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系統識別號 U0007-1704200714545919
論文名稱(中文) 大鼠體內L-3-羥基丁酸之分析及其在心臟之作用
論文名稱(英文) Determination of L-3-Hydroxybutyrate in rats and its effects on rat heart
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 93
學期 2
出版年 94
研究生(中文) 蔡屹喬
研究生(英文) Yih-Chiao Tsai
學號 D88010014
學位類別 博士
語文別 中文
口試日期
論文頁數 71頁
口試委員 指導教授-李仁愛
中文關鍵字 L-3-羥基丁酸  衍生化  心肌細胞 
英文關鍵字 ketone bodies  L-3-Hydroxybutyrate  derivatization  enantiomeric separation  column-switching HPLC  glucose utilization  cardiomyocytes 
學科別分類
中文摘要 D-3-Hydroxybutyrate(D-3HB)為體內之ketone bodies中含量最高者,且被作為研究ketone bodies之作用的主要目標。相反地,一般認為L-3-hydroxybutyrate(L-3HB)並非生物體內源生之ketone body,或許是基於其尚有爭議的代謝途徑;以及目前不瞭解其生成來源。在本論文中以螢光衍生化法搭配High-Performance Liquid Chromatography(HPLC)所開發之分析方法已能證實rat serum中的確有L-3HB的存在。Serum中之total 3HB經NBD-PZ衍生化後先經由一ODS column將之分離,隨後以兩支CHIRALCEL OD-RH串聯之chiral columns進行chiral separation。Rat serum並以D-3-hydroxybutyryl dehydrogenase處理作為對照組,以驗證所分離之3HB的真實性。實驗結果顯示serum中含有L-3HB,其與D-3HB的濃度分別為3.98(3.61%)與106.20 ?M(96.39%)。在以此分析方法成功地證實L-3HB存在後,我們再進一步應用於rat體內各組織中D-與L-3HB的分佈情形。 分取rat之腦、肝、心、及腎的研磨後均質液,經分析後發現heart中含有特殊高量的L-3HB;為所有檢測的組織中最特別者。D/L-3HB之比例在正常與diabetes mellitus(DM)時有所不同,其比例(D/L)分別為66/37與87/13。此變化可能是造成glucose代謝能力受影響的原因之一。當投予5 mM 之D-3HB於medium中,cardiomyocytes的glucose代謝會降低至控制組的61%,但給予同等劑量之L-3HB並未對細胞之glucose代謝造成任何影響,此結果反映著L-3HB與其他ketone bodies不同;並非作為提供能量的物質。此外,D-3HB抑制glucose代謝的作用可被另行添加之L-3HB阻斷;且L-3HB回復glucose代謝的作用會隨著其濃度增加而升高。藉由測量cardiomyocytes代謝D-、L-、與(D+L)-3HB能力的結果發現,D-與L-3HB同時存在的情況下反而會加速D-3HB的代謝;且可發現L-3HB的生成。由此推測L-3HB會刺激D-3HB進行interconversion生成L-3HB;使D-3HB的代謝不再回復產生acetyl CoA。實驗結果顯示L-3HB有別於D-3HB,為維持正常glucose utilization的重要物質,其可能在ketone bodies與glucose的代謝之間扮演著調節的角色。
英文摘要 While D-3-Hydroxybutyrate (D-3HB) is usually the major ketone body which was under intensive investigation, little attention had been paid to L-3-hydroxybutyrate (L-3HB). It had been considered nonexistent physiologically, perhaps due to its dubious metabolic route and lack of knowledge about its origin. In the present study, we proved that L-3HB is an original substance in rat serum by applying fluorescence derivatization and a column-switching high-performance liquid chromatography (HPLC) system as the analysis technique. Total 3HB in rat serum derivatized by 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was separated by an ODS column, and was confirmed by verifying the disappearance of the total 3HB peak after pretreating rat serum with D-3-hydroxybutyryl dehydrogenase (D-3HB dehydrogenase). A switching valve was used to simultaneously introduce isolated (D+L)-3HB to the enantiomeric separation by two CHIRALCEL OD-RH columns connected in tandem. An L-isomer was found to accompany the D-isomer, which were quantified to be 3.98 (3.61%) and 106.20 ?M (96.39%), respectively. Using the present analytical method, the dubious interpretation of the existence of L-3HB was clarified. Subsequently, distribution of D- and L-3HB in rat brain, liver, heart, and kidney homogenates were measured. The results showed that an enriched amount of L-3HB is present in rat hearts. The ratio would be changed from 66/34 to 87/13 (D/L) in normal and diabetic states, respectively. The altered D/L ratio may contribute to the reduction in glucose utilization by cardiomyocytes. Glucose utilization of cardiomyocytes with 5 mM of D-3HB was decreased to 61% of the control, but no interfering was observed when D-3HB was replaced with L-3HB, suggesting L-3HB is not utilized as the energy fuel as other ketone bodies are. In addition, the reduced glucose utilization caused by D-3HB could gradually recover in a dose-dependent manner with administration of additional L-3HB. Determination on metabolism of D-, L-, and (D+L)-3HB by cardiomyocytes showed cells had increased D-3HB metabolism when (D+L)-3HB was administered, and re-generation of L-3HB was found under the circumstance. It was speculated that in the presence of L-3HB, D-3HB might go through interconversion to generate L-3HB rather than being oxidized to acetyl CoA. The results suggest that it is a necessity of taking L-3HB together with D-3HB when it comes to glucose utilization. A physiological role is proposed for L-3HB as an important substrate which regulates the metabolism between glucose and ketone bodies.
論文目次 目 錄 目 錄 I 附圖目錄 III 附表目錄 IV 縮 寫 表 V 摘 要 VI Abstract VIII 第一章 緒 論 1 1.1 Ketone bodies的生成及代謝 1 1.2 Ketone bodies於病理狀態下的堆積 4 1.3 Ketone bodies的生理作用 4 1.4 L-3-hydroxybutyrate的研究歷史 5 1.5 研究目的 6 第二章 Enantiomeric separation of D- and L-3HB之分析方法 8 2.1 前言 8 2.2 實驗設計及進行 10 2.2.1 衍生化的進行 10 2.2.2 衍生化條件 10 2.2.3. HPLC之分析條件 11 2.2.4 Chiral separation 12 2.3 實驗結果與討論 13 第三章 Rat體內之D-及L-3HB的分析 20 3.1 前言 20 3.2 實驗設計及進行 22 3.2.1 Rat serum中D-與L-3HB的分析 22 3.2.2 以D-3HB dehydrogenase確認rat serum中之(D+L)-3HB 22 3.2.3 分析方法之確效試驗 23 3.2.4 Brain、liver、heart、及kidney內D-及L-3HB的分析 23 3.3 實驗結果與討論 25 3.3.1 Rat serum中(D+L)-3HB之分析及驗證 25 3.3.2 Rat serum中L-3HB的檢驗與D-及L-3HB的定量 26 3.3.3 Rat內各組織之D-與L-3HB的分析 27 3.3.4 L-3HB生成機制之探討 30 第四章 D-與L-3HB對心肌細胞使用glucose的影響 36 4.1 前言 36 4.2 實驗設計及進行 40 4.2.1 動物實驗 40 4.2.2 Cardiomyocytes之分離與培養 40 4.2.3 D-與L-3HB對cardiomyocytes之glucose utilization的影響 41 4.2.4 Cardiomyocytes utilize D-、L-、及(D+L)-3HB之調查 41 4.2.5 統計分析 42 4.3 實驗結果與討論 43 4.3.1 Normal 與 DM rat heart homogenates中D-與L-3HB之分析 43 4.3.2 D-與L-3HB 對心肌細胞代謝glucose的不同影響 44 4.3.3 Cardiomycytes對D-、L-、與DL-3HB的不同代謝能力 46 4.3.4 L-3HB生成機制的回顧 47 第五章 結 論 56 參考文獻 58
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