May J M, Qu Z C, Mendiratta S
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303, USA.
Arch Biochem Biophys. 1998 Jan 15;349(2):281-9. doi: 10.1006/abbi.1997.0473.
Ascorbic acid can recycle alpha-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle alpha-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of alpha-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of alpha-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte alpha-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of alpha-tocopherol from both cells and from alpha-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas alpha-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects alpha-tocopherol in the cell membrane by a direct recycling mechanism.
抗坏血酸可在脂质双层和微团中从生育酚自由基回收α-生育酚，但尚未证明这种回收能跨细胞膜发生。在本研究中，我们调查了细胞内抗坏血酸盐在完整人红细胞和红细胞影中保护及回收α-生育酚的能力。在用氮氧化物Tempol处理使细胞内抗坏血酸盐耗竭80%的红细胞中，2,2'-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)和铁氰化物氧化α-生育酚的程度均比未耗竭抗坏血酸盐的细胞更大。相反，在用脱氢抗坏血酸加载使细胞内抗坏血酸盐浓度增加的红细胞中，两种氧化剂导致的α-生育酚损失均比对照细胞少。细胞外抗坏血酸氧化酶不能阻止对AAPH诱导的α-生育酚氧化的保护作用，这表明保护作用是由于细胞内而非细胞外抗坏血酸盐。用卵磷脂脂质体孵育红细胞也会产生氧化应激，导致脂质体中的脂质过氧化并耗尽红细胞α-生育酚，从而导致溶血。红细胞加载抗坏血酸盐可延迟脂质体氧化，并减少细胞及加载α-生育酚的脂质体中α-生育酚的损失。当将红细胞影重新封闭使其含有抗坏血酸盐，并使其受到红细胞影外AAPH产生的自由基攻击时，在1小时的孵育过程中，囊泡内抗坏血酸盐完全耗尽，而α-生育酚仅在抗坏血酸盐大量氧化后才减少。这些结果表明，红细胞内的抗坏血酸盐通过直接回收机制保护细胞膜中的α-生育酚。
