Ascorbic acid spares alpha-tocopherol and prevents lipid peroxidation in cultured H4IIE liver cells

Ascorbic acid, or vitamin C, can recycle alpha-tocopherol in lipid bilayers, but even sparing of alpha-tocopherol has not been a consistent finding in intact cells. Therefore, we tested the ability of ascorbate loading to spare alpha-tocopherol and to prevent lipid peroxidation of cultured H4IIE rat liver cells. Although alpha-tocopherol was undetectable in H4IIE cells, its cell content was increased by overnight incubation with alpha-tocopherol in culture. Cells incubated with ascorbate 2-phosphate accumulated ascorbate to concentrations as high as 0.6 mM after overnight loading, but also released ascorbate into the medium. Ascorbate loading of alpha-tocopherol-treated cells spared alpha-tocopherol in a concentration-dependent manner during overnight incubation. Lipid peroxidative damage, measured as a decrease in fluorescence of cell-bound cis-parinaric acid, was decreased in cells loaded with either alpha-tocopherol or ascorbate 2-phosphate, and showed an additive effect. These results suggest that ascorbate loading of H4IIE cells spares cellular alpha-tocopherol and either directly or through recycling of alpha-tocopherol prevents lipid peroxidative damage due to oxidant stress in culture.