESULTS: The phase-transition temperature was significantly higher (4°C) for the Md compared with the Mn of age-matched donors with no history of dry-eye symptoms. Most (82%) of the phase-transition temperatures measured for Md were above the values for Mn. The small change in the transition temperature was amplified in the average lipid order (stiffness) at 33.4°C. The average lipid order at 33.4°C for Md was significantly higher (30%, P = 0.004) than for Mn. The strength of lipid-lipid interactions was 72% higher for Md than for Mn. The ability of one lipid to influence the melting of adjacent lipids is termed cooperativity. There were no significant differences between Mn and Md in phase-transition cooperativity, nor was there a difference between Mn and Md in the minimum order or maximum order that Mn and Md achieved at very low and very high temperatures, respectively. The model wax studies showed that the phase transition of complex mixtures of natural lipids was set by the level of unsaturation. A double bond decreased the phase-transition temperature by approximately 40°C. The addition of a second CH CH moiety decreased the phase-transition temperature by approximately 19°C. Unsaturated waxes were miscible with saturated waxes. When a saturated wax was mixed with an unsaturated one, the saturated wax disproportionately increased the phase transition of the mixture by approximately 30°C compared with the saturated wax alone. Cholesterol ester had little effect on the phase-transition temperature of the waxes. Model studies indicated that changes in the amount of lipid saturation, rather than the amount of cholesterol esters, could be a factor in the observed conformational changes. CONCLUSIONS: Meibum lipid compositional changes with meibomian gland dysfunction reflect changes in hydrocarbon chain conformation and lipid-lipid interaction strength. Spectroscopic techniques are useful in studying the lipid-lipid interactions and conformation of lipid from individual patients. (ClinicalTrials.gov number, NCT00803452.).