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Lack of antioxidative properties of vitamin C and pyruvate in cultured retinal pigment epithelial cells

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Abstract

Background

Oxidative damage to the retinal pigment epithelium might be involved in the pathogenesis of age related macular degeneration. Thus antioxidative protection represents a rationale for a causative therapy or prophylaxis. The aim of the present study is to evaluate antioxidative properties of vitamin C and pyruvate at retinal pigment epithelial (RPE) cells exposed to oxidative stress.

Methods

The ability of vitamin C and pyruvate to quench hydroxyl radicals was tested using the di-hydro-rhodamine (DHR) assay. Cells of the human RPE cell line ARPE-19 were exposed for 8 min to hydroxyl radicals generated by the Fenton reaction from 2.25 mM H2O2 and 30 μM Fe3+-nitrilo-tri-acetate. This was done in the absence and presence of 0.3–3.0 mM pyruvate and vitamin C, respectively. Cell survival was analysed by vitality staining (life-dead-assay) and expressed as cell survival ratio. A survival ratio <1.0 indicates cell loss.

Results

At concentrations from 0.1 to 1.0 mM vitamin C and pyruvate quench hydroxyl radicals in the DHR assay in absence of living matter. In the presence of 0.1– 0.3 mM vitamin C and pyruvate, ARPE-19 showed a reduced survival ratio (0.87±0.01 to 0.89±0.02 after 6 h) which was not the case at the higher concentrations between 1 and 3 mM. The exposure of ARPE-19 cells to hydroxyl radicals reduced the survival ratio to 0.92±0.02. At concentrations at which vitamin C and pyruvate exert toxic effects, a potentiation of radical induced cell death can be observed (survival ratio 0.79±0.02 and 0.82±0.03, respectively). Higher concentrations of vitamin C or pyruvate had no explicit protective effect to the hydroxyl radical induced damage.

Discussion

Although vitamin C and pyruvate are potent hydroxyl radical quenchers in vitro they failed to protect cultured ARPE-19 cells from oxidative stress induced cell death. In contrast, when applying the scavengers at low concentrations a potentiation of cell damage was observed.

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Acknowledgements

This work was supported by a grant (F-135-1) from the Forschungsförderungsfond Medizin from the Medizinsiche Fakultät, Universitätsklinikum Hamburg-Eppendorf (O. Zeitz) and the Deutsche Forschungsgemeinschaft DFG STR 480/8-2 (O-Strauss).

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Authors and Affiliations

  1. Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde, Martinistr. 52, 20246, Hamburg, Germany

    Oliver Zeitz & Gisbert Richard

  2. Universitätsklinikum Hamburg-Eppendorf, Experimentelle Ophthalmologie, Hamburg, Germany

    Oliver Zeitz, Lars Schlichting & Olaf Strauß

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  1. Oliver Zeitz
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  2. Lars Schlichting
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  3. Gisbert Richard
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  4. Olaf Strauß
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Correspondence to Oliver Zeitz.

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Oliver Zeitz and Lars Schlichting contributed equally.

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Zeitz, O., Schlichting, L., Richard, G. et al. Lack of antioxidative properties of vitamin C and pyruvate in cultured retinal pigment epithelial cells. Graefe's Arch Clin Exp Ophthalmol 245, 276–281 (2007). https://doi.org/10.1007/s00417-006-0384-5

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  • DOI: https://doi.org/10.1007/s00417-006-0384-5

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