Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles

Abstract

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. 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. The tear fluid covers the anterior surface of the cornea and serves critical functions in maintaining the homeostasis of the ocular surface. Tears hydrate and lubricate the mucous membranes constituting the ocular surface, supply nourishment to the avascular corneal epithelium, and provide a smooth optical surface essential for visual acuity. The drainage of tears via the lacrimal puncta flushes contaminants and irritants out of the eye, thereby functioning as a first line of defense for the anterior eye against invading pathogens (1Zhou L. Beuerman R.W. Tear analysis in ocular surface diseases.Prog. Retin. Eye Res. 2012; 31: 527-550Crossref PubMed Scopus (182) Google Scholar, 2Rantamäki A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google Scholar). The typical volume of tears in normal eyes ranges from 3.4 to 10.7 μl per eye (3Scherz W. Doane M.G. Dohlman C.H. Tear volume in normal eyes and keratoconjunctivitis sicca.Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 1974; 192: 141-150Crossref PubMed Scopus (73) Google Scholar). Despite its small volume, tears represent a biological fluid of immense complexities with a wide array of proteins/peptides, electrolytes, lipids, and small molecule metabolites contributed by distinct sources (1Zhou L. Beuerman R.W. Tear analysis in ocular surface diseases.Prog. Retin. Eye Res. 2012; 31: 527-550Crossref PubMed Scopus (182) Google Scholar). The precise balance of these various metabolites is crucial in ensuring proper physiological function and maintaining biophysical integrity of the precorneal tear film. Perturbations in this delicate equilibrium may be manifested in various ocular conditions such as dry eye syndrome (DES) and blepharitis (1Zhou L. Beuerman R.W. Tear analysis in ocular surface diseases.Prog. Retin. Eye Res. 2012; 31: 527-550Crossref PubMed Scopus (182) Google Scholar, 4Zhou L. Beuerman R.W. Chan C.M. Zhao S.Z. Li X.R. Yang H. Tong L. Liu S. Stern M.E. Tan D. Identification of tear fluid biomarkers in dry eye syndrome using iTRAQ quantitative proteomics.J. Proteome Res. 2009; 8: 4889-4905Crossref PubMed Scopus (231) Google Scholar, 5Tong L. Zhou L. Beuerman R.W. Zhao S.Z. Li X.R. Association of tear proteins with Meibomian gland disease and dry eye symptoms.Br. J. Ophthalmol. 2011; 95: 848-852Crossref PubMed Scopus (87) Google Scholar). Recent decades have witnessed tremendous progress in the systematic profiling of proteins as well as small molecule metabolites present in tears (1Zhou L. Beuerman R.W. Tear analysis in ocular surface diseases.Prog. Retin. Eye Res. 2012; 31: 527-550Crossref PubMed Scopus (182) Google Scholar, 6Zhou L. Zhao S.Z. Koh S.K. Chen L. Vaz C. Tanavde V. Li X.R. Beuerman R.W. In-depth analysis of the human tear proteome.J. Proteomics. 2012; 75: 3877-3885Crossref PubMed Scopus (236) Google Scholar, 7Green-Church K.B. Nichols K.K. Kleinholz N.M. Zhang L. Nichols J.J. 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Ophthalmol. Vis. Sci. 2008; 49: 3779-3789Crossref PubMed Scopus (140) Google Scholar). The presence of wax esters and in tears using D. Foulks G.N. Yappert M.C. D. of human tear 2007; PubMed Scopus Google Scholar). J.T. Zhao Z. Willcox M.D.P. Blanksby S.J. Mitchell T.W. and of tear phospholipids and in the of and Ophthalmol. Vis. Sci. 2010; 51: PubMed Scopus Google the relative abundance of species of and in human tears. The lipid composition by reported a of A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google Scholar, of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google Scholar). to and A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google reported the presence of and in tears. most of these on lipid in a comprehensive lipidome with details to the complexities of the various lipid in tears is from the by the abundance great complexities of lipid species in the analysis of tears has been by the of a systematic of tear using different collection be of immense for the ocular in the of collection to human tears as a fluid for we report for the and comparative of human tears and meibum from Our that human tears more than 600 lipid species from 17 distinct lipid in than μl per for the first the presence of a novel lipid cholesteryl in the human tears and have impact on the existent tear film model. systematic of the tear with different techniques analysis and and to of tears and meibum. The with the Eye for the study be in the from and the for the by the number to the of the of for human in this The have been reported (10Lam S.M. Tong L. Yong S.S. Li B. Chaurasia S.S. Shui G. Wenk M.R. Meibum lipid composition in Asians with dry eye disease.PLoS ONE. 2011; 6: e24339Crossref PubMed Scopus (137) Google Scholar). tears using μl The collection to a of with collection and The in the tear and to with the the in tears by μl of the using to the eye with the to and their Tears by the as Tears from the with to lipids and the with the tear and tears from eyes of the and and the in Meibum as (10Lam S.M. Tong L. Yong S.S. Li B. Chaurasia S.S. Shui G. Wenk M.R. Meibum lipid composition in Asians with dry eye disease.PLoS ONE. 2011; 6: e24339Crossref PubMed Scopus (137) Google Scholar). Lipids for with μl in a for and μl of to The out by the and the and using lipid mass The first of the using with Lipids with μl of in a and to and on for for and the The with μl of and μl of The and using lipid mass Meibum as (10Lam S.M. Tong L. Yong S.S. Li B. Chaurasia S.S. Shui G. Wenk M.R. Meibum lipid composition in Asians with dry eye disease.PLoS ONE. 2011; 6: e24339Crossref PubMed Scopus (137) Google Scholar). lipid species using in a MS for and of lipid for of for mass by out μl of for of cholesteryl and such that the concentrations for mass the of the various lipid lipids using with a trap mass as G. S.M. J. Wenk M.R. lipidome human and are lipids biomarkers for ONE. 2011; 6: PubMed Scopus Google Scholar). of lipid of lipids by normal out using a with the and for comparative analysis of various lipid species by to and from Lipids and from and with for and lipid from using quantitative lipid to their for species using as species using as I.A. Wojtowicz J.C. Molai M. Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum.J. Lipid Res. 2009; 50: 2471-2485Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). lipids and and using a of G. Yang H. Wenk M.R. analysis of using with mass to and 2010; 6: PubMed Scopus Google Scholar). of the lipids out on a using a of for and species using and as analysis of validated using as Using the of as relative to the species using as of using for with fatty fatty using and using the V. J. of wax species by ionisation mass 2010; PubMed Scopus Google Scholar). The mass for analysis of have been reported S.M. Tong L. B. Wenk M.R. Shui G. and sensitive profiling of tear wax species using with tandem mass PubMed Scopus Google Scholar). MS of lipid out using with mass using a and a as the of μl of a with the as a of for a of of lipid of lipids by out using a with the and MS and in and mass of than the and using ionization as with and as G. I.A. Tan Wenk M.R. analysis in lipid by to a for 2011; PubMed Scopus Google Scholar). we that for a wide in the of in due to with the G. I.A. Tan Wenk M.R. analysis in lipid by to a for 2011; PubMed Scopus Google the of in to in the analysis of The of lipids in tears using different collection techniques using with a to that tears using accurate of the lipid the ocular surface, we a is the lipid in the per the as a the of tears by the represent a of the concentrations of lipid metabolites in the tear that lipids in the first of the with the to in with the the collection the abundance of lipid The to in the mass analysis of the lipid in the mass that the the tear lipids and that the for tears for lipid to the of we to and lipids from the first to of the of the of the as from that lipids, the nonpolar lipids such as and the as as the of the tears. the of by the the of the tear lipids, as the first of lipids the Furthermore, lipid from a of the the in for of lipid the of from the of the with the collection for with and of the first of the with the lipids in a that the concentrations of lipids from the in the tear using a lipid of different lipid with concentrations that the relative abundance of various lipid in human tears. The lipid first and the in and to different concentrations of tear in concentrations of the tear lipids for and lipid and mass out on the first of the the of lipid and their relative concentrations in the lipid that the of the of lipids in the lipid the that the first of the of to provide a tear lipidome that of the lipid in the tear with The of tear collection is for in lipid has been that on the of tears be to in collection of Ophthalmol. Full Text PDF PubMed Scopus Google we the quantitative and in tear lipids using different been on the lipid tears using and I.A. On the lipid composition of human meibum and tears: comparative analysis of nonpolar lipids.Invest. Ophthalmol. Vis. Sci. 2008; 49: 3779-3789Crossref PubMed Scopus (140) Google Scholar). the different techniques for tear collection on the and composition of lipids The of tear collection the of lipids and to be the predominant nonpolar lipid and the major lipid in collection of lipid in tears using be to the of tears with this The detection of lipid limiting of lipids in tears be to the different of detection using for the various lipid in the lipid composition of tears using a a from that with a from the of lipids in the is in with a study that reported tear proteome using the M. E. B. of the as to tear Eye Res. 2011; PubMed Scopus Google Scholar). On the of the tear lipid using the we the for tear analysis in due to the of tear lipids that this Furthermore, the is a in dry eye and be with relative from tear for the of tear collection confers to the ocular of with the a tear the current lipid species in human tears in a using tear from first to The lipid composition of tear fluid from the been validated in from a of with a in the tear ocular surface disease and for corneal and The lipidomic composition of tear fluid a for with very demonstrating the of and the of to be for in dry eye for analysis of tear from with lacrimal mass for of lipids in tears. of major species from lipid of and are using the with Our the complexities of the human tear fluid of the nonpolar lipid of and the such as and the and as well as and a of amphiphilic first reported in meibum by and Foulks I.A. Wojtowicz J.C. Molai M. Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum.J. Lipid Res. 2009; 50: 2471-2485Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The tear in this study are a from a report by (10Lam S.M. Tong L. Yong S.S. Li B. Chaurasia S.S. Shui G. Wenk M.R. Meibum lipid composition in Asians with dry eye disease.PLoS ONE. 2011; 6: e24339Crossref PubMed Scopus (137) Google and of A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google Scholar, J.T. Zhao Z. Willcox M.D.P. Blanksby S.J. Mitchell T.W. and of tear phospholipids and in the of and Ophthalmol. Vis. Sci. 2010; 51: PubMed Scopus Google Scholar, of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google Scholar). and have been reported in tears meibum we the of in tears to and from reported and A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google Scholar, of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google Scholar, J.T. Identification and of the phospholipids in normal and dry eye tears by mass spectrometry.Invest. Ophthalmol. Vis. Sci. 2006; PubMed Scopus Google Scholar). we report the relative abundance of species in the tear fluid using the we on meibum (10Lam S.M. Tong L. Yong S.S. Li B. Chaurasia S.S. Shui G. Wenk M.R. Meibum lipid composition in Asians with dry eye disease.PLoS ONE. 2011; 6: e24339Crossref PubMed Scopus (137) Google of major lipid in human and using MS presence via using MS presence via using to be of in of to in of and of to in the of detection in their the of detection in their are as of lipids with on human tear using the presence of in tears been on of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google Scholar, Tear a array of lipid Eye Res. 14: PubMed Scopus Google mass analysis of tears has the presence of in tears. to we of phospholipids in tears. be due to the of in the analysis of tears by A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google as species in the On the and of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google in the for analysis of of in due to of The of in the by and of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google with the of phospholipids in tear have the ionization of to J.T. Zhao Z. Willcox M.D.P. Blanksby S.J. Mitchell T.W. and of tear phospholipids and in the of and Ophthalmol. Vis. Sci. 2010; 51: PubMed Scopus Google and and of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google reported to be than the of detection in their we the presence of in tear relative abundance using MS presence via using to be of in of to in of and the of detection in their in a are as of lipids with on human tear using the presence of in tears been on of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google Scholar, Tear a array of lipid Eye Res. 14: PubMed Scopus Google mass analysis of tears has the presence of in tears. to we of phospholipids in tears. be due to the of in the analysis of tears by A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google as species in the On the and of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google in the for analysis of of in due to of The of in the by and of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google with the of phospholipids in tear have the ionization of to J.T. Zhao Z. Willcox M.D.P. Blanksby S.J. Mitchell T.W. and of tear phospholipids and in the of and Ophthalmol. Vis. Sci. 2010; 51: PubMed Scopus Google and and of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google reported to be than the of detection in their we the presence of in tear relative abundance particular to tear film structural a of amphiphilic lipid and to and of the tear the presence of in human with as the most species of lipids to for the predominant species in tears to be species of and their species via their different using various of phospholipids and their with A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google Scholar, of phospholipids in human tears and tear Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus (58) Google a major of the with A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google we of and in human tears using the in and of species in human tears that of distinct and different from the species reported by A.H. Seppänen-Laakso T. Oresic M. Jauhiainen M. Holopainen J.M. Human tear fluid lipidome: from composition to function.PLoS ONE. 2011; 6: e19553Crossref PubMed Scopus (100) Google Scholar). with I.A. On the lipid composition of human meibum and tears: comparative analysis of nonpolar lipids.Invest. Ophthalmol. Vis. Sci. 2008; 49: 3779-3789Crossref PubMed Scopus (140) Google Scholar, D. Foulks G.N. Yappert M.C. D. of human tear 2007; PubMed Scopus Google and in human tears as the predominant nonpolar lipid in in current study is the quantitative of the of and in the human tears and meibum and Using as S.M. Tong L. B. Wenk M.R. Shui G. and sensitive profiling of tear wax species using with tandem mass PubMed Scopus Google we that of the lipids in the human meibum is in with a by I.A. Arciniega J.C. Lu H. Molai M. Evaluation and quantitation of intact wax esters of human meibum by gas-liquid chromatography-ion trap mass spectrometry.Invest. Ophthalmol. Vis. Sci. 2012; 53: PubMed Scopus Google Scholar). Furthermore, we and the comprehensive of species in human tears than in human meibum of to that human tears a of with than the nonpolar components of meibum that with I.A. On the lipid composition of human meibum and tears: comparative analysis of nonpolar lipids.Invest. Ophthalmol. Vis. Sci. 2008; 49: 3779-3789Crossref PubMed Scopus (140) Google Scholar). On the J.T. and of major lipids in normal and dry eye tear by electrospray tandem mass PubMed Scopus Google reported the presence of a such as and in the human tears. The the of the for tears for lipid and the different tear collection in eye in of and of lipids comprehensive lipidome of the human tear fluid and the relative abundance of various novel lipid amphiphiles in tears and The compositional details of the human tear lipidome reported herein have major on ocular surface as well as tear film the physiological functions of the novel lipids in human is to the the ocular surface and the critical of ocular homeostasis in maintaining precise insights be from the tear fluid of proteins such as and the (1Zhou L. Beuerman R.W. Tear analysis in ocular surface diseases.Prog. Retin. Eye Res. 2012; 31: 527-550Crossref PubMed Scopus (182) Google Scholar). line with has been reported to the of J.J. J. of in 10: PubMed Scopus Google to and in the E. D. L. Identification of as in the Biol. PubMed Scopus Google Scholar). the physiological of the tear to with that of the tear a of metabolites with of with corneal by human tear PubMed Scopus Google Scholar). The that phospholipids as amphiphilic of the tear film lipid has been due to the of phospholipids in human the predominant source of lipids for tears (10Lam S.M. Tong L. Yong S.S. Li B. Chaurasia S.S. Shui G. Wenk M.R. Meibum lipid composition in Asians with dry eye disease.PLoS ONE. 2011; 6: e24339Crossref PubMed Scopus (137) Google Scholar, I.A. The Meibomian Retin. Eye Res. 2009; PubMed Scopus Google Scholar). Our current analysis of the tear lipidome that phospholipids a of the tear The of phospholipids and sphingolipids in the human tear fluid with the meibum has for understanding of tear film On the the detection of a of in the tear fluid has a to the tear film has been to with phospholipids in a to in lipid and membranes M. J. The of on and in PubMed Scopus Google Scholar). physiological the of is from a molecule a amphiphilic comprising a G. J. and biological function as lipid in the of the human 1974; PubMed Scopus Google Scholar). the physiological of in tears is be a for the amphiphilic sublayer of the tear film on its biophysical as a of biological membranes G. J. and biological function as lipid in the of the human 1974; PubMed Scopus Google Scholar). Furthermore, and represent the lipid that with tear is a of tear film S.M. Tong L. B. Yong S.S. Wenk M.R. Shui G. analysis of human tear fluid reveals lipid in dry eye Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). Our analysis of the human tear lipidome has insights to ocular surface and tear film with to composition of the amphiphilic lipid sublayer of the tear film. have that the tear fluid is in phospholipids and sphingolipids relative to the and that a of phospholipids and sphingolipids is in tears to the amphiphilic lipid have as a novel for this lipid The of various techniques collection thereby on tears. Furthermore, we the of using for tear collection for lipid confers in of lipid with a wide of and is to with