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Jianzhong CHEN, Kelly K Nichols, Jason J Nichols; Untargeted Mass Spectrometry Analysis of Lipids in Human Tears. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3285. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The tear film lipid layer (TFLL) is thought to reduce evaporation of tears. Alterations to the TFLL may lead to evaporative dry eye. However, tear film lipid composition, compared to meibum analysis, remains controversial. To better understand the TFLL composition, we analyzed the lipidome of human tears collected using two methods with an untargeted mass spectrometry approach.
Tears from individual eyes of ten subjects were collected into microcapillary tubes of either glass or polytetrafluoroethylene (PTFE). Lipids of tears were extracted with the Folch method by mixing tears with chloroform, methanol, and water in the ratio of 8:4:3. The lower phase of the mixture, which contained lipids, was further diluted with an equal volume of methanol along with an additive of ammonium hydroxide. The resultant solutions were analyzed on a high-resolution mass spectrometer (TripleTOF 5600, SCIEX, MA) by direct infusion and electrospray ionization. MS and MS/MSall data were acquired in positive and negative ion modes.
The lipid profiles between the two collection methods are consistent, although the signal-to-noise ratios for lipid peaks were much higher (more lipids) for tear samples collected with PTFE tubes. The tear lipids detected in MS include wax esters, cholesteryl esters, diesters, triacylglycerols, O-acyl-ω-hydroxyl fatty acids, consistent with meibum. In addition, phosphatidylcholines (PCs) and sphingomyelins (SMs) were detected in MS/MSall. Interestingly, the normalized peak intensities for PCs and SMs appeared to be much higher for tears collected in glass tubes compared to PTFE tubes, typically more than ten-fold. These PCs and SMs were probably present predominantly in the aqueous layer instead of the TFLL since glass tubes collected a much greater volume of the aqueous layer due to the hydrophilic (water-loving) nature of glass. In contrast, PTFE is hydrophobic (water-hating) and therefore the PTFE tubes collected a much lower volume of tears (typically < 0.1 µL) and a higher ratio of lipids, primarily hydrophobic lipids with a negligible aqueous portion of the tears.
Lipid collection and analysis from tears is augmented with PTFE tubes, yet differences in the lipidome indicate PCs and SMs are likely negligible in the TFLL and could reside in the aqueous/protein-lipid carrier component of the tears.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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