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Anne McMahon, Igor A. Butovich; Toward Quantitation and Characterization of Ceramides in Human Meibum and Tear Film. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3741.
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© ARVO (1962-2015); The Authors (2016-present)
Hyperkeratinization of Meibomian gland ducts, observed in certain types of dry eye disease (DE), could change meibum composition, in part due to increased production of ceramides (Cer). However, neither the normal levels of Cer in healthy meibum, nor their chemical structures, have been reported to date. Thus, the goals of this study were to evaluate the Cer presence in normal meibum, and, should any Cer be found, determine their normal concentration ranges.
Samples of human meibum were collected from healthy volunteers with no signs of any ocular disorders. Meibum was dissolved in a chloroform-methanol=2:1 (vol/vol) solvent mixture to produce a 0.1mg/mL sample stock solution. Samples were analyzed using high performance liquid chromatography with mass spectrometric detection of analytes (HPLC-MS). Reverse phase (RP) and normal phase (NP) HPLC were both used in the analyses. The NP separation of lipids was achieved on a Phenomenex’s Diol column in a hexane-iso-propanol-acetic acid solvent mixture. The RP HPLC was conducted using a Hypersil Gold C18 column and an iso-propanol:acetonitrile:ammonium formate solvent mixture. Cer were monitored in positive ion mode. Lipid samples extracted from mouse skin were used as positive controls for Cer and their more complex derivatives. The analytes were quantified using calibration curves obtained for authentic standards of typical Cer (Sigma product C-2137 and authentic standards of individual Cer from various manufacturers).
Authentic Cer were mostly detected as (M - H2O + H)+ ions. The instrument response was found to be linear in a range from, at least, 5 ng Cer to 1000 ng Cer/injection. The low limit of detection (LLOD) of Cer in these experiments was in the sub-nanogram range. Meibum amounts up to 11,000 ng/injection were analyzed. Skin Cer of various kinds were observed and structurally characterized. Those included normal and acylated ceramides, among others. Meibomian samples showed no Cer in amounts above LLOD, even at the highest column loads (11µg meibum/injection).
Thus, Cer are not among the normally detectable components of meibum from healthy persons. Their content in normal meibum, if any, was estimated to be well below 0.01% (w/w), i.e. fewer than one molecule of Cer per 10,000 (or so) molecules of other meibomian lipids. It remains to be seen if this ratio goes up in patients with hyperkeratinized Meibomian gland ducts. The data further shows that there is no significant contribution of ceramides from other ocular surfaces to meibum in healthy patients.
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