Meibomian gland dysfunction (MGD) is a commonly diagnosed ophthalmic condition that contributes to dry eye disease. The meibum secretion itself constitutes the external lipid layer of the tear film that inhibits evaporation of the internal aqueous layer. Among the varied lipids identified in human meibum, free fatty acids (FAs) have been shown to differentially express in MGD-affected patients. (O-acyl)-w-hydroxyFAs (OAHFAs), which downregulate as dry eye disease becomes more severe, have even been posited as potential biomarkers for MGD.
In a recent study, Mori et al. (2014) investigated an alternative rapid identification method because gas chromatography is unsuitable for the analysis of meibum lipids, which lack the volatility required for conventional extraction and derivatization techniques.1 To do this, the research team manufactured in-house OAHFA standards and collected human meibum samples from 82 patients reporting clinical indicators of MGD. Using a Dionex Ultimate 3000 liquid chromatography system, they separated FAs and OAHFAs for spectral analysis by mass spectrometry (MS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). For platform control, they relied upon Xcalibur software (version 2.2, Thermo Scientific). The researchers used full-scan MS (m/z 180 to 550) for FA analytes and full-scan MS (m/z 400 to 1,000), followed by data-dependent MS, for OAHFA analytes. They turned to LCQUAN Quantitative software (version 7.2, Thermo Scientific) to profile FAs based on retention time and theoretical accurate mass, and the Lipid Search database to identify OAHFAs.
Using the compiled data, the team semi-quantitatively revealed the variety of FAs and OAHFAs present in MGD-affected human meibum. Overall, they identified 161 molecular species. Of these, 100 were long-chain FAs containing 12 to 37 carbons, which is a marked increase in comparison with other detection methods. The remaining 61 species were long-chain OAHFAs containing 34 to 56 carbons. The researchers note that a wider mass range could result in identifications beyond the 37-carbon threshold for FAs but indicate that this study is the first report of large numbers of very-long-chain species in human meibum. Given the rare nature of species longer than 35 carbons in blood, skin or cell samples, this finding confirms the unusual lipid composition of meibum. Interestingly, the team also detected odd-numbered carbon-chain FAs, which previously have been linked to bacteria. Mori et al. recommend further investigation into a potential link between this finding and possible bacterial infection in the eyelids of MGD patients.
Because human meibum is a complex matrix with no perfect internal standard available, a stable, sensitive platform is imperative for gathering semi-quantitative data. For this reason, the team injected standard solution before and after each of the 82 samples, resulting in analysis times of 12 and 15 hours for FAs and OAHFAs, respectively. They calculated the area value for each species in order to ascertain the stability and sensitivity of the Q Exactive mass spectrometer. They observed that the quadrupole filter was effective for preventing interference, allowing the team to target the selected mass range for both types of analytes. They also note that there was no distinguishable shift in retention time over the course of the study. The results demonstrate that, despite the lack of internal standard, it was possible to compare the lipid response of each analyte between samples, indicating excellent sensitivity and stability in the platform.
Mori et al. offer this rapid identification method with broad applications for lipid profiling. They propose further studies be conducted to reveal the precise quantification of FAs and OAHFAs in both MGD-affected and normal meibum samples. They also posit that further development of their method could enable research into the ways that aging, disease and nutritional variation influence the lipid composition of human meibum.
1. Mori, N., et al. (2014) “Rapid identiﬁcation of fatty acids and (O-acyl)–hydroxy fatty acids in human meibum by liquid chromatography/high-resolution mass spectrometry,” Journal of Chromatography A, 1347 (pp. 129–36).
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.