Role of ascorbic acid in transferrin-independent reduction and uptake of iron by U-937 cells

The role of ascorbic acid in transferrin-independent ferric iron reduction and uptake was evaluated in cultured U-937 monocytic cells. Uptake of Fe-55 by U-937 cells was doubled by 100 mu M extracellular ascorbate, and by pre -incubation of cells with 100 mu M dehydroascorbic acid, the two-electron-o xidized form of ascorbate. Reduction of extracellular ferric citrate also w as enhanced by loading the cells with dehydroascorbic acid. Dehydroascorbic acid was taken up rapidly by the cells and reduced to ascorbate, such that the latter reached intracellular concentrations as high as 6 mM. However, some ascorbate did escape the cells and could be detected at concentrations of up to 1 mu M in the incubation medium. Further, addition of ascorbate o xidase almost reversed the effects of dehydroascorbic acid on both Fe-55 up take and ferric citrate reduction. Thus, it is likely that extracellular as corbate reduced ferric to ferrous iron, which was then taken up by the cell s. This hypothesis also was supported by the finding that during loading wi th ferric citrate, only extracellular ascorbate increased the pool of intra cellular ferrous iron that could be chelated with cell-penetrant ferrous ir on chelators. In contrast to its inhibition of ascorbate-dependent ferric i ron reduction, ascorbate oxidase was without effect on ascorbate-dependent: reduction of extracellular ferricyanide. This indicates that the cells use different mechanisms for reduction of ferric iron and ferricyanide. Theref ore, extracellular ascorbate derived from cells can enhance transferrin-ind ependent iron uptake by reducing ferric to ferrous iron, but intracellular ascorbate neither contributes to this reduction nor modifies the redox stat us of intracellular free iron. BIOCHEM PHARMACOL 57;11:1275-1282, 1999. (C) 1999 Elsevier Science Inc.