May J M, Qu Z C, Whitesell R R, Cobb C E
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA.
Free Radic Biol Med. 1996;20(4):543-51. doi: 10.1016/0891-5849(95)02130-2.
Human erythrocytes regenerate ascorbate from its oxidized product, dehydroascorbate. The extent to which such ascorbate recycling occurs by a GSH-dependent mechanism was investigated. In the presence of glucose, erythrocytes took up over 90% of extracellular [14C]dehydroascorbate and rapidly converted it to [14C]ascorbate, which was trapped within the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake and reduction of dehydroascorbate by glucose-depleted erythrocytes coordinately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by L-lactate. Conversely, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which cells were able to reduce extracellular ferricyanide. Monoascorbyl free radical was formed during the reduction of extracellular ferricyanide, indicating that one electron transfer steps were involved in this process. In GSH-depleted cells, addition of L-lactate as an energy source for glycolysis-dependent NADH regeneration did cause a partial recovery of the ability of cells to reduce ferricyanide. However, in resealed erythrocyte ghosts containing either 4 mM GSH or 400 mu M NADH, only the GSH-containing ghosts supported regeneration of ascorbate from added dehydroascorbate. These results suggest that in human erythrocytes ascorbate regeneration from dehydroascorbate is largely GSH dependent, and that it occurs through either enzymatic or nonenzymatic reactions not involving the monoascorbyl free radical.
人类红细胞可从其氧化产物脱氢抗坏血酸再生抗坏血酸。本研究调查了这种抗坏血酸循环通过谷胱甘肽（GSH）依赖性机制发生的程度。在葡萄糖存在的情况下，红细胞摄取了超过90%的细胞外[14C]脱氢抗坏血酸，并迅速将其转化为[14C]抗坏血酸，后者被困在细胞内。脱氢抗坏血酸的摄取和还原与单抗坏血酸自由基中间体的产生无关。葡萄糖缺乏的红细胞对脱氢抗坏血酸的摄取和还原协同降低了红细胞内的GSH水平并提高了GSSG浓度。这种效应可被D-葡萄糖逆转，但不能被L-乳酸逆转。相反，细胞内GSH的消耗降低了细胞将脱氢抗坏血酸循环再生为抗坏血酸的能力，这反映在细胞还原细胞外铁氰化物的程度上。在还原细胞外铁氰化物的过程中形成了单抗坏血酸自由基，表明该过程涉及单电子转移步骤。在GSH缺乏的细胞中，添加L-乳酸作为糖酵解依赖性NADH再生的能量来源确实导致细胞还原铁氰化物的能力部分恢复。然而，在含有4 mM GSH或400 μM NADH的重封红细胞空壳中，只有含有GSH的空壳支持从添加的脱氢抗坏血酸再生抗坏血酸。这些结果表明，在人类红细胞中，从脱氢抗坏血酸再生抗坏血酸在很大程度上依赖于GSH，并且它通过不涉及单抗坏血酸自由基的酶促或非酶促反应发生。
