Specificity of ascorbate analogs for ascorbate transport - Synthesis and detection of [I-125]-6-deoxy-6-iodo-1-ascorbic acid and characterization of its ascorbate-specific transport properties

Cellular ascorbic acid accumulation occurs in vitro by two distinct mechani sms: transport of ascorbate itself or transport and subsequent intracellula r reduction of its oxidized product, dehydroascorbic acid. It is unclear wh ich mechanism predominates in vivo, An easily detectable compound resemblin g ascorbate but not dehydroascorbic acid could be a powerful tool to distin guish the two transport activities. To identify compounds, 21 ascorbate ana logs were tested for inhibition of ascorbate or dehydroascorbic acid transp ort in human fibroblasts. The most effective analogs, competitive inhibitor s of ascorbate transport with K-i values of 3 mu M, were 6-deoxy-6-bromo-, 6-deoxy-6-chloro-, and 6-deoxy-6-iodo-L-ascorbate, No analog inhibited dehy droascorbic acid transport. Using substitution chemistry, [I-125]6-deoxy-6- iodo-L-ascorbate (1.4 x 10(4) mCi/mmol) was synthesized. HPLC detection met hods were developed for radiolabeled and nonradiolabeled compounds, and tra nsport kinetics of both compounds were characterized. Transport was sodium- dependent, inhibited by excess ascorbate, and similar to that of ascorbate, Transport of oxidized ascorbate and oxidized 6-deoxy-6-iodo-L-ascorbate wa s investigated using Xenopus laevis oocytes expressing glucose transporter isoform GLUT1 or GLUT3, Oxidation of ascorbate or its analog in media incre ased uptake of ascorbate in oocytes by 6-13-fold compared with control but not that of 6-deoxy-6-iodo-L-ascorbate. Therefore, 6-deoxy-6-iodo-L-ascorba te, although an effective inhibitor of ascorbate transport, either in its r educed or oxidized form was not a substrate for dehydroascorbic acid transp ort. Thus, radiolabeled and nonradiolabeled 6-deoxy-6-iodo-L-ascorbate prov ide a new means for discriminating dehydroascorbic acid and ascorbate trans port in ascorbate recycling.