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Mass Spectrometric Analysis of Meibomian Gland Lipids

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Mass Spectrometry-Based Lipidomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2306))

Abstract

The precorneal tear film keeps the eye surface moist and helps to maintain normal eye function. The outermost lipid layer of the tear film, which attenuates tear film evaporation, contains meibum secreted from the meibomian gland. Most meibum lipids are neutral, including wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs), along with some polar lipids including free fatty acids (FFAs), O-acyl-ω-hydroxy fatty acids (OAHFAs), and trace phospholipids. Detection of neutral lipids by mass spectrometry (MS) is challenging due to interference from impurities, particularly when working with minute-volume meibum samples. Here, we describe procedures for sample preparation and MS analysis of these elusive meibum lipids that can be used to examine dry eye disease mechanisms. Because the method described here minimizes impurity peaks for lipids generally, neutral and otherwise, it may be applied to high-sensitivity analysis of other biological samples.

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References

  1. Ohashi Y, Dogru M, Tsubota K (2006) Laboratory findings in tear fluid analysis. Clin Chim Acta 369(1):17–28. https://doi.org/10.1016/j.cca.2005.12.035

    Article  CAS  PubMed  Google Scholar 

  2. Willcox MDP, Argüeso P, Georgiev GA, Holopainen JM, Laurie GW, Millar TJ, Papas EB, Rolland JP, Schmidt TA, Stahl U, Suarez T, Subbaraman LN, Uçakhan OÖ, Jones L (2017) TFOS DEWS II tear film report. Ocul Surf 15(3):366–403. https://doi.org/10.1016/j.jtos.2017.03.006

    Article  PubMed  PubMed Central  Google Scholar 

  3. Wolff E (1946) The muco-cutaneous junction of the lid margin and the distribution of the tear fluid. Trans Ophthalmol Soc UK 66:291–308

    Google Scholar 

  4. Hisatake K, Tanaka S, Aizawa Y (1993) Evaporation rate of water in a vessel. J Appl Phys 73 (11):7395-7401. https://doi.org/10.1063/1.354031

  5. Tomlinson A, Doane MG, McFadyen A (2009) Inputs and outputs of the lacrimal system: review of production and evaporative loss. Ocul Surf 7(4):186–198. https://doi.org/10.1016/S1542-0124(12)70186-6

    Article  PubMed  Google Scholar 

  6. Tsubota K, Nakamori K (1995) Effects of ocular surface area and blink rate on tear dynamics. Arch Ophthalmol 113 (2):155–158. https://doi.org/10.1001/archopht.1995.01100020037025

  7. Goto E, Endo K, Suzuki A, Fujikura Y, Matsumoto Y, Tsubota K (2003) Tear evaporation dynamics in normal subjects and subjects with obstructive meibomian gland dysfunction. Invest Ophthalmol Vis Sci 44(2):533–539. https://doi.org/10.1167/iovs.02-0170

    Article  PubMed  Google Scholar 

  8. Chen J, Keirsey JK, Green KB, Nichols KK (2017) Expression profiling of nonpolar lipids in meibum from patients with dry eye: a pilot study. Invest Ophthalmol Vis Sci 58(4):2266–2274. https://doi.org/10.1167/iovs.16-20902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Foulks GN (2007) The correlation between the tear film lipid layer and dry eye disease. Surv Ophthalmol 52(4):369–374. https://doi.org/10.1016/j.survophthal.2007.04.009

    Article  PubMed  Google Scholar 

  10. Knop E, Knop N, Millar T, Obata H, Sullivan DA (2011) The international workshop on meibomian gland dysfunction: report of the subcommittee on anatomy, physiology, and pathophysiology of the meibomian gland. Invest Ophthalmol Vis Sci 52(4):1938–1978. https://doi.org/10.1167/iovs.10-6997c

    Article  PubMed  PubMed Central  Google Scholar 

  11. Butovich IA, Uchiyama E, McCulley JP (2007) Lipids of human meibum: mass-spectrometric analysis and structural elucidation. J Lipid Res 48(10):2220–2235

    Article  CAS  Google Scholar 

  12. Chen J, Green-Church KB, Nichols KK (2010) Shotgun lipidomic analysis of human meibomian gland secretions with electrospray ionization tandem mass spectrometry. Invest Ophthalmol Vis Sci 51(12):6220–6231. https://doi.org/10.1167/Iovs.10-5687

    Article  PubMed  PubMed Central  Google Scholar 

  13. Lam SM, Tong L, Duan X, Petznick A, Wenk MR, Shui G (2014) Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles. J Lipid Res 55(2):289–298. https://doi.org/10.1194/jlr.M044826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chen J, Green KB, Nichols KK (2013) Quantitative profiling of major neutral lipid classes in human meibum by direct infusion electrospray ionization mass spectrometry. Invest Ophthalmol Vis Sci 54(8):5730–5753. https://doi.org/10.1167/iovs.12-10317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Brown SH, Kunnen CM, Duchoslav E, Dolla NK, Kelso MJ, Papas EB, Lazon de la Jara P, Willcox MD, Blanksby SJ, Mitchell TW (2013) A comparison of patient matched meibum and tear lipidomes. Invest Ophthalmol Vis Sci 54(12):7417–7424. https://doi.org/10.1167/iovs.13-12916

    Article  CAS  PubMed  Google Scholar 

  16. Ende M, Spiteller G (1982) Contaminants in mass spectrometry. Mass Spectrom Rev 1(1):29–62. https://doi.org/10.1002/mas.1280010105

    Article  CAS  Google Scholar 

  17. Keller BO, Sui J, Young AB, Whittal RM (2008) Interferences and contaminants encountered in modern mass spectrometry. Anal Chim Acta 627(1):71–81. https://doi.org/10.1016/j.aca.2008.04.043

    Article  CAS  PubMed  Google Scholar 

  18. Murphy RC, Fitzgerald M, Barkley RM (2008) Neutral lipidomics and mass spectrometry. In: Griffiths WJ (ed) Metabolomics, metabonomics and metabolite profiling. The Royal Society of Chemistry, Cambridge, pp 161–194

    Google Scholar 

  19. Butovich IA (2013) Tear film lipids. Exp Eye Res 117:4–27. https://doi.org/10.1016/j.exer.2013.05.010

    Article  CAS  PubMed  Google Scholar 

  20. Chen J, Panthi S (2019) Lipidomic analysis of meibomian gland secretions from the tree shrew: identification of candidate tear lipids critical for reducing evaporation. Chem Phys Lipids 220:36–48. https://doi.org/10.1016/j.chemphyslip.2019.01.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen J, Keirsey J, Basso K, Nichols KK (2015) Differentially expressed non-polar lipids in human meibum of dry eye disease. Invest Ophthalmol Vis Sci 56(7):342–342

    CAS  Google Scholar 

  22. Postnikoff CK, Chen J, Keirsey J, Basso K, Nichols KK (2015) Altered regulation of expressed polar meibum lipids in dry eye disease. Invest Ophthalmol Vis Sci 56(7):343–343

    Google Scholar 

  23. Chen J, Nichols KK, Wilson L, Barnes S, Nichols JJ (2019) Untargeted lipidomic analysis of human tears: a new approach for quantification of O-acyl-omega hydroxy fatty acids. Ocul Surf 17(2):347–355. https://doi.org/10.1016/j.jtos.2019.02.004

    Article  PubMed  PubMed Central  Google Scholar 

  24. Ngo W, Chen J, Panthi S, Nichols KK, Nichols JJ (2018) Comparison of collection methods for the measure of human meibum and tear film-derived lipids using mass spectrometry. Curr Eye Res 43(10):1244–1252. https://doi.org/10.1080/02713683.2018.1501803

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Camera E, Ludovici M, Galante M, Sinagra JL, Picardo M (2010) Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry. J Lipid Res 51(11):3377–3388. https://doi.org/10.1194/Jlr.D008391

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kalužíková A, Vrkoslav V, Harazim E, Hoskovec M, Plavka R, Buděšínský M, Bosáková Z, Cvačka J (2017) Cholesteryl esters of ω-(O-acyl)-hydroxy fatty acids in vernix caseosa. J Lipid Res 58(8):1579–1590. https://doi.org/10.1194/jlr.M075333

    Article  PubMed  PubMed Central  Google Scholar 

  27. Stransky K, Valterova I, Kofronova E, Urbanova K, Zarevucka M, Wimmer Z (2011) Non-polar lipid components of human cerumen. Lipids 46(8):781–788. https://doi.org/10.1007/s11745-011-3564-y

    Article  CAS  PubMed  Google Scholar 

  28. Rijpstra WIC, Reneerkens J, Piersma T, Sinninghe Damsté JS (2007) Structural identification of the β-hydroxy fatty acid-based diester preen gland waxes of shorebirds. J Nat Prod 70(11):1804–1807. https://doi.org/10.1021/np070254z

    Article  CAS  PubMed  Google Scholar 

  29. Iven T, Herrfurth C, Hornung E, Heilmann M, Hofvander P, Stymne S, Zhu LH, Feussner I (2013) Wax ester profiling of seed oil by nano-electrospray ionization tandem mass spectrometry. Plant Methods 9(1):24. https://doi.org/10.1186/1746-4811-9-24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Haworth KM, Nichols JJ, Thangavelu M, Sinnott LT, Nichols KK (2011) Examination of human meibum collection and extraction techniques. Optom Vis Sci 88(4):525–533. https://doi.org/10.1097/OPX.0b013e318214ac0f

    Article  PubMed  PubMed Central  Google Scholar 

  31. Nichols KK, Ham BM, Nichols JJ, Ziegler C, Green-Church KB (2007) Identification of fatty acids and fatty acid amides in human meibomian gland secretions. Invest Ophthalmol Vis Sci 48(1):34–39

    Article  Google Scholar 

  32. Abelson MBO, Supna (2006) Treating dysfunctional Meibomian glands. Rev Ophthalmol

    Google Scholar 

  33. Kawai S (1966) Discussion on decomposition of chloroform. Yakugaku Zasshi 86(12):1125–1132. https://doi.org/10.1248/yakushi1947.86.12_1125

    Article  CAS  PubMed  Google Scholar 

  34. Tsujikawa K, Segawa H, Kuwayama K, Yamamuro T, Kanamori T, Iwata YT, Ohmori T (2020) Phosgene in deteriorated chloroform: presumptive cause of production of 3,4-dimethyl-5-phenyl-2-oxazolidones in methamphetamine. Forensic Toxicol 38(2):475–480. https://doi.org/10.1007/s11419-019-00517-7

    Article  CAS  Google Scholar 

  35. Maudens KE, Wille SMR, Lambert WE (2007) Traces of phosgene in chloroform: consequences for extraction of anthracyclines. J Chromatogr B Analyt Technol Biomed Life Sci 848(2):384–390. https://doi.org/10.1016/j.jchromb.2006.10.073

    Article  CAS  PubMed  Google Scholar 

  36. Green-Church KB, Butovich I, Willcox M, Borchman D, Paulsen F, Barabino S, Glasgow BJ (2011) The international workshop on meibomian gland dysfunction: report of the subcommittee on tear film lipids and lipid-protein interactions in health and disease. Invest Ophthalmol Vis Sci 52(4):1979–1993. https://doi.org/10.1167/iovs.10-6997d

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kunnen CM, Brown SH, Lazon de la Jara P, Holden BA, Blanksby SJ, Mitchell TW, Papas EB (2016) Influence of meibomian gland expression methods on human lipid analysis results. Ocul Surf 14(1):49–55. https://doi.org/10.1016/j.jtos.2015.10.001

    Article  PubMed  Google Scholar 

  38. Chen J, Nichols KK (2018) Comprehensive shotgun lipidomics of human meibomian gland secretions using MS/MSall with successive switching between acquisition polarity modes. J Lipid Res 59(11):2223–2236. https://doi.org/10.1194/jlr.D088138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1):497–509

    Article  CAS  Google Scholar 

  40. Wang GD, Cole RB (1994) Effect of solution ionic-strength on analyte charge-state distributions in positive and negative-ion electrospray mass-spectrometry. Anal Chem 66(21):3702–3708

    Article  CAS  Google Scholar 

  41. Chernushevich IV, Loboda AV, Thomson BA (2001) An introduction to quadrupole-time-of-flight mass spectrometry. J Mass Spectrom 36(8):849–865. https://doi.org/10.1002/jms.207

    Article  CAS  PubMed  Google Scholar 

  42. Ferrer I, Thurman EM (2007) Importance of the electron mass in the calculations of exact mass by time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 21(15):2538–2539. https://doi.org/10.1002/rcm.3102

    Article  CAS  PubMed  Google Scholar 

  43. Wangkarn S, Soisungnoen P, Rayanakorn M, Grudpan K (2005) Determination of linear alkylbenzene sulfonates in water samples by liquid chromatography–UV detection and confirmation by liquid chromatography–mass spectrometry. Talanta 67(4):686–695. https://doi.org/10.1016/j.talanta.2005.03.011

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by the National Institutes of Health Grant P30 EY003039.

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Authors and Affiliations

  1. Department of Optometry and Vision Science, The University of Alabama at Birmingham, Birmingham, AL, USA

    Jianzhong Chen

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  1. Jianzhong Chen
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Editors and Affiliations

  1. Mass Spectrometry Resource Division of Endocrinology Metabolism, and Lipid Research Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

    Fong-Fu Hsu

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Chen, J. (2021). Mass Spectrometric Analysis of Meibomian Gland Lipids. In: Hsu, FF. (eds) Mass Spectrometry-Based Lipidomics. Methods in Molecular Biology, vol 2306. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1410-5_11

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  • DOI: https://doi.org/10.1007/978-1-0716-1410-5_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1409-9

  • Online ISBN: 978-1-0716-1410-5

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