大剂量维生素E补充对大鼠生物大分子氧化损伤及细胞功能影响的研究
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摘要
目的 维生素E(VE)是人体所需的重要脂溶性维生素和营养性抗氧化剂，但大剂量补充是否具有不良影响尚未见到明确报道，本研究拟通过大剂量维生素E补充从生物大分子氧化损伤及细胞功能方面探讨大剂量维生素E的影响。
方法 实验动物选用Wistar大鼠48只按体重随机分成4组，对照组给予普通大鼠饲料，三个干预组均喂饲添加了大剂量α-生育酚乙酸酯的饲料，其中维生素E的含量分别为500 IU／kg、2000 IU／kg、7500 IU／kg，补充时间为8周。实验结束时将大鼠安乐处死，收集血样和尿样进行抗氧化能力及细胞活性分析。抗氧化能力分析主要包括DNA氧化(H_2O_2诱导)损伤、血清超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)含量分析及尿中O~6-甲基鸟嘌呤含量分析。DNA氧化损伤采用“彗星”电泳技术进行分析，尿O~6-甲基鸟嘌呤含量分析采用高效毛细管电泳法，SOD等抗氧化酶及MDA用生化试剂盒进行测定。细胞功能分析主要包括红细胞膜流动性和外周血淋巴细胞转化率测定。红细胞膜流动性测定采用荧光偏振法，应用四甲基偶氮唑盐(MTT)法培养大鼠外周血淋巴细胞并测定淋巴细胞转化率，观察大剂量维生素E对免疫细胞增殖活性的影响。
结果 补充较大剂量VE能显著增强大鼠机体抗氧化能力及细胞增殖和膜流动的功能活性。研究结果显示：当VE剂量为500IU／kg时，与对照组(75IU／kgVE)相比，大鼠血清VE水平提高了69.2％，SOD活性显著升高(P＜0.05)，提高了11％；血清和红细胞膜中GSH-Px活性均明显升高，分别提高了5.4％(P＜0.05)和101.5％(P＜0.01)；血清MDA水平明显降低，比对照组下降了34％(P＜0.01)。DNA损伤分析结果显示由10μmol／L过氧化氢诱发的DNA氧化损伤均显著降低(P＜0.05)以及尿中O~6-甲基鸟嘌呤排出量也呈现明显下降(P＜0.05)。细胞功能分析结果显示：补充500IU／kg VE 8周后，大鼠红细胞膜流动性和淋巴细胞转化率均比对照组显著提高(P＜0.01，P＜0.05)。
但是，长期补充过大剂量VE可引起生物大分子损伤和细胞功能降低。当饲料中维生素E的剂量进一步增加为2000 IU／kg、7500 IU／kg(分别为大鼠需要量的27倍、100倍)时，与对照组(75IU／kgVE)相比，大鼠血清VE浓度分别
中文摘要
提高了93.7%、n7.4%，而SOD活性却分别下降了17%、18%(尸<0.05，尸<
0.01);彗星电泳结果显示，10拜mol/L过氧化氢诱发的DNA氧化损伤明显加重
(尸<0.05，尸<0.01)。细胞功能分析结果显示:补充75。。IU/kgVES周后，大
鼠淋巴细胞转化率比对照组显著降低(尸<0.05)，而2000 IU/kgVE对细胞转
化率没有影响(尸>.05)，对细胞增殖功能既没有产生保护作用，也没有产生损
伤作用。红细胞膜流动性在这两个过大剂量补充组均未见改善。
结论①so0IU/kg维生素E可以增强血清、细胞膜GSH一Px、SOD等抗氧
化酶的活性，降低血清中脂质过氧化终产物MDA水平;同时，补充该剂量维生
素E可降低DNA诱发氧化损伤及自发烷化损伤水平;提高大鼠红细胞膜流动
性和淋巴细胞转化率。②饲料中维生素E进一步增高为Zoo0lu/kg、7500IU/
kg时，可降低血清SOD活性，并且加重较大剂量过氧化氢诱发的DNA氧化损
伤;7500IU/kgVE可降低淋巴细胞转化率。而GSH一Px、MDA、红细胞膜流动
性等则未见改善。
总之，较大剂量维生素E可提高机体的抗氧化能力，并且改善细胞增殖及
膜流动等细胞活性。但是，过大剂量维生素E则会引起生物大分子损伤和细胞
功能下降，并且随着剂量增大，这种损伤作用可能更加严重。
Objectives Vitamin E is an essential fat-soluble vitamin that has antioxidant activities. But it had not been reported definitely whether supplementation of high dose vitamin E would cause adverse effects. In this in vivo study we investigated the influence of different levels of high dose vitamin E on oxidative damage of macromolecule and cell function to determine its effect when used excessively.
Methods 48 Wistar rats , 60~90g body weight(half male and half female) were randomly divided into 4 groups based on body weight. The control group was fed with normal rat diet which contented vitamin E 75 IU/kg and the other three interfered groups were supplemented with different levels of high dose all-rac-a-tocopheryl acetate enriched diets which contented vitamin E 500 IU/kg,2000 IU/kg,7500IU/kg,respectively. The period of the trial was about 8 weeks. Urine of each rat was collected one week before the end of the trail. At the end of the trial, the rat will be died without pain by drug anesthesia, and samples of the whole blood were collected for analysis. Antioxidation activity analysis included DNA oxidative damage induced by H2O2, activities of serum superoxide dismutase (SOD), glutatathione peroxidase (GSH-Px) content of malanydiadehyde(MDA) and the concerntration of O6-methylguanine in urine. The comet assay or the alkaline single cell gel electrophoresis (SCGE) assay was used to measure the DNA oxi
dative damage. High performance capillary zone electrophoresis was used to determine O6-methylguanine. SOD, GSH-Px and MDA were measured by biochemical methods with the kits. Cell function a-nalysis included the erythrocyte membrane fluidity and the lymphocyte transformation rate . The method of fluorescence polarization was used to measure membrane fluidity and MTT was used to detected the lymphocyte transformation rate which reflected proliferation activity of immune cell.
Results Relatively high dose vitamin E could significantly increase antioxidation activity and cell function detected by lymphocyte proliferation and membrane fluidity . Compared with the control group, 500IU/kg VE increased serum VE by 69. 2%, serum SOD activity by 11% , serum and membrane GSH-Px activities by 5. 4% and 101. 5% while serum MDA decreased by 34%. 500IU/kg VE significantly lowered DNA oxidative damage
induced by 10 tmol/L H2O2 and urine O6-methylguanine was also decreased. After 500IU/ kg VE supplementation with 8 weeks , the erythrocyte membrane fluidity and lymphocyte transformation rate were significantly increased.
It worth notion that long term supplementation with too excessive vitamin E would damage macrobiomolecule and decrease cell function. 2000 IU/kg,7500 IU/kg vitamin E increased serum VE by 93. 7%, 117. 4% respectively. But serum SOD activities were decreased by 17% and 18% respectively and DNA oxidative damages induced by 10 mol/L H2O2 were both aggravated in this two groups. At the same time, lymphocyte transformation rate of the rat supplemented with 7500 lU/kg VE for 8 weeks was significantly decreased.
Conclusion (1)500 lU/kg vitamin E could increase the activities of antioxidant enzymes such as SOD,GSH-Px and decrease the formation of serum MDA. DNA oxidative damage induced by H2O2 and DNA alkyl damage were decreased by 500 lU/kg VE . As to cell function, 500 lU/kg VE significantly improved membrane fluidity and promoted lymphocyte transformation. (2)2000 IU/kg,7500 IU/kg vitamin E decreased serum SOD activities and aggravated DNA oxidative damages induced by H2O2. Lymphocyte transformation rate was decreased by 7500 IU/kg VE.
Above all, relatively high dose vitamin E could increase antioxidation activity of the body and increase cell function indexes such as membrane fluidity and lymphocyte proliferation rate. However, too excessive vitamin E would induce macrobiomolecule damage and decrease cell function.
方法 实验动物选用Wistar大鼠48只按体重随机分成4组，对照组给予普通大鼠饲料，三个干预组均喂饲添加了大剂量α-生育酚乙酸酯的饲料，其中维生素E的含量分别为500 IU／kg、2000 IU／kg、7500 IU／kg，补充时间为8周。实验结束时将大鼠安乐处死，收集血样和尿样进行抗氧化能力及细胞活性分析。抗氧化能力分析主要包括DNA氧化(H_2O_2诱导)损伤、血清超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)含量分析及尿中O~6-甲基鸟嘌呤含量分析。DNA氧化损伤采用“彗星”电泳技术进行分析，尿O~6-甲基鸟嘌呤含量分析采用高效毛细管电泳法，SOD等抗氧化酶及MDA用生化试剂盒进行测定。细胞功能分析主要包括红细胞膜流动性和外周血淋巴细胞转化率测定。红细胞膜流动性测定采用荧光偏振法，应用四甲基偶氮唑盐(MTT)法培养大鼠外周血淋巴细胞并测定淋巴细胞转化率，观察大剂量维生素E对免疫细胞增殖活性的影响。
结果 补充较大剂量VE能显著增强大鼠机体抗氧化能力及细胞增殖和膜流动的功能活性。研究结果显示：当VE剂量为500IU／kg时，与对照组(75IU／kgVE)相比，大鼠血清VE水平提高了69.2％，SOD活性显著升高(P＜0.05)，提高了11％；血清和红细胞膜中GSH-Px活性均明显升高，分别提高了5.4％(P＜0.05)和101.5％(P＜0.01)；血清MDA水平明显降低，比对照组下降了34％(P＜0.01)。DNA损伤分析结果显示由10μmol／L过氧化氢诱发的DNA氧化损伤均显著降低(P＜0.05)以及尿中O~6-甲基鸟嘌呤排出量也呈现明显下降(P＜0.05)。细胞功能分析结果显示：补充500IU／kg VE 8周后，大鼠红细胞膜流动性和淋巴细胞转化率均比对照组显著提高(P＜0.01，P＜0.05)。
但是，长期补充过大剂量VE可引起生物大分子损伤和细胞功能降低。当饲料中维生素E的剂量进一步增加为2000 IU／kg、7500 IU／kg(分别为大鼠需要量的27倍、100倍)时，与对照组(75IU／kgVE)相比，大鼠血清VE浓度分别
中文摘要
提高了93.7%、n7.4%，而SOD活性却分别下降了17%、18%(尸<0.05，尸<
0.01);彗星电泳结果显示，10拜mol/L过氧化氢诱发的DNA氧化损伤明显加重
(尸<0.05，尸<0.01)。细胞功能分析结果显示:补充75。。IU/kgVES周后，大
鼠淋巴细胞转化率比对照组显著降低(尸<0.05)，而2000 IU/kgVE对细胞转
化率没有影响(尸>.05)，对细胞增殖功能既没有产生保护作用，也没有产生损
伤作用。红细胞膜流动性在这两个过大剂量补充组均未见改善。
结论①so0IU/kg维生素E可以增强血清、细胞膜GSH一Px、SOD等抗氧
化酶的活性，降低血清中脂质过氧化终产物MDA水平;同时，补充该剂量维生
素E可降低DNA诱发氧化损伤及自发烷化损伤水平;提高大鼠红细胞膜流动
性和淋巴细胞转化率。②饲料中维生素E进一步增高为Zoo0lu/kg、7500IU/
kg时，可降低血清SOD活性，并且加重较大剂量过氧化氢诱发的DNA氧化损
伤;7500IU/kgVE可降低淋巴细胞转化率。而GSH一Px、MDA、红细胞膜流动
性等则未见改善。
总之，较大剂量维生素E可提高机体的抗氧化能力，并且改善细胞增殖及
膜流动等细胞活性。但是，过大剂量维生素E则会引起生物大分子损伤和细胞
功能下降，并且随着剂量增大，这种损伤作用可能更加严重。
Objectives Vitamin E is an essential fat-soluble vitamin that has antioxidant activities. But it had not been reported definitely whether supplementation of high dose vitamin E would cause adverse effects. In this in vivo study we investigated the influence of different levels of high dose vitamin E on oxidative damage of macromolecule and cell function to determine its effect when used excessively.
Methods 48 Wistar rats , 60~90g body weight(half male and half female) were randomly divided into 4 groups based on body weight. The control group was fed with normal rat diet which contented vitamin E 75 IU/kg and the other three interfered groups were supplemented with different levels of high dose all-rac-a-tocopheryl acetate enriched diets which contented vitamin E 500 IU/kg,2000 IU/kg,7500IU/kg,respectively. The period of the trial was about 8 weeks. Urine of each rat was collected one week before the end of the trail. At the end of the trial, the rat will be died without pain by drug anesthesia, and samples of the whole blood were collected for analysis. Antioxidation activity analysis included DNA oxidative damage induced by H2O2, activities of serum superoxide dismutase (SOD), glutatathione peroxidase (GSH-Px) content of malanydiadehyde(MDA) and the concerntration of O6-methylguanine in urine. The comet assay or the alkaline single cell gel electrophoresis (SCGE) assay was used to measure the DNA oxi
dative damage. High performance capillary zone electrophoresis was used to determine O6-methylguanine. SOD, GSH-Px and MDA were measured by biochemical methods with the kits. Cell function a-nalysis included the erythrocyte membrane fluidity and the lymphocyte transformation rate . The method of fluorescence polarization was used to measure membrane fluidity and MTT was used to detected the lymphocyte transformation rate which reflected proliferation activity of immune cell.
Results Relatively high dose vitamin E could significantly increase antioxidation activity and cell function detected by lymphocyte proliferation and membrane fluidity . Compared with the control group, 500IU/kg VE increased serum VE by 69. 2%, serum SOD activity by 11% , serum and membrane GSH-Px activities by 5. 4% and 101. 5% while serum MDA decreased by 34%. 500IU/kg VE significantly lowered DNA oxidative damage
induced by 10 tmol/L H2O2 and urine O6-methylguanine was also decreased. After 500IU/ kg VE supplementation with 8 weeks , the erythrocyte membrane fluidity and lymphocyte transformation rate were significantly increased.
It worth notion that long term supplementation with too excessive vitamin E would damage macrobiomolecule and decrease cell function. 2000 IU/kg,7500 IU/kg vitamin E increased serum VE by 93. 7%, 117. 4% respectively. But serum SOD activities were decreased by 17% and 18% respectively and DNA oxidative damages induced by 10 mol/L H2O2 were both aggravated in this two groups. At the same time, lymphocyte transformation rate of the rat supplemented with 7500 lU/kg VE for 8 weeks was significantly decreased.
Conclusion (1)500 lU/kg vitamin E could increase the activities of antioxidant enzymes such as SOD,GSH-Px and decrease the formation of serum MDA. DNA oxidative damage induced by H2O2 and DNA alkyl damage were decreased by 500 lU/kg VE . As to cell function, 500 lU/kg VE significantly improved membrane fluidity and promoted lymphocyte transformation. (2)2000 IU/kg,7500 IU/kg vitamin E decreased serum SOD activities and aggravated DNA oxidative damages induced by H2O2. Lymphocyte transformation rate was decreased by 7500 IU/kg VE.
Above all, relatively high dose vitamin E could increase antioxidation activity of the body and increase cell function indexes such as membrane fluidity and lymphocyte proliferation rate. However, too excessive vitamin E would induce macrobiomolecule damage and decrease cell function.
引文
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38 Cheng WH, Valentine BA, Lei XG. High Levels of Dietary Vitamin E Do Not Replace Celluar Glutathione Peroxidase in Protecting Mice from Acute Oxidative Stress. J Nutr. 1999;129:1951～1957
39 李锋，李宣海，程五凤，谢良民．补充VE、Se对大鼠肝纤维化和抗氧化功能影响的研究．营养学报．2003，25(1)：60～64
40 Andersen HR,Andersen O. Effects of dietary alpha-tocopherol and beta-carotene on lipid peroxidation induced by methyl mercuric chloride in mice. Pharmacol Toxicol 1993 ;73(4) :192～201
41 Huang HY, Helzlsouer KJ, Appel LJ. The effects of vitamin C and vitamin E on oxi-
dative DNA damage: results from a randomized controlled trial. Cancer Epidemiol Biomarkers Prey. 2000;9(7):647～652
42 Huang HY, Appel LJ, Croft KD, Miller ER 3rd, Mori TA, Puddey IB. Effects of vitamin C and vitamin E on in vivo lipid peroxidation :results of a randomized controlled trial. Am J Clin Nutr . 2002:76:549～555
43 Chao JC, Huang CH, Wu SJ, Yang SC, Chang NC, Shieh MJ, Lo PN. Effects of bete-carotene, vitaminC and vitamin E on antioxidant status in hyperlopidemic smokers. J Mutr Biochem. 2002;13(7):427～434
44 Schmidt MC, Askew EW, Roberts DE, Prior RL, Ensign WY Jr, Hesslink RE Jr. Oxidative stress in humans training in a cold,moderate altitude enviroment and their response to a phytochmical antioxidant supplement. Wilderness Environ Med. 2002; 13(2): 94～105
45 Claycombe KJ,Meydani SN. Vitamin E and genome stability. Mutat Res. 2001;475(1-2): 37～44
46 Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA. 1993;90(17) :7915～7922
47 Halliwell B, Aruoma OI. ,DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems,FEBS Lett. 1991;281(1-2):9～19
48 Metzger Z, Hoffeld JT. Macrophage-mediated suppression:Evidence for participation of murine lmyphocyte proliferation. J Immumol. 1980,124(2) : 983～988
49 Schraufstatter I, Hyslop PA, Jackson JH, Cochrane CG. Oxidant-induced DNA damage of target cells. J Clin Invest. 1988;82(3):1040～1050
50 Factor VM, Laskowska D, Jensen MR, Woitach JT, Popescu NC, Thorgeirsson SS. Vitamin E reduces chromosomal damage and inhibits hepatic tumor formation in a transgenic mouse model. Proc Natl Acad Sci USA. 2000,97(5) :2196～2201
51 Antunes LM,Takahashi CS. Effects of high doses of vitamins C and E against doxorubicin-induced chromosomal damage in Wistar rat bone marrow cells. Murat Res. 1998; 419 (1-3) :137～143
52 Brennan LA, Morris GM, Wasson GR, Hannigan BM, Barnett YA. The effect of vitamin C or vitamin E supplementation on basal and H_2O_2-induced DNA damage in human lymphocytes. British Journal of Nutrition. 2000 ;84(2) : 195～202
53 Sun M, Sakakibara H, Ashida H, Danno G, Kanazawa K. Dietary antixidants fail in protection against oxidative genetic damage in in vitro evaluation. Biosci Biotechnol Biochern 2000 ;64(11):2395～2401
54 Tsuda S, Matsusaka N ,Ueno S, Susa N, Sasaki YF. The influence of antioxodants on cigarette smoke-induced DNA single-strand breaks in mouse organs: a preliminary
study with the alkaline single cell gel electrophoresis assay. Toxicol Sci. 2000 ;54(1) :104～109
55 Royack GA, Nguyen MP, Tong DC, Poot M, Oda D. Response of human oral epithelial cells to oxidative damage and the effect of vitamin E. Oral Oncol. 2000;36 (1): 37～41A
56 Slamenova D, Horvathova E, Kosikova B, Ruzekova L, Labaj J. Detection of lifnin biopolymer and vitamin E-stimulated reduction of DNA strand breaks in H_2O_2 and MNNG-treated mammalian cells by the comet assay. Nutr Cancer. 1999;33(1):88～94
57 Georgiadis P, Samoli E, Kaila S, Katsouyanni K, Kyrtopoulos SA. Ubiquitous presence of O~6-methylguanine in human peripheral and cord blood DNA. Cancer Epidemiology Biomarkers & Prevention. 2000;9(3):299～305
58 Patel JM, Block ER. The effect of oxidant gases on membrane fluidity and fuctionin pulmonary endothelial cells. Free Rad Biol Med. 1988;4(2) :121～125
59 Kamada T, Otsuji S. Lower levels of erythrocyte membrane fluidity in diabetic patients. Diabetic. 1983,32 : 585
60 石学香，马爱国，梁惠，张秀珍，徐宏伟，杜卫．补充抗氧化营养素对青年人群抗氧化能力的影响．营养学报．2002，24(3)：307～308
61 Pathania V, Syal N,Pathak CM, Khanduja kl. Changes in rat alveolar macrophageal antioxidant defense and reactive oxygen species release by high dietary vitamin E. J Nutr Sci Vitaminol (Tokyo) 1998; 44(4) : 491～502
62 Thomas SR, Neuzil J, Stocker R. Cosupplemetation with coenzyme Q prevents the prooxidant effect of α-tocopherol and increases the resistance of LDL to transition metal-depent oxidation initiation. Arterioscler. Thromb. Vasc. Biol. 1996;16(5):687～696
63 Bowry VW, Ingold KU, Stocker R. Vitamin E in human low-density lipoprotein. When and how this antioxidant becomes a pro-oxidant. Biochem J. 1992 ;288: 341～344
64 Keaney JF Jr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Low-doseα-tocopherol improves and high-doseα-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest. 1994;93:844～851
65 Cameron IL, Munoz J, Barnes CJ, Hardman WE. High dietary level of synthetic vitamin E on lipid peroxidation, membrane fatty acid compisition and cytotoxity in breast cancer xenograft and in mouse host tissue. Cancer Cell Int. 2003;3(1):3
66 Fenech M, Morley AA. The effect of donor age on spontaneous and induced micronuclei. Mutation Research. 1985 ; 148: 99～105
67 Woodson K, Triantos S . Long-term alpha-tocopherol supplementation is associated with lower serum vascular endothelial growth factor levels. Anticancer Res. 2002 ;22 (1A): 375～378
68 Engelhart MJ, Geerlings MI et. al. Dietary intake of antioxidants and risk of Alzheimer disease. JAMA . 2002;287(24):3223～3229
69 杨杰，钱亦华，胡海涛，张樟进，王唯析，胡晓丹．维生素E对Alzheimer病模型大鼠的影响．中国老年学杂志，1998，18(2)：95～97
2 Konopacka M, Widel M, Rzeszowska-Wolny J. Modifying effect of vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in mouse cells. Murat Res. 1998;417(2-3):85～94
3 Lee CY, Man-Fan Wan J. Vitamin E Supplementation Imoproves Cell-Mediated Immunity and Oxidative Stress of Asian Men and Women. J Nutr. 2000;130(12):2932～2937
4 Donnelly ET, MeClure N, Lewis SE. The effect of ascorbate and alpha-tocopherol sup-plementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis. 1999;14(5): 505～512
5 Lii CK,Ko YJ, Chiang MT,Sung WC,Chen HW. Effect of dietary vitamin Eon antioxidant status and antioxidant enzyme activities in Spragkue-Dawley rats. Nutr Cancer. 1998;32(2):95～100
6 Woodson K, Triantos S, Hartman T, Taylor PR, Virtamo J, Albanes D. Long-term alpha-tocopherol supplementation is associated with lower serum vascular endothelial growth factor levels. Anticancer Res. 2002;22 (1A) : 375～378
7 Meydani SN. Vitamin E enhancement of T cell-mediated function in healthy elderly: mechanisms of action. Nutr Rev. 1995;53(4 Pt 2):S52～56
8 Meydani SN, Meydani M, Blumberg J B, Leka LS, Siber G, Loszewski R, Thompson C, Pedrosa MC, Diamond RD, Stollar BD. Vitamin E supplementation and in vivo immune response in healthy elderly subjects. A randomized controlled trial. JAMA . 1997;277(17): 1380～1386
9 Giakoustidis D, Papageorgiou G, Iliadis S, Kontos N, Kostopoulou E, Papachrestou A, Tsantilas D, Spyridis C, Takoudas D, Botsoglou N, Dimitriadou A, Giakoustidis E. Intramuscular administration of very high dose of α-tocopherol protects liver from sever ischemia/reperfusion injury. World J Surg. 2002;26 (7): 872～877
10 Meagher EA, Barry OP, Lawson JA, Rokach J, FitzGerald GA. Effects of vitamin E on lipid peroxidation in healthy persons. JAMA. 2001;285(9):1178～1182
11 Brown KM, Mortice PC, Duthie GG. Erythrocyte vitamin E AND plasma ascorbate concentrations in relation to erythrocyte perixidation in smokers snd nonsmokers:dose response to vitamin Esupplementation. Am J Clin Nutr. 1997;65(2):496～502
12 Graat JM,Schouten EG, Kok FJ. Effect of daily vitamin E and multivitamin-mineral supplementation on acute respiratory tract infections in elderly persons:a randomized controlled trial. JAMA . 2002;288(6) :715～721
13 Eder K, Flader D, Hirche F, Brandsch C. Excess dietary vitamin E lowers the activities of ntioxidative enzymes in erythrocytes of rats fed salmon oil. J Nutr . 2002;132(11) :3400～3404
14 Record IR, Jannes M. Iron,alpha-tocpherol,oxidative damage and micronucleus formation in rat splenocytes. Cancer Lett. 2000;160(2) : 125～131
15 Boomershine KH, Shelton PS, Boomershine JE. Vitamin E in the treament of tardive dyskinesia. Ann Pharmacother. 1999;33(11):1195～1202
16 Kakizaki S, Takagi H, Fukusato T, Toyoda M, Horiguchi N, Sato K, Takayama H, Nagamine T, Mori M. Effect of alpha-tocopherol on hepatocarcinogenesis in transforming growth factor-alpha(TGF-alpha)transgenic mice treated with diethylnitrosamice. Int J Vitam Nutr Res. 2002 ;71(5) :261～267
17 Tarin JJ, Perez-Albala S, Pertusa JF, Cano A. Oral administration of pharmacological doses of vitamins C and E reduces reproductive fitness and impairs the ovarian and uterine functions of female mice. Theriogenology. 2002; 57 (5): 1539～1550
18 Miyauvhi M,Nakamura H. Promoting effects of combined antioxidant and sodium nitrite treatment on forestomach carcinogenesis in rats after initation with N-methyl-N'-nitro-N-nitrosoguanidiine. Czncer Lett. 2002;178 (1): 19～24
19 Blasiak J, Gloc E, Wozniak K, Mlynarski W, Stolarska M, Skorski T, Majsterek I. Genotoxicity of idarubicin and its modulation by vitamins C and E and amifostine. Chem Biol Interact 2002;140 (1): 1～18
20 王小红，江洪，夏明珠，杨益寿．单细胞凝胶电泳技术的研究进展及其应用．国外医学临床生物化学与检验学分册．2001，22(1)：5～7
21 马爱国，藏金林，宋凤荣．SCGE，SCE和染色体畸变分析法用于DNA损伤与修复检测的对比研究．癌变·畸变·突变．2001，13(3)：154～157
22 马爱国，刘四朝．不同剂量维生素C对DNA氧化损伤影响的研究．营养学报．2001，23(1)：12～15
23 聂松青，礴蕙卿，林克椿．蜂王精对大鼠红细胞膜流动性的影响．北京医学院学报．1983，15：249～252
24 Lowry OH, Rosebourough NJ, Farr AL. Protein measurement with the folin phenol reagent. J-Biol-Chem. 1951;193:265
25 张秀珍，沈芳兰，王建华．牛磺酸对红细胞膜流动性的影响．青岛医学院学报．1996，32(3)：106～108
26 梁惠，杜卫，张秀珍．高效毛细管电泳法在检测生物样品中O~6-甲基鸟嘌呤的应用．青岛大学学报．1999，12(3)：95～96
27 Thomas SR ,Neuzil J,Mohr D, Stocker R . Coantioxidants make alpha-tocopherol an efficient antioxidant for low-density lipoprotein. Am J Clin Nutr. 1995,62 (suppl) :1357s～1364s
28 Hsiao G ,Teng CM, Wu CL, KO FN. Marchantin H as a natural antioxidant and free radical scavenger. Arch Biochem Biophys. 1996,334(1) : 18～26
29 Di Mascio PD ,Murphy ME, Sies H. Antioxidant defense systems:the role of carotenoids,tocopherols and thiols. Am J Clin Nutr. 1991;53: 194s～200s
30 Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Lab investig. 1982;47(5):412～426
31 Slater TF,Cheeseman KH ,Davies MJ ,Proudfoot K, Xin W. Free radical mechanisms in relation to tissue injury. Proc Nutr Soc. 1987;46(1);1～12
32 Kagan VE. Tocopherol stabilizes membrane against phospholipase A,free fatty acids, and lysophopholipids. Ann N Y Acad Sci. 1989 ; 570: 121～135
33 Nagel E, Meyer zu Vilsendorf A, Battles M, Pichlmayr R. Antioxidative vitamins in prevetion of ischemia/reperfusion injury. Int J Vit Nutr Res. 1997;67(5) :298～306
34 Azzi A, Stocker A. Vitamin E:non-antioxidant roles. Prog Lipid Res. 2000;39(3): 231～255
35 Azzi A, Breyer I, Feher M, Pastori M, Ricciarelli R, Spycher S, Staffieri M, Stocker A, Zimmer S, Zingg JM. Specific cellular responses to alpha-tocopherol. J Nutr. 2000; 130(7) : 1649～1652
36 Barja G, Cadenas S, Rojas C, Perez-Campo R, Lopez-Torres M, Prat J, Pamplona R. Effect of dietary vitamin E levels on fatty acid profiles and nonenzymatic lipid peroxidation in the guinea pig liver. Lipids. 1996;31(9):963-970
37 Kapps H, Diplock AT. Tolerance and safety of vitamin E: a toxicological position report. Free Radic Biol Med. 1992,13:55～74
38 Cheng WH, Valentine BA, Lei XG. High Levels of Dietary Vitamin E Do Not Replace Celluar Glutathione Peroxidase in Protecting Mice from Acute Oxidative Stress. J Nutr. 1999;129:1951～1957
39 李锋，李宣海，程五凤，谢良民．补充VE、Se对大鼠肝纤维化和抗氧化功能影响的研究．营养学报．2003，25(1)：60～64
40 Andersen HR,Andersen O. Effects of dietary alpha-tocopherol and beta-carotene on lipid peroxidation induced by methyl mercuric chloride in mice. Pharmacol Toxicol 1993 ;73(4) :192～201
41 Huang HY, Helzlsouer KJ, Appel LJ. The effects of vitamin C and vitamin E on oxi-
dative DNA damage: results from a randomized controlled trial. Cancer Epidemiol Biomarkers Prey. 2000;9(7):647～652
42 Huang HY, Appel LJ, Croft KD, Miller ER 3rd, Mori TA, Puddey IB. Effects of vitamin C and vitamin E on in vivo lipid peroxidation :results of a randomized controlled trial. Am J Clin Nutr . 2002:76:549～555
43 Chao JC, Huang CH, Wu SJ, Yang SC, Chang NC, Shieh MJ, Lo PN. Effects of bete-carotene, vitaminC and vitamin E on antioxidant status in hyperlopidemic smokers. J Mutr Biochem. 2002;13(7):427～434
44 Schmidt MC, Askew EW, Roberts DE, Prior RL, Ensign WY Jr, Hesslink RE Jr. Oxidative stress in humans training in a cold,moderate altitude enviroment and their response to a phytochmical antioxidant supplement. Wilderness Environ Med. 2002; 13(2): 94～105
45 Claycombe KJ,Meydani SN. Vitamin E and genome stability. Mutat Res. 2001;475(1-2): 37～44
46 Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA. 1993;90(17) :7915～7922
47 Halliwell B, Aruoma OI. ,DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems,FEBS Lett. 1991;281(1-2):9～19
48 Metzger Z, Hoffeld JT. Macrophage-mediated suppression:Evidence for participation of murine lmyphocyte proliferation. J Immumol. 1980,124(2) : 983～988
49 Schraufstatter I, Hyslop PA, Jackson JH, Cochrane CG. Oxidant-induced DNA damage of target cells. J Clin Invest. 1988;82(3):1040～1050
50 Factor VM, Laskowska D, Jensen MR, Woitach JT, Popescu NC, Thorgeirsson SS. Vitamin E reduces chromosomal damage and inhibits hepatic tumor formation in a transgenic mouse model. Proc Natl Acad Sci USA. 2000,97(5) :2196～2201
51 Antunes LM,Takahashi CS. Effects of high doses of vitamins C and E against doxorubicin-induced chromosomal damage in Wistar rat bone marrow cells. Murat Res. 1998; 419 (1-3) :137～143
52 Brennan LA, Morris GM, Wasson GR, Hannigan BM, Barnett YA. The effect of vitamin C or vitamin E supplementation on basal and H_2O_2-induced DNA damage in human lymphocytes. British Journal of Nutrition. 2000 ;84(2) : 195～202
53 Sun M, Sakakibara H, Ashida H, Danno G, Kanazawa K. Dietary antixidants fail in protection against oxidative genetic damage in in vitro evaluation. Biosci Biotechnol Biochern 2000 ;64(11):2395～2401
54 Tsuda S, Matsusaka N ,Ueno S, Susa N, Sasaki YF. The influence of antioxodants on cigarette smoke-induced DNA single-strand breaks in mouse organs: a preliminary
study with the alkaline single cell gel electrophoresis assay. Toxicol Sci. 2000 ;54(1) :104～109
55 Royack GA, Nguyen MP, Tong DC, Poot M, Oda D. Response of human oral epithelial cells to oxidative damage and the effect of vitamin E. Oral Oncol. 2000;36 (1): 37～41A
56 Slamenova D, Horvathova E, Kosikova B, Ruzekova L, Labaj J. Detection of lifnin biopolymer and vitamin E-stimulated reduction of DNA strand breaks in H_2O_2 and MNNG-treated mammalian cells by the comet assay. Nutr Cancer. 1999;33(1):88～94
57 Georgiadis P, Samoli E, Kaila S, Katsouyanni K, Kyrtopoulos SA. Ubiquitous presence of O~6-methylguanine in human peripheral and cord blood DNA. Cancer Epidemiology Biomarkers & Prevention. 2000;9(3):299～305
58 Patel JM, Block ER. The effect of oxidant gases on membrane fluidity and fuctionin pulmonary endothelial cells. Free Rad Biol Med. 1988;4(2) :121～125
59 Kamada T, Otsuji S. Lower levels of erythrocyte membrane fluidity in diabetic patients. Diabetic. 1983,32 : 585
60 石学香，马爱国，梁惠，张秀珍，徐宏伟，杜卫．补充抗氧化营养素对青年人群抗氧化能力的影响．营养学报．2002，24(3)：307～308
61 Pathania V, Syal N,Pathak CM, Khanduja kl. Changes in rat alveolar macrophageal antioxidant defense and reactive oxygen species release by high dietary vitamin E. J Nutr Sci Vitaminol (Tokyo) 1998; 44(4) : 491～502
62 Thomas SR, Neuzil J, Stocker R. Cosupplemetation with coenzyme Q prevents the prooxidant effect of α-tocopherol and increases the resistance of LDL to transition metal-depent oxidation initiation. Arterioscler. Thromb. Vasc. Biol. 1996;16(5):687～696
63 Bowry VW, Ingold KU, Stocker R. Vitamin E in human low-density lipoprotein. When and how this antioxidant becomes a pro-oxidant. Biochem J. 1992 ;288: 341～344
64 Keaney JF Jr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Low-doseα-tocopherol improves and high-doseα-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest. 1994;93:844～851
65 Cameron IL, Munoz J, Barnes CJ, Hardman WE. High dietary level of synthetic vitamin E on lipid peroxidation, membrane fatty acid compisition and cytotoxity in breast cancer xenograft and in mouse host tissue. Cancer Cell Int. 2003;3(1):3
66 Fenech M, Morley AA. The effect of donor age on spontaneous and induced micronuclei. Mutation Research. 1985 ; 148: 99～105
67 Woodson K, Triantos S . Long-term alpha-tocopherol supplementation is associated with lower serum vascular endothelial growth factor levels. Anticancer Res. 2002 ;22 (1A): 375～378
68 Engelhart MJ, Geerlings MI et. al. Dietary intake of antioxidants and risk of Alzheimer disease. JAMA . 2002;287(24):3223～3229
69 杨杰，钱亦华，胡海涛，张樟进，王唯析，胡晓丹．维生素E对Alzheimer病模型大鼠的影响．中国老年学杂志，1998，18(2)：95～97