臺灣博碩士論文加值系統

維生素 E 為動物體內最重要的抗氧化劑，體內維生素 E 含量受到組織保留能力、氧化需求及代謝速率各層面所影響。前人研究指出炸油餵食會降低大鼠體內維生素 E 的營養狀況，但是至今其影響機制未明，本研究目的即欲探討炸油飲食影響維生素 E 營養狀況的可能機制。實驗一以二倍維生素E補充飼料，觀察炸油對維生素 E代謝機制之影響，採用 21 隻離乳 Wistar 雄鼠，依飼料不同分成三組：控制組 (CO組，新鮮油組) 和兩組炸油組 (FO組和FOE組)，其中的炸油來源為新鮮大豆油以 205±5℃ 油炸巧果 24 小時所製得；已知維生素 E 吸收率會因為炸油飲食而下降，因此特別設計 FOE 組，在炸油飼料中額外添加 50 ppm 維生素 E。大鼠飼養六週後於犧牲前收集 24 小時尿液以供 a-生育醇 (�娗-TOH) 代謝產物 (2,5,7,8- tetramethyl- 2-(2’-carboxyethyl)-6-
hydroxychroman ; a-CEHC) 之分析。FO 組和FOE組血漿 �娗-TOH濃度顯著低於CO組 (P＜0.05)，但肝臟 �娗-TOH濃度則以 FO 組顯著最低，FOE 組次之 (P＜0.05)。而肝臟 TBARS (Thiobarbituric acid- reactive substances) 含量與Catalase (CAT) 蛋白質含量皆以兩炸油組顯著高於CO組；且炸油組的 CAT 蛋白質含量約增為控制組的三倍之多。肝臟CYP3A2蛋白質表現以FO組顯著最高 (P＜0.05) ， FOE組次之。尿液中 a-CEHC 的排出量以 FO 組顯著低於其餘兩組 (P＜0.05)。MDR和a-TTP蛋白質含量於三組間並無顯著差異。實驗二以注射方式給予維生素E，觀察炸油對維生素E代謝機制之影響，並且額外分析CYP3A與PXR mRNA表現。炸油組肝臟 �娗-TOH含量顯著較低、TBARS 含量與 CAT蛋白質含量則顯著較高，此結果皆與實驗一相似 (P＜0.05)，炸油組 PXR mRNA 表現量顯著高於新鮮油組 (P＜0.05)，其下游基因CYP3A不論是蛋白質或是mRNA之表現皆不受炸油所影響，但兩個實驗皆指出炸油會顯著誘導CYP4A1蛋白質的表現。綜合本實驗結果，炸油餵食顯著誘導肝臟中PXR mRNA的表現量，但並未促進尿液中 a-CEHC 排出量以及肝中 a-TTP與 MDR 之蛋白質表現

Vitamin E is the most important lipid-soluble antioxidant in the body . The previous study showed that the retention of dietary vitamin E in rat tissues was affected by dietary frying oil , but the mechanism is still unclear . The purpose of this study is to investigate the effects of oxidized frying oil on the expression of protein and genes relative to vitamin E metabolism in rat liver. In experiment 1 , three groups of male Wistar weanling rats were fed the control diet (CO , containing 15% fresh soyben oil ) or test diets (FO and FOE , containing 15% frying oil ) . Frying oil was obtained by frying dough sheet in fresh soybean oil at 205±5℃ for four 6-periods . The FOE diet contained 100ppm vitamin E which is about 2 fold of normal vitamin E levels in control diet . After 6 weeks feeding , rats were then moved into metabolic cage to collect urine for 24 hours . Urine samples were analyzed for a-CEHC ( 2,5,7,8 - tetramethyl -2- (2’-carboxyethyl) - 6 -hydroxychroman) content . At the end of the feeding period , the rats were sacrificed and blood collected and liver were excised for various biochemical analysis. The plasma a-tocopherol concentration were significantly lower in FO and FOE groups than that of CO group . Liver a-tocopherol levels were significantly lowest in FO group among the three groups . FO and FOE groups showed significantly higher liver TBARS concentration when compared with the CO group ; The CAT protein levels in livers of rats fed FO and FOE diet showed significantly 3 fold higher than that of CO group . The amount of urine a-CEHC in FO groups were significantly lower than that of CO and FOE groups . All the protein levels of ��-TTP , CYP3A1 and MDR protein in liver had no significant difference among the three groups . The frying oil diet could promote the lipid proxidation and induce the protein levels of CAT in liver ; but did not alter the protein levels of other proteins relative to vitamin E metabolism . In experiment 2 , rats were divided into 2 groups (oxidized frying oil , O ; vitamin E-stripped fresh oil , C) and a-tocopherol were also collected . The results of liver a-tocopherol , TBARS and protein levels of CAT were similar to that of experiment 1 . The O group showed a significantly increase in liver PXR mRNA expression . The mRNA expression of CYP3A , the downstream gene of PXR , showed no significant difference between the two groups . In conclusion , the mRNA expression levels of PXR were induced by oxidized frying oil in rat liver . The excretion amount of urine�na-CEHC , and the protein levels of a-TTP and MDR in rat liver were not influeuced by oxidized frying oil feeding

中文摘要..................................................I
英文摘要 ...............................................III
目錄…………………………………………………………………….V
表目錄……………………….………………………….……..…… XI
圖目錄 ……………………………………………………………… XIII
縮寫表…………………………………………………….……..…. XIV

第一章 緒言……………………………………………………………1
第一節 前言…………………………………………………………...1
第二節 文獻回顧……………………………………………………..2
一、維生素E介紹 ………………………………………………….…2
(一) 維生素E的弁?.………………………………………………..2
(二) 維生素E的吸收、儲存及運送………………………………….3
(三) 維生素E的排泄及代謝途徑……………………………………6
(四) 維生素E營養狀況指標………………………………………….9
二、炸油…………………………………………………………………9
(一) 油脂在油炸的化學變化………………………………………….9
(二) 炸油飲食對動物體之影響………………………………………10
(三) 炸油飲食與動物體內維生素E之相關研究…………………….11
三、研究動機………………………………………………………. .12

第二章 材料與方法……………………………………………..….13
一、實驗假說與設計……………………………………………..…13
實驗一：以二倍維生素E補充飼料，觀察炸油對維生素E
代謝機制之影響
實驗二：以注射方式給予維生素E，觀察炸油對維生素E
代謝機制之影響
二、炸油的製備………………………………………………………17
三、脫除維生素E之新鮮黃豆油的製備…………………………17
四、試驗飼料的配製………………………………………………..18
五、動物飼養…………………………………………………………19
六、動物犧牲…………………………………………………………20
七、尿液分析…………………………………………………………20
(一) 樣品收集………………………………………………………..20
(二) 尿液肌酸酐分析……………………………………………….21
(三) 尿液��-CEHC含量分析……………………………………….22
八、血液分析…………………………………………………………24
(一) 血漿膽固醇含量測定…………………………………………24
(二) 血漿三酸甘油酯含量測定……………………………………25
(三) 血漿白蛋白含量測定…………………………………………26
(四) 血漿維生素E含量測定……………………………………….26
九、肝臟分析………………………………………………………….28
(一) 樣品處理………………………………………………………..28
(二) 肝臟維生素E含量測定………………………………………29
(三) 肝藏CYP3A1、CYP3A2、CYP4A1、��-TTP、MDR1、CAT
、SOD1和GPx1蛋白質含量測定…………………………….30
(四) 肝臟TBARS含量測定……………………………….……….37
十、油脂品質分析…………………………………….……...……38
(一) UV233吸光值測定.………………………………….…………38
(二) 酸價測定………………………………………………………..39
(三) 羰基價測定……………………………………………………..40
(四) 過氧化物之含量測定…………………………………………42
(五) 油脂維生素E含量測定………………………………………43
十一、肝臟PXR、CYP3A mRNA含量…………………………...44
(一) 總RNA之抽取………………………………………….……..44
(二) RNA電泳…………………………………………………….…46
(三) 半定量逆轉錄聚合酶連鎖反應（RT-PCR）………….…...47
(四) DNA電泳……………………………………………………….48
十二、統計分析.............................................49

第三章結果………………………………………………………..….50
第一節實驗一:以二倍維生素E補充飼料，觀察炸油對維生素E
代謝機制之影響
一、油脂分析……………………………………………………….…50
二、體重變化……………………………………………………….…50
三、飼料利用效率………………………………………………….…51
四、組織重量與相對組織重量………………………………….….51
五、血漿白蛋白、脂質含量…………………………………………51
六、 血漿、肝臟維生素 E 與 TBARS 含量…………………………52
七、尿液肌酸酐、��-CEHC含量…………………………………....52
八、肝臟 CYP蛋白質含量：CYP4A1、CYP3A1、CYP3A2…........53
九、肝臟 ��-TTP蛋白質含量……………………………………….53
十、肝臟MDR1蛋白質含量………………………………………..53
十一、肝臟CAT、SOD和GPx蛋白質含量……………………..54

第二節 實驗二: 以注射方式給予維生素E，觀察炸油對維生素
E代謝機制之影響
一、油脂分析………………………………………………………….54
二、體重變化………………………………………………………….54
三、飼料利用效率…………………………………………………….55
四、組織重量與相對組織重量…………………………..………….55
五、血漿白蛋白、脂質含量…………………………………………55
六、血漿、肝臟維生素 E 與TBARS含量……………………………55
七、尿液肌酸酐、��-CEHC含量分析…………………………….….56
八、肝臟CYP蛋白質含量：CYP4A1、CYP3A1、CYP3A2…………56
九、肝臟CAT、SOD和GPx蛋白質含量測定………………………57
十、肝臟PXR、 CYP3A1及 CYP3A2 mRNA含量………………57

第四章 討論………………………………………………………….58
一、油脂品質與動物生長………..…………………..………….…58
二、炸油飲食對大鼠肝臟維生素E代謝機制之影響…………………59

第五章 結論………………………………………………………….67
參考文獻………..…………………………………………….………69
附錄……………………………………………………………………108
附圖1…………………………………………………………………108
附圖2…………………………………………………………………109

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