Marta C. Yappert

Topical Azithromycin Therapy of Meibomian Gland Dysfunction: Clinical response and lipid alterations

Purpose: Meibomian gland dysfunction (MGD) is a common clinical problem that is often associated with evaporative dry eye disease. Alterations of the lipids of the meibomian glands have been identified in several studies of MGD. This prospective, observational, open-label clinical trial documents the improvement in both clinical signs and symptoms of disease and spectroscopic behavior of the meibomian gland lipids after therapy with topical azithromycin ophthalmic solution. Methods: Subjects with symptomatic MGD were recruited. Signs of MGD were evaluated with a slit lamp. Symptoms of MGD were measured by the response of subjects to a questionnaire. Meibum lipid, lipid-lipid interaction strength, and conformation and phase transition parameters were measured using Fourier transform infrared spectroscopy. Results: In subjects with clinical evidence of MGD, changes in ordering of the lipids and resultant alteration of phase transition temperature were identified. Topical therapy with azithromycin relieved signs and symptoms and restored the lipid properties of the meibomian gland secretion toward normal. Conclusions: Improvement in phase transition temperature of the meibomian gland lipid with the determined percent trans rotomer composition of the lipid strongly suggests that the ordering of the lipid molecules is altered in the disease state (MGD) and that azithromycin can improve that abnormal condition toward normal in a manner that correlates with clinical response to therapy.

Lipids and the ocular lens

The unusually high levels of saturation and thus order contribute to the uniqueness of human lens membranes. In addition, and unlike in most biomembranes, most of the lens lipids are associated with proteins, thus reducing their mobility. The major phospholipid of the human lens is dihydrosphingomyelin. Found in significant quantities only in primate lenses, particularly human ones, this lipid is so extremely stable that it was reported to be the only lipid remaining in a frozen mammoth 40,000 years after its death. Unusually high levels of cholesterol add peculiarity to the composition of lens membranes. Beyond the lateral segregation of lipids into dynamic domains known as rafts, the high abundance of cholesterol in the human lens leads to the formation of patches of pure cholesterol. Changes in human lens lipid composition with age and disease as well as differences among species are greater than those observed for any other biomembrane. The relationships among lens membrane composition, structure, and lipid conformation reviewed in this article are unique to the mammalian lens and offer exciting insights into lens membrane function. This review focuses on findings reported over the last two decades that demonstrate the uniqueness of mammalian lens membranes regarding their morphology and composition. Becaue the membranes of human lenses do undergo the most dramatic changes with age and cataractogenesis, the final sections of this review address our current knowledge of the unusual composition and organization of adult human lens membranes with and without opacification. Finally, the questions that still remain to be answered are presented.

Lipid Composition, Membrane Structure Relationships in Lens and Muscle Sarcoplasmic Reticulum Membranes

Membrane lipid composition varies in different tissues and species. Since a defined lipid composition is essential to the function of many membranes, the relationship between membrane lipid composition and structure was determined using infrared and Raman spectroscopy in four membranes containing a calcium pump: rabbit fast and slow twitch muscle sarcoplasmic reticulum and human and bovine lens fiber cell membranes. We found that membrane sphingolipid and phosphatidylcholine content were correlated to a decrease and increase, respectively, in the infrared lipid CH2 symmetric stretching band frequency. We interpret the change in frequency as a change in lipid hydrocarbon chain structural order. This was confirmed by Raman order parameters. The high degree of hydrocarbon chain saturation found in the variable amide chains of sphingolipids is likely to account for this correlation. Lipid phase transition temperature and cooperativity also correlated to sphingolipid and phosphatidylcholine content, and are the forces defining the order in at physiological temperature in the samples studied. Ca(2+)-ATPase caused an increase in the CH2 symmetric stretching frequency in fast twitch muscle sarcoplasmic reticulum (interpreted as an increase in hydrocarbon chain disorder), but had no effect on slow twitch muscle sarcoplasmic reticulum lipid hydrocarbon chain structure. In the natural systems studied, we find that it is the lipid hydrocarbon chain saturation that defines lipid hydrocarbon chain order.

Topical Azithromycin and Oral Doxycycline Therapy of Meibomian Gland Dysfunction: A Comparative Clinical and Spectroscopic Pilot Study

Purpose: Meibomian gland dysfunction (MGD) is a common clinical problem that is often associated with evaporative dry eye disease. Alterations of the lipids of the meibomian glands have been identified in several studies of MGD. This prospective, observational, open-label clinical trial documents the improvement in both clinical signs and symptoms of disease as well as spectroscopic characteristics of the meibomian gland lipids after therapy with topical azithromycin ophthalmic solution and oral doxycycline treatment. Methods: Subjects with symptomatic MGD were recruited. Signs of MGD were evaluated with a slit lamp. Symptoms of MGD were measured by the response of subjects to a questionnaire. Meibum lipid–lipid interaction strength, conformation, and phase transition parameters, and meibum protein content were measured using Fourier transform infrared spectroscopy and principal component analysis. Terpenoids, short-chain CH3 moieties, lipid oxidation, wax, cholesterylesters and glycerides were measured with a proton nuclear magnetic resonance (1H-NMR) spectrometer. Results: Topical therapy with azithromycin and oral therapy with doxycycline relieved signs and symptoms and restored the lipid properties of the meibomian gland secretion toward normal. Compared with 4 weeks of azithromycin treatment reported in our previous study, oral doxycycline treatment was slightly less effective in improving foreign body sensation and the signs of plugging and secretion. In subjects with clinical evidence of MGD, changes in ordering of the lipids and phase transition temperature were brought closer to normal with azithromycin treatment than doxycycline treatment. Treatment with doxycycline but not azithromycin restored the Fourier transform infrared spectroscopy–principal component analysis scores and relative area of the 1H-NMR resonance at 1.26 ppm. Both doxycycline and azithromycin treatment restored the levels of the relative areas of the 1H-NMR resonance at 5.2 and 7.9 ppm to normal levels. The levels of meibum protein and meibum lipid oxidation were not influenced by azithromycin or doxycycline treatment. Conclusions: The mechanism of action of doxycycline may be different from that of azithromycin in therapy of MGD. It is notable that when carotenoids in meibum are low, as in MGD, the tear film is unstable and patients have the signs and symptoms of dry eyes. When carotenoids are restored with azithromycin and doxycycline treatment, tear film stability is restored and patients no longer have the signs and symptoms of dry eyes.

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.).

Factors affecting evaporation rates of tear film components measured in vitro.

Objectives: With increasing age and in patients affected with dry-eye symptoms, the human tear film becomes more unstable and exhibits shorter tear break-up times. We examined whether the inclusion of proteins and lipids to water affected the evaporation rates measured in vitro and could account for the lower rates reported previously from in vivo measurements. The impact of temperature, air flow, and humidity on the evaporation rate of tears was measured in vitro. Methods: Lipid-protein interactions were measured using fluorescence spectroscopy and in vitro rates of evaporation were performed gravimetrically. Results: Human reflex tears evaporated at a rate similar to that of water. A temperature increase from 25°C to 34°C caused a threefold increase in the evaporation rates of tears in still air. Further increases were observed under a current of dry air. Wax, mucin, lysozyme, or &bgr;-lactoglobulin did not influence significantly the rates of evaporation measured in vitro. In contrast, lipid layered on the surface resulted in a 23% decrease in the rates. Conclusions: Environmental factors affect evaporation rates significantly and should be carefully controlled when performing in vivo measurements. The presence of a lipid layer lowers evaporation rates. The significantly lower rates of evaporation of tears measured in vivo suggest that with the lipid layer intact, the high reserve capacity of the lacrimal gland to provide both unstimulated and stimulated tear flow is more than enough to compensate for evaporative loss. However, with dry eye, increased rates of evaporation and decreased lacrimal tear flow could result in decreased break-up times.

Changes in human meibum lipid with meibomian gland dysfunction using principal component analysis

Changes in the phase transition temperatures and conformation of human meibum lipid with age and meibomian gland dysfunction have been quantified but with analysis of less than 1% of the infrared spectral range. The remaining 99% of the spectral range was analyzed with principal component analysis (PCA) and confirms our previous studies and reveal further insights into changes that occur in meibum with age and disease. Infrared spectra of meibum from 41 patients diagnosed with meibomian gland dysfunction (Md) and 32 normal donors (Mn) were measured. Principal component analysis (PCA) was used to quantify the variance among the spectra and meibum protein was quantified using the infrared carbonyl and amide I and II bands. A training set of spectra was used to discriminate between Mn and Md with an accuracy of 93%. This shows that certain spectral regions (eigenvectors) contain compositional and structural information about the changes that occur with the principal component (variable), meibomian gland dysfunction. The spectral features of the major eigenvector indicate that Md contains more protein and relatively less CH(3) and cis = CH band intensity compared to Mn. The amount of protein was confirmed from relative infrared band intensities. Our study supports the idea that compositional differences result in meibum that is less fluid and more viscous with meibomian gland dysfunction so that less lipid flows out of the meibomian gland orifice as observed clinically. This study also demonstrates the power of the combination of infrared spectroscopy and PCA as a diagnostic tool that discriminates between Mn and Md.

Characterization of Human Meibum Lipid using Raman Spectroscopy

Purpose: Raman spectroscopy could potentially be used as a diagnostic tool for the detection of meibomian gland dysfunction by measuring the composition, conformation, and amount of meibum lipid on the lid margin. Toward this goal and to test our studies of meibum lipid using infrared spectroscopy, we measured the Raman spectra of samples from human donors with and without meibomian gland dysfunction. Materials and Methods: Human meibum was expressed from the eyelids and collected on a spatula. Meibum was placed onto an AgCl window for analysis using a Raman spectrometer. Results: Raman spectra of human meibum closely resembled that of wax. The Raman band intensity ratio, 1303/1267 cm−1, was used to calculate a saturation level CH2/=CH of 12 for meibum from normal donors and 11.8 for meibum from donors with meibomian gland dysfunction. Human meibum was found to fit well in the linear relationships between lipid saturation and lipid order at physiological temperature and between lipid saturation and the lipid phase transition temperature. The CH stretching band region predominates the Raman spectra of human meibum. If sample temperature could be controlled, the CH stretching bands could be used to calculate the lipid structural order in terms of trans and gauche rotomer levels. Raman spectra indicate that carotenoid-like bands decrease from the equivalent of about 90 μg carotenoid per gram of meibum wax at 19 years of age to about 0 at 80 years of age in samples from patients with meibomian gland dysfunction. The carotenoids may serve as antioxidants. Conclusions: Principal component analysis shows that a component containing carotenoid-like bands may be a promising spectral feature that could be used to distinguish differences between the Raman spectra of donors from normal and those with meibomian gland dysfunction. This study demonstrates the wealth of structural and compositional information that Raman spectra of human meibum provide.

Physical Changes in Human Meibum with Age as Measured by Infrared Spectroscopy

Both lipids and mucins contribute to the stability of the tear film and lipids may inhibit tears from evaporating. Younger people have lower lipid viscosity, higher lipid volume, and a lower rate of tear evaporation. Since age-related changes in human meibum composition and conformation have never been investigated, as a basis for the study of lipid-associated changes with meibomian gland dysfunction, we used the power of infrared spectroscopy to characterize hydrocarbon chain conformation and packing in meibum from humans without dry eye symptoms in relation to age and sex. Meibum from normal human donors ranging in age from 3 to 88 years was studied. Meibum phase transitions were quantified by fitting them to a 4-parameter 2-state sigmoidal equation. Human meibum order and phase transition temperatures decrease with age and this trend may be attributed to lipid compositional changes. If meibum has the same thermodynamic properties on the surface of the tears as it does on the lid margin, a decrease in lipid-lipid interaction strength with increasing age could decrease the stability of tears since lipid-lipid interactions on the tear surface must be broken for the tear film to break up. This study also serves as a foundation to examine meibum conformational differences in meibum from people with meibomian gland dysfunction.

Analysis of the Composition of Lipid in Human Meibum from Normal Infants, Children, Adolescents, Adults, and Adults with Meibomian Gland Dysfunction Using 1H-NMR Spectroscopy

PURPOSE This study represents a first step toward the evaluation of possible compositional differences in meibum from normal donors (Mn) and donors with meibomian gland dysfunction (Md) by (1)H-NMR spectroscopy. The results highlight the applicability of (1)H-NMR spectroscopy for the quantitative analysis of waxes, cholesteryl esters, and glycerides in meibum lipid (ML). METHODS Meibum was obtained from 41 normal donors and 51 donors with meibomian gland dysfunction (MGD). (1)H-NMR spectroscopy was used to quantify the amount of waxes, glycerides, and cholesteryl esters in human meibum. RESULTS The relative amount of cholesteryl esters in Mn increased with age and was 40% (P < 0.05) lower in Md. Interestingly, the relative levels of cholesteryl esters in infant meibum were comparable to those in Md. The relative amounts of glycerides were not affected significantly by age or MGD. CONCLUSIONS The changes in cholesteryl ester could be used as a molecular marker for MGD and could potentially be applied to follow the efficacy of drug therapy in the treatment of MGD. The similarity of the levels of cholesteryl esters in infant meibum and Md suggests that the relative amounts of these meibum components alone are unlikely to be responsible for the increased stability of the infant tear film and decreased stability of the tear film with MGD. This study reveals the complexity of human MLs and the changes that occur with age and disease. Understanding the factors that lead to such variations is of utmost relevance in the design of effective therapies.