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C. Beadle, R.W. Browne, P.E. Spoerri, A. Ozarowski, J. Busik, S. Mohr, D. Armstrong, M.B. Grant; Free Radical Derived Oxidants, Lipid Hydroxyperoxides, Lipid Hydroxides and Polyunsaturated Fatty Acids in Response to High and Low Glucose in Cultured Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3890.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Hyperglycemia is implicated in altered retinal vascular function associated with diabetic retinopathy. Oxidative stress is an increase in the steady state levels of free radicals and may result from increased generation and/or decreased antioxidant defense mechanisms. In this study, we measured generation of glucose-induced free radical derived oxidants (FRDO) and identified specific lipid hydroperoxides (LHP), lipid hydroxide (LOH) metabolites and polyunsaturated fatty acids (PUFA) using HPLC. We used human retinal endothelial cells (HRECs) and microvascular porcine endothelial cells (PECs) grown in high and low glucose (GLU) and compared to galactose (GAL) or fructose (FRU) medium. Methods: Free radicals were assayed in cells exposed to low (5mM) and high (25mM) glucose for 7 days. FRDO was assayed by fluorescence spectroscopy and by Electron Spin Resonance (ESR) using TEMPO-am, TEMPO, MGD, and DMPO. For identification of specific LHP, LOH metabolites and PUFA precursor substrates, cell pellets from PECs were extracted and the lipids separated by HPLC. LHP and LOH were monitored at 234-236 nm and PUFA at 215 nm using UV/Vis photoiode array technology. Results: Intracellular radical production from HRECs and PECs in high and low GLU media were similar. FRDO signals were observed after low and high GLU and were similar. Linoleic acid (18:2) was the major PUFA in each cell type, and increased after GLU and GAL. No LHP were detected in either cell type. HRECs produced 13-HpODE and PECs produced 9-HpODE regioisomer. FRU had no effect. Conclusion: HRECs and microvascular PECs in vitro have similar FRDO levels and lipid peroxidation profiles for low and high GLU. There were species differences in the PUFA/LHP/LOH profiles and especially following GLU or GAL. Radicals generated within endothelial cells by GLU or GAL, damaged membrane lipids and crucial endothelium protective mechanisms could reflect differences in uptake, metabolism and/or level of antioxidant defense systems. Increased levels of aqueous/lipid soluble antioxidants, as well as enzymes such as superoxide dismutase , catalase and glutathione peroxidase may protect the retina from sudden increases in oxidative stress.
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