Vitamin C transport in oxidized form across the rat blood-retinal barrier

PURPOSE. To elucidate the mechanisms of vitamin C transport across the blood - retinal barrier (BRB) in vivo and in vitro. METHODS. [C-14] Dehydroascorbic acid (DHA) and [C-14] ascorbic acid (AA) transport in the retina across the BRB were examined using in vivo integration plot analysis in rats, and the transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line ( TR-iBRB2) as an in vitro model of the inner BRB. RESULTS. The apparent influx permeability clearance ( K in) per gram of retina of [C-14] DHA and [C-14] AA was found to be 2.44 x 10(3) muL/(min . g retina) and 65.4 muL/(min . g retina), respectively. In the retina and brain, the K in of [C-14] DHA was approximately 38 times greater than that of [C-14] AA, whereas there was no major difference in the heart. The K in of [C-14] DHA in the retina was eight times greater than that in the brain. HPLC analysis revealed that most of the vitamin C accumulated in AA form in the retina. These results suggest that vitamin C is mainly transported in DHA form across the BRB and accumulates in AA form in the rat retina. In an in vitro uptake study in TR-iBRB2 cells, the initial uptake rate of [C-14] DHA was 37 times greater than that of [C-14] AA, which is in agreement with the results of the in vivo study. [C-14] DHA uptake by TR-iBRB2 cells took place in an Na+-independent and concentration-dependent manner with a K-m of 93.4 muM. This process was inhibited by substrates and inhibitors of glucose transporters. [C-14] DHA uptake was inhibited by D-glucose in a concentration-dependent manner with a 50% inhibition concentration of 5.56 mM. Quantitative real-time PCR and immunostaining analyses revealed that expression of GLUT1 and - 3 was greater than that of the Na+-dependent L-ascorbic acid transporter (SVCT)-2 in TR-iBRB2 cells. CONCLUSIONS. Vitamin C is mainly transported across the BRB as DHA mediated through facilitative glucose transporters and accumulates as AA in the rat retina.