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J. Biol. Chem., Vol. 280, Issue 7, 5211-5220, February 18, 2005

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6-Bromo-6-deoxy-L-ascorbic Acid

AN ASCORBATE ANALOG SPECIFIC FOR Na⁺-DEPENDENT VITAMIN C TRANSPORTER BUT NOT GLUCOSE TRANSPORTER PATHWAYS^*

Christopher P. Corpe, Je-Hyuk Lee, Oran Kwon, Peter Eck, Jayan Narayanan, Kenneth L. Kirk, and Mark Levine¶

From the Molecular and Clinical Nutrition Section, Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, 20892-1372 and the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland, 20892-0820

Vitamin C intracellular accumulation is mediated by Na⁺-dependent vitamin C transporters SVCT1 and -2 and dehydroascorbic acid transporters GLUT1 and -3. It is unclear which pathways dominate in vivo. As a new step to resolve this issue, we identified and tested 6-bromo-6-deoxy-L-ascorbic acid as a specific candidate for SVCTs. In high performance liquid chromatography and electron paramagnetic resonance analyses, the reduced compounds ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid were similar. The oxidized products 6-bromo-6-deoxy dehydroascorbic acid (BrDHA) and dehydroascorbic acid (DHA) had comparable stabilities, based on reduction recoveries. Upon expression of GLUT1 or GLUT3 in Xenopus oocytes, BrDHA was neither transported nor bound, in contrast to robust transport of DHA. The findings were not explained by differences in the oocyte reduction of DHA and BrDHA because lysed oocytes reduced both compounds equally. Further, there was no transport of the reduced compound, 6-bromo-6-deoxy-L-ascorbic acid, by GLUT1 or GLUT3. As a prerequisite for investigating 6-bromo-6-deoxy-L-ascorbic acid transported by SVCTs, SVCT2 transport activity in oocytes was enhanced 14-fold by construction and use of a vector that added a fixed poly(A) tail to the 3' end of cRNA. For SVCT1 and SVCT2 expressed in oocytes, similar K_m and V_max values were observed for ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid. In human fibroblasts, predicted to have SVCT-mediated ascorbate accumulation, K_m and V_max values were again comparable for ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid. Using activated human neutrophils, predicted to have ascorbate accumulation mediated predominantly by DHA and GLUT transporters, 6-bromo-6-deoxy-L-ascorbic acid accumulation was <1% of accumulation when compared with ascorbic acid. We conclude that 6-bromo-6-deoxy-L-ascorbic acid is the first transport substrate identified as completely specific for SVCTs, but not GLUTs, and provide a new strategy to determine the contribution of each pathway to ascorbate accumulation.

Received for publication, November 15, 2004

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Molecular and Clinical Nutrition Section, Digestive Diseases Branch, Bldg. 10, Rm. 4D52 – MSC 1372. Tel.: 301-402-5588; Fax: 301-402-6436; E-mail: markl{at}intra.niddk.nih.gov.

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