Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction.
ABSTRACT: 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.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.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.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.).
Project description:Meibomian glands that are embedded in tarsal plates of human eyelids, and sebaceous glands found in the skin, including that of eyelids, are two related types of holocrine glands that produce lipid-rich secretions called meibum and sebum. Pervasive ocular disorders, such as Meibomian gland dysfunction and dry eye, have been linked to changes in the lipid composition of meibum. However, in most described cases the changes were either small, or random, or insufficiently characterized on the molecular level. Here, we present results of comprehensive lipidomic analyses of meibum, tears and sebum of a patient whose secretions were highly abnormal (abnormal meibum, tears and sebum, or AMTS, patient). The lipidomes were characterized on the level of individual lipid species using ultra-high performance liquid chromatography and high resolution mass spectrometry. The major differences between the AMTS patient and normal age- and gender-matched subjects included, among others, severely diminished pools of normal meibomian lipids such as wax esters and cholesteryl esters in meibum and tears, a 2x increase in total cholesteryl esters to wax esters ratio, their skewed molecular profiles, a ~3x increase in free cholesterol to cholesteryl esters ratio, and, most importantly, a 20x to 30x increase in the triglicerides fraction over the norm. Sebum of the AMTS patient was also highly abnormal lacking major wax esters. Notably, the routine blood lipid panel test of the AMTS patient showed no abnormalities. The data imply that the AMTS patient had a severe, previously unreported, metabolic disorder that affected meibogenesis in Meibomian glands and sebogenesis in sebaceous glands. This is, to the best of our knowledge, a first observation of the condition that we have termed High Triglycerides/Low Waxes (HTLW) syndrome.
Project description:A lipid layer consisting of meibum lipids exists in the tear film and functions in preventing dry eye disease. Although the meibum lipids include diverse lipid classes, the synthesis pathway and role of each class remain largely unknown. Here, we created single and double knockout (KO and DKO, respectively) mice for the two acyl-CoA wax alcohol acyltransferases (<i>Awat1</i> and <i>Awat2</i>) and investigated their dry eye phenotypes and meibum lipid composition. <i>Awat2</i> KO and DKO mice exhibited severe dry eye with meibomian gland dysfunction, whereas <i>Awat1</i> KO mice had mild dry eye. In these mice, specific meibum lipid classes were reduced: (<i>O</i>-acyl)-ω-hydroxy fatty acids and type 1ω wax diesters in <i>Awat1</i> KO mice, wax monoesters and types 1ω and 2ω wax diesters in <i>Awat2</i> KO mice, and most of these in DKO mice. Our findings reveal that Awat1 and Awat2 show characteristic substrate specificity and together produce diverse meibum lipids.
Project description:Secretions from meibomian glands located within the eyelid (commonly known as meibum) are rich in nonpolar lipid classes incorporating very-long (22-30 carbons) and ultra-long (>30 carbons) acyl chains. The complex nature of the meibum lipidome and its preponderance of neutral, nonpolar lipid classes presents an analytical challenge, with typically poor chromatographic resolution, even between different lipid classes. To address this challenge, we have deployed differential mobility spectrometry (DMS)-MS to interrogate the human meibum lipidome and demonstrate near-baseline resolution of the two major nonpolar classes contained therein, namely wax esters and cholesteryl esters. Within these two lipid classes, we describe ion mobility behavior that is associated with the length of their acyl chains and location of unsaturation. This capability was exploited to profile the molecular speciation within each class and thus extend meibum lipidome coverage. Intriguingly, structure-mobility relationships in these nonpolar lipids show similar trends and inflections to those previously reported for other physicochemical properties of lipids (e.g., melting point and phase-transition temperatures). Taken together, these data demonstrate that differential ion mobility provides a powerful orthoganol separation technology for the analysis of neutral lipids in complex matrices, such as meibum, and may further provide a means to predict physicochemical properties of lipids that could assist in inferring their biological function(s).
Project description:<h4>Purpose</h4>The purpose of this investigation was to better understand lipid composition in human meibum.<h4>Methods</h4>Intact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation.<h4>Results</h4>The absolute amount (?mol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 ± 4% and 40 ± 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 ± 2% and 25 ± 1% for CEs and 14 ± 1% and 86 ± 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 ?,? Type II DEs, and 21 ? Type I-St DEs.<h4>Conclusions</h4>Major neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive.
Project description:The mechanism of performance of hydrocarbon waxes used as external lubricants in typical PVC pipe formulations is not completely understood or defined. This study will benefit pipe manufacturers to tailor PVC processing conditions and wax addition levels to enhance the value of waxes with different molecular architectures in Sn-stabilized PVC formulations. A selection of waxes was comprehensively analyzed to understand how their structural and morphological differences impact the fusion behavior of a Sn-stabilized PVC compound. Results showed a high probability of fusion for waxes with melting points between 65 and 80 °C and a kinematic viscosity (at 135 °C) of less than 5 cSt across all experimental temperatures and concentration levels tested. The general probability of fusion increased with increasing temperature and decreasing wax concentration. Wax compositional differences only had a large impact on PVC fusion at lower experimental temperatures and high wax concentrations. The stable time and onset of degradation were found to be sensitive to experimental temperature but less so to wax concentration. High melting waxes had the lowest fusion torque but a narrow operating window in terms of concentration and temperature for a successful fusion to occur. Overall, fully refined paraffin waxes had the widest operating window and were less sensitive to a change in the wax concentration and experimental temperature than the rest of the waxes that were tested.
Project description:Cuticular waxes are essential for the well-being of all plants, from controlling the transport of water and nutrients across the plant surface to protecting them against external environmental attacks. Despite their significance, our current understanding regarding the structure and function of the wax film is limited. In this work, we have formed representative reconstituted wax film models of controlled thicknesses that facilitated an ex vivo study of plant cuticular wax film properties by neutron reflection (NR). Triticum aestivum L. (wheat) waxes were extracted from two different wheat straw samples, using two distinct extraction methods. Waxes extracted from harvested field-grown wheat straw using supercritical CO2 are compared with waxes extracted from laboratory-grown wheat straw via wax dissolution by chloroform rinsing. Wax films were produced by spin-coating the two extracts onto silicon substrates. Atomic force microscopy and cryo-scanning electron microscopy imaging revealed that the two reconstituted wax film models are ultrathin and porous with characteristic nanoscale extrusions on the outer surface, mimicking the structure of epicuticular waxes found upon adaxial wheat leaf surfaces. On the basis of solid-liquid and solid-air NR and ellipsometric measurements, these wax films could be modelled into two representative layers, with the diffuse underlying layer fitted with thicknesses ranging from approximately 65 to 70 Å, whereas the surface extrusion region reached heights exceeding 200 Å. Moisture-controlled NR measurements indicated that water penetrated extensively into the wax films measured under saturated humidity and under water, causing them to hydrate and swell significantly. These studies have thus provided a useful structural basis that underlies the function of the epicuticular waxes in controlling the water transport of crops.
Project description:Lipids secreted by the meibomian glands (MGs) of the eyelids are essential to the protection of the eye's surface. An altered meibum composition represents the primary cause of evaporative dry eye disease (DED). Despite the critical importance of the meibum, its biosynthetic pathways and the roles of individual lipid components remain understudied. Here, we report that the genetic deletion of Acyl-CoA:wax alcohol acyltransferase 2 (AWAT2) causes the obstruction of MGs and symptoms of evaporative DED in mice. The lipid composition of the meibum isolated from Awat2<sup>-/-</sup> mice revealed the absence of wax esters, which was accompanied by a compensatory overproduction of cholesteryl esters. The resulting increased viscosity of meibum led to the dilation of the meibomian ducts, and the progressive degeneration of the MGs. Overall, we provide evidence for the main physiological role of AWAT2 and establish Awat2<sup>-/-</sup> mice as a model for DED syndrome that can be used in studies on tear film-oriented therapies.
Project description:Solid deposition during production, transport, and storage of crude oils leads to significant technical problems and economic losses for the oil and gas industry. The thermodynamic equilibrium between high-molecular-weight components of crude oil, such as asphaltenes, resins, and waxes, is an important parameter for the stability of crude oil. Once the equilibrium is disturbed due to variations in temperature, pressure, and oil composition during production, the solubility of high-molecular-weight waxes decreases. This results in a decrease in the wax appearance temperature (WAT) and the deposit of wax onto solid surfaces. On the other hand, under these conditions, asphaltenes do not interact sufficiently with the resins/waxes and tend to flocculate among themselves and form asphaltene nanoaggregates. The role of waxes during the asphaltene aggregation and deposition has not been appropriately explained yet. The objective of this research study is to describe the interaction of asphaltenes and waxes and subsequently address the specific example of an asphaltenic oil commingled with a wax inhibitor-containing oil during the combination of different oil streams. It is a crucial building block for the development of a suitable and cost-effective strategy for the handling of wax/asphaltene associated flow assurance problems. In this work, the quartz crystal microbalance (QCM) technique has been used for the first time to investigate the effect of waxes and related chemicals, which are used to mitigate wax deposition, on asphaltene aggregation and deposition phenomena. Asphaltene onset point and asphaltene deposition rate have been monitored using QCM at high pressure-high temperature (HPHT) conditions. This study confirms that the different wax inhibitor chemistries result in significant differences in the pour point decrease and viscosity profiles in crude oil. Different wax inhibitors also showed different outcomes regarding the asphaltene deposition tendency. A comprehensive modeling study has also been conducted for mechanistic investigation of experimental results. In this regard, the perturbed chain statistical associating fluid theory equation of state (PC-SAFT EoS) was utilized to model the systems.
Project description:Previous studies on ablation of several key genes of meibogenesis related to fatty acid elongation, omega oxidation, and esterification into wax esters have demonstrated that inactivation of any of them led to predicted changes in the meibum lipid profiles and caused severe abnormalities in the ocular surface and Meibomian gland (MG) physiology and morphology. In this study, we evaluated the effects of <i>Soat1</i> ablation that were expected to cause depletion of the second largest class of Meibomian lipids (ML)-cholesteryl esters (CE)-in a mouse model. ML of the <i>Soat1-</i>null mice were examined using liquid chromatography high-resolution mass spectrometry and compared with those of <i>Soat1<sup>+/-</sup></i> and wild-type mice. Complete suppression of CE biosynthesis and simultaneous accumulation of free cholesterol (Chl) were observed in <i>Soat1-</i>null mice, while <i>Soat1<sup>+/-</sup></i> mutants had normal Chl and CE profiles. The total arrest of the CE biosynthesis in response to <i>Soat1</i> ablation transformed Chl into the dominant lipid in meibum accounting for at least 30% of all ML. The <i>Soat1-</i>null mice had clear manifestations of dry eye and MG dysfunction. Enrichment of meibum with Chl and depletion of CE caused plugging of MG orifices, increased meibum rigidity and melting temperature, and led to a massive accumulation of lipid deposits around the eyes of <i>Soat1</i>-null mice. These findings illustrate the role of <i>Soat1</i>/SOAT1 in the lipid homeostasis and pathophysiology of MG.
Project description:Natural edible waxes mixed with plant oils, containing high levels of unsaturated fatty acids (FAs), are known as oleogels. Oleogels are used for replacing saturated FAs in animal-derived food with unsaturated FAs. However, the health effects of edible waxes are not yet clearly defined. The purpose of this study was to investigate the effect of FAs and natural waxes on the adipogenesis in 3T3-L1 cells. The 3T3-L1 cells were differentiated and treated with FAs and waxes. These FAs [Palmitic acid (PA), Stearic acid (SA), Oleic acid (OA), Linoleic acid (LA), and Alpha-linolenic acid (ALA)] and waxes [beeswax (BW) and carnauba wax (CW)] were prepared at varying concentrations, and cell toxicity, triglyceride accumulation, lipid droplets size, and distribution inside of cells were determined. Adipogenic gene expression including PPAR?, FASN, C/EBP?, SREBP-1, and CPT-1 was determined. Results showed that increasing the concentration of FAs and waxes led to a decrease in the adipocyte cells viability and metabolic performance. SA showed the highest level of triglyceride accumulation (p<0.05), whereas ALA showed the lowest (p<0.05). Both BW and CW at 3.0 ppm showed significantly higher lipid accumulation than in the control and other groups (p<0.05). ALA had significantly downregulated adipogenic gene expression levels, excluding those of CPT-1, compared to the other treatment groups (p<0.05). Moreover, BW demonstrated similar adipogenic gene expression levels as ALA compared to CW. Consequently, ALA and BW may have health benefits by reducing adipogenesis and can be used in processed meat.