Abstract
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 MS(n)). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MS(n) detection of the analytes (HPLC API MS(n)). The major precursor ions were isolated and subjected to further sequential fragmentation in MS(n) 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 - H(2)O + H](+)) and oleic acid (OA; m/z 283; [M + H](+)) were found. The other major compounds of the general molecular formula C(n)H(2n-2)O(2) 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(n)H(2n-4)O(2) and C(n)H(2n)O(2). 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 MS(n) 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.
Highlights
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 retention times (RTs) of the lipid standards were used to map the corresponding lipid classes that were expected to be observed in MGS
MGS produced only one major elution peak whose RT was identical to those of standard wax ester (WE), cholesteryl ester (Chl-E), and TAG (Fig. 2) The peak showed a wide range of MS signals (Fig. 2, insert A), which were further analyzed using the direct infusion method
Summary
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 HPLC API MS experiments with human MGS produced clear evidence of the presence of very hydrophobic compounds similar to 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-2oleoyl-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-phosphatidylglycerol; PPPE, 1,2-dipalmitoylphosphatidylethanolamine; RT, retention time; SAPI, 1-stearoyl-2-arachidonoyl-phosphatidylinositol; SOPS, 1stearoyl-2-oleoylphosphatidylserine; SS, stearyl stearate; SSPC-D9, 1,2distearoyl-sn-glycero-3-phospho-[(CD3)3N1]-choline; TAG, triacylglycerol; TFLL, tear film lipid layer; TM, trimyristin; TP, tripalmitin; WE, wax ester. It was suggested that animals (rabbits, in particular) are poor models of human TFLL and DES [16, 17]
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