GM-CSF increases the accumulation of ascorbic acid by HL-60 neutrophils. (A) HPLC separation of dehydroascorbic acid generated by treating a sample of ascorbic acid with ascorbic acid oxidase. The arrows indicate the elution positions of dehydroascorbic acid (DHA) and ascorbic acid (AA). (B) HPLC separation of a commercial preparation of ascorbic acid after treatment with 1 mmol/L DTT. (C) HPLC separation of a cellular extract from cells incubated with 100 μmol/L dehydroascorbic acid. (D) HPLC separation of a cellular extract from cells incubated with 10 mmol/L dehydroascorbic acid. Data (A through D) represent the result of one of three similar experiments. (E) Accumulation of ascorbic acid. Cells were incubated for 30 minutes in the absence (○) or in the presence (•) of 0.5 nmol/L GM-CSF and afterwards the accumulation of ascorbic acid as a function of different concentrations of dehydroascorbic acid was measured for 10 minutes. Data are expressed as the ratio of the intracellular concentration of ascorbic acid to the extracellular concentration of dehydroascorbic acid. Data represent the mean ± SD of four samples and correspond to one of three similar experiments. (F) Double reciprocal plot of the substrate dependence for ascorbic acid accumulation. Cells were incubated for 30 minutes in the absence (○) or in the presence (•) of 0.5 nmol/L GM-CSF and accumulation of ascorbic acid as a function of different concentrations of dehydroascorbic acid was measured at 10 minutes. Data represent the mean of four samples and correspond to one of three similar experiments.