Abstract

Human myeloid leukemia cells (HL-60) transport only the oxidized form of vitamin C (dehydroascorbic acid) and accumulate the vitamin in the reduced form, ascorbic acid. We performed a detailed study of the role of glutathione in the intracellular trapping/accumulation of ascorbic acid in HL-60 cells. Uptake studies using HL-60 cells depleted of glutathione by treatment with L-buthionine-(S,R) sulfoximine and diethyl maleate, revealed no changes in the cells' ability to transport dehydroascorbic acid and accumulate ascorbic acid. Similar transport and accumulation rates were obtained using HL-60 cells containing intracellular glutathione concentrations from 6 mM to 1 microM. HL-60 cells, containing as little as 5 microM glutathione, were able to accumulate up to 150 mM ascorbic acid intracellularly when incubated with dehydroascorbic acid. Glutathione was capable of reducing dehydroascorbic acid by a direct chemical reaction, but only when present in a greater than 10-fold stoichiometric excess over dehydroascorbic acid. The accumulation of ascorbic acid by HL-60 cells was strongly temperature-dependent and was very inefficient at 16 degrees C. On the other hand, the direct chemical reduction of dehydroascorbic acid by excess glutathione proceeded efficiently at temperatures of 16 degrees C. Our data indicate that glutathione-dependent reductases in HL-60 cells are not responsible for the ability of these cells to accumulate millimolar concentrations of ascorbic acid. These findings indicate that alternative enzymatic mechanisms are involved in the cellular reduction of dehydroascorbic acid.