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研究生(中)周育如
研究生(英)Yu-Ru Chou
論文名稱(中)膳食維生素B2含量對於長期高硒攝取時大白鼠麩胱甘太代謝之影響
論文名稱(英)Effects of Dietary Vitamin B2 Levels on the Metabolism of Glutathione during Long-term Excessive Selenium Consumption in Rats.
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指導教授(中)楊駱菲莉 博士
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關鍵字(中)高硒攝取 維生素B2 麩胱甘太
關鍵字(英)Excessive Selenium Consumption Vitamin B2 Excessive Selenium Consumption
摘要(中)輔仁大學八十八學年度第二學期碩士論文摘要 研究所別:食品營養學系碩士班 研究生:周育如 論文名稱:膳食維生素B2含量對於長期高硒攝取時大白鼠麩胱甘太代謝之影響 指導教授:楊駱菲莉 博士 摘 要 本研究之主要目的於探討長期(12週)給予不同硒濃度(0.1、3、5 mg Se/kg diet,以sodium selenite形式供應),及不同濃度維生素B2(3、6、12 mg vit. B2/kg diet)的飼料,對雄性Wistar大白鼠體內glutathione(GSH)和硒的代謝及維生素B2營養狀況之影響。結果顯示:長期高硒代謝顯著消耗肝臟GSH含量,提高glutathione disulfide(GSSG)含量,使GSH/GSSG比值下降。紅血球雖適應性的提高GSH總含量,仍不足以應付高硒代謝下GSH之持續損耗,導致GSSG含量增加,以致無法維持紅血球之GSH/GSSG比值。飼料維生素B2含量提高至12 mg vit. B2/kg diet(AIN-93 G diet之2倍)時,仍無法提高肝臟和紅血球中GSH含量及GSH/GSSG比值。肝臟中有關GSH代謝的酵素方面:隨飼料硒含量增加,肝臟γ-glutamylcysteine synthetase(γ-GCS)和glutathione reductase(GR)活性均顯著提高;維生素B2含量多寡對二酵素活性並沒有影響。長期高硒給予顯著提高肝臟中glutathione-S-transferase活性;隨著飼料維生素B2含量增加,此酵素活性顯著下降。肝臟硒含量隨著飼料硒濃度增加而上升,在高硒組(3、5 mg Se/kg diet)發現,隨著維生素B2含量增加,肝臟硒含量呈現上升的情形。長期高硒攝取下,飼料中僅給予3 mg vit. B2/kg diet的維生素B2(低於AIN-93 G diet之50%),造成大白鼠體重下降、脫毛、肝病變和邊緣性維生素B2缺乏的趨勢。綜合以上結果可知:長期給予大白鼠高硒飲食,會干擾GSH氧化還原代謝之平衡,進而可能提高細胞之氧化壓力。給予較正常量高的維生素B2(12 mg vit. B2/kg diet)時有利於動物之生長,但無法維持正常維生素B2營養狀況及提高肝臟GSH/Se比值。推測:長期高硒代謝下,維生素B2在硒的還原代謝過程中可能扮演著雙重不同的角色。
摘要(英)Effects of Dietary Vitamin B2 Levels on the Metabolism of Glutathione during Long-term Excessive Selenium Consumption in Rats. Yu-Ru Chou Abstract The purpose of this study was to investigate the effect of dietary vitamin B2 levels on glutathione metabolism of rats during long term Se consumption. After 1 week of adaptation on normal diet (0.1 mg Se/kg diet, 6 mg vit. B2/kg diet), 80 weanling male Wistar rats were randomly assigned to receive diets containing 3 levels of dietary Se (0.1, 3, 5 mg Se/kg diet) and 3 levels of dietary vitamin B2 (3, 6, or 12 mg vit. B2/kg diet). Results indicated that hepatic glutathione (GSH) concentration and GSH/GSSG ratio decreased significantly whereas glutathione disulfide (GSSG) increased significantly due to long-term excessive Se ingestion. Although the erythrocytes adapted to elevate total GSH content, the erythrocyte GSSG was continuously increased resulting in depressed GSH/GSSG ratio by long-term high Se ingestion. However, offering diet with 12 mg vit. B2/kg diet (about 2 folds of vit. B2 content of AIN-93G diet) did not further elevate hepatic and erythrocyte GSH and GSH/GSSG ratio. In terms of activities of hepatic GSH related enzymes, γ-glutamylcysteine synthetase (γ-GCS) and glutathione reductase (GR) activities increased significantly with elevated dietary Se concentration. The levels of vitamin B2 did not elicit significant changes in the activities of the γ-GCS and GR. Long term high Se consumption significantly elevated glutathione-S-transferase activity, which was reduced by dietary vitamin B2 levels. Hepatic Se concentration increased significantly with elevated dietary Se concentration. And hepatic Se concentration increased with elevated dietary vitamin B2 concentration during long-term excessive Se ingestion. Three mg vit. B2 of per kg diet (about 50% of vit. B2 content in AIN-93G diet) caused growth restriction , hair loss, liver damage and marginal vitamin B2 deficiency during excessive Se intake. In summary, long term excessive Se consumption (5 mg Se/kg diet, 12 weeks) resulted in significant disturbance in GSH redox metabolism and probably raised cellular oxidative stress. However, 12 mg vit. B2/kg diet of dietary vitamin B2 level failed to maintain adequate nutriture of vitamin B2 and to relieve Se toxicity.
論文目次目 錄 中文摘要…………………………………………………………………….Ⅰ 英文摘要…………………………………………………………………….Ⅱ 目錄……………………………………………………………………………IV 表目錄…………………………………………………………………………....IX 圖目錄……………………………………………………………………....XI 第一章前言……………………………………………………….….1 第二章文獻回顧………………………………………………….…….3 一、硒的簡介…………….…………………… ………………...3 (一)硒的吸收………………………………………………………….….3 (二)硒的運送………………………………………………….…….4 (三)硒的代謝………………………………………………………….….4 (四)硒的排泄…………………………………………………….….5 (五)含硒蛋白質………………………………………………….….6 1.Glutathione Peroxidase….…………………………………..6 2.Iodothyronine 5’-deiodinase………………………………7 3.Selenoprotein P…………………………………………………7 4.其他含硒蛋白質………………………………………………….8 (六)硒與免疫…………………………………………………… ...8 (七)硒與癌症………………………………………………………...……9 二、胱甘?之簡介…………………...………………………....11 (一)GSH之吸收與分佈………………………………………………12 (二)GSH之合成與代謝………………………………………………14 (三)GSH之生化功能…………………………………………………15 1.參與硒的代謝…………………………………………………..15 2.抗氧化之功能………………………………………………….16 3.解毒功能……………………………………………………….17 4.胺基酸的儲存和運送………………………………………….18 三、高硒給予對GSH代謝之影響…………………………………..19 四、維生素B2對硒和GSH代謝之影響……………………………….22 (一)維生素B2之吸收……………………………………………….23 (二)維生素B2之儲存和運送……………………………………….23 (三)維生素B2之代謝……………………………………………….24 (四)維生素B2之排泄……………………………………………….26 (五)維生素B2評估營養狀況之方法……………………………….27 1.尿液測量法…………………………………………………….27 2.Erthyrocyte glutathione reductase activity coefficient…………27 (六)核黃素的生化功能……………………………………………..28 五、實驗目的與假說……………………………………………….29 第三章材料與方法…………………………………………………...31 一、實驗設計……………………………………………………….31 二、動物飼養環境……………………………………………………….32 三、飼料配製…………………………………………………………….33 (一)飼料成份及配方……………………………………………….….33 (二)飼料製備程序………………………………………………….….34 四、樣本的收集與處理………………………………………………….34 (一)血液……………………………………………………………...34 (二)肝臟……………………………………………………………...35 五、分析項目與方法…………………………………………………….35 (一)肝臟、紅血球和血漿中GSH和GSSG含量…………………….35 (二)肝臟γ-glutamylcysteine synthetase(γ-GCS)活性……….…...37 (三)肝臟中glutathione reductase(GR)活性…………………………37 (四)肝臟中glutathione-S-transferase(GST)活性……………………37 (五)紅血球中glutathione reductase activity coefficienet之測定………38 (六)飼料、肝臟、血漿與紅血球中硒含量……………………….……38 (七)酵素蛋白質定量……………………………………………….……39 (八)代謝實驗……………………………………………………….……39 六、統計分析……………………………………………………………...39 第四章結果……………………………………………………………….……40 一、組織中GSH與GSSG含量 分析…………………………………...40 (一)HPLC測定GSH和GSSG…..……………………………………...40 (二)肝臟GSG、GSSG含量和GSH/GSSG比值………………………42 (三)紅血球GSG、GSSG含量和GSH/GSSG比值……………………43 (四)血漿GSG、GSSG含量和GSH/GSSG比值………………………47 二、肝臟中與GSH代謝相關之酵素分析…………………………….53 (一)肝臟之γ-GCS活性………………………………………….……..53 (二)肝臟之GR活性……………………………………………………53 (三)肝臟之GST活性………………………………………………...54 三、紅血球中EGR活性係數………………………………………………54 四、組織中硒含量…………………………………………………………..55 (一)肝臟中硒含量…………………………………………………..55 (二)腎臟中硒含量…………………………………………………..55 (三)紅血球中硒含量………………………………………………..61 五、肝臟中 GSH/Se比值…………………………………………………..61 六、硒的吸收率……………………………………………………………..61 七、實驗期間動物生長情形……………………………………………….62 (一)飼料攝食量…………………………………………………….62 (二)體重變化…………………………………………………………62 (三)動物外觀…………………………………………………………70 (四)肝臟體重相對百分比……………………………………………70 (五)腎臟與體重相對百分比…………………………………………70 八、飼料中硒含量分析……………………………………………………76 九、飼料維生素B2和甲硫胺酸含量之計算……………………….…….76 第五章討論……………………………………………………………………78 一、長期高硒攝取時維生素B2對肝臟中GSH代謝相關酵素活性 之影響………………………………………………………………...78 (一)glutathione reductase(GR)………………………………78 (二)γ-glutamylcysteine synthetase(γ-GCS)………………79 (三)glutathione-S-transferase(GST)…………………………80 二、長期高硒攝取時維生素B2對組織GSH、GSSG含量及GSH/GSSG 比值之影響…………………………………………………………..80 (一)肝臟…………………………………………………………...81 (二)紅血球………………………………………………………...82 (三)血漿…………………………………………………………...82 三、長期高硒攝取時維生素B2對紅血球EGR活性係數之影響 ……………………………………………………………………...83 四、長期高硒攝取時維生素B2對組織中硒含量之影響………………84 (一)肝臟、腎臟…………………………………………………..84 (二)紅血球………………………………………………………..85 五、驗期間動物生長和健康狀況之討論…………………….……86 (一)體重變化及硒中毒現象………………………………………..86 (二)動物器官體重百分比…………………………………………..87 (三)硒的吸收………………………………………………………..87 (四)GSH/Se比值………………………………………………………88 第六章結論…………………………………………………………...90 參考文獻…………………………………………………………………...92 附錄一、肝臟、紅血球和血漿中GSH和GSSG之測定方法………………101 附錄二、肝臟γ-glutamylcysteine synthetase(γ-GCS)活性 之測定方法…………………………………………………………..107 附錄三、肝臟中glutathione reductase(GR)活性之測定方 法………..109 附錄四、肝臟中glutathione-S-transferase(GST)活性之測定方法……..111 附錄五、紅血球中glutathione reductase activity coefficienet之測定 ..113 附錄六、硒含量之測定方法……………………….……………………..115 附錄七、蛋白質之測定方法……………………………………………...119 附錄八、代謝實驗………………………………………………………...121
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