Douglas Borchman

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.

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

PURPOSEnInstability 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.nnnMETHODSnChanges 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnMeibum 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.).

The dual effect of oxidation on lipid bilayer structure.

Sphingomyelin membranes were prepared with different levels of oxidative damage caused bytert-butyl hydroperoxide (TBH). Temperature-induced changes in membrane hydrocarbon chain packing (phase transitions) were monitored using infrared spectroscopy. Lipid phase transition characteristics were evaluated from thermodynamic parameters fitted to the experimental transition curve data. At temperatures below the lipid phase transition Tc, hydrocarbon chains pack in an ordered state whereas above the Tc the hydrocarbonchains pack in a disordered state. Compared to the non-oxidized control, the packing of the hydrocarbon chains of mildly oxidized sphingomyelin (<10 nmol TBH/mg lipid) was no different at all temperatures below the Tc, and was more ordered above the Tc. The hydrocarbon chains of strongly oxidized sphingomyelin (>10nmol TBH/mg lipid) were more disordered at temperatures above and below the Tc compared to the control samples. These results suggest that lipid oxidation has a dual effect on lipid order. A more ordered or disordered state may result depending on the degree of oxidation and the state of lipid order prior to oxidation. These results could be important for explaining the structural changes in oxidized membranes high in sphingomyelin such as those found in the ocular lens and liver plasma membranes.

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

PURPOSEnThis 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).nnnMETHODSnMeibum 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnThe 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.

Differences in Human Meibum Lipid Composition with Meibomian Gland Dysfunction Using NMR and Principal Component Analysis

PURPOSEnNuclear magnetic resonance (NMR) spectroscopy has been used to quantify lipid wax, cholesterol ester terpenoid and glyceride composition, saturation, oxidation, and CH₂ and CH₃ moiety distribution. This tool was used to measure changes in human meibum composition with meibomian gland dysfunction (MGD).nnnMETHODSn(1)H-NMR spectra of meibum from 39 donors with meibomian gland dysfunction (Md) were compared to meibum from 33 normal donors (Mn).nnnRESULTSnPrincipal component analysis (PCA) was applied to the CH₂/CH₃ regions of a set of training NMR spectra of human meibum. PCA discriminated between Mn and Md with an accuracy of 86%. There was a bias toward more accurately predicting normal samples (92%) compared with predicting MGD samples (78%). When the NMR spectra of Md were compared with those of Mn, three statistically significant decreases were observed in the relative amounts of CH₃ moieties at 1.26 ppm, the products of lipid oxidation above 7 ppm, and the =CH moieties at 5.2 ppm associated with terpenoids.nnnCONCLUSIONSnLoss of the terpenoids could be deleterious to meibum since they exhibit a plethora of mostly positive biological functions and could account for the lower level of cholesterol esters observed in Md compared with Mn. All three changes could account for the higher degree of lipid order of Md compared with age-matched Mn. In addition to the power of NMR spectroscopy to detect differences in the composition of meibum, it is promising that NMR can be used as a diagnostic tool.

Changes in Human Meibum Lipid Composition with Age Using Nuclear Magnetic Resonance Spectroscopy

PURPOSEnHuman tear film stability decreases with increasing age. In this study, the changes in meibum composition were measured in search of markers of tear film instability.nnnMETHODSn(1)H NMR nuclear magnetic resonance (NMR) spectra of 43 normal donors aged 1 to 88 years were acquired.nnnRESULTSnCompared with meibum from adolescents and adults, meibum from infants and children contains less CH(3) and C═C groups and an increased aldehyde-to-lipid hydroperoxide ratio.nnnCONCLUSIONSnIt is reasonable that tear film stability is higher in infants than in adults. Their meibum contains less CH(3) and C═C groups and higher levels of protein, and as a result, the lipid is more ordered because of the tighter and stronger lipid-lipid interactions. For water to evaporate, it must first pass through the tight lipid-lipid barrier. For tears to break up, lipid-lipid interactions must be broken. It is reasonable that because the lipid-lipid interactions are stronger in infants and childrens tears compared with those of adolescents and adults, the tear film in the younger groups is more stable and provides a better barrier to evaporation than does the tear film of adults. Lipid saturation could be the critical feature in meibum that stabilizes tears in infants.

Age and regional structural characterization of lipid hydrocarbon chains from human lenses by infrared, and near-infrared raman, spectroscopies

Age related changes in the lipid composition of human lens membranes have been documented and could be responsible for alterations in the function of lens membranes. To establish age related lipid composition-membrane structure relationships, we have examined spectroscopically the hydrocarbon chain structure of lipid membranes from human lens cortex and nucleus 0–95 years of age. Lipid membranes were extracted from human lenses using a monophasic methanolic extraction. The lipid composition of these membranes was determined by 31P-NMR and has already been reported. Fourier transform near-infrared Raman and Fourier transform infrared spectroscopies were used to determine human lens lipid structure. Lipid compositional differences were related to membrane structure. The frequency corresponding to the CH2 symmetric stretching band was found to increase with age in lipid samples from all regions of the lens. The frequency was used to estimate lipid hydrocarbon order. Lipid order was found to increase with age and was not significantly different for lipids extracted from the cortex compared to those from the nucleus. These results were confirmed qualitatively by comparing the height of the 2880 cm−1 band with the height of the 2850 cm−1 Raman band. Increased lipid order with age was also confirmed by the analysis of the C(SINGLE BOND)C stretching bands. Lipid hydrocarbon chain order increased linearly with increasing sphingomyelin content and decreased linearly with increasing phosphatidylcholine content. This trend, similar to that observed in other types of membranes, suggests that these two lipids may play a role in modulating lipid order.

Thermodynamic Phase Transition Parameters of Human Lens Dihydrosphingomyelin

Dihydrosphingomyelin (DHS) is the major phospholipid in the human lens. The influence of this phospholipid on membrane structure and function is not known. In this study we used infrared spectroscopy to determine the thermodynamic and molecular structural properties of the hydrocarbon chains of DHS membranes isolated from human lenses. The phase transition temperature of human lens DHS was 9 degrees C higher than for bovine brain sphingomyelin membranes and 14 and 7 degrees C higher than human lens cortical and nuclear membranes, respectively. This increase in the phase transition temperature results in 20% increase in lipid order at 36 degrees C in comparison to that of native membranes and bovine brain sphingomyelin. DHS is likely to provide structural order to the hydrocarbon chain region and upholds the integrity of native membranes under oxidative conditions.