(54.236.58.220) 您好!臺灣時間:2021/02/28 08:23
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:蔡屹喬
研究生(外文):Yih-Chiao Tsai
論文名稱: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(外文):Determination of L-3-Hydroxybutyrate in rats and its effects on rat heart
指導教授:李仁愛
指導教授(外文):Jen-Ai Lee
學位類別:博士
校院名稱:臺北醫學大學
系所名稱:藥學系
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:71
中文關鍵詞:L-3-羥基丁酸衍生化心肌細胞
外文關鍵詞:ketone bodiesL-3-Hydroxybutyratederivatizationenantiomeric separationcolumn-switching HPLCglucose utilizationcardiomyocytes
相關次數:
  • 被引用被引用:0
  • 點閱點閱:99
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
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。
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.
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
1. Lehninger AL, Nelson DL, Cox MM: Principles of Biochemistry Second Edition. New York, Worth Publishers, 1993
2. Laffel L: Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes/metabolism research and reviews 15:412-426, 1999
3. Kashiwaya Y, Takeshima T, Mori N, Nakashima K, Clarke K, Veech RL: D--Hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease. Proceedings of the National Academy of Sciences of the United States of America 97:5440-5444, 2000
4. Greene AE, Todorova MT, Seyfried TN: Perspectives on the metabolic management of epilepsy through dietary reduction of glucose and elevation of ketone bodies. Journal of Neurochemistry 86:529-537, 2003
5. Zou Z, Sasaguri S, Rajesh KG, Suzuki R: dl-3-Hydroxybutyrate administration prevents myocardial damage after coronary occlusion in rat hearts. American Journal of Physiology - Heart & Circulatory Physiology 283:H1968-1974, 2002
6. Oshida Y, Iwao N, Ohsawa I, Sato J, Nakao T, Sato Y: Effect of insulin on intramuscular 3-hydroxybutyrate levels in diabetic rats. Hormone & Metabolic Research 30:70-71, 1998
7. Isales CM, Min L, Hoffman WH: Acetoacetate and -hydroxybutyrate differentially regulate endothelin-1 and vascular endothelial growth factor in mouse brain microvascular endothelial cells. Journal of Diabetes & its Complications 13:91-97, 1999
8. Zdzisinska B, Filar J, Paduch R, Kaczor J, Lokaj I, Kandefer-Szerszen M: The influence of ketone bodies and glucose on interferon, tumor necrosis factor production and NO release in bovine aorta endothelial cells. Veterinary Immunology & Immunopathology 74:237-247, 2000
9. Chen V, Wagner G, Spitzer JJ: Regulation of substrate oxidation in isolated myocardial cells by -hydroxybutyrate. Hormone & Metabolic Research 16:243-247, 1984
10. Klee CB, Sokoloff L: Changes in D(-)--hydroxybutyric dehydrogenase activity during brain maturation in the rat. Journal of Biological Chemistry 242:3880-3883, 1967
11. Edmond J: Ketone bodies as precursors of sterols and fatty acids in the developing rat. Journal of Biological Chemistry 249:72-80, 1974
12. Swiatek KR, Dombrowski GJ, Chao KL, Chao HL: Metabolism of L- and D-3-hydroxybutyrate by rat liver during development. Biochemical Medicine 25:160-167, 1981
13. Swiatek KR, Dombrowski GJ, Chao KL: The metabolism of D- and L-3-hydroxybutyrate in developing rat brain. Biochemical Medicine 31:332-346, 1984
14. Webber RJ, Edmond J: Utilization of L(+)-3-hydroxybutyrate, D(-)-3-hydroxybutyrate, acetoacetate, and glucose for respiration and lipid synthesis in the 18-day-old rat. Journal of Biological Chemistry 252:5222-5226, 1977
15. Reed WD, Ozand PT: Enzymes of L-(+)-3-hydroxybutyrate metabolism in the rat. Archives of Biochemistry & Biophysics 205:94-103, 1980
16. Lincoln BC, Des Rosiers C, Brunengraber H: Metabolism of S-3-hydroxybutyrate in the perfused rat liver. Archives of Biochemistry & Biophysics 259:149-156, 1987
17. Scofield RF, Brady PS, Schumann WC, Kumaran K, Ohgaku S, Margolis JM, Landau BR: On the lack of formation of L-(+)-3-hydroxybutyrate by liver. Archives of Biochemistry & Biophysics 214:268-272, 1982
18. Rho JM, Anderson GD, Donevan SD, White HS: Acetoacetate, acetone, and dibenzylamine (a contaminant in L-(+)--hydroxybutyrate) exhibit direct anticonvulsant actions in vivo. Epilepsia 43:358-361, 2002
19. Donevan SD, White HS, Anderson GD, Rho JM: Voltage-dependent block of N-methyl-D-aspartate receptors by the novel anticonvulsant dibenzylamine, a bioactive constituent of L-(+)--hydroxybutyrate. Epilepsia 44:1274-1279, 2003
20. Ahuja S: Chiral Separations: An Overview. In Chiral separations by liquid chromatography Ahuja S, Ed. Washington, D.C., American Chemical Society, 1991, p. 1-3
21. Imai K, Uzu S, Kanda S, Baeyens WRG: Availability of fluorogenic reagents having a benzofurazan structure in the biosciences. Analytica Chimica Acta 290:3-20, 1994
22. Fukushima T, Santa T, Homma H, Al-Kindy SM, Imai K: Enantiomeric separation and detection of 2-arylpropionic acids derivatized with [(N,N-dimethylamino)sulfonyl]benzofurazan reagents on a modified cellulose stationary phase by high-performance liquid chromatography. Analytical Chemistry 69:1793-1799, 1997
23. Yashima E, Okamoto Y: Chiral discrimination on polysaccharides derivatives. Bulletin of the Chemical Society of Japan 68:3289-3307, 1995
24. Guo X, Fukushima T, Li F, Imai K: Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography. The Analyst 127:480-484, 2002
25. Aboul-Enein HY, Ali I, Gubitz G, Simons C, Nicholls PJ: HPLC enantiomeric resolution of novel aromatase inhibitors on cellulose- and amylose-based chiral stationary phases under reversed phase mode. Chirality 12:727-733, 2000
26. Yang X, Fukushima T, Santa T, Homma H, Imai K: Enantiomeric separation and sensitive detection of propranolol, metoprolol and atenolol derivatized with a fluorogenic reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N- dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), on cellulose chiral columns in the reversed-phase mode. The Analyst 122:1365-1369, 1997
27. Aboul-Enein HY, Ali I: Studies on the effect of alcohols on the chiral discrimination mechanisms of amylose stationary phase on the enantioseparation of nebivolol by HPLC. Journal of Biochemical & Biophysical Methods 48:175-188, 2001
28. Lee JA, Tsai YC, Chen HY, Wang CC, Chen SM, Fukushima T, Imai K: Fluorimetric determination of D-lactate in urine of normal and diabetic rats by column-switching high-performance liquid chromatography. Analytica Chimica Acta 534:185-191, 2005
29. Al-Kindy S, Santa T, Fukushima T, Homma H, Imai K: Enantiomeric determination of amines by high-performance liquid chromatography using chiral fluorescent derivatization reagents. Biomedical Chromatography 12:276-280, 1998
30. Hawkins RA, Williamson DH, Krebs HA: Ketone-body utilization by adult and suckling rat brain in vivo. Biochemical Journal 122:13-18, 1971
31. Ruell PA, Gass GC: Enzymatic measurement of 3-hydroxybutyrate in extracts of blood without neutralization. Annals of Clinical Biochemistry 28:183-184, 1991
32. Forsey RG, Reid K, Brosnan JT: Competition between fatty acids and carbohydrate or ketone bodies as metabolic fuels for the isolated perfused heart. Canadian Journal of Physiology & Pharmacology 65:401-406, 1987
33. Ichihara H, Fukushima T, Imai K: Enantiomeric determination of D- and L-lactate in rat serum using high-performance liquid chromatography with a cellulose-type chiral stationary phase and fluorescence detection. Analytical Biochemistry 269:379-385, 1999
34. Paterson P, Sheath J, Taft P, Wood C: Maternal and foetal ketone concentrations in plasma and urine. Lancet 1:862-865, 1967
35. Edmond J: Energy metabolism in developing brain cells. Canadian Journal of Physiology & Pharmacology 70:S118-129, 1992
36. Caamano GJ, Sanchez-Del-Castiool MA, Linares A, Garcia-Peregrin E: In vivo lipid and amino acid synthesis from 3-hydroxybutyrate in 15-day-old chick. Archives Internationales de Physiologie et de Biochimie 98:217-224, 1990
37. Ferrier B, Martin M, Janbon B, Baverel G: Transport of -hydroxybutyrate and acetoacetate along rat nephrons: a micropuncture study. American Journal of Physiology 262:F762-769, 1992
38. Briffeuil P, Thu TH, Lammerant J, Kolanowski J: Increased ketone utilization by the kidney reduces renal lactate uptake but does not affect tubular sodium reabsorption. Metabolism: Clinical & Experimental 42:766-771, 1993
39. Barac-Nieto M: Renal hydroxybutyrate and acetoacetate reabsorption and utilization in the rat. American Journal of Physiology 249:F40-48, 1985
40. Ikeda T, Ishimura M, Terasawa H, Ochi H, Ohtani I, Fujiyama K, Hoshino T, Tanaka Y, Mashiba H: Uptake of ketone bodies in perfused hindquarter and kidney of starved, thyrotoxic, and diabetic rats. Proceedings of the Society for Experimental Biology & Medicine 203:55-59, 1993
41. Poole RC, Halestrap AP: Transport of lactate and other monocarboxylates across mammalian plasma membranes. American Journal of Physiology 264:C761-782, 1993
42. Wang X, Levi AJ, Halestrap AP: Substrate and inhibitor specificities of the monocarboxylate transporters of single rat heart cells. American Journal of Physiology 270:H476-484, 1996
43. Pinson A, Desgres J, Heller M: Partial and incomplete oxidation of palmitate by cultured beating cardiac cells from neonatal rats. Journal of Biological Chemistry 254:8331-8335, 1979
44. Brownsey RW, Boone AN, Allard MF: Actions of insulin on the mammalian heart: metabolism, pathology and biochemical mechanisms. Cardiovascular Research 34:3-24, 1997
45. Zorzano A, Sevilla L, Camps M, Becker C, Meyer J, Kammermeier H, Munoz P, Guma A, Testar X, Palacin M, Blasi J, Fischer Y: Regulation of glucose transport, and glucose transporters expression and trafficking in the heart: studies in cardiac myocytes. American Journal of Cardiology 80:65A-76A, 1997
46. Lewandowski ED, White LT: Pyruvate dehydrogenase influences postischemic heart function. Circulation 91:2071-2079, 1995
47. Stanley WC, Lopaschuk GD, McCormack JG: Regulation of energy substrate metabolism in the diabetic heart. Cardiovascular Research 34:25-33, 1997
48. Randle PJ, Garland PB, Hales CN, Newsholme EA: The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1:785-789, 1963
49. Fischer Y, Bottcher U, Eblenkamp M, Thomas J, Jungling E, Rosen P, Kammermeier H: Glucose transport and glucose transporter GLUT4 are regulated by product(s) of intermediary metabolism in cardiomyocytes. Biochemical Journal 321:629-638, 1997
50. Newsholme EA, Randle PJ, Manchester KL: Inhibition of the phosphofructokinase reaction in perfused rat heart by respiration of ketone bodies, fatty acids and pyruvate. Nature 193:270-271, 1962
51. McGarry JD: What if Minkowski had been ageusic? An alternative angle on diabetes. Science 258:766-770, 1992
52. Chen TM, Goodwin GW, Guthrie PH, Taegtmeyer H: Effects of insulin on glucose uptake by rat hearts during and after coronary flow reduction. American Journal of Physiology - Heart & Circulatory Physiology 273:H2170-2177, 1997
53. Siess EA: Stimulation by 3-hydroxybutyrate of pyruvate carboxylation in mitochondria from rat liver. European Journal of Biochemistry 152:131-136, 1985
54. Hu CM, Chen YH, Chiang MT, Chau LY: Heme oxygenase-1 inhibits angiotensin II-induced cardiac hypertrophy in vitro and in vivo. Circulation 110:309-316, 2004
55. Grinblat L, Pacheco Bolanos LF, Stoppani AO: Decreased rate of ketone-body oxidation and decreased activity of D-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats. Biochemical Journal 240:49-56, 1986
56. Stern JR, Campillo AD, Lehninger AL: Enzymatic racemization of -hydroxybutyryl-S-CoA and the stereospecificity of enzymes of the fatty acid cycle. Journal of the American Chemical Society 77:1073-1074, 1955
57. Kashiwaya Y, Sato K, Tsuchiya N, Thomas S, Fell DA, Veech RL, Passonneau JV: Control of glucose utilization in working perfused rat heart. Journal of Biological Chemistry 269:25502-25514, 1994
58. King LM, Sidell RJ, Wilding JR, Radda GK, Clarke K: Free fatty acids, but not ketone bodies, protect diabetic rat hearts during low-flow ischemia. American Journal of Physiology - Heart & Circulatory Physiology 280:H1173-1181, 2001
59. Izumi Y, Ishii K, Katsuki H, Benz AM, Zorumski CF: -Hydroxybutyrate fuels synaptic function during development. Histological and physiological evidence in rat hippocampal slices. Journal of Clinical Investigation 101:1121-1132, 1998
60. Webber RJ, Edmond J: The in vivo utilization of acetoacetate, D-(-)-3-hydroxybutyrate, and glucose for lipid synthesis in brain in the 18-day-old rat. Evidence for an acetyl-CoA bypass for sterol synthesis. Journal of Biological Chemistry 254:3912-3920, 1979
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
系統版面圖檔 系統版面圖檔