Glutathione Improves the Antioxidant Activity of Vitamin C in Human Lens and Retinal Epithelial Cells: Implications for Vitreous Substitutes.

ABSTRACT

PURPOSE: Tissues in the eye are particularly susceptible to oxidative damage due to light exposure. While vitamin C (ascorbic acid) has been noted as a vital antioxidant in the vitreous humor, its physiological concentration (1-2 mM) has been shown to be toxic to retinal and lens epithelial cells in in vitro cell culture. We have explored adding vitamin C to hydrogel vitreous substitutes as a potential therapeutic to prevent oxidative damage to intraocular tissues after vitrectomy. However, vitamin C degrades rapidly even when loaded at high concentrations, limiting its long-term effectiveness. Glutathione, another antioxidant found abundantly in the lens at concentrations of 2-10 mM, was proposed to be used in conjunction with vitamin C. METHODS: Cell viability and reactive oxygen species activity of human retinal and lens epithelial cells treated with various combinations of vitamin C, glutathione, hydrogen peroxide, and a hydrogel vitreous substitute were determined using CellTiter-Glo luminescent cell viability assay and dichlorofluorescein assay, respectively. The vitamin C remaining in hydrogel vitreous substitute or glutathione-vitamin C solutions was determined using a microplate reader at 265 nm wavelength, compared against standard solutions with known concentrations. RESULTS: Glutathione protected the lens and retinal cells from the negative effect of vitamin C on cell viability and prolonged the antioxidant effect of vitamin C in vitro. While the detected reading of pure vitamin C solution decreased rapidly from 100% to 10% by 3 days, glutathione provided a significant extension to vitamin C stability, with 70% remaining after 14 days when the glutathione was used at physiological concentrations found in the lens (2-10 mM). CONCLUSIONS: These results indicate glutathione might be an effective addition to vitamin C in intraocular implants, including potential vitreous substitutes, and warrants additional studies on the effectiveness of the vitamin C - glutathione combination in preventing oxidative stress post-vitrectomy.