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J. C. Wojtowicz, M. Molai, I. A. Butovich; Comparative Lipidomic Analysis of Mono- and Polyunsaturated Lipids Found in Human Meibum and Aqueous Tears Using HPLC-UV/MS/MS. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4255.
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© ARVO (1962-2015); The Authors (2016-present)
The human ocular surface is protected by tear film (TF) and its outermost part - TF lipid layer (TFLL). TFLL is believed to be largely formed from Meibomian gland (MG) secretions (meibum). However, the overall compositions of TF and TFLL are not well established. Thus, the goal of our project was to compare the nonpolar lipids present in meibum (samples T1) with aqueous tears (AT) collected from the lower tear menisci of normal volunteers using microcapillaries (T2) and Schirmer test strips (T3).
Samples T1 were squeezed from MG and collected using a spatula. Up to 1 mg of dry meibum/volunteer was collected. Samples T2 were ~10 µL/volunteer. Quantitation of samples T3 was done gravimetrically and by Schirmer scaling (on average, 9 mg/eye, wet weight, and 17 mm, wetting distance). Intact lipid species were analyzed by HPLC-UV/MS/MS. Their fatty acid (FA) content was evaluated after transesterification of the entire lipid pool.
Samples T2 and T3 were similar to each other, but fairly different from samples T1. The lipid composition of samples T1 demonstrated very little inter-donor variability. Samples T1 and T3 were compared and found to be similar, except for the substantially larger presence of free cholesterol (Ch) and lower molecular weight (e.g. C14 to C20 FA-based) wax and Ch esters (WE and CE) in samples T2 and T3. Structural assignments of the compounds of WE and CE groups were confirmed by analyzing their transesterification products. Interestingly, WE in all of the samples had very long chain C16 to C30 fatty alcohols (FAl) and mostly C18-type FA, while FA of CE ranged from C18 to C34. The observed degree of unsaturation of the FA and FAl did not exceed 4 no matter the chain length. An attempt was made to identify their oxidized derivatives by using HPLC-MS in combination with UV-Vis spectrophotometry. A range of oxidation products was observed in samples T1 and T3. Some of the compounds were identified as hydroxylated polyunsaturated FA (PUFA), e.g. linolenic acid, and hydroxylated derivatives of PUFA-based WE. No epoxy-derivatives of nonpolar lipids were found in the samples.
Meibum is a source of very long chain unsaturated FA and FAl-based lipids for the TF. High content of low-melting mono- and polyunsaturated lipids in meibum and AT is, apparently, needed to maintain proper fluidity of TF. Our data demonstrate that nonpolar lipids in both AT and meibum may undergo lipid peroxidation. The presence of oxidized lipids in freshly expressed meibum suggests that their oxidation happens, at least in part, in a controlled environment inside MG, and may continue after they have been excreted from MG onto the ocular surface.
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