Comparative HPLC-MSn analysis of canine and human meibomian lipidomes: many similarities, a few differences

The aim of this study was to evaluate the lipidome of meibomian gland secretions in canines (cMGS) – a common pet and laboratory animal – and to compare it with that of human MGS (hMGS), to determine whether canines could be used as a valid experimental animal model in studies of the biochemistry and physiology of the human ocular surface in general, and of the Meibomian glands in particular. The MGS of both species were evaluated using HPLC in combination with atmospheric pressure chemical ionization ion trap mass spectrometry. The main lipid classes found in cMGS were very long chain cholesteryl esters, wax esters, (O-acyl)-omega-hydroxy fatty acids (OAHFA), and cholesteryl esters of OAHFA. The lipidomes of cMGS and hMGS were found to be qualitatively similar, which implies similar biosynthetic and biodegradation pathways in canines and humans. However, some quantitative differences between the two were observed

Evaluation and Quantitation of Intact Wax Esters of Human Meibum by Gas-Liquid Chromatography-Ion Trap Mass Spectrometry

PURPOSE. Wax esters (WE) of human meibum are one of the largest group of meibomian lipids. Their complete characterization on the level of individual intact lipid species has not been completed yet. We obtained detailed structural information on previously uncharacterized meibomian WE. METHODS. Intact WE were separated and analyzed by means of high-temperature capillary gas-liquid chromatography (GLC) in combination with low voltage (30 eV) electron ionization ion trap mass spectrometry (ITMS). 3D (mass-to-charge ratio [m/z] versus lipid sample weight versus signal intensity) calibration plots were used for quantitation of WE. RESULTS. We demonstrated that GLC-ITMS was suitable for analyzing unpooled/underivatized WE collected from 14 individual donors. More than 100 of saturated and unsaturated WE (SWE and UWE, respectively) were detected. On average, UWE represented about 82% of the total WE pool. About 90% of UWE were based on oleic acid, while less than 10% were based on palmitoleic acid. The amounts of poly-UWE were <3% of their mono-UWA counterparts. SWE were based primarily on C 16 -C 18 fatty acids (FA) in overall molar ratios of 22:65:13. A pool of C 16:0 -FA was comprised of a 20:80 (mol/ mol) mixture of straight chain and iso-branched isomers, while the corresponding ratio for C 18:0 -FA was 43:57. Interestingly, C 17:0 -FA was almost exclusively branched, with anteiso-and isoisomers found in a ratio of 93:7. CONCLUSIONS. GLC-ITMS can be used successfully to analyze more than 100 individual species of meibomian WE, which were shown to comprise 41 6 8% (wt/wt) of meibum, which made them the largest group of lipids in meibum. (Invest Ophthalmol Vis Sci. 2012;53:3766-3781 1 This secretion has a critical role in protecting the surface of the human eye from desiccation by mixing with aqueous tears (produced by lacrimal glands) and forming the outermost part of the tear film layer, the tear film lipid layer. This lipid-rich layer ''seals'' the underlaying aqueous layer of the tear film, keeping the ocular surface moist, which is critical for its health and good vision. 2,3 Over a period of more than four decades, meibomian WE have been evaluated in a number of studies of animals and humans (for comprehensive reviews of earlier findings, see the most recent reviews on the topic 4-7 ). However, for the purpose of our study, only information related directly to human meibum will be discussed. Earlier, human meibomian WE from normal (nondry eye) and dry eye subjects were studied using gas (or gasliquid) chromatography with flame ionization detection (GC-FID and GLC-FID), and GC-and GLC-mass spectrometry (GCand GLC-MS). MATERIALS AND METHODS Samples of meibum were collected using a protocol described previously 13 and the amounts of collected meibum were measured gravimetrically. All sample collection procedures that involved human subjects were approved by the UT Southwestern Institutional Review Board, and were conducted in accordance with the Declaration of From th

Adsorption of Human Tear Lipocalin to Human Meibomian Lipid Films

PURPOSE. Tear lipocalin (Tlc) is a major lipid binding protein in tears and is thought to have an important role in stabilizing the Meibomian lipid layer by transferring lipids to it from the aqueous layer or ocular surface, or by adsorbing to it directly. These possible roles have been investigated in vitro using human Tlc. METHODS. Tlc was purified from human tears by size exclusion chromatography followed by ion exchange chromatography. Three additional samples of the Tlc were prepared by lipidation, delipidation, and relipidation. The lipids extracted from the purified Tlc were analyzed by HPLC-MS followed by fragmentation. Adsorption of these different forms of Tlc to a human Meibomian lipid film spread on the surface of an artificial tear buffer in a Langmuir trough were observed by recording changes in the pressure with time (⌸-T profile) and monitoring the appearance of the film microscopically. These results were compared with similar experiments using a bovine Meibomian lipid film. RESULTS. The results indicated that Tlc binds slowly to a human Meibomian lipid film compared with lysozyme or lactoferrin, even at 37°C. The adsorption of Tlc to a human Meibomian lipid film was very different from its adsorption to a bovine Meibomian lipid film, indicating the nature of the lipids in the film is critical to the adsorption process. Similarly, the different forms of Tlc had quite distinct adsorption patterns, as indicated both by changes in ⌸-T profiles and the microscopic appearance of the films. CONCLUSIONS. It was concluded that human Tlc was capable of adsorbing to and penetrating into a Meibomian lipid layer, but this process is very complex and depends on both the types of lipids bound to Tlc and the lipid complement comprising the Meibomian lipid film. (Invest Ophthalmol Vis Sci. 2009;50: 140 -151

On the lipid composition of human meibum and tears: comparative analysis of nonpolar lipids,”

PURPOSE. To qualitatively compare the nonpolar lipids present in meibomian gland (MG) secretions (samples T1) with aqueous tears (AT) collected from the lower tear menisci of healthy, non-dry eye volunteers using either glass microcapillaries (samples T2) or Schirmer test strips (samples T3). METHODS. Samples T1 to T3 were analyzed with the use of high-pressure liquid chromatography/positive ion mode atmospheric pressure chemical ionization mass spectrometry. Where possible, the unknown lipids were compared with known standards. RESULTS. Samples T1 had the simplest lipid composition among all the tested specimens. Samples T2 and T3 were similar to each other but were noticeably different from samples T1. In addition to all the compounds detected in samples T1, lower molecular weight wax esters and other compounds were found in samples T2 and T3. No appreciable amounts of fatty acid amides (e.g., oleamide), ceramides, or monoacyl glycerols were routinely detected. The occasionally observed minor signals of oleamide (m/z 282) in samples T3 were attributed to the contamination of the samples with common plasticizers routinely found in plastic ware extractives and organic solvents. CONCLUSIONS. The MG is a prominent source of lipids for the tear film. However, it would have been a mistake to exclude from consideration other likely sources of lipids such as conjunctiva, cornea, and tears produced by the lacrimal glands. These data showed that lipids in AT are more complex than MG secretions, which necessitates more cautious interpretation of the functions of the latter in the tear film. (Invest Ophthalmol Vis Sci. 2008;49:3779 -3789) DOI:10.1167/ iovs.08-1889 H uman tears are an extremely complex mixture of lipids, carbohydrates, proteins, peptides, salts, and other lowand high-molecular-weight compounds. Watery secretions known as aqueous tears (AT) are produced by lacrimal glands and are secreted onto the ocular surface through lacrimal ducts to form the bulk of the tear film (TF). The meibomian glands (MG) located on the margins of eyelids produce oily lipid secretions (MGs) that, after being mixed with the AT, also contribute to the TF. MGs are believed to form what is known as the tear film lipid layer (TFLL). 1-3 The TFLL lies on top of the TF in immediate contact with the surrounding air. The MGs have been proposed to be critical for maintaining the structural integrity of the TFLL 3 and its ability to protect the ocular surface from losing water because of evaporation of the TF. 5-7 DE affects millions of people, ranging-depending on the severity of condition-from 0.5% of the general population 8 to more than 30%, 9 ; most are elderly, women, and those who live in adverse climates. Therefore, finding correlations between the lipid composition of the MGs and the development of DE has been the focus of attention of DE researchers for decades. Surprisingly, comprehensive lipidomic analyses of the AT and the TFLL have not been completed, and no direct experiments have been conducted to verify the proposed structures of the TF and TFLL. Considering the very small amounts of material that can be collected from an individual human eye without harming the donor (a few microliters of the AT 10 and 1 mg or less of the MGs 11,12 ), the diversity of lipid species found in humans, 11,12 To our surprise, the overall lipid composition human normal MGs in humans was different from the one reported in earlier publications. MATERIALS AND METHODS Materials and Reagents Lipid standards were purchased from Nu-Chek Prep, Inc. (Elysian, MN) and Avanti Polar Lipids (Alabaster, AL). Other reagents were from Sigma-Aldrich (St. Louis, MO). HPLC-grade solvents were products of Burdick & Jackson (Muskegon, MI). Mass spectra were captured on an ion trap spectrometer (LCQ Deca XP Max MS n ; Thermo Fisher Scientific, Waltham, MA) using data system software (Xcalibur; Thermo Fisher Scientific). Chromatographic experiments were performed on a Waters HPLC system (Alliance 2695 HPLC Separations Module; Waters Corp., Milford, MA) interfaced to the mass spectrometer. Melting ranges of lipids were determined with a melting point apparatus (Optimelt MPA100; Stanford Research Systems, Sunnyvale, CA). Firepolished glass microcapillaries, manufactured by Baxter Healthcare Corp. (Deerfield, IL) and standardized Schirmer tear test strips (Alcon Laboratories, Inc., Fort Worth, TX) were used to collect tears

Dynamic Changes in the Gene Expression Patterns and Lipid Profiles in the Developing and Maturing Meibomian Glands

Meibomian glands (MGs) and their holocrine secretion—meibum—play crucial roles in the physiology of the eye, providing protection from environmental factors and desiccation, among other functions. Importantly, aging was implicated in the deterioration of the morphology and functions of MGs, and the quantity and quality of meibum they produce, leading to a loss of its protective properties, while the meibum of young individuals and experimental animals provide ample protection to the eye. Currently, the molecular mechanisms of meibum biosynthesis (termed meibogenesis) are not fully understood. To characterize the physiological changes in developing and maturing MGs, we studied the lipidomes and transcriptomes of mouse MGs ranging from newborns to adults. The results revealed a gradual increase in the critical genes of meibogenesis (such as Elovl3, Elovl4, Awat2, and Soat1, among others) that positively correlated with the biosynthesis of their respective lipid products. The MG transcriptomes of young and adult mice were also analyzed using single-cell RNA sequencing. These experiments revealed the existence of multiple unique populations of MG cells (meibocytes, epithelial cells, and others) with specific combinations of genes that encode meibogenesis-related proteins, and identified clusters and subclusters of cells that were tentatively classified as meibocytes at different stages of differentiation/maturation, or their progenitor cells. A hypothesis was formulated that these cells may produce different types of lipids, and contribute differentially to the Meibomian lipidome