Compositional Analysis of Wax Esters in Human Meibomian Gland Secretions by Direct Infusion Electrospray Ionization Mass Spectrometry

Wax esters (WE) are one of the predominant lipid types in human meibomian gland secretions (meibum) and account for 40–50 % of total meibum lipids. Recently, we managed to quantify 51 isomeric groups of intact WE in normal human meibum samples by direct infusion electrospray ionization mass spectrometry (ESI–MS), with each WE peak in the MS spectrum corresponding to one isomeric group (Chen et al, Invest Ophthalmol Vis Sci 54(8):5730–53, 2013). However, the information of the isomeric composition in each group was not obtained. In this study, tandem mass spectrometry (MS/MS) was applied to quantify relative amounts of these isomers using the intensities of the corresponding diagnostic ions after appropriate correction and normalization. This data was combined with the previous obtained mole fraction of each isomeric group to total WE in human meibum to determine the corresponding percentage of each isomer. A total of 23 of the most abundant WE peaks of different molecular weights (corresponding to 85.3 % of the total amount of WE) in human meibum were studied and resulted in quantification of 92 WE species. The quantitative information of composition of WE in human meibum will help better understand their role in the tear film.     

Liquid Chromatography–Mass Spectrometric Analysis of Lipids Present in Human Meibomian Gland Secretions

The purpose of the study was to qualitatively characterize the major lipid species present in human meibomian gland secretions (MGS) by means of high-performance liquid chromatography with atmospheric pressure ionization mass spectrometric detection of the analytes (NP HPLC-MS). Two different NP HPLC-MS methods have been developed to analyze lipid species that were expected to be present in MGS. The first method was optimized for the analysis of relatively nonpolar lipids [wax esters (WE), di- and triacyl glycerols (DAG and TAG), cholesterol (Chl) and its esters (Chl-E), and ceramides (Cer)], while the second method was designed to separate and detect phospholipids. The major lipid species in MGS were found to be WE, Chl-E, and TAG. A minor amount of free Chl (less then 0.5% of the Chl-E fraction) was detected in MGS. No appreciable amounts of DAG and Cer were found in MGS. The second NP HPLC-MS method, capable of analyzing model mixtures of authentic phospholipids (e.g. phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin) in submicrogram/mL concentrations, showed little or no presence of these species in the MGS samples. These observations suggest that MGS are a major source of the nonpolar lipids of the WE and Chl-E families for the tear film lipid layer (TFLL), but not of the previously reported phospholipid components of the TFLL.     

Depletion of Cholesteryl Esters Causes Meibomian Gland Dysfunction-Like Symptoms in a Soat1-Null Mouse Model

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 Soat1 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 Soat1-null mice were examined using liquid chromatography high-resolution mass spectrometry and compared with those of Soat1^+/− and wild-type mice. Complete suppression of CE biosynthesis and simultaneous accumulation of free cholesterol (Chl) were observed in Soat1-null mice, while Soat1^+/− mutants had normal Chl and CE profiles. The total arrest of the CE biosynthesis in response to Soat1 ablation transformed Chl into the dominant lipid in meibum accounting for at least 30% of all ML. The Soat1-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 Soat1-null mice. These findings illustrate the role of Soat1/SOAT1 in the lipid homeostasis and pathophysiology of MG.     

Fatty Acid Methyl Ester Profiles of Bat Wing Surface Lipids

Sebocytes are specialized epithelial cells that rupture to secrete sebaceous lipids (sebum) across the mammalian integument. Sebum protects the integument from UV radiation, and maintains host microbial communities among other functions. Native glandular sebum is composed primarily of triacylglycerides (TAG) and wax esters (WE). Upon secretion (mature sebum), these lipids combine with minor cellular membrane components comprising total surface lipids. TAG and WE are further cleaved to smaller molecules through oxidation or host enzymatic digestion, resulting in a complex mixture of glycerolipids (e.g., TAG), sterols, unesterified fatty acids (FFA), WE, cholesteryl esters, and squalene comprising surface lipid. We are interested if fatty acid methyl ester (FAME) profiling of bat surface lipid could predict species specificity to the cutaneous fungal disease, white nose syndrome (WNS). We collected sebaceous secretions from 13 bat spp. using Sebutape^® and converted them to FAME with an acid catalyzed transesterification. We found that Sebutape^® adhesive patches removed ~6× more total lipid than Sebutape^® indicator strips. Juvenile eastern red bats (Lasiurus borealis) had significantly higher 18:1 than adults, but 14:0, 16:1, and 20:0 were higher in adults. FAME profiles among several bat species were similar. We concluded that bat surface lipid FAME profiling does not provide a robust model predicting species susceptibility to WNS. However, these results provide baseline data that can be used for lipid roles in future ecological studies, such as life history, diet, or migration.     

The occurrence of long chain α,ω-diols in the lipids of steer and human meibomian glands

A group of long chain α,ω-diols (C₂₉ to C₃₄) has been identified in the lipids of steer and human meibomian gland excreta (meibum). These new lipids were isolated from the steer meibum unsaponifiables. Proof of structure was provided by 1) the column chromatographic behavior and TLC of the diols and their diacetates; 2) GLC on glass capillary columns; 3) fragmentation patterns in GC-MS; 4) NMR data, and 5) ozonolysis studies of the unsaturates. Chain types for the steer sample were 51% straight monoenes, 8.5% straight saturates, 39% iso and anteiso saturates and 1.5% iso and anteiso unsaturates. GC for the human sample gave straight monoenes 83%, straight saturates 8%, and iso plus anteiso saturates 9%. Close correspondence of the α,ω-diol chain lengths and types with meibum ω-hydroxy fatty acids suggests a biochemical precursor relationship.     

Double-bond patterns of fatty acids and alcohols in steer and human meibomian gland lipids

Ozonolysis studies of the monoenes of the fatty chain types in lipids of steer meibomian gland excreta (meibum) have confirmed earlier structural assignments based on gas liquid chromatography (GLC) retention data and have assisted in assigning complete structures to a group of recently identified ω-hydroxy fatty acids. The ω-hydroxy acids include straight-chain monoenoic acids (85%), saturated anteiso and iso acids (13%), monoenoic acids of the latter group (1%) and, finally, saturates of the normal monoenoic acids (1%). All the fatty chains of meibum can be biosynthesized by a unified process of chain buildup to primary chain lengths of 12∶0–20∶0 for the straight evens, with 16∶0 predominating, 13∶0–21∶0 for the straight odds with 17∶0 predominating, i16∶0 to i28∶0 for the iso and ai17∶0 to ai29∶0 for the anteiso chain types; then Δ9 desaturation of each of these chain types; and finally chain elongation of 1–10 C₂ units. Some chain degradation may also occur. The meibum lipid components involved are unsubstituted fatty acids, α-OH fatty acids, ω-OH fatty acids, fatty alcohols and some other lipid components incompletely characterized. The carbon skeletons are straight even, straight odd, iso and anteiso except that the α-OH fatty acids are only straight even and straight odd and these chains are not elongated. All fatty chains are almost entirely saturated and monoenoic, the polyenes occurring in only trace amounts. Biosynthesis of the fatty chains of human meibum evidently occurs similarly, except that considerably more 18∶0 than 16∶0 fatty acids are built up by the fatty acid synthetase, before desaturation and extension.     

Rapid Quantitative Analysis of Lipids Using a Colorimetric Method in a Microplate Format

A colorimetric sulfo-phospho-vanillin (SPV) method was developed for high throughput analysis of total lipids. The developed method uses a reaction mixture that is maintained in a 96-well microplate throughout the entire assay. The new assay provides the following advantages over other methods of lipid measurement: (1) background absorbance can be easily corrected for each well, (2) there is less risk of handling and transferring sulfuric acid contained in reaction mixtures, (3) color develops more consistently providing more accurate measurement of absorbance, and (4) the assay can be used for quantitative measurement of lipids extracted from a wide variety of sources. Unlike other spectrophotometric approaches that use fluorescent dyes, the optimal spectra and reaction conditions for the developed assay do not vary with the sample source. The developed method was used to measure lipids in extracts from four strains of microalgae. No significant difference was found in lipid determination when lipid content was measured using the new method and compared to results obtained using a macro-gravimetric method.     

Interactions of Meibum and Tears with Mucomimetic Polymers: A Hint towards the Interplay between the Layers of the Tear Film

Recent clinical findings suggest that mucomimetic polymers (MMP) can alter not only the texture of the aqueous tear but also the spreading and structure of the tear film (TF) lipid layer, thereby allowing for their synchronized performance in vivo. Thus, we aimed to evaluate in vitro (i) the capability of pharmaceutically applicable MMP to ensure the formation of post-evaporative ferning patterns (a characteristic feature of the “healthy” tear colloid) and (ii) the MMP interactions with human meibum films accessed in the course of blink-like deformations via Langmuir surface balance and Brewster angle microscopy (BAM). Four MMP were used- hyaluronic acid (HA), cross-linked hyaluronic acid (CHA), carboxymethyl cellulose (CMC) and gellan gum (GG)- at the concentrations of 0.0001%, 0.001%, 0.01%, 0.05% and 0.1%. Significant differences were observed in the MMP fern formation capability: CHA (≥0.001%) > HA (≥0.01%) = CMC (≥0.01%) > GG (≥0.05%). All MMP affected the spreading of meibum, with BAM micrographs revealing thickening of the films. CHA was particularly efficient, showing concentration-dependent enhancement of tear ferning and of meibomian layer structure, surfactant properties and viscoelasticity. Thus, endogenous and exogenous MMP may play key roles for the concerted action of the TF layers at the ocular surface, revealing novel routes for TF-oriented therapeutic applications.     

Wax Ester Analysis of Bats Suffering from White Nose Syndrome in Europe

The composition of wax esters (WE) in the fur of adult greater mouse–eared bats (Myotis myotis), either healthy or suffering from white nose syndrome (WNS) caused by the psychrophilic fungus Pseudogymnoascus destructans, was investigated by high‐resolution mass spectrometry analysis in the positive ion mode. Profiling of lipid classes showed that WE are the most abundant lipid class, followed by cholesterol esters, and other lipid classes, e.g., triacylglycerols and phospholipids. WE abundance in non‐polar lipids was gender‐related, being higher in males than in females; in individuals suffering from WNS, both male and female, it was higher than in healthy counterparts. WE were dominated by species containing 18:1 fatty acids. Fatty alcohols were fully saturated, dominated by species containing 24, 25, or 26 carbon atoms. Two WE species, 18:1/18:0 and 18:1/20:0, were more abundant in healthy bats than in infected ones.     

Determination of <Emphasis Type="Italic">c</Emphasis>9,<Emphasis Type="Italic">t</Emphasis>11-CLA in major human plasma lipid classes using a combination of methylating methodologies

Isomeric CLA exhibit several significant biological activities in animals and humans and are easily isomerized to their corresponding t,t-CLA isomers during methylation with various acid-catalyzed reagents. To minimize such isomerization and provide a valid quantification of human plasma CLA content, several methylation methods were tested. Plasma neutral lipid, nonesterified FA (NEFA), and polar lipid classes were separated into the following fractions: (i) cholesteryl ester (CE, 1.2 mg/12 mL, 37.5% lipids), (ii) TAG (0.8 mg/12 mL, 25% lipids), (iii) NFFA (0.2 mg/12 mL, 6.2% lipids), (iv) MAG/DAG/cholesterol (0.3 mg/12 mL, 9.4% lipids), and (v) phospholipid (PL, 0.5 mg/20 mL, 15.6% lipids). Data showed that c9,t11-CLA found in TAG, MAG/DAG/cholesterol, and PL fractions were converted to methyl esters with sodium methoxide within 2 h at 55°C. However, the c9,t11-CLA in the CE fraction could not be completely converted to methyl esters by sodium methoxide/acetylchloride in methanol or methanolic KOH; instead, CE was treated with sodium methoxide and methyl acetate in diethyl ether for 1 h. NEFA were converted to methyl esters with trimethylsilyldiazomethane (TMSDAM). All reaction mixtures were monitored by TLC prior to GLC analysis. The highest enrichment of c9,t11-18∶2 (% FA) was in TAG (0.31%), followed by CE (0.14%) and PL (0.13%). The above methylation methods were then applied to a small subset (n=10) of nonfasting plasma lipid fractions to confirm the applicability of these data. Results from this subset of samples also indicated that the greatest enrichment of c9,t11-CLA was present in the TAG fraction (0.39%), followed by CE (0.27%) and PL (0.22%). These data indicate that different plasma fractions have different c9,t11-CLA contents.     

Lipid compounds in secretions of fishing bat,Noctilio leporinus (Chiroptera: Noctilionidae)

The distinctive odor ofNoctilio leporinus arises from oily secretions found beneath the wings in the subaxillary region. Analysis of secretions by gas chromatography-mass spectrometry identified 372 lipid compounds. Differences in number and chemical composition of glycolipids suggest that secretions of males from the same roost are more similar to each other than to other males or females. Differences in number and chemical composition of nonpolar lipids indicate that secretions of males are more similar to each other than to females. Since secretions differ between sexes, information on sexual identity and reproductive condition may be communicated. Individually unique lipid compositions further suggest that bats may be recognizable by their odor within the roost and while flying.     

Confirmation of the Presence of Squalene in Human Eyelid Lipid by Heteronuclear Single Quantum Correlation Spectroscopy

¹H-NMR spectroscopy has been used to quantify squalene in meibum and sebum. Squalene has many beneficial properties and its loss on the surface of skin upon ultraviolet light exposure or in the tear film with dry eye could be detrimental. In this study, we confirm the NMR proton resonance assignments of squalene, squalene in human meibum, and in human eyelid lipid using heteronuclear single quantum correlation spectroscopy. Our results confirm the presence of squalene in eyelid lipid. We speculate that the squalene in eyelid lipid could be secreted from sebaceous glands. The NMR resonances between 5.2 and 5.0 ppm, characteristic of the =CH of squalene, were resolved in the spectrum of human meibum and used to estimate that 1 % or less of squalene is present in meibum. However, the resonance assignments of squalene in meibum were not confirmed. The characteristics of squalene including its anti-inflammatory, antioxidant, and antibacterial qualities suggest that the presence of a squalene film is beneficial. Its loss in human meibum from patients with dry eye could be detrimental and contribute to the symptoms observed in these patients.     

A DC electrochemical method for studying the inhibition of metal corrosion by chromate-containing paint

A DC potentiodynamic curve method is used to observe the effect of a pigmented organic coating on the passivity of a metal substrate. In this method, a painted metal coupon, which is scribed to expose metal, functions as the working electrode (WE) of an electrochemical cell; the cell includes a graphite bar counter electrode (CE), a saturated calomel reference electrode (RE), and aqueous electrolyte. Increasingly oxidizing potentials are imposed on the WE by a potentiostat, and current between the WE and CE is measured at each applied potential. The resulting current–voltage (I–V) curve indicates effects of the coating on the metal's open circuit potential and passivity. The passivating effect of a chromate-containing primer paint on mild steel is demonstrated and compared to the passivity of 316 stainless steel.     

Autoimmune Epithelitis and Chronic Inflammation in Sjögren’s Syndrome-Related Dry Eye Disease

Autoimmune epithelitis and chronic inflammation are one of the characteristic features of the immune pathogenesis of Sjögren’s syndrome (SS)-related dry eye disease. Autoimmune epithelitis can cause the dysfunction of the excretion of tear fluid and mucin from the lacrimal glands and conjunctival epithelia and meibum from the meibomian glands. The lacrimal gland and conjunctival epithelia express major histocompatibility complex class II or human leukocyte antigen-DR and costimulatory molecules, acting as nonprofessional antigen-presenting cells for T cell and B cell activation in SS. Ocular surface epithelium dysfunction can lead to dry eye disease in SS. Considering the mechanisms underlying SS-related dry eye disease, this review highlights autoimmune epithelitis of the ocular surface, chronic inflammation, and several other molecules in the tear film, cornea, conjunctiva, lacrimal glands, and meibomian glands that represent potential targets in the treatment of SS-related dry eye disease.     

Ethanol-Modified Supercritical Carbon Dioxide Extraction of the Bioactive Lipid Components of <Emphasis Type="Italic">Camelina sativa</Emphasis> Seed

Camelina sativa seed is an underutilized oil source that attracts a growing interest, but it requires more research on its composition and processing. Its high omega‐3 content and growing demand for clean food processing technologies make conventional oil extraction less attractive. In this study, the effect of extraction methods on the bioactive lipid composition of the camelina seed lipid was investigated, and its bioactive lipid composition was modified at the extraction stage using ethanol‐modified supercritical carbon dioxide (SC‐CO₂). Ethanol‐modified SC‐CO₂ extractions were carried out at varying temperatures (50 and 70 °C), pressures (35 and 45 MPa), and ethanol concentrations (0–10%, w/w), and were compared to SC‐CO₂, cold press, and hexane extraction. The highest total lipid yield (37.6%) was at 45 MPa/70 °C/10% (w/w) ethanol. Phospholipids and phenolic content increased significantly with ethanol‐modified SC‐CO₂ (p < 0.05). SC‐CO₂ with 10% (w/w) ethanol concentration selectively increased phosphatidylcholine (PC) content. Apparent solubility of camelina seed lipids in SC‐CO₂, determined using the Chrastil model, ranged from 0.0065 kg oil/kg CO₂ (35 MPa/50 °C) to 0.0133 kg oil/kg CO₂ (45 MPa/70 °C). Ethanol‐modified SC‐CO₂ extraction allowed modification of the lipid composition that was not possible with the conventional extraction methods. This is a promising green method for extraction and fractionation of camelina seed lipids to separate and enrich its bioactives.     

Effects of dietary cholesterol and triglycerides on lipid concentrations in liver, plasma, and bile

Dietary cholesterol (CHL) and triglycerides (TG) can influence plasma, hepatic, and biliary lipid composition, but effects on lipids in these three compartments during the early stages of CHL gallstone formation have not been studied in parallel. We fed prairie dogs diets containing one of four tes oils (safflower, coconut, olive, or menhaden) at either 5 or 40% of calories, in the presence of 0 or 0.34% CHL, for 3 wk. In the absence of dietary CHL, increases in dietary TG produced 50–200% increases in the concentrations of biliary CHL and hepatic cholesteryl ester (CE), while the concentrations of hepatic free CHL (FC) as well as plasma FC and CE remained relatively unchanged. Increasing dietary CHL to 0.34% resulted in increases in hepatic FC of approximately 50% for all four fats regardless of whether they were supplied at 5 or 40% of calories. CHL supplementation caused more pronounced increases in biliary CHL (200–400%), hepatic CE (50–200%), plasma FC (up to 100%), and plasma CE (up to 150%), and these increases were exacerbated by concurrent supplementation of dietary fat and CHL (biliary CHL: 300–700%; hepatic CE: 100–250%; plasma FC: up to 165%; plasma CE: 100–350%). These results indicate that enhanced secretion of biliary CHL and, to a lesser extent, increased synthesis of hepatic CE, may be primary mechanisms for maintaining the hepatic FC pool. Furthermore, dietary CHL and high levels of fat intake are independent risk factors for increasing biliary CHL concentrations, and adverse effects on lipid concentrations in plasma and bile tend to be exacerbated by ingestion of diets rich in both fat and CHL.     

From coffee industry waste materials to skin‐friendly products with improved skin fat levels

Spent coffee grounds (SCG), which are the residue obtained from the treatment of coffee with hot water or steam, can be used for industrial applications, due to the high content in lipids. The cosmetic products might be a suitable application for these types of residues because the barrier properties of the stratum corneum (SC) are largely dependent on the intactness of the lipid lamellae that surrounds the corneocytes. The purpose of this work was to assess the feasibility of using the lipid fraction of SCG extracted with supercritical carbon dioxide in the development of new cosmetic formulations with improved skin lipids (sebum) and hydration. The use of spent coffee lipid extract in cosmetic industry seems to be a suitable approach to recycle the wastes from coffee industry. Emulsion containing 10% of the lipid fraction of SCG (SpentCofOil cream) presented promising characteristics in the improvement of sebum skin levels with a good acceptance by consumers when compared to an emulsion containing 10% w/w of green coffee oil (GreenCofOil cream) and a placebo without coffee oil (NoCofOil cream). Practical applications: In this work, the authors develop and characterize a cream containing 10% of the lipid fraction of SCG extracted with supercritical carbon dioxide with improved skin lipids (sebum) and hydration.     

Glycolipids improve lutein bioavailability and accumulation in eyes in mice

Intestinal carotenoid absorption is greatly affected by dietary factors. In this study, it was hypothesized that lipids with varying functional groups may influence differentially lutein bioavailability. Hence, the influence of glyco‐, phospho‐, neutral, crude (mixture of lipids) lipids, or mixed micelles (control) on the percent lutein micellarization in vitro and its postprandial plasma, liver, and eye response in mice were investigated. Results show that the percent micellarization of lutein with crude lipids and glycolipids were higher (91.4 and 45.7%) than control, while no significant difference was found between phospho‐ and neutral lipids. The mean plasma response of lutein was higher for crude‐ (6 times), glyco‐ (3 times), phospho‐ (2.7 times), and neutral (2 times) lipid than control (12.4 ± 1.18 nmol/mL 8 h^?1) group. Lutein levels (pmol/g) in liver were higher in crude (7.4 ± 1) and phospho‐ (3.6 ± 0.8) lipid groups while in eyes it was higher in glyco‐ (54.0) and neutral (21.2) lipid groups than control. The influential effect of glyco‐ and phospholipids may be due to smaller micellar size (glyco‐upto 3.43 µm, phospho‐ upto 5.78 µm) than the neutral lipids (upto 66 µm). Ingestion of lutein with glycolipid or phospholipids may improve lutein bioavailability. Practical applications: The findings of the present study will be useful in nutritional and biomedical applications for feeding lutein with specific lipid combinations to achieve enhanced lutein absorption. Specifically, feeding diet/emulsion with lutein and glyco‐ and phospholipid combination may reduce the risk of macular degeneration, owing to the influential effect of these lipids on intestinal absorption of lutein.     

Fatty Acid Profile of Sunshine Bass: II. Profile Change Differs Among Fillet Lipid Classes

Fatty acid (FA) profile of fish tissue mirrors dietary FA profile and changes in a time-dependent manner following a change in dietary FA composition. To determine whether FA profile change varies among lipid classes, we evaluated the FA composition of fillet cholesteryl esters (CE), phospholipids (PL), and triacylglycerols (TAG) of sunshine bass (SB, Morone chrysops x M. saxatilis) raised on feeds containing fish oil or 50:50 blend of fish oil and coconut, grapeseed, linseed, or poultry oil, with or without implementation of a finishing period (100% FO feed) prior to harvest. Each lipid class was associated with a generalized FA signature, irrespective of nutritional history: fillet PL was comprised largely of saturated FA (SFA), long-chain polyunsaturated FA (LC-PUFA), and total n-3 FA; fillet TAG was higher in MC-PUFA and total n-6 FA; and fillet CE was highest in monounsaturated FA (MUFA). Neutral lipids reflected dietary composition in a near-direct fashion; conversely, PL showed evidence of selectivity for MC- and LC-PUFA. Shorter-chain SFA were not strongly reflected within any lipid fraction, even when dietary availability was high, suggesting catabolism of these FA. FA metabolism in SB is apparently characterized by a division between saturated and unsaturated FA, whereby LC-PUFA are preferentially incorporated into tissues and SFA are preferentially oxidized for energy production. We demonstrated provision of SFA in grow-out feeds for SB, instead MC-PUFA which compete for tissue deposition, meets energy demands and allows for maximum inclusion of LC-PUFA within fillet lipids.