EFFICIENT TRANSPORT AND ACCUMULATION OF VITAMIN-C IN HL-60 CELLS DEPLETED OF GLUTATHIONE

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 glu tathione by treatment with L-buthionine-(S,R) sulfoximine and diethyl maleate, revealed no changes in the cells' ability to transport dehydr oascorbic acid and accumulate ascorbic acid. Similar transport and acc umulation rates were obtained using HL-60 cells containing intracellul ar glutathione concentrations from 6 mM to 1 mu M. HL-60 cells, contai ning as little as 5 mu M glutathione, were able to accumulate up to 15 0 mM ascorbic acid intracellularly when incubated with dehydroascorbic acid. Glutathione was capable of reducing dehydroascorbic acid by a d irect chemical reaction, but only when present in a greater than 10-fo ld stoichiometric excess over dehydroascorbic acid. The accumulation o f ascorbic acid by HL-60 cells was strongly temperature-dependent and was very inefficient at 16 degrees C. On the other hand, the direct ch emical reduction of dehydroascorbic acid by excess glutathione proceed ed efficiently at temperatures of 16 degrees C. Our data indicate that glutathione-dependent reductases in HL-60 cells are not responsible f or the ability of these cells to accumulate millimolar concentrations of ascorbic acid. These findings indicate that alternative enzymatic m echanisms are involved in the cellular reduction of dehydroascorbic ac id.