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B.J. Glasgow, O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov; Tear Lipocalin (TL) Scavenges Lipids From the Corneal Surface . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2421.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The cornea is coated with membrane spanning and secreted mucins that create a hydrophilic surface. Apical shedding of squamous epithelium and ectodomain shedding of mucins as well as minor trauma may leave areas of corneal epithelium exposed (Dartt DA, Exp Eye Res 2004;78:173–185, Gipson IK, Exp Eye Res 2004;78:379–388). Lipid contamination of these areas could create an unwettable surface. TL the principle lipid binding protein in tears, has been shown to scavenge lipids from hydrophobic surfaces (IOVS 1999;40:3100–7). The hypothesis that TL can remove contaminating fatty acids and phospholipids from the human corneal surface was tested. Methods: TL was purified from pooled human tear samples by size exclusion and ion exchange chromatographies (ibid). Tears without TL were reconstituted from eluted fractions of Sephadex G–100 (ibid). SDS PAGE confirmed the resultant mixture was depleted of TL. Fresh and formalin–fixed human eyes, were obtained from exenteration specimens. Fluorescent analogs, 5.3 mM, of fatty acid (16–(9–anthroyloxy)palmitic acid, 16AP) or phospholipid (NBDC6 HPC), Molecular Probes, were applied uniformly to the corneal epithelial surface. Excess lipid was removed by gentle rinsing in buffer (10mM sodium phosphate, 100 mM NaCl, pH 7.4). The corneas, in quartz cuvettes were overlayed with solutions of buffer, tears, TL (70 µM), or tears depleted of TL. Entry of 16AP bound to protein in solution was monitored by fluorescence, em=450nm, (ex=361nm), until saturation was achieved. For NBDC6HPC, ex=420nm, em=524 nm. Tears used in the experiments were fractionated by size exclusion to determine the component of tears associated with fluorescence. Histopathology was performed to verify the presence of corneal epithelium. Results: 16AP and NBDC6HPC demonstrate enhanced fluorescence and a blue shift of the emission peak when protein is bound. Significant enhancement of fluorescence for 16AP and NBDC6HPC was evident in solutions incubated with whole tears and purified TL but not with tears depleted of TL for fixed corneas. Similar results were obtained for both lipids in fresh human corneas. After the experiment, size exclusion fractions of tears showed that the fluorescence component co–eluted with TL. Conclusions: Lipids may stick to the cornea surface in which mucins are depleted. These data provide evidence that TL, a potent binding protein, scavenges lipids from the human corneal surface and delivers them into aqueous phase of tears. TL may have an important role in removing lipids from the cornea surface where mucins are depleted to maintain the wettability and integrity of the ocular surface.
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