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

Abstract

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.