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Journal of Lipid Research, Vol. 48, 2220-2235, October 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Lipids of human meibum: mass-spectrometric analysis and structural elucidation¹

Igor A. Butovich², Eduardo Uchiyama and James P. McCulley

Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9057

¹ The results of this study were presented in part at the 2006 and 2007 annual meetings of the Association for Research in Vision and Ophthalmology, Inc., in Fort Lauderdale, FL.

Published, JLR Papers in Press, July 12, 2007.

² To whom correspondence should be addressed. e-mail: igor.butovich{at}utsouthwestern.edu

The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MSⁿ). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MSⁿ detection of the analytes (HPLC API MSⁿ). The major precursor ions were isolated and subjected to further sequential fragmentation in MSⁿ experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M – H₂O + H]⁺) and oleic acid (OA; m/z 283; [M + H]⁺) were found. The other major compounds of the general molecular formula C_nH_2n-2O₂ were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas C_nH_2n-4O₂ and C_nH_2nO₂. Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MSⁿ analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components.

Supplementary key words tear film lipid layer • human meibomian gland • mass spectrometry • wax esters • phospholipids • oleamide

Abbreviations: APCI, atmospheric pressure chemical ionization; API, atmospheric pressure ionization; BO, behenyl oleate; Cer, ceramide; Chl, cholesterol; Chl-E, cholesteryl ester; Chl-O, cholesteryl oleate; DAG; diacylglycerol; DES, dry eye syndrome; DP, 1,2-dipalmitoylglycerol; ESI, electrospray ionization; HPA, n-hexane-propan-2-ol-acetic acid; HPLC, high-pressure liquid chromatography; MAG, monoacylglycerol; MC, methanol-chloroform; MG, meibomian gland; MGS, meibomian gland secretions; MS, mass spectrometry; NP-HPLC, normal-phase HPLC; OA, oleic acid; OAm, oleamide; PC, phosphatidylcholine; PL, phospholipid; POPA, 1-palmitoyl-2-oleoyl-phosphatidic acid; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-phosphatidylglycerol; PPPE, 1,2-dipalmitoylphosphatidylethanolamine; RT, retention time; SAPI, 1-stearoyl-2-arachidonoyl-phosphatidylinositol; SOPS, 1-stearoyl-2-oleoylphosphatidylserine; SS, stearyl stearate; SSPC-D₉, 1,2-distearoyl-sn-glycero-3-phospho-[(CD₃)₃N⁺]-choline; TAG, triacylglycerol; TFLL, tear film lipid layer; TM, trimyristin; TP, tripalmitin; WE, wax ester

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