Identification, quantification and comparison of major non‐polar lipids in normal and dry eye tear lipidomes by electrospray tandem mass spectrometry

Abstract

Millions of individuals suffer from a health condition known as keratoconjunctivitis sicca (KCS, also known as 'dry eye'). Studies have indicated that the lipids in the tear film layer, which covers the outer portion of the eye, may be directly correlated with the existence of dry eye syndrome. By identifying and comparing the major, non-polar lipids in normal eye tears with a dry eye model, it may be possible to identify a symptom of, or a contributing factor to, dry eye. Electrospray tandem mass spectrometry (ES-MS/MS) was used to identify and compare the non-polar lipids, detected as lithium adducts, from normal and dry eye tear samples obtained from rabbits. A limited number of normal human tear samples were also examined for lipid content, and a close resemblance to rabbit was observed. Three distinct regions were delineated in the ES mass spectra of the non polar lipids, m/z 20-500, 500-800 and 800-1100. A common feature noted among identified lipid components was a glycerol backbone with fatty acyl substituents attached. Product ion spectra were obtained for lithiated monoacyl-, 1,2-and 1,3-diacyl-and triacylglyceride standards. Newly proposed structures and fragmentation pathways for the major product ions are presented for the 1,2-and 1,3-diglycerides, and also for the monoglyceride. New approaches to distinguishing asymmetric 1,2-diglycerides and 1,2-from 1,3-diglycerides are proposed. For the rabbit tear samples, the m/z 20-500 range contains monoester diols with empirical formulas C n H 2n O 4 , the m/z 500-800 range includes diesters with empirical formulas C n H 2n−2 O 5 and the m/z 800-1100 range contains triesters with empirical formulas C n H 2n−4 O 6 . Also found in the extracts were three isoprene acetals (terpenoids).

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

“…This class of compounds corresponded to an earlier preliminary observation stating that human tears contain a new range of compounds with higher polarity than the nonpolar components of meibum that coelute with authentic DAGs ( 13 ). On the other hand, Ham et al ( 31 ) had also previously reported the presence of a few DAG species, such as DAG16:0/16:0 and DAG 16:0/18:0, in the human tears.…”

Resolvin D1, protectin D1, and related docosahexaenoic acid-derived products: Analysis via electrospray/low energy tandem mass spectrometry based on spectra and fragmentation mechanisms

“…[cc ϩ H] ions are generated with the shift of H from the hydroxy or hydroperoxy to the ␥ position in segment cc through a ␤-ene rearrangement if the segment cc has a ␤ double bond, an ␣ vinyl single bond, and an allylic hydrogen (for 20-HDHA and 17-HDHA) or conjugatedtriene (for 17 cc in RvD1 and PD1, 12 cc of LTB 4 [19], 15 cc of LXA 4 [19] that can facilitate the migration of electrons from the ␤ double bond to the ␥-bond and convert the ␣ vinyl single bond to an ␣ allylic single bond. Examples are ions m/z 277 from RvD1 ( Figure 1 and Scheme 3), 261 from PD1 ( Figure 2, Scheme 4), 245 from 17-HpDHA (Figure 4 and Scheme 6), 195 from LTB 4 and 251 from LXA 4 [27,29], as well as ions 285 from 20-HDHA, 245 from 17-HDHA, 205 from 14-HDHA, and 165 from 11-HDHA (Table 1, Supplemental Figure 1 and Scheme 5).…”

“…They also developed an analytical method and conducted stereochemistry studies of all the HDHAs produced by human platelets and rat brain homogenates using chiral LC-thermospray-MS and GC/MS (electron-impact ionization) [11]. Low-energy ionization primarily generates molecular (or pseudo-molecular) ions for collision-induced dissociation (CID) MS/MS analysis, through which the MS/MS spectra obtained are used widely to identify and elucidate the structures of lipid mediators derived from polyunsaturated fatty acids [12][13][14][15][16][17][18][19][20]. The lowenergy CID of eicosanoids includes charge-remote and charge-directed fragmentations [12,21], many of which occur through "␣-hydroxy-␤-ene like rearrangement" as referred to by Murphy, i.e., ␣-cleavage of the carbonOcarbon bond (␣ position to hydroxy group), facilitated by a double bond (ene) in the ␤ position [22].…”

Cholesteryl esters as a depot for very long chain fatty acids in human meibum

“…The lipid composition of TFLL is critical for maintaining the stability of the preocular tear film, deterioration of which has been associated with various ocular diseases and pathological conditions. Human meibum has been shown to have a large number of diverse lipid species, including wax esters (WE) (3, 5-7, 12, 16-20, 25, 26), cholesteryl esters (Chl-E) (3,5,7,11,12,18,20,25,26), mono-, di-, and triacyl glycerols (3, 5-7, 14, 16-18, 20, 21, 25, 26), hydrocarbons (6), free fatty acids (31), sterols (1, 3-7, 9, 11-13, 16-20), phospholipids (2,6,7,15,19,21,22,25,26), ceramides (6,21), and many other poorly identified lipid compounds. Historically, the main methods used to characterize meibomian lipids were TLC (3-5, 15, 20-22), gas-liquid chromatography (6-10, 13), and gas chromatography-electron ionization GC-MS (11, 15, 20-22, 30, 31), HPLC with UV detection (15,(20)(21)(22), infrared spectrometry (27,28), and, more recently, various types of modern mass spectrometry (MS) techniques with or without HPLC (16,17,19,23,25,26).…”