高糖環境下維生素E對低密度脂蛋白糖化與氧化之影響

The Effect of Vitamin E on Glycation and Oxidation ofLow Density Lipoprotein in High Glucose Concentration

王慧芳(Hui-Fang Wang)

指導教授 ： 黃士懿 博士

臺北醫學大學/公共衛生暨營養學院/保健營養學研究所/碩士(2001年)

摘要

糖尿病患長期處於高血糖環境下，葡萄糖自動氧化及蛋白質糖化作用過程會引發自由基的產生，增加糖尿病患體內之氧化壓力。研究顯示自由基攻擊血液低密度脂蛋白及糖化作用的修飾可能是引發糖尿病患產生血管性併發症之主因。研究亦證實維生素E可降低氧化壓力，降低低密度脂蛋白 (LDL) 對氧化的感受性，有利於延緩併發症的發生。本研究以分別以體外試驗及動物實驗模式探討，體外試驗將低密度脂蛋白 (LDL) 以不同濃度葡萄糖溶液 (0、5、25 & 50 mM glucose) 、在有無添加脂質過氧化物MDA及有無富含維生素E處理下，於不同時間點 (第0、1、3 & 7天) 進行糖化程度、TBARs測定、離體銅離子誘導後氧化延遲時間試驗、維生素E含量、洋菜膠電泳試驗，以探討維生素E對脂蛋白糖化及氧化之影響。另以動物實驗進行不同劑量維生素E投與對糖尿病鼠LDL糖化及氧化的影響。結果顯示：native LDL在高糖環境 (25、50 mM) 及伴隨MDA處理處理下，會增加LDL糖化程度、LDL縮短氧化延遲時間、增加TBARs值及降低LDL維生素 E含量，且隨著實驗天數增加有更顯著差異。維生素E添加後，LDL維生素E含量較添加前高，添加維生素E之LDL，顯著延長氧化延遲時間、降低TBARs值及增加LDL維生素 E含量。此外，1.1%洋菜膠電泳試驗結果顯示，無添加維生素E之LDL隨著實驗天數增加，葡萄糖濃度，伴隨MDA的添加，其在電泳膠片上有較大的移動度。添加維生素E後，各組間則無顯著差異。動物實驗模式中以STZ誘發老鼠產生糖尿病，投予不同維生素E含量 (1x、5x、10x及20x) 飼料，結果顯示，糖尿病誘發經餵食實驗飲食四週後，糖尿病組禁食血糖值及糖化血色素均較誘發前及空白組高。血脂質部分，E 10x組有較低總膽固醇濃度、高及低密度脂蛋白膽固醇濃度。E 5x組與E 10x組LDL氧化延遲時間均顯著較E 1x組延長。E 10x組有顯著較低TBARs值。E 5x組及E 20x組有顯著較低LDL維生素E含量。本研究得知LDL處於高葡萄糖濃度下，會增加糖化作用並引發自由基作用，增加對氧化的感受性；而維生素E的添加，可藉著降低LDL之氧化壓力，降低對氧化的感受性，降低LDL被氧化所修飾之機會。

關鍵字

維生素E ； 低密度脂蛋白 ； 糖化 ； 氧化

並列摘要

Reactive oxygen species, which can be generated during glucose autoxidation and during protein glycation, may be response for increased oxidative stress in diabetes patients with high blood glucose concentration. It may be the major reason that reactive oxygen species-modified and glycated of protein, especially in collagen and low density lipoprotein (LDL) has been implicated in mediating diabetic complications (for example: hyperliperemia). Numerous studies have documented that a-tocopherol could decrease the susceptibility of LDL to oxidation both in vivo and in vitro. The present study was undertaken to investigate the effect of a-tocopherol on LDL glycation and oxidation, we test the effect of a-tocopherol on LDL oxidative susceptibility and glycation, after enrichment of plasma with α-tocopherol in vitro and also different doses of a-tocopherol supplementation in streptozotocin (STZ)-induced diabetic rat. In vitro study, we incubated LDL in different concentration of glucose (0、5、25 & 50 mM) with and without malondialdehyde (MDA), and with and without a-tocopherol enriched, sampling in different time points (Day 0、1、3 & 7). We measured the glycation degree of LDL, LDL TBARs test, LDL oxidized lag time, the concentration of a-tocopherol on LDL and 1.1% agarose gel electrophoresis of LDL. The animal study, we test the effect with different doses of a-tocopherol after in vivo supplement in STZ-induced diabetic rat. The in vitro result showed that native LDL with high glucose treatment and with MDA treatment had higher glycation degree of LDL, MDA production of LDL, and shorter LDL oxidized lag time, the lower concentration of a-tocopherol of LDL. After a-tocopherol enriched, LDL had more content of a-tocopherol. The a-tocopherol enriched LDL with high glucose showed a lower glycation degree of LDL, MDA production and longer LDL oxidized lag time. In 1.1% agarose gel electrophoresis of native LDL showed increasing electrophoretic mobility under high glucose concertration and with MDA. After a-tocopherol enriched, it showed no difference between all treatments. In animal study, after STZ-induced and 4-week supplementation, the diabetic rat had higher fasting blood glucose and hemoglobin A1C (HbA1C) than control group. E 10x group had lower total cholesterol concentration, HDL-cholesterol and LDL-cholesterol compared with E 1x group. In E 10x group, a-tocopherol supplement clearly decreased the oxidative susceptibility of LDL as evidenced by prolongation of the lag phase of LDL oxidation and a lower MDA production. In conclusion, LDL readily increased oxidative stress under high glucose concentration and elevated the oxidative susceptibility of LDL. a-Tocopherol supplementations decreases individual oxidative stress, declines the oxidative susceptibility of LDL, and avert the molecular modification of LDL.

並列關鍵字

Vitamin E ； low density lipoprotein ； glycation ； oxidatin

參考文獻

參考文獻 英文部分: Aoki, Y., Yanagisawa, Y., Yazaki, K., Oguchi, H., Kiyosawa, K. and Furuta, S. (1992) Protective effect of vitamin E supplementation on increased thermal stability of collagen in diabetic rats. Diabetologia. 35:913-6.

Bioship, N., Schorach, C.J. and Wales, J.K. (1985) The effect of vitamin C supplementation on diabetic hyperlipidemia: a double-blind, crossover study. Diabetic Med. 2:121-4.

Ceriello, A., Giugliano, D., Quatraro, A., Donzella, C., Dipalo, G. and. Lefebvre, P.J. (1991) Vitamin E reduction of protein glycosylation in diabetes: New prospect for prevention of diabetic complications? Diabetes Care. 14:68-72.

Ceriello, A., Bortoltti, N., Falleti, E., Crescentini, A., Tonutti, L., Motz, E., Russo, A., Bartoli, E., Lizzo, S. and Taboga, C. (1997) Total radical-trapping antioxidant parameter in NIDDM patients. Diabetes Care 20:194-7.

Chan, A.C. (1998) Vitamin E and atherosclerosis. J. Nutr. 128:1593-6.