May James M, Qu Zhi-chao, Cobb Charles E
Department of Medicine and Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303, USA.
J Biol Chem. 2004 Apr 9;279(15):14975-82. doi: 10.1074/jbc.M312548200. Epub 2004 Jan 29.
Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.
从其氧化形式回收抗坏血酸有助于在人体红细胞中维持该维生素。为了确定从抗坏血酸自由基和脱氢抗坏血酸回收的相对贡献，我们研究了暴露于铁氰化物跨膜氧化应激的红细胞。铁氰化物既用于诱导跨细胞膜的氧化应激，也用于量化抗坏血酸的回收。红细胞将铁氰化物还原，产生细胞内抗坏血酸自由基，其浓度随着细胞内抗坏血酸增加而饱和，并且在用葡萄糖孵育的细胞中随时间持续存在。铁氰化物还产生脱氢抗坏血酸，其在细胞和孵育培养基中积累的浓度远高于自由基的浓度，尤其是在没有葡萄糖的情况下。铁氰化物刺激的从脱氢抗坏血酸回收抗坏血酸依赖于细胞内谷胱甘肽（GSH），但当GSH被马来酸二乙酯严重消耗时，以细胞内抗坏血酸为代价仍能很好地维持。这可能反映了不依赖于GSH的持续自由基还原。红细胞溶血产物显示出NAD和NADPH依赖性的抗坏血酸自由基还原。后者部分归因于硫氧还蛋白还原酶。溶血产物中GSH依赖性的脱氢抗坏血酸还原，既是直接的又是酶依赖性的，大于自由基还原酶活性，但表观亲和力较低。总之，这些结果表明存在一个有效的两层系统，其中抗坏血酸自由基的高亲和力还原足以去除未处于氧化应激的细胞可能遇到的低浓度自由基，并在更严重氧化应激条件下由一个高容量系统作为后备来还原脱氢抗坏血酸。
