May J M, Qu Z C, Whitesell R R
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303, USA.
Biochemistry. 1995 Oct 3;34(39):12721-8. doi: 10.1021/bi00039a031.
The role of ascorbate transport and metabolism in the response of human erythrocytes to an extracellular oxidant stress was investigated. Rates of entry and exit of [14C]dehydroascorbate from erythrocytes were more than 10-fold greater than those of [14C]ascorbate. Both the reduced and oxidized forms of the vitamin were transported largely by the glucose transporter. Inside erythrocytes, dehydroascorbate was converted to ascorbate, increasing intracellular ascorbate concentrations 2-3-fold over those in the medium. In such ascorbate-loaded cells, the membrane-impermeant oxidant ferricyanide induced a transmembrane oxidation of intracellular ascorbate to dehydroascorbate. The latter escaped the cells on the glucose transporter, which resulted in a halving of the net entry of [14C]dehydroascorbate in the presence of ferricyanide. Treatment of ascorbate-loaded cells with H2O2 and Cu2+ also oxidized ascorbate and induced efflux of [14C]dehydroascorbate. Ferricyanide-dependent intracellular oxidation of ascorbate resulted in a corresponding reduction of extracellular ferricyanide, which served as an integrated measure of ascorbate recycling. Ferricyanide reduction was proportional to the loading concentration of dehydroascorbate and was enhanced when loss of dehydroascorbate from cells was decreased, either by blockade of the glucose transporter or by concentrating the cells. Selective depletion of cellular ascorbate lowered rates of ferricyanide reduction by two-thirds, suggesting that ascorbate rather than NADH is the major donor for the transmembrane ferricyanide oxidoreductase activity. On the basis of the ascorbate-dependent rate of ferricyanide reduction, erythrocytes at a 45% hematocrit can regenerate the ascorbic acid present in whole blood every 3 min. Erythrocyte ascorbate recycling may thus contribute more to the antioxidant reserve of blood than is evident from plasma ascorbate concentrations alone.
研究了抗坏血酸盐转运和代谢在人类红细胞对细胞外氧化应激反应中的作用。[14C]脱氢抗坏血酸盐进出红细胞的速率比[14C]抗坏血酸盐的速率大10倍以上。维生素的还原形式和氧化形式主要通过葡萄糖转运体进行转运。在红细胞内部，脱氢抗坏血酸盐被转化为抗坏血酸盐，使细胞内抗坏血酸盐浓度比培养基中的浓度增加2至3倍。在这种抗坏血酸盐负载的细胞中，膜不透性氧化剂铁氰化物诱导细胞内抗坏血酸盐跨膜氧化为脱氢抗坏血酸盐。后者通过葡萄糖转运体逸出细胞，这导致在存在铁氰化物的情况下[14C]脱氢抗坏血酸盐的净进入量减半。用H2O2和Cu2+处理抗坏血酸盐负载的细胞也会氧化抗坏血酸盐并诱导[14C]脱氢抗坏血酸盐外流。铁氰化物依赖性的细胞内抗坏血酸盐氧化导致细胞外铁氰化物相应减少，这作为抗坏血酸盐循环利用的综合指标。铁氰化物还原与脱氢抗坏血酸盐的负载浓度成正比，并且当通过阻断葡萄糖转运体或浓缩细胞减少脱氢抗坏血酸盐从细胞中的损失时会增强。细胞内抗坏血酸盐的选择性消耗使铁氰化物还原速率降低三分之二，这表明抗坏血酸盐而非NADH是跨膜铁氰化物氧化还原酶活性的主要供体。根据抗坏血酸盐依赖性的铁氰化物还原速率，血细胞比容为45%的红细胞每3分钟可以再生全血中存在的抗坏血酸。因此，红细胞抗坏血酸盐循环利用对血液抗氧化储备的贡献可能比仅从血浆抗坏血酸盐浓度中所显示的更为显著。
