Human tear fluid lipidome: from composition to function

Abstract

We have explored human aqueous tear fluid lipidome with an emphasis to identify the major lipids. We also address the physiological significance of the lipidome. The tears were analysed using thin layer chromatographic, enzymatic and mass spectrometric techniques. To emphasize the physiological aspect of the lipidome, we modelled the spreading of the non-polar tear fluid lipids at air-water interface in macroscopic scale with olive oil and egg yolk phosphatidylcholine. Based on enzymatic analysis the respective concentrations of choline-containing lipids, triglycerides, and cholesteryl esters were 48±14, 10±0, and 21±18 µM. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry analysis showed that phosphatidylcholine and phosphatidylethanolamine were the two most common polar lipids comprising 88±6% of all identified lipids. Triglycerides were the only non-polar lipids detected in mass spectrometric analysis i.e. no cholesteryl or wax esters were identified. The spreading experiments show that the presence of polar lipids is an absolute necessity for a proper spreading of non-polar tear fluid lipids. We provide evidence that polar lipids are the most common lipid species. Furthermore, we provide a physiological rationale for the observed lipid composition. The results open insights into the functional role of lipids in the tear fluid and also aids in providing new means to understand and treat diseases of the ocular surface.

Figure 1. Western blot showing the negative cellular contamination of the tear samples.

Four differing tear fluid pools P1–P4 were applied into the wells of SDS-PAGE gel. Equal amount of protein was loaded in each well. CEC, corneal epithelial cell lysate.

Figure 2. TLC of non-polar lipids.

TF, tear fluid; Std, standard solution containing free cholesterol (FC), triglycerides (TG), and cholesteryl oleate (CE). The arrow shows the direction of the eluent flow.

Figure 3. Two-dimensional map view of UPLC-MS data.

The first dimension shows the retention times of the separated lipids and the second dimension reveals the detected lipid ions.

Figure 4. Molar percentage of the five most abundant lipid classes among the identified tear fluid lipids.

The proportions are based on the UPLC-MS data. Cer, Ceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; TG, triglyceride.

Figure 5. A photo sequence of olive oil spreading.

(A) Blank mica and (B) eggPC monolayer on mica at π = 30 mN/m. Time points indicate the reading of the contact angles in second-scale. See Materials and Methods for more details.

Figure 6. Spreading of olive oil: contact angle vs. time.

(▪) Blank mica and (□) eggPC monolayer on mica at π = 30 mN/m. See Materials and Methods for more details.