Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome

As current diagnostic markers for dry eye syndrome (DES) are lacking in both sensitivity and specificity, a pressing concern exists to develop activity markers that closely align with the principal axes of disease progression. In this study, a comprehensive lipidomic platform designated for analysis of the human tear lipidome was employed to characterize changes in tear lipid compositions from a cohort of 93 subjects of different clinical subgroups classified based on the presence of dry eye symptoms and signs. Positive correlations were observed between the tear levels of cholesteryl sulfates and glycosphingolipids with physiological secretion of tears, which indicated the possible lacrimal (instead of meibomian) origin of these lipids. Notably, we found wax esters of low molecular masses and those containing saturated fatty acyl moieties were specifically reduced with disease and significantly correlated with various DES clinical parameters such as ocular surface disease index, tear breakup time, and Schirmer''s I test (i.e., both symptoms and signs). These structure-specific changes in tear components with DES could potentially serve as unifying indicators of disease symptoms and signs. In addition, the structurally-specific aberrations in tear lipids reported here were found in patients with or without aqueous deficiency, suggesting a common pathology for both DES subtypes.     

Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: lipidome meets proteome

In the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerols (TG) and steryl esters (SE) are stored in so-called lipid particles/droplets (LP) as biologically inert form of fatty acids and sterols. To understand LP structure and function in more detail we investigated the molecular equipment of this compartment making use of mass spectrometric analysis of lipids (TG, SE, phospholipids) and proteins. We addressed the question whether or not lipid and protein composition of LP influence each other and performed analyses of LP from cells grown on two different carbon sources, glucose and oleate. Growth of cells on oleate caused dramatic cellular changes including accumulation of TG at the expense of SE, enhanced the amount of glycerophospholipids and strongly increased the degree of unsaturation in all lipid classes. Most interestingly, oleate as a carbon source led to adaptation of the LP proteome resulting in the appearance of several novel LP proteins. Localization of these new LP proteins was confirmed by cell fractionation. Proteomes of LP variants from cells grown on glucose or oleate, respectively, were compared and are discussed with emphasis on the different groups of proteins detected through this analysis. In summary, we demonstrate flexibility of the yeast LP lipidome and proteome and the ability of LP to adapt to environmental changes.     

Interaction of ceramides and tear lipocalin

The distribution of lipids in tears is critical to their function. Lipids in human tears may retard evaporation by forming a surface barrier at the air interface. Lipids complexed with the major lipid binding protein in tears, tear lipocalin, reside in the bulk (aqueous) and may have functions unrelated to the surface. Many new lipids species have been revealed through recent mass spectrometric studies. Their association with lipid binding proteins has not been studied. Squalene, (O-acyl) omega-hydroxy fatty acids (OAHFA) and ceramides are examples. Even well-known lipids such as wax and cholesteryl esters are only presumed to be unbound because extracts of protein fractions of tears were devoid of these lipids. Our purpose was to determine by direct binding assays if the aforementioned lipids can bind tear lipocalin. Lipids were screened for ability to displace DAUDA from tear lipocalin in a fluorescence displacement assay. Di- and tri-glycerides, squalene, OAHFA, wax and cholesterol esters did not displace DAUDA from tear lipocalin. However, ceramides displaced DAUDA. Apparent dissociation constants for ceramide-tear lipocalin complexes using fluorescent analogs were measured consistently in the submicromolar range with 3 methods, linear spectral summation, high speed centrifugal precipitation and standard fluorescence assays. At the relatively small concentrations in tears, all ceramides were complexed to tear lipocalin. The lack of binding of di- and tri-glycerides, squalene, OAHFA, as well as wax and cholesterol esters to tear lipocalin is consonant with residence of these lipids near the air interface.     

Biophysical investigations of the structure and function of the tear fluid lipid layers and the effect of ectoine. Part B: Artificial lipid films

The tear fluid lipid layer is present at the outermost part of the tear film which lines the ocular surface and functions to maintain the corneal surface moist by retarding evaporation. Instability in the structure of the tear fluid lipid layer can cause an increased rate of evaporation and thus dry eye syndrome. Ectoine has been previously shown to fluidize lipid monolayers and alter the phase behavior. In the current study we have investigated the effect of ectoine on the artificial tear fluid lipid layer composed of binary and ternary lipid mixtures of dipalmitoyl phosphatidylcholine (DPPC), cholesteryl esters and tri-acyl-glycerols. The focus of our study was mainly the structural and the biophysical aspects of the artificial tear fluid lipid layer using surface activity studies and topology analysis. The presence of ectoine consistently causes an expansion of the pressure–area isotherm indicating increased intermolecular spacing. The topology studies showed the formation of droplet-like structures due to the addition of ectoine only when tri-acyl-glycerol is present in the mixture of DPPC and chol-palmitate, similar to the natural meibomian lipids. Consequently, the hypothesis of an exclusion of tri/di-acyl-glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed. A model describing the effect of ectoine on meibomian lipid films is further presented which may have an application for the use of ectoines in eye drops as a treatment for the dry eye syndrome.     

Lipids including cholesteryl linoleate and cholesteryl arachidonate contribute to the inherent antibacterial activity of human nasal fluid

Mucosal surfaces provide first-line defense against microbial invasion through their complex secretions. The antimicrobial activities of proteins in these secretions have been well delineated, but the contributions of lipids to mucosal defense have not been defined. We found that normal human nasal fluid contains all major lipid classes (in micrograms per milliliter), as well as lipoproteins and apolipoprotein A-I. The predominant less polar lipids were myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid, cholesterol, and cholesteryl palmitate, cholesteryl linoleate, and cholesteryl arachidonate. Normal human bronchioepithelial cell secretions exhibited a similar lipid composition. Removal of less-polar lipids significantly decreased the inherent antibacterial activity of nasal fluid against Pseudomonas aeruginosa, which was in part restored after replenishing the lipids. Furthermore, lipids extracted from nasal fluid exerted direct antibacterial activity in synergism with the antimicrobial human neutrophil peptide HNP-2 and liposomal formulations of cholesteryl linoleate and cholesteryl arachidonate were active against P. aeruginosa at physiological concentrations as found in nasal fluid and exerted inhibitory activity against other Gram-negative and Gram-positive bacteria. These data suggest that host-derived lipids contribute to mucosal defense. The emerging concept of host-derived antimicrobial lipids unveils novel roads to a better understanding of the immunology of infectious diseases.     

Meibum lipid composition in Asians with dry eye disease

Background

Previous lipidomic analyses of the human meibum had largely focused on individuals from non-Asian populations, despite the higher prevalence of dysfunctional tear syndrome (DTS) observed across Asia. Information pertaining to the alterations in lipid profiles in relation to DTS onset and progression is also lacking and warrants comprehensive experimental analysis.

Methodologies/Principal Findings

We examined the meibum lipidome of 27 DTS patients and 10 control subjects for a total of 256 lipid species from 12 major lipid classes, including cholesteryl ester (CE), wax ester (WE), triacylglyceride (TAG), (O-acyl)-ω-hydroxy fatty acid (OAHFA), glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG) and sphingolipids (sphingomyelin, SM; ceramide, Cer; glucosylceramide, GluCer; dihexosylceramide, DihexCer). Neutral lipids were analysed using high-performance liquid-chromatography coupled with mass spectrometry (HPLC/MS) and tandem mass spectrometry (MS/MS) was used for the qualitative and quantitative analysis of polar lipid species. DTS patients were classified into three severity groups (i.e. mild, moderate and severe) based on the ocular surface disease index (OSDI). A significantly lower level of TAG (p<0.05) was observed in patients under the moderate category compared to the mild category. Notably, a number of OAHFA species displayed consistently decreasing levels that correlate with increasing disease severity. An attempt was also made to investigate the changes in meibum lipid profiles of DTS patients compared to normal individuals classified based on OSDI score. Several unsaturated TAG and PC species were found at significantly higher levels (p<0.05) in patients than controls.

Conclusion

The current study presents, for the first time, a comprehensive lipidome of meibum from individuals of an Asian ethnicity, which can potentially offer new insights into the higher prevalence of DTS observed amongst Asian populations. This study also represents an attempt towards identification of lipid species in meibum which could serve as marker for DTS.     

Molecular organization of the tear fluid lipid layer

The tear fluid protects the corneal epithelium from drying out as well as from invasion by pathogens. It also provides cell nutrients. Similarly to lung surfactant, it is composed of an aqueous phase covered by a lipid layer. Here we describe the molecular organization of the anterior lipid layer of the tear film. Artificial tear fluid lipid layers (ATFLLs) composed of egg yolk phosphatidylcholine (60 mol %), free fatty acids (20 mol %), cholesteryl oleate (10 mol %), and triglycerides (10 mol %) were deposited on the air-water interface and their physico-chemical behavior was compared to egg-yolk phosphatidylcholine monolayers by using Langmuir-film balance techniques, x-ray diffraction, and imaging techniques as well as in silico molecular level simulations. At low surface pressures, ATFLLs were organized at the air-water interface as heterogeneous monomolecular films. Upon compression the ATFLLs collapsed toward the air phase and formed hemispherelike lipid aggregates. This transition was reversible upon relaxation. These results were confirmed by molecular-level simulations of ATFLL, which further provided molecular-scale insight into the molecular distributions inside and dynamics of the tear film. Similar type of behavior is observed in lung surfactant but the folding takes place toward the aqueous phase. The results provide novel information of the function of lipids in the tear fluid.     

Phospholipid transfer protein is present in human tear fluid

The human tear fluid film consists of a superficial lipid layer, an aqueous middle layer, and a hydrated mucin layer located next to the corneal epithelium. The superficial lipid layer protects the eye from drying and is composed of polar and neutral lipids provided by the meibomian glands. Excess accumulation of lipids in the tear film may lead to drying of the corneal epithelium. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. To gain insight into the formation of tear film, we investigated whether PLTP and CETP are present in human tear fluid. Tear fluid samples were collected with microcapillaries. The presence of PLTP and CETP was studied in tear fluid by Western blotting, and the PLTP concentration was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. Size-exclusion and heparin-affinity chromatography assessed the molecular form of PLTP. PLTP is present in tear fluid, whereas CETP is not. Quantitative assessment of PLTP by ELISA indicated that the PLTP concentration in tear fluid, 10.9 +/- 2.4 microg/mL, is about 2-fold higher than that in human plasma. PLTP-facilitated phospholipid transfer activity in tears, 15.1 +/- 1.8 micromol mL(-)(1) h(-)(1), was also significantly higher than that measured in plasma. Inactivation of PLTP by heat treatment (+58 degrees C, 60 min) or immunoinhibition abolished the phospholipid transfer activity in tear fluid. Size-exclusion chromatography of tear fluid indicated that PLTP eluted in a position corresponding to a size of 160-170 kDa. Tear fluid PLTP was quantitatively bound to Heparin-Sepharose and could be eluted as a single peak by 0.5 M NaCl. These data indicate that human tear fluid contains catalytically active PLTP protein, which resembles the active form of PLTP present in plasma. The results suggest that PLTP may play a role in the formation of the tear film by supporting phospholipid transfer.     

Concerted action of human carboxyl ester lipase and pancreatic lipase during lipid digestion in vitro: importance of the physicochemical state of the substrate

The pancreatic enzyme carboxyl ester lipase (CEL) has been shown to hydrolyse a large number of different esters, including triacylglycerols, cholesteryl esters and retinyl esters with an absolute requirement for bile salts. Some of the lipids that are substrates for CEL can also be hydrolysed by pancreatic lipase. In order to investigate the relative roles of human CEL and pancreatic lipase, the two enzymes were incubated on a pH-stat with isotope-labelled lipid substrate mixtures in physicochemical forms resembling the state of the dietary lipids in human intestinal contents. In the first set of experiments, cholesteryl oleate (CO) and retinyl palmitate (RP) were solubilised in an emulsion of triolein (TO) stabilised by egg phosphatidylcholine and bile salts. Lipase (always added together with its cofactor, colipase) hydrolysed TO, with monoolein and oleic acid as end-products, whereas CEL alone could not hydrolyse TO in the presence of phosphatidylcholine (PC). Lipase alone did not hydrolyse CO or RP, but CEL did hydrolyse these esters if lipase was present. Release of [3H]glycerol from labelled TO increased only slightly if CEL was added compared to lipase alone, suggesting that monoolein hydrolysis was slow under these conditions. In the second set of experiments, CO and RP were dissolved in bile salt/monoolein/oleic acid dispersions with varying bile salt concentrations. CEL hydrolysed CO and RP more rapidly in a system with a high bile salt concentration containing mixed micelles than in a system with a low bile salt concentration, where the lipids were dispersed in the form of mixed micellar and non-micellar aggregates; both types of aggregate have been reported to exist in human intestinal contents. In conclusion, these data suggest that the main function of CEL under physiological conditions is to hydrolyse cholesteryl and retinyl esters, provided that the triacylglycerol oil phase is hydrolysed by pancreatic lipase, which probably causes a transfer of the substrate lipids of CEL from the oil emulsion phase to an aqueous bile salt/lipolytic product phase. Depending on the bile salt/lipolytic product ratio, the substrate will reside in either micellar or non-micellar lipid aggregates, of which the micellar state is preferred by CEL.     

Lipidomics as a Principal Tool for Advancing Biomedical Research

Lipidomics,which targets at the construction of a comprehensive map of lipidome comprising the entire lipid pool within a cell or tissue,is currently emerging as an independent discipline at the interf...     

Properties of cholesteryl esters in pure and mixed monolayers

The surface properties of cholesteryl palmitate, stearate, linoleate, linolenate, arachidonate, and acetate were investigated. Long-chain esters were not surface-active and force-area (pi-A) isotherms were not obtained. Unsaturated cholesteryl esters were oxidized at the air-water interface and these oxidized lipids gave expanded pi-A isotherms. Cholesteryl acetate had an equilibrium spreading pressure of 14.0 dynes/cm and formed a stable monolayer indistinguishable from cholesterol below that surface pressure. Cholesteryl linoleate formed mixed monolayers with surface-active lipids, and the amount of cholesteryl linoleate in the monolayer depended both on its solubility in the other lipid and on the surface pressure. Even at moderate surface pressures cholesteryl linoleate was extruded from the monolayer into a bulk phase. Cholesteryl acetate exhibited the well-known condensing effect of cholesterol in mixed monolayers with egg lecithin.     

Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic

Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that approximately 10-20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1-2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic.     

High density lipoprotein efficiently accepts surface but not internal oxidised lipids from oxidised low density lipoprotein

ObjectiveOxidised low density lipoprotein (oxLDL) contributes to atherosclerosis, whereas high density lipoprotein (HDL) is known to be atheroprotective due, at least in part, to its ability to remove oxidised lipids from oxLDL. The molecular details of the lipid transfer process are not fully understood. We aimed to identify major oxidised lipid species of oxLDL and investigate their transfer upon co-incubation with HDL with varying levels of oxidation.Approach and resultsA total of 14 major species of oxidised phosphatidylcholine and oxidised cholesteryl ester from oxLDL were identified using an untargeted mass spectrometry approach. HDL obtained from pooled plasma of normolipidemic subjects (N = 5) was oxidised under mild and heavy oxidative conditions. Non-oxidised (native) HDL and oxidised HDL were co-incubated with oxLDL, re-isolated and lipidomic analysis was performed. Lipoprotein surface lipids, oxidised phosphatidylcholines and oxidised cholesterols (7-ketocholesterol and 7β-hydroxycholesterol), but not internal oxidised cholesteryl esters, were effectively transferred to native HDL. Saturated and monounsaturated lyso-phosphatidylcholines were also transferred from the oxLDL to native HDL. These processes were attenuated when HDL was oxidised under mild and heavy oxidative conditions. The impaired capacities were accompanied by an increase in a ratio of sphingomyelin to phosphatidylcholine and a reduction in phosphatidylserine content in oxidised HDL, both of which are potentially important regulators of the oxidised lipid transfer capacity of HDL.ConclusionsOur study has revealed the differential transfer efficiency of surface and internal oxidised lipids from oxLDL and their acceptance onto HDL. These capacities were modulated when HDL was itself oxidised.     

Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma

In a previous study we demonstrated that highly purified lipid-transfer protein facilitated the transfer of triglyceride, cholesteryl ester, and phosphatidylcholine between plasma lipoproteins. It remained unclear, however, whether these lipids were transferred by independent sites on the lipid-transfer protein. To address this point, we have studied the protein-mediated transfer of triglyceride, cholesteryl ester, and phosphatidylcholine as a function of the concentration and lipid composition of donor and acceptor lipoproteins. Lipoproteins labeled in vitro, reconstituted lipoproteins of defined lipid composition, and phosphatidylcholine liposomes with or without triglyceride and/or cholesteryl ester have been used to investigate the inter-relationships of lipids transferred by the lipid-transfer protein. In studies of initial (less than or equal to 10-13%) transfer, we found that, although absolute transfer rates were affected, the ratio of cholesteryl ester to triglyceride transferred was independent of donor and acceptor lipoprotein concentrations and acceptor lipoprotein lipid composition. With reconstituted lipoproteins as donor, we demonstrated that this ratio was linearly related to the ratio of cholesteryl ester to triglyceride in the donor particle; the sum of triglyceride and cholesteryl ester transferred remained constant and independent of the lipid composition of the donor. Experiments with intact lipoproteins labeled in vitro and with small unilamellar vesicles in the presence and absence of p-chloromercuriphenylsulfonate, confirmed the interdependence of triglyceride and cholesteryl ester transfer. In contrast, under all assay conditions, no correlation was found between the amount of phosphatidylcholine transferred and the transfer of triglyceride and/or cholesteryl ester. We conclude that triglyceride and cholesteryl ester compete for transfer and that the extent of transfer for each lipid is determined by its relative concentration in the donor particle, whereas phosphatidylcholine transfer is independent of triglyceride and cholesteryl ester transfer. The data also strongly support the conclusion that lipid transfer protein promotes both the exchange and net transfer of triglyceride and cholesteryl ester and that the net transfer process proceeds by a reciprocal exchange of triglyceride and cholesteryl ester without net transfer of core lipid between lipoproteins.     

Enhancement of non-polar lipid transfer reaction through stabilization of substrate lipid particles with apolipoproteins

Transfer of lipids was studied between human plasma low density lipoproteins (LDL) and triolein particles coated with an egg phosphatidylcholine monolayer, with diameter of 27 +/- 4 nm. The lipid particles were unstable and seemed to aggregate to LDL when incubated with LDL either in the presence or the absence of bovine serum albumin. Human apolipoproteins A-I, A-II, C-II, C-III, and E stabilized the lipid particles and completely prevented this process. Cholesterol rapidly appeared in the lipid particles to reach homogeneous distribution among the phospholipid surfaces of LDL and the lipid particles regardless of whether apolipoproteins were present or absent. Cholesteryl ester spontaneously appeared in the lipid particles to some extent in the absence of the apolipoproteins, and human plasma lipid transfer protein enhanced this reaction only to a very limited extend. When the lipid particles were stabilized with the apolipoproteins, spontaneous cholesteryl ester transfer was minimized and the lipid transfer protein catalyzed the transfer of cholesteryl ester markedly. There was no specific difference among the apolipoproteins in stabilizing the particles and enhancing the transfer reaction. Reciprocal decrease in volume of triglyceride was observed at the same time in the lipid particles until the relative content of cholesteryl ester in the cores of LDL was the same as in the lipid particles. The kinetics of the cholesteryl ester and triglyceride transfer was consistent with the model that the reaction is bidirectional in equilibrium and takes both non-polar lipids as substrate in a single pool.     

Skin lipids from Saudi Arabian birds

Skin lipids play an important role in the regulation of cutaneous water loss (CWL). Earlier studies have shown that Saudi desert birds exhibit a tendency of reduced CWL than birds from temperate environment due to adaptive changes in composition of their skin lipids. In this study, we used thin-layer chromatography (TLC) for separation and detection of non-polar and polar lipids from the skin of six bird species including sooty gull, brown booby, house sparrow, Arabian waxbill, sand partridge, and laughing dove. The lipids were separated and detected on Silica gel G coated TLC plates and quantified by using densitometric image analysis. Rf values of the non-polar lipids were as follows: cholesterol (0.29), free fatty acids (0.58), triacylglycerol (0.69), fatty acids methyl esters (0.84) and cholesterol ester (0.97). Rf values for the polar lipids were: cerebroside (0.42), ceramide (0.55) and cholesterol (0.73). The results showed the abundance of fatty acids methyl esters (47.75–60.46%) followed by triacylglycerol (12.69–24.14%). The remaining lipid compositions were as follows: cholesterol (4.09–13.18%), ceramide (2.18–13.27%), and cerebroside (2.53–12.81%). In conclusion, our findings showed that TLC is a simple and sensitive method for the separation and quantification of skin lipids. We also reported a new protocol for lipid extraction using the zirconia beads for efficient disruption of skin tissues. This study will help us better understand the role of skin lipids in adaptive physiology towards adverse climatic conditions.     

Blood Plasma Lipidome: Opportunities in the Early Diagnostics of Preeclampsia

Russian Journal of Bioorganic Chemistry - Blood plasma lipidome analysis is a mass spectrometric (MS) evaluation of molecular types of lipids, sometimes in combination with high performance liquid...     

Avanti lipid tools: Connecting lipids,technology, and cell biology

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein–lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.