Purpose: To establish whether the oxidation of ascorbic acid (AA) in the aqueous humor may contribute to maintain the high concentration of AA of the anterior eye tissues during oxidative stress.
Methods: Primary cultures of bovine lens epithelial cells (BLEC) were incubated in a medium with the concentration of AA, glutathione (GSH), and cysteine (Cys) found in the aqueous humor. The intracellular concentration of AA was measured over time and compared to that of cultures maintained in the same medium but exposed to UV light.
Results: The uptake of extracellular AA by BLEC was 0.09+/-0.01 nmol/mg protein/min, but rose to 0.53+/-0.02 nmol/mg protein/min when cell cultures were exposed to a dose of UV-C light (178 microW/cm2) capable of oxidizing extracellular AA but not intracellular AA. Under the same conditions, intracellular AA was oxidized when cell monolayers were treated with 1,3-bis(2-Chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase and thioredoxin reductase. The uptake of the oxidized form of AA, dehydroascorbic acid (DHAA), was approximately 7 times quicker than that of AA; the uptake and intracellular reduction of DHAA to AA was inhibited by depleting intracellular GSH. Extracellular glucose inhibited the uptake of DHAA by BLEC, and the extent of this inhibition was dependent on the concentration of extracellular glucose.
Conclusions: During mild oxidative stress, AA contained in aqueous humor may provide tissues of the anterior eye with a source of DHAA which is readily taken up and converted to intracellular AA, while intracellular AA is maintained in the reduced status by intracellular antioxidant systems. Since glucose inhibited DHAA uptake, this protective mechanism could be impaired in diabetes.