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Yousef A Alghamdi, Mitchell Martinez, Faisal Khattab, Sanjoy K Bhattacharya, Richard K Lee; Secreted Phospholipids of the Organ Cultured Cornea, Iris and Lens. Invest. Ophthalmol. Vis. Sci. 2014;55(13):391.
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To compare different subtypes of phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol,) secreted by organ cultured cornea, iris, and lens from human donors.
Human donor eyes were collected from the Lions Eye Bank, Miami, Florida adhering to the Tenets of the Declaration of Helsinki. Eyes were carefully dissected to isolate lens, cornea and iris which were incubated in serum free culture medium for up to 72 hours. Secreted lipids in the culture media was collected every 12 hours and stored at -80C until further use. Total collected media after incubation was subjected to lipid extraction using the Bligh and Dyer method. All organ cultures were also subjected to an assessment of trypan blue exclusion cell viability assay and assessment of pyruvate kinase activity at the end of incubation period. Cultured organs with less than 90% viability were excluded from further lipid analyses. Phospholipids were identified and quantified using established class specific mass spectrometry settings (ion mode and collision energy) with appropriate class-specific lipid standards on a TSQ Quantum Access Max mass spectrometer. A Triversa Nanomate was used for direct infusion with optimized parameters.
Organ cultured secretions of cornea, iris and lens contain all four classes of phospholipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine albeit to varying quantities. In the secretions of these organ cultures, most notable was the low presence of phosphatidylcholine as a percent of total phospholipids. On a relative basis, the amount of secreted lipids were approximately 10 fold less than that assayed directly from the tissues or isolated cells from these tissues.
All four classes of phospholipids were found in organ culture secretions of cornea, iris and lens with phosphatidylcholines being the lowest by relative amounts to other phospholipid classes.
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