Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry.
Saville Jennifer T,Zhao Zhenjun,Willcox Mark D P,Ariyavidana Manjula A,Blanksby Stephen J,Mitchell Todd W
Experimental eye research
Meibum is believed to be the major source of tear film lipids, which are vital in the prevention of excess evaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established. While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometric studies have not detected them. In this study we use electrospray ionisation tandem mass spectrometry to investigate the presence of phospholipids in meibum and provide comparison to the phospholipid profile of tears. Lipids were extracted from human meibum and tear samples using standard biphasic methods and analysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. A total of 35 choline-containing phospholipids were identified in meibum and the profile of these was similar to that observed in tears, suggesting tear lipids are derived from meibum. The results shown here highlight the need for a combination of optimised techniques to enable the identification of the large range of lipid classes in meibum.
10.1016/j.exer.2010.12.012
A simple and reproducible method for quantification of human tear lipids with ultrahigh-performance liquid chromatography-mass spectrometry.
Acera Arantxa,Pereiro Xandra,Abad-Garcia Beatriz,Rueda Yuri,Ruzafa Noelia,Santiago Carlos,Barbolla Iratxe,Duran Juan A,Ochoa Begoña,Vecino Elena
Molecular vision
Purpose:The purpose was to select a simple and reproducible method for lipid measurements of human tears with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). Two sample preparation procedures were evaluated and compared: the Bligh and Dyer (BD) liquid-liquid extraction method with chloroform and methanol and protein precipitation with isopropanol (IPA). Methods:Reproducibility and recovery efficiencies of 20 non-endogenous internal lipid standards were tested in 10-µl tear samples from healthy subjects. The lipid coverage and the simplicity of execution were also assessed. Lipid profiles of the tear extracts were acquired with UHPLC-MS, uhpland the lipids were identified using SimLipid software. Results:Both methods were robust producing good lipid coverage and reproducibility and high recovery efficiencies. The two protocols identified a 69-feature tear lipidome that covered 11 lipid classes from six different lipid categories. The main differences in recovery were due to the intrinsic lipid selectivity of each solvent. Although both methods were similarly efficient in recovering -acyl-ω-hydroxy fatty acid (OAHFAs) and non-polar lipids, polar lipids were more efficiently recovered with IPA precipitation, which, in turn, exhibited higher reproducibility. In addition, IPA precipitation is automatable and simpler than the BD approach. Conclusions:IPA precipitation is an excellent procedure for extracting lipids from small tear volumes for quantitative large-scale, untargeted lipid profiling, which may be useful for identifying lipid biomarkers in tears from patients with different ocular surface pathologies, allowing personalized therapies to be designed.
Comparison of tear lipid profile among basal, reflex, and flush tear samples.
Rohit Athira,Stapleton Fiona,Brown Simon H J,Mitchell Todd W,Willcox Mark D P
Optometry and vision science : official publication of the American Academy of Optometry
PURPOSE:To determine whether tear collection by flushing the ocular surface with saline (flush tears) or collection by stimulation (reflex tears) can be used as an alternative to basal tear collection for the identification and quantification of lipids in the tear film. METHODS:Tear samples were collected from 10 participants with no history of ocular surface disease or contact lens wear. Up to 10 μl of basal, reflex, and flush tear samples were collected from each eye using a microcapillary tube on three occasions with the order of methods randomized and allowing at least 24 hours between each collection method. Lipids were quantified from each tear sample using nano-electrospray ionization tandem mass spectrometry. RESULTS:Total lipids significantly differed in their concentration (pmol/μl) and mole % with each collection technique. Cholesterol esters [mean % (SE)] formed the major component of the total lipidome in basal [54.8% (3.1)], reflex [35.7% (6.4)], and flush [33.0% (3.1)] tear samples. However, the mole % of each lipid class substantially varied with each tear collection method. Nonpolar lipids, including cholesterol, wax esters, and triacylglycerols, dominated the tear lipidome in basal [92.8% (1.9)], reflex [71.8% (7.9)], and flush [83.6% (3.8)] tear samples. However, the mole % of phospholipids in reflex [27.5% (8.1)] and flush [15.8% (3.8)] tear samples was higher (p = 0.005) than that in basal tears [5.4% (2.0)]. CONCLUSIONS:Flush or reflex tears did not have similar lipid profiles in either concentration or in mole % to basal tears. It is recommended that basal tears are used for tear lipid analysis as the reflex or flush tears contain very low levels of most lipid components.
10.1097/OPX.0000000000000411
Clinical and biochemical tear lipid parameters in contact lens wearers.
Rohit Athira,Willcox Mark D P,Brown Simon H J,Mitchell Todd W,Stapleton Fiona
Optometry and vision science : official publication of the American Academy of Optometry
PURPOSE:Alterations in tear film lipids may be important in modulating discomfort during contact lens wear. This study investigates associations between clinical and biological components of the lipid layer and seeks to determine the effect of lipid supplementation on contact lens wear comfort. METHODS:Participants were grouped into symptomatic (n = 10) and asymptomatic (n = 10) contact lens wearers according to the Contact Lens Dry Eye Questionnaire. After 6 hours of lens wear, noninvasive surface drying time (NISDT) and the lipid layer grade were assessed using a Tearscope. Basal tears were collected using a microcapillary tube and assayed for concentration and activity of the secretory phospholipase A2 enzyme and concentration of the lipid aldehyde malondialdehyde. Mass spectrometry was used to characterize the tear lipidome. In the second phase, a liposomal spray (Tears Again, BioRevive) or a saline spray was sprayed over the upper eyelids of each subject during their down gaze and during lens wear. Noninvasive surface drying time and ocular comfort were obtained soon after spraying and again at 2 and 6 hours after the initial spray. Statistical tests included the Student t test, repeated-measures analysis of variance, and the Pearson correlation test where appropriate. RESULTS:Noninvasive surface drying time was lower (p = 0.01) in symptomatic (4.5 ± 0.6 seconds) than in asymptomatic (9.9 ± 3.1 seconds) contact lens wearers. The mole percentage of wax esters in the total lipidome increased with NISDT (R = 0.70, p = 0.01). Secretory phospholipase A2 enzyme activity in tears was associated with higher levels of malondialdehyde (R = 0.65, p = 0.01) and shorter NISDT (R = 0.84, p = 0.001). Noninvasive surface drying time reduced over the time course for the saline spray (p = 0.01) but did not reduce until the 6-hour time point with the liposomal spray. With liposomal spray, NISDT was higher (p = 0.03) immediately after instillation compared with 6 hours later (9.5 ± 1.9 vs. 5.2 ± 2.1 seconds). A longer NISDT was associated with improved ocular comfort for those using the liposomal spray (R = 0.25, p = 0.005) but not with saline. CONCLUSIONS:Degraded lipids and a lower mole percentage of wax esters in the tear film may be associated with a lower NISDT. Lipid supplements may improve ocular comfort during lens wear by increasing NISDT.
10.1097/OPX.0000000000000420
Rapid identification of fatty acids and (O-acyl)-ω-hydroxy fatty acids in human meibum by liquid chromatography/high-resolution mass spectrometry.
Mori Naoto,Fukano Yasufumi,Arita Reiko,Shirakawa Rika,Kawazu Kouichi,Nakamura Masatsugu,Amano Shiro
Journal of chromatography. A
We report a rapid liquid chromatography/quadrupole Orbitrap Fourier transform mass spectrometry (LC-FTMS) method for identifying free fatty acids (FAs) and (O-acyl)-ω-hydroxyFAs (OAHFAs) in human meibum without derivatization. Meibum is a lipid-rich secretion and an important component of the tear film lipid layer. FAs are commonly detected by gas chromatography (GC) or GC/MS after methyl ester derivatization. We developed high-throughput lipid profiling using LC-FTMS and lipid identification software, Lipid Search, without derivatization and applied the method to human meibum. Chromatographic separation was performed on a C18 column. We selected negative electrospray ionization [M-H](-), and applied high-resolution full scan mode to FA analysis and data-dependent MS(2) mode to OAHFA analysis. High-resolution Orbitrap MS proved to be an excellent tool for the rapid analysis of lipids from meibum, and 100 FA and 61 OAHFA molecular species were detected. The analysis times were 12 and 16.5min, respectively. Very long chain FAs (up to C37) and OAHFAs (up to C56) were also detected. The results clearly showed that retention time correlates with the number of double bonds and carbon chains. This LC/MS method can be applied to the identification of FAs and OAHFAs in human meibum.
10.1016/j.chroma.2014.04.082
Integrated Lipidomics and Metabolomics Analysis of Tears in Multiple Sclerosis: An Insight into Diagnostic Potential of Lacrimal Fluid.
Cicalini Ilaria,Rossi Claudia,Pieragostino Damiana,Agnifili Luca,Mastropasqua Leonardo,di Ioia Maria,De Luca Giovanna,Onofrj Marco,Federici Luca,Del Boccio Piero
International journal of molecular sciences
Metabolomics based on mass spectrometry represents an innovative approach to characterize multifactorial diseases, such as multiple sclerosis (MuS). To date, the most important biomarker source for MuS diagnosis is the cerebrospinal fluid. However, an important goal for research is to identify new molecules in more easily accessible biological fluids. A very interesting biofluid in MuS is represented by tears, considered as an intermediate fluid between the cerebrospinal fluid and serum. In this work, we developed a merged strategy for the analysis of lipids containing choline by Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS), as well as for the targeted analysis of free carnitine, acylcarnitines and aminoacids by direct infusion mass spectrometry. Samples for both metabolomics and lipidomics approaches were obtained in a single extraction procedure from tears of patients affected by MuS and healthy controls. Tear lipidomics showed 30 phospholipids significantly modulated and, notably, many sphingomyelins resulted lower in MuS. Moreover, the metabolomics approach carried out both on tears and serum highlighted the diagnostic potential of specific aminoacids and acylcarnitines. In conclusion, the metabolic profiling of tears appears to reflect the pathological conditions of the central nervous system, suggesting that the molecular repository of tears can be considered as a source of potential biomarkers for MuS.
10.3390/ijms20061265
Detection of Lipid Mediators of Inflammation in the Human Tear Film.
Panthi Shyam,Chen Jianzhong,Wilson Landon,Nichols Jason J
Eye & contact lens
PURPOSE:Lipid mediators of inflammation are a group of signaling molecules produced by various cells under physiological conditions and modulate the inflammatory process during various pathologic conditions. Although eicosanoids and F2-isoprostanes are recognized lipid mediators of inflammation, there is no consensus yet on the extraction and mass spectrometry (MS) method for their analysis in individual human tear samples. Thus, the aim of this study was to develop an optimal method for extraction of lipid mediators of inflammation in the tear film and evaluate MS techniques for their analysis. METHODS:Basal tears were collected from each eye of 19 subjects using glass microcapillaries. Lipid extraction was performed using either varying concentrations of acidified methanol, a modified Folch method, or solid-phase extraction. Initially, an untargeted analysis of the extracts was performed using SCIEX TripleTOF 5600 mass spectrometer to identify any lipid mediators of inflammation (eicosanoids) and later a targeted analysis was performed using the SCIEX 6500 Qtrap to identify and quantify prostaglandins and isoprostanes. Mass spectra and chromatograms were analyzed using Peakview, XCMS, and Multiquant software. RESULTS:Prostaglandins and isoprostanes were observed and quantified using the Qtrap mass spectrometer under multiple reaction monitoring (MRM) mode after solid-phase extraction. Extraction with acidified methanol along with the Folch method produced cleaner spectra during MS with the Triple time of flight (TOF) mass spectrometer. Lipid mediators of inflammation were not observed in any of the tear samples using the Triple TOF mass spectrometer. CONCLUSIONS:Solid-phase extraction may be the method of choice for extraction of prostaglandins and isoprostanes in low volumes of tears. The SCIEX Qtrap 6500 in MRM mode may be suitable to identify and quantify similar lipid mediators of inflammation.
10.1097/ICL.0000000000000551
Integrated Tear Proteome and Metabolome Reveal Panels of Inflammatory-Related Molecules via Key Regulatory Pathways in Dry Eye Syndrome.
Chen Xueli,Rao Jun,Zheng Zhi,Yu Yan,Lou Shang,Liu Liping,He Qinsi,Wu Luhua,Sun Xinghuai
Journal of proteome research
Dry eye syndrome (DES) is a growing public health concern with a high global prevalence; however, the fundamental processes involved in its pathogenic mechanisms remain poorly understood. In the present study, we applied nanoscale liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (nanoLC/Q-TOF-MS/MS) and ultraperformance LC/Q-TOF-MS/MS technologies on tear samples obtained from 18 dry eye patients and 19 healthy controls for integrated proteomic and metabolomic analyses. Overall, 1031 tear proteins were detected, while 190 proteins were determined to be significantly expressed in dry eye patients. Further functional analysis suggested that various biological processes were highly expressed and involved in the pathogenesis of DES, especially immune and inflammatory processes. In total, 156 named metabolites were identified, among which 34 were found to be significantly changed in dry eye patients. The results highlighted the key elements, especially inflammatory-related proteins and metabolites that played important roles in the development of DES. Further, the regulatory roles of primary pathways, including complement and coagulation cascades, glycolysis/gluconeogenesis, and amino acid metabolism, were also identified as processes involved in DES. Collectively, our work not only provided insight into the potential biomarkers of DES for diagnostic and prognostic purposes but extended our knowledge of the physiopathology of this syndrome.
10.1021/acs.jproteome.9b00149
Cyclic Change of Fatty Acid Composition in Meibum During the Menstrual Cycle.
Suzuki Tomo,Fujiwara Satoshi,Kinoshita Shigeru,Butovich Igor A
Investigative ophthalmology & visual science
Purpose:To evaluate the fatty acid (FA) composition in the meibum of pre- and postmenopausal women and age-matched men. Methods:This prospective study involved 24 healthy subjects; six premenopausal women in their 30s with a regular menstrual cycle (young-female [yF] group), six postmenopausal women in their 60s (elderly-female [eF] group), and 12 age-matched men (i.e., young-male [yM] and elderly-male [eM] groups, respectively). The menstrual cycle was divided into six phases (phase I-VI). Meibum was obtained from the meibomian gland orifices via a Daviel spoon, and its FA composition was then analyzed via gas chromatography mass spectrometry (GC-MS). Principal component analysis (PCA) was performed on the GC-MS results. Results:The mean FA composition of all subjects was 40% saturated FAs (SFA) and 60% unsaturated FAs (UFAs). The PCA results of all groups indicated two categories (PC1 [77.5%] and PC2 [12.4%]); one consisting of yF-group samples of mainly phase II and III and the other consisting of the yF-group samples of the rest of the cycle, as well as from eF-group, yM-group, and eM-group samples. Each group had a distinctive nature. The FAs that most contributed to PC1 were C14:0, C16:0, and C18:0 in a positive correlation, and C18:1n9 in a negative correlation. Conclusions:FA composition noticeably changes during the menstrual cycle and is somewhat affected by sex and age. The ratio of SFAs (C16:0, C18:0) to mono-UFAs (C18:1n9) in the FA composition might have an impact on the lipid quality of meibum, thus suggesting alteration of its melting temperature and viscosity.
10.1167/iovs.18-26390
Tear metabolite changes in keratoconus.
Karamichos D,Zieske J D,Sejersen H,Sarker-Nag A,Asara John M,Hjortdal J
Experimental eye research
While efforts have been made over the years, the exact cause of keratoconus (KC) remains unknown. The aim of this study was to identify alterations in endogenous metabolites in the tears of KC patients compared with age-matched healthy subjects. Three groups were tested: 1) Age-matched controls with no eye disease (N = 15), 2) KC - patients wearing Rigid Gas permeable lenses (N = 16), and 3) KC - No Correction (N = 14). All samples were processed for metabolomics analysis using LC-MS/MS. We identified a total of 296 different metabolites of which >40 were significantly regulated between groups. Glycolysis and gluconeogenesis had significant changes, such as 3-phosphoglycerate and 1,3 diphosphateglycerate. As a result the citric acid cycle (TCA) was also affected with notable changes in Isocitrate, aconitate, malate, and acetylphosphate, up regulated in Group 2 and/or 3. Urea cycle was also affected, especially in Group 3 where ornithine and aspartate were up-regulated by at least 3 fold. The oxidation state was also severely affected. Groups 2 and 3 were under severe oxidative stress causing multiple metabolites to be regulated when compared to Group 1. Group 2 and 3, both showed significant down regulation in GSH-to-GSSG ratio when compared to Group 1. Another indicator of oxidative stress, the ratio of lactate - pyruvate was also affected with Groups 2 and 3 showing at least a 2-fold up regulation. Overall, our data indicate that levels of metabolites related to urea cycle, TCA cycle and oxidative stress are highly altered in KC patients.
10.1016/j.exer.2015.01.007
Characterization of human reflex tear proteome reveals high expression of lacrimal proline-rich protein 4 (PRR4).
Perumal Natarajan,Funke Sebastian,Wolters Dominik,Pfeiffer Norbert,Grus Franz H
Proteomics
In-depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC-ESI-MS/MS strategy for label-free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC-ESI-MS/MS and targeted-MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline-rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland-specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha-1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface.
10.1002/pmic.201400239
ω-3 Tear Film Lipids Correlate With Clinical Measures of Dry Eye.
Walter Scott D,Gronert Karsten,McClellan Allison L,Levitt Roy C,Sarantopoulos Konstantinos D,Galor Anat
Investigative ophthalmology & visual science
PURPOSE:ω-3 and ω-6 polyunsaturated fatty acids modulate inflammatory processes throughout the body through distinct classes of lipid mediators that possess both proinflammatory and proresolving properties. The purpose of this cross-sectional study was to explore the relationship between lipid profiles in human tears and dry eye (DE) symptoms and signs. METHODS:Forty-one patients with normal eyelid and corneal anatomy were prospectively recruited from a Veterans Administration Hospital over 18 months. Symptoms and signs of DE were assessed, and tear samples was analyzed by mass spectrometry-based lipidomics. Statistical analyses comparing the relationship between tear film lipids and DE included Pearson/Spearman correlations and t-tests. RESULTS:Arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were present in more than 90% of tear film samples. The ratio of ω-6 (AA) to ω-3 (DHA+EPA) fatty acids was correlated with multiple measures of tear film dysfunction (tear breakup time, Schirmer 2 scores, and corneal staining; all P < 0.05). Arachidonic acid-derived prostaglandin E2 was detected in the majority of samples and correlated with low tear osmolarity, meibomian gland plugging, and corneal staining. CONCLUSIONS:Both ω-3 and ω-6 lipid circuits are activated in the human tear film. The ratio of ω-6:ω-3 tear lipids is elevated in DE patients in proportion to the degree of tear film dysfunction and corneal staining. Metabolic deficiency of ω-3 tear film lipids may be a driver of chronic ocular surface inflammation in DE.
10.1167/iovs.16-19131
Identification and Profiling of Specialized Pro-Resolving Mediators in Human Tears by Lipid Mediator Metabolomics.
English Justin T,Norris Paul C,Hodges Robin R,Dartt Darlene A,Serhan Charles N
Prostaglandins, leukotrienes, and essential fatty acids
Specialized pro-resolving mediators (SPM), e.g. Resolvin D1, Protectin D1, Lipoxin A₄, and Resolvin E1 have each shown to be active in ocular models reducing inflammation. In general, SPMs have specific agonist functions that stimulate resolution of infection and inflammation in animal disease models. The presence and quantity of SPM in human emotional tears is of interest. Here, utilizing a targeted LC-MS-MS metabololipidomics based approach we document the identification of pro-inflammatory (Prostaglandins and Leukotriene B₄) and pro-resolving lipid mediators (D-series Resolvins, Protectin D1, and Lipoxin A₄) in human emotional tears from 12 healthy individuals. SPMs from the Maresin family (Maresin 1 and Maresin 2) were not present in these samples. Principal Component Analysis (PCA) revealed gender differences in the production of specific mediators within these tear samples as the SPMs were essentially absent in these female donors. These results indicate that specific SPM signatures are present in human emotional tears at concentrations known to be bioactive. Moreover, they will help to further appreciate the mechanisms of production and action of SPMs in the eye, as well as their physiologic roles in human ocular disease resolution.
10.1016/j.plefa.2017.01.004
Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry.
Pieragostino Damiana,Agnifili Luca,Cicalini Ilaria,Calienno Roberta,Zucchelli Mirco,Mastropasqua Leonardo,Sacchetta Paolo,Del Boccio Piero,Rossi Claudia
International journal of molecular sciences
Dry eye disease (DED) is a multifactorial disorder of the ocular surface unit resulting in eye discomfort, visual disturbance, and ocular surface damage; the risk of DED increases with age in both sexes, while its incidence is higher among females caused by an overall hormonal imbalance. The role of androgens has recently investigated and these hormones were considered to have a protective function on the ocular surface. In order to correlate DED to tear steroid levels, a robust, specific, and selective method for the simultaneous quantification of cortisol (CORT), corticosterone (CCONE), 11-deoxycortisol (11-DECOL), 4-androstene-3,17-dione (ADIONE), testosterone (TESTO), 17α-hydroxyprogesterone (17-OHP), and progesterone (PROG) was developed and applied for the analysis of tear samples. The method involves a simple extraction procedure of steroids from tears collected on Schirmer strips, followed by a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. In total, tear samples from 14 DED female patients and 13 healthy female controls were analysed and, CORT, ADIONE, and 17-OHP response levels resulted significantly decreased in dry eye patients respect to controls. The receiver operating characteristic (ROC) curve obtained by the combination of these three steroids (AUC = 0.964) demonstrated the good diagnostic power of the differential tear steroids in identifying DED. In conclusion, the present method made it possible, for the first time, to study steroid profiling directly in tear fluid.
10.3390/ijms18071349
Compositional Analysis of Wax Esters in Human Meibomian Gland Secretions by Direct Infusion Electrospray Ionization Mass Spectrometry.
Chen Jianzhong,Green Kari B,Nichols Kelly K
Lipids
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.
10.1007/s11745-016-4183-4
Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.
Lam Sin Man,Tong Louis,Duan Xinrui,Petznick Andrea,Wenk Markus R,Shui Guanghou
Journal of lipid research
The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-ω-hydroxyfatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model.
10.1194/jlr.M044826
Comprehensive shotgun lipidomics of human meibomian gland secretions using MS/MS with successive switching between acquisition polarity modes.
Journal of lipid research
The lipid composition of human meibomian gland secretions (meibum) has been analyzed using both targeted and untargeted mass spectrometric approaches, each of which has its advantages and disadvantages. Herein we report the results of shotgun lipidomic profiling of human meibum using a new approach that combines the advantages of targeted and untargeted analyses to yield highly sensitive and comprehensive profiles. Samples containing an estimated 7-13 µg (8-16 nL) of human meibum lipids were analyzed using MS/MS, an untargeted approach for MS/MS. Using MS/MS with ESI and successive polarity switching, we obtained tandem mass spectra in both modes at every 1 Da step for all ions in the / 200-1,200 range. In approximately 12 min, a total of 2 MS spectra and 2,000 MS/MS spectra were acquired for each sample, from which targeted analysis information was extracted. This approach allowed for the comprehensive and highly sensitive detection of meibum lipids, including species low in abundance. Altogether, more than 600 unique lipid molecular species were identified in meibum, 3 times more than previously reported in untargeted analyses of meibum samples. This untargeted MS and MS/MS approach may be extended to other biological systems for the detection of lipids with sensitivity comparable to targeted analysis.
10.1194/jlr.D088138
Association between very long chain fatty acids in the meibomian gland and dry eye resulting from n-3 fatty acid deficiency.
Tanaka Hideko,Harauma Akiko,Takimoto Mao,Moriguchi Toru
Prostaglandins, leukotrienes, and essential fatty acids
In our previously study, we reported lower tear volume in with an n-3 fatty acid deficient mice and that the docosahexaenoic acid and total n-3 fatty acid levels in these mice are significantly reduced in the meibomian gland, which secretes an oily tear product. Furthermore, we noted very long chain fatty acids (≥25 carbons) in the meibomian gland. To verify the detailed mechanism of the low tear volume in the n-3 fatty acid-deficient mice, we identified the very long chain fatty acids in the meibomian gland, measured the fatty acid composition in the tear product. Very long chain fatty acids were found to exist as monoesters. In particular, very long chain fatty acids with 25-29 carbons existed for the most part as iso or anteiso branched-chain fatty acids. n-3 fatty acid deficiency was decreased the amount of meibum secretion from meibomian gland without change of fatty acid composition. These results suggest that the n-3 fatty acid deficiency causes the enhancement of evaporation of tear film by reducing oily tear secretion along with the decrease of meibomian gland function.
10.1016/j.plefa.2015.02.004
Expression Profiling of Nonpolar Lipids in Meibum From Patients With Dry Eye: A Pilot Study.
Chen Jianzhong,Keirsey Jeremy K,Green Kari B,Nichols Kelly K
Investigative ophthalmology & visual science
Purpose:The purpose of this investigation was to characterize differentially expressed lipids in meibum samples from patients with dry eye disease (DED) in order to better understand the underlying pathologic mechanisms. Methods:Meibum samples were collected from postmenopausal women with DED (PW-DED; n = 5) and a control group of postmenopausal women without DED (n = 4). Lipid profiles were analyzed by direct infusion full-scan electrospray ionization mass spectrometry (ESI-MS). An initial analysis of 145 representative peaks from four classes of lipids in PW-DED samples revealed that additional manual corrections for peak overlap and isotopes only slightly affected the statistical analysis. Therefore, analysis of uncorrected data, which can be applied to a greater number of peaks, was used to compare more than 500 lipid peaks common to PW-DED and control samples. Statistical analysis of peak intensities identified several lipid species that differed significantly between the two groups. Data from contact lens wearers with DED (CL-DED; n = 5) were also analyzed. Results:Many species of the two types of diesters (DE) and very long chain wax esters (WE) were decreased by ∼20% in PW-DED, whereas levels of triacylglycerols were increased by an average of 39% ± 3% in meibum from PW-DED compared to that in the control group. Approximately the same reduction (20%) of similar DE and WE was observed for CL-DED. Conclusions:Statistical analysis of peak intensities from direct infusion ESI-MS results identified differentially expressed lipids in meibum from dry eye patients. Further studies are warranted to support these findings.
10.1167/iovs.16-20902
Tear eicosanoids in healthy people and ocular surface disease.
Ambaw Yohannes Abere,Chao Cecilia,Ji Shanshan,Raida Manfred,Torta Federico,Wenk Markus R,Tong Louis
Scientific reports
Meibomian gland (MG) dysfunction is the leading cause of evaporative dry eye and it leads to inflammation of the ocular surface. Eicosanoids may be involved in inflammation of dry eye. This study aimed to profile tear eicosanoid levels in healthy individuals and those with MG dysfunction, and to examine if these levels are associated with clinical factors and expressibility of MG. Forty participants with MG dysfunction and 30 healthy controls were recruited in this study. Clinical signs of MG dysfunction were assessed, and tear lactoferrin concentration was evaluated. Tear eicosanoids were extracted from Schirmer's strips and analyzed using mass spectrometry. We were able to quantify 38 tear eicosanoids and levels were increased in older individuals. In participants with MG dysfunction, higher 5-HETE, LTB, 18-HEPE, 12-HEPE and 14-HDoHE were associated with poorer MG expressibility. The eicosanoids PGF, 18-HEPE, 20-HDoHE and 17-HDoHE were elevated with increased corneal staining; higher 5-HETE, LTB were associated with lower tear lactoferrin levels. The receiver-operating-characteristics analysis shows higher levels of 5-HETE, LTB and 18-HEPE were able to predict poor expressibility of MGs. In conclusion, tear eicosanoid levels are age-dependent and specific eicosanoids may be indicators of clinical obstruction of MG or the severity of ocular surface damage.
10.1038/s41598-018-29568-3
Comparison of Collection Methods for the Measure of Human Meibum and Tear Film-Derived Lipids Using Mass Spectrometry.
Ngo William,Chen Jianzhong,Panthi Shyam,Nichols Kelly K,Nichols Jason J
Current eye research
Purpose/aim: To assess the effectiveness of polytetrafluoroethylene (PTFE) tubes in the collection of human tears and meibum. MATERIALS AND METHODS:This was a prospective study that enrolled 10 healthy human subjects. Both the tear film and meibum were sampled using PTFE tubes in the right eye of all subjects. In the left eyes, either 5-µL or 1-µL glass microcapillary tubes were used to collect tears, and 0.5-µL glass microcapillary tubes were used to collect meibum. The lipids from the samples were extracted and analyzed using mass spectrometry (SCIEX TripleTOF 5600, Framingham, MA, USA). The absolute peak intensities of the omega-acyl hydroxy fatty acids (OAHFA), cholesterol esters (CE), and wax esters (WE) obtained for both methods were summed and compared between collection methods. RESULTS:A total of 10 subjects completed the study (five female, mean age: 35.7 ± 7.9 years). Using the mass spectrometer output, the median (first quartile, third quartile) summed intensity units of OAHFA, CE, and WE collected associated with tears using PTFE were 516 (125, 1315), 7946 (2571, 19,915), and 38,892 (139,630, 174,082), all of which were significantly higher (all p ≤ 0.04) than those collected from glass microcapillaries (91 (41, 408), 2463 (1389, 6042), and 11,109 (7465, 37,371), respectively). The median summed intensity units of OAHFA, CE, and WE associated with meibum (1958 (1417, 3502), 11,726 (8434, 87,691), and 84,771 (52,657, 206,050), respectively) using PTFE were not significantly different (all p ≥ 0.13) than those associated with glass microcapillaries (1502 (699, 4407), 10,781 (3287, 38,205), and 77,381 (26,590, 178,213), respectively). CONCLUSIONS:PTFE tubes, which are thought to be lipophilic, were associated with more measurable lipids from the tear film than glass microcapillaries. There was no difference between collection methods in lipid profiles when used with meibum.
10.1080/02713683.2018.1501803
Influence of Meibomian Gland Expression Methods on Human Lipid Analysis Results.
Kunnen Carolina M E,Brown Simon H J,Lazon de la Jara Percy,Holden Brien A,Blanksby Stephen J,Mitchell Todd W,Papas Eric B
The ocular surface
PURPOSE:To compare the lipid composition of human meibum across three different meibum expression techniques. METHODS:Meibum was collected from five healthy non-contact lens wearers (aged 20-35 years) after cleaning the eyelid margin using three meibum expression methods: cotton buds (CB), meibomian gland evaluator (MGE) and meibomian gland forceps (MGF). Meibum was also collected using cotton buds without cleaning the eyelid margin (CBn). Lipids were analyzed by chip-based, nano-electrospray mass spectrometry (ESI-MS). Comparisons were made using linear mixed models. RESULTS:Tandem MS enabled identification and quantification of over 200 lipid species across ten lipid classes. There were significant differences between collection techniques in the relative quantities of polar lipids obtained (P<.05). The MGE method returned smaller polar lipid quantities than the CB approaches. No significant differences were found between techniques for nonpolar lipids. No significant differences were found between cleaned and non-cleaned eyelids for polar or nonpolar lipids. CONCLUSION:Meibum expression technique influences the relative amount of phospholipids in the resulting sample. The highest amounts of phospholipids were detected with the CB approaches and the lowest with the MGE technique. Cleaning the eyelid margin prior to expression was not found to affect the lipid composition of the sample. This may be a consequence of the more forceful expression resulting in cell membrane contamination or higher risk of tear lipid contamination as a result of reflex tearing.
10.1016/j.jtos.2015.10.001