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M.C. Yappert, R. Estrada, L. Huang, D. Borchman, A. Hitt, J.R. Reddan; Choline–Phospholipid Biogenesis in Human Lens Epithelial Cells by MALDI–MS . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1863.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The current work was designed to test the ability of MALDI–MS to track the synthesis of both phosphatidylcholines (PCs) and sphingomyelins (SMs) in human lens epithelial cells, and to provide information on the relative rates of biogenesis of these two phospholipids (PLs). Methods: The human lens epithelial cell line FHL124 was cultured in a mixture of KGM and M199 (1:4), 0.050 mg/ml gentamicin and 10% fetal bovine serum. Control experiments were carried out with and without addition of exogenous choline (0.10 mM). A similar protocol was followed using deuterated (d–9) choline. Phospholipids were analyzed after total confluence was reached both in situ and in vitro, after extraction with methanol. Two matrices, 2,5–dihydroxybenzoic acid and para–nitroaniline were used to acquire MALDI–TOF positive–and negative–ion mass spectra, respectively. Results:Positive–ion spectra acquired in situ and in vitro showed PCs with multiple acyl chain lengths but the most abundant was PC(34:1). Only SM(16:0) was detected in significant quantities. Negative–ion spectral data revealed the presence of significant amounts of phosphatidylinositols (PIs), particularly PI(38:4), as well as phosphatidylethanolamines and corresponding plasmalogens with high levels of unsaturation. Phosphatidylserines (PSs), such as PS(36:1) and PS(34:1), were also detected. When the cells were supplemented with d–9 choline, the newly synthesized lipids could be readily distinguished because their masses differ by 9 Da from the non–deuterated PLs. The ratio of deuterated versus non–deuterated PCs was twice higher than that measured for SMs. The slower uptake of exogenous choline by SMs suggests that the transfer of the choline from PCs to SMs may control the rate of synthesis of SMs. Conclusions: This study demonstrates the power of MALDI–MS as a tool for tracking the synthesis of PLs, even in intact cells and without radioactive labels. This methodology can be applied to investigate the possible roles of PLs and their metabolites during mitosis, differentiation and senescence of lens epithelial cells.
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