Quantification of human sebum on skin and human meibum on the eye lid margin using Sebutape®, spectroscopy and chemical analysis.

PURPOSE: The purpose of this study was to determine if the variability in the amount of lid margin meibum is from donor-to-donor or from day-to-day variations and to determine if meibum from donors with meibomian gland dysfunction (MGD) had altered levels of casual eyelid meibum or skin sebum. METHODS: Lipid absorbent Sebutape(®) was used to collect sebum or meibum. Samples were collected from six donors without dry eye and 21 donors with MGD. Lipid absorbed to Sebutape(®) was quantified using infrared and visible absorbance spectroscopy. RESULTS: The amount of sebum from donors with MGD and donors without MGD was not significantly different. The amount of casual meibum from normal donors was 50% lower than that for donors with MGD using the spectroscopic assay, but was not significantly different using the chemical assay. The frequency and bandwidth of the infrared carbonyl band from sebum samples was significantly higher than that for meibum samples which indicates the carbonyls are in a different "dielectric" environment. The average relative standard deviation for the casual level of meibum and the level of sebum suggests that the 49% relative standard deviation of casual meibum measured once for each subject using a meibometer may have been due to day-to-day variations and not necessarily due to variations between individuals. The values measured using two different assays were correlated and therefore reliable. CONCLUSIONS: The idea that tear film instability is associated with the quantity of lid margin lipid is not supported by this study because the quantity did not change with MGD. The amount of forehead sebum was not a bio-marker for MGD. Sebutape(®) is an excellent vehicle to remove tenths of a milligram of meibum from the eyelid and sebum from skin for experimental analysis.

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.).