RESOLUTION OF THE FACILITATED TRANSPORT OF DEHYDROASCORBIC ACID FROM ITS INTRACELLULAR ACCUMULATION AS ASCORBIC-ACID

We performed a detailed kinetic analysis of the uptake of dehydroascor bic acid by HL-60 cells under experimental conditions that enabled the differentiation of dehydroascorbic acid transport from the intracellu lar reduction/accumulation of ascorbic acid. Immunoblotting and immuno localization experiments identified GLUT1 as the main glucose transpor ter expressed in the HL-60 cells. Kinetic analysis allowed the identif ication of a single functional activity involved in the transport of d ehydroascorbic acid in the HL-60 cells. Transport was inhibited in a c ompetitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars. A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated w ith the intracellular reduction of dehydroascorbic acid to ascorbic ac id and is not directly involved in the transport of dehydroascorbic ac id via GLUT1. Transport of dehydroascorbic acid by HL-60 ells was inde pendent of the presence of external Na+, whereas the intracellular acc umulation of ascorbic acid was found to be a Na+-sensitive process. Th us, the transport of dehydroascorbic acid via glucose transporters is a Na+-independent process which is kinetically and biologically separa ble from the reduction of dehydroascorbic acid to ascorbic acid and it s subsequent intracellular accumulation.