跳到主要內容

臺灣博碩士論文加值系統

(3.236.50.201) 您好!臺灣時間:2021/08/06 07:38
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:黃于華
研究生(外文):Yu-Hua Huang
論文名稱:高效能液相層析法配合電化學偵測器為定量全血中還原型及氧化型麩胱甘肽之最佳方法
論文名稱(外文):HPLC with electrochemical detector, a good choice for the quantification of reduced and oxidized glutathione in whole blood samples
指導教授:趙崇義趙崇義引用關係
指導教授(外文):Tsun-Yee Chiu
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
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:80
中文關鍵詞:麩胱甘肽高效能液相層析法毛細管電泳法螢光法
外文關鍵詞:Glutathionehigh performance liquid chromatographycapillary electrophoresisFluorometric method
相關次數:
  • 被引用被引用:0
  • 點閱點閱:280
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
麩胱甘肽(Glutathione)是一種非蛋白硫醇,不但可以保護細胞抵抗外來及體內產生的氧化壓力及傷害,而且在整個抗氧化系統中扮演很重要的角色, GSH / GSSG 的比值為體內重要的氧化還原狀態的指標。在之前的文獻報導指出急性心肌梗塞(acute myocardial infarction, AMI)的病人比健康者承受較高的氧化壓力。所以本實驗主要有兩個研究方向,一為發展出一個靈敏度及穩定性高且可同時偵測細胞中及細胞外GSH 與GSSG 含量的方法,因此在本實驗中比較高效能液相層析法-電化學偵測技術(high performance liquid chromatography - electrochemical detector, HPLC-ECD)、毛細管電泳法(capillary electrophoresis, CE)及螢光法(Fluorometric method)來偵測GSH與GSSG 含量。由結果得知使用電化學電位偵測器的高效能液相層析法其靈敏度、回收率及再現性都比毛細管電泳法及螢光法高;另一目的為應用靈敏度及穩定性高的高效能液相層析法來偵測急性心肌梗塞病患與健康者其全血中GSH與GSSG 含量,結果指出急性心肌梗塞病患其GSH濃度與GSH/GSSG 比值明顯比健康者低,此研究結果與文獻報告相符,即急性心肌梗塞病患比健康者承受較高的氧化壓力。
Glutathione is a non-protein thiol which plays an important role in antioxidant system. It can protect the cells from oxidative stress. The ratio of GSH/GSSG can be regarded as an important parameter for the intracellular redox status. Several methods have been described for the quantitation of GSH. Patients with acute myocardial infarction (AMI) have been reported to have higher oxidative stress than normal individuals. The objectives of this research are two folds. One is to define a more sensitive and reliable method for the simultaneous detection of tracellular and extracellular levels of GSH and GSSG. In this study, high performance liquid chromatography (HPLC) with electrochemical detector, capillary electrophoresis (CE) and fluoromertic methods were compared for GSH and GSSG determination. The sensitivity for the detection of GSH and GSSG by coularray multichannel electrochemical detection system was more sensitive than CE and fluoromertic methods. The second objective of this research is to apply this sensitive method to detect the whole blood levels of GSH and GSSG in patients with AMI. Our data indicate that GSH concentration and the ratio of GSH/GSSG are significantly lower in AMI patients. Such finding supports the notion that patients with AMI have higher oxidative stress than normal controls.
第一章 序論
1.1 研究背景及相關文獻 1
1.1.1 麩胱甘肽(Glutathione) 1
1.1.2 偵測全血中氧化型與還原型麩胱甘肽含量之方法 4
1.1.2.1高效能液相層析-電化學偵測技術(High Performance Liquid Chromatography - electrochemical detection,HPLC-ECD) 4
1.1.2.2 毛細管電泳法(Capillary electrophoresis ,CE) 6
1.1.2.3 螢光法(Fluorometric method) 7
1.1.3 麩胱甘肽與疾病之關連性 9
1.2 實驗計畫目標 12
第二章 材料與方法
2.1 實驗材料 13
2.1.1 儀器 13
2.1.2 材料 13
2.1.3 藥品 14
2.1.4 試劑配置 14

2.1.4.1 高效能液相層析法(HPLC) 14
2.1.4.2 毛細管電泳法(Capillary electrophoresis) 15
2.1.4.3 螢光法(Fluorometric method) 15
2.1.5 檢體來源與採集 16
2.2 實驗方法
2.2.1 檢體處理 18
2.2.2 高效能液相層析法-電化學偵測技術(HPLC-ECD) 18
2.2.3 毛細管電泳法(Capillary electrophoresis) 19
2.2.4 螢光法(Fluorometric method) 20
2.2.5 靈敏度 20
2.2.6 穩定性 21
2.2.7 回收率 21
2.2.8 統計方法與繪圖軟體 21
第三章 結果
3.1 HPLC-ECD、毛細管電泳法及螢光法測量全血中GSH、GSSG濃度 22
3.1.1 高效能液相層析法-電化學偵測技術 22
3.1.2 毛細管電泳法 22

3.1.3 螢光法 23
3.2比較HPLC-ECD、毛細管電泳法及螢光法其靈敏度、穩定性和回收率 24
3.2.1 靈敏度 24
3.2.2 再現性 25
3.2.3 回收率 26
3.3 HPLC-ECD、毛細管電泳法及螢光法偵測GSH、GSSG及其ratio之相關性 27
3.4以HPLC-ECD測量健檢者與AMI病患全血中GSH及GSSG含量 28
第四章 討論
4.1 HPLC-ECD偵測全血中GSH 及 GSSG之含量 43
4.2毛細管電泳法偵測全血中GSH 及 GSSG之含量 44
4.3螢光法偵測全血中GSH 及 GSSG之含量 45
4.4 HPLC-ECD、毛細管電泳法及螢光法其靈敏度、再現性和回收率之比較 46
4.5 HPLC-ECD、毛細管電泳法及螢光法之相關性 49


4.6以HPLC-ECD測量健檢者與AMI病患全血中GSH及GSSG含量 50
4.7比較健檢者與AMI病患全血中GSH及GSSG含量 51
參考文獻 54
附錄 65
1.Santangelo F, Witko-Sarsat V, Drueke T, Descamps-Latscha B. Restoring glutathione as a therapeutic strategy in chronic kidney disease. Nephrol Dial Transplant. 2004;19:1951-1955.
2.Meister A. Glutathione metabolism and its selective modification. J Biol Chem. 1988;263:17205-17208.
3.Altomare E, Vendemiale G, Albano O. Hepatic glutathione content in patients with alcoholic and non alcoholic liver diseases. Life Sci. 1988;43:991-998.
4.Deneke SM, Fanburg BL. Regulation of cellular glutathione. Am J Physiol. 1989;257:L163-173.
5.Wu D, Meydani SN, Sastre J, Hayek M, Meydani M. In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects. J Nutr. 1994;124:655-663.
6.Zinellu A, Sotgia S, Usai MF, Chessa R, Deiana L, Carru C. Thiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detection. Electrophoresis. 2005;26:1963-1968.
7.Anderson ME. Glutathione: an overview of biosynthesis and modulation. Chem Biol Interact. 1998;111-112:1-14.
8.Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res. 1999;31:273-300.
9.Carlucci F, Tabucchi A, Biagioli B, et al. Capillary electrophoresis in the evaluation of ischemic injury: simultaneous determination of purine compounds and glutathione. Electrophoresis. 2000;21:1552-1557.
10.Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983;52:711-760.
11.Meister A. On the enzymology of amino acid transport. Science. 1973;180:33-39.
12.Camera E, Picardo M. Analytical methods to investigate glutathione and related compounds in biological and pathological processes. J Chromatogr B Analyt Technol Biomed Life Sci. 2002;781:181-206.
13.Richman PG, Meister A. Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem. 1975;250:1422-1426.
14.Misra I, Griffith OW. Expression and purification of human gamma-glutamylcysteine synthetase. Protein Expr Purif. 1998;13:268-276.
15.Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem. 1979;254:7558-7560.
16.Griffith OW, Meister A. Glutathione: interorgan translocation, turnover, and metabolism. Proc Natl Acad Sci U S A. 1979;76:5606-5610.
17.Richman PG, Orlowski M, Meister A. Inhibition of gamma-glutamylcysteine synthetase by L-methionine-S-sulfoximine. J Biol Chem. 1973;248:6684-6690.
18.Ronzio RA, Meister A. Phosphorylation of methionine sulfoximine by glutamine synthetase. Proc Natl Acad Sci U S A. 1968;59:164-170.
19.Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev. 1979;59:527-605.
20.Winterbourn CC, Metodiewa D. The reaction of superoxide with reduced glutathione. Arch Biochem Biophys. 1994;314:284-290.
21.Pastore A, Federici G, Bertini E, Piemonte F. Analysis of glutathione: implication in redox and detoxification. Clin Chim Acta. 2003;333:19-39.
22.Filosa S, Fico A, Paglialunga F, et al. Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene-deleted mouse embryonic stem cells subjected to oxidative stress. Biochem J. 2003;370:935-943.
23.Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human disease. Biomed Pharmacother. 2003;57:145-155.
24.Hwang C, Sinskey AJ, Lodish HF. Oxidized redox state of glutathione in the endoplasmic reticulum. Science. 1992;257:1496-1502.
25.Liu MJ, Li JX, Lee KM, Qin L, Chan KM. Oxidative stress after muscle damage from immobilization and remobilization occurs locally and systemically. Clin Orthop Relat Res. 2005:246-250.
26.Senthil S, Veerappan RM, Ramakrishna Rao M, Pugalendi KV. Oxidative stress and antioxidants in patients with cardiogenic shock complicating acute myocardial infarction. Clin Chim Acta. 2004;348:131-137.
27.Chillemi R, Zappacosta B, Simpore J, Persichilli S, Musumeci M, Musumeci S. Hyperhomocysteinemia in acute Plasmodium falciparum malaria: an effect of host-parasite interaction. Clin Chim Acta. 2004;348:113-120.
28.Brigelius-Flohe R. Tissue-specific functions of individual glutathione peroxidases. Free Radic Biol Med. 1999;27:951-965.
29.De Chiara B, Bigi R, Campolo J, et al. Blood glutathione as a marker of cardiac allograft vasculopathy in heart transplant recipients. Clin Transplant. 2005;19:367-371.
30.Coral K, Raman R, Rathi S, et al. Plasma homocysteine and total thiol content in patients with exudative age-related macular degeneration. Eye. 2005.
31.Reid M, Jahoor F. Methods for measuring glutathione concentration and rate of synthesis. Curr Opin Clin Nutr Metab Care. 2000;3:385-390.
32.Richie JP, Jr., Lang CA. The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection. Anal Biochem. 1987;163:9-15.
33.Hiraku Y, Murata M, Kawanishi S. Determination of intracellular glutathione and thiols by high performance liquid chromatography with a gold electrode at the femtomole level: comparison with a spectroscopic assay. Biochim Biophys Acta. 2002;1570:47-52.
34.Parmentier C, Wellman M, Nicolas A, Siest G, Leroy P. Simultaneous measurement of reactive oxygen species and reduced glutathione using capillary electrophoresis and laser-induced fluorescence detection in cultured cell lines. Electrophoresis. 1999;20:2938-2944.
35.Lakritz J, Plopper CG, Buckpitt AR. Validated high-performance liquid chromatography-electrochemical method for determination of glutathione and glutathione disulfide in small tissue samples. Anal Biochem. 1997;247:63-68.
36.Muscari C, Pappagallo M, Ferrari D, et al. Simultaneous detection of reduced and oxidized glutathione in tissues and mitochondria by capillary electrophoresis. J Chromatogr B Biomed Sci Appl. 1998;707:301-307.
37.Jin W, Li W, Xu Q. Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection. Electrophoresis. 2000;21:774-779.
38.Patterson JW, Lazarow A. Determination of glutathione. Methods Biochem Anal. 1955;2:259-278.
39.Grassetti DR, Murray JF, Jr. The use of 2,2'-dithiodipyridine in the determination of glutathione and of triphosphopyridine nucleotide by enzymatic cycling. Anal Biochem. 1967;21:427-434.
40.Mapson LW. The estimation of oxidized glutathione. Biochem J. 1953;55:714-717.
41.Jocelyn PC. The reduction of oxidized glutathione in erythrocyte haemolysates in pernicious anaemia. Biochem J. 1960;77:363-368.
42.Martin H, Mc IH. Glutathione, oxidized and reduced, in the brain and in isolated cerebral tissue. Biochem J. 1959;71:275-280.
43.Srivastava SK, Beutler E. Permeability of normal and glucose-6-phosphate dehydrogenase deficient erythrocytes to glutathione. Biochem Biophys Res Commun. 1967;28:659-664.
44.Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74:214-226.
45.Cohn VH, Lyle J. A fluorometric assay for glutathione. Anal Biochem. 1966;14:434-440.
46.Browne RW, Armstrong D. Reduced glutathione and glutathione disulfide. Methods Mol Biol. 1998;108:347-352.
47.Gambhir JK, Lali P, Jain AK. Correlation between blood antioxidant levels and lipid peroxidation in rheumatoid arthritis. Clin Biochem. 1997;30:351-355.
48.Pirmohamed M, Williams D, Tingle MD, et al. Intracellular glutathione in the peripheral blood cells of HIV-infected patients: failure to show a deficiency. Aids. 1996;10:501-507.
49.Samiec PS, Drews-Botsch C, Flagg EW, et al. Glutathione in human plasma: decline in association with aging, age-related macular degeneration, and diabetes. Free Radic Biol Med. 1998;24:699-704.
50.Degl'Innocenti D, Rosati F, Iantomasi T, Vincenzini MT, Ramponi G. GSH system in relation to redox state in dystrophic skin fibroblasts. Biochimie. 1999;81:1025-1029.
51.Bonnefont-Rousselot D, Lacomblez L, Jaudon M, et al. Blood oxidative stress in amyotrophic lateral sclerosis. J Neurol Sci. 2000;178:57-62.
52.Cecchi C, Latorraca S, Sorbi S, et al. Gluthatione level is altered in lymphoblasts from patients with familial Alzheimer's disease. Neurosci Lett. 1999;275:152-154.
53.Hadi Yasa M, Kacmaz M, Serda Ozturk H, Durak I. Antioxidant status of erythrocytes from patients with cirrhosis. Hepatogastroenterology. 1999;46:2460-2463.
54.Lou MF, Dickerson JE, Jr. Protein-thiol mixed disulfides in human lens. Exp Eye Res. 1992;55:889-896.
55.Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J. 2000;16:534-554.
56.Kleinman WA, Richie JP, Jr. Status of glutathione and other thiols and disulfides in human plasma. Biochem Pharmacol. 2000;60:19-29.
57.Lang CA, Mills BJ, Mastropaolo W, Liu MC. Blood glutathione decreases in chronic diseases. J Lab Clin Med. 2000;135:402-405.
58.Hernanz A, Fernandez-Vivancos E, Montiel C, Vazquez JJ, Arnalich F. Changes in the intracellular homocysteine and glutathione content associated with aging. Life Sci. 2000;67:1317-1324.
59.Asensi M, Sastre J, Pallardo FV, et al. Ratio of reduced to oxidized glutathione as indicator of oxidative stress status and DNA damage. Methods Enzymol. 1999;299:267-276.
60.Herzenberg LA, De Rosa SC, Dubs JG, et al. Glutathione deficiency is associated with impaired survival in HIV disease. Proc Natl Acad Sci U S A. 1997;94:1967-1972.
61.Ryan TJ, Antman EM, Brooks NH, et al. 1999 update: ACC/AHA guidelines for the management of patients with acute myocardial infarction. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Management of Acute Myocardial Infarction). J Am Coll Cardiol. 1999;34:890-911.
62.Russo C, Olivieri O, Girelli D, et al. Anti-oxidant status and lipid peroxidation in patients with essential hypertension. J Hypertens. 1998;16:1267-1271.
63.Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000;87:840-844.
64.Berry C, Brosnan MJ, Fennell J, Hamilton CA, Dominiczak AF. Oxidative stress and vascular damage in hypertension. Curr Opin Nephrol Hypertens. 2001;10:247-255.
65.Wu L, Noyan Ashraf MH, Facci M, et al. Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system. Proc Natl Acad Sci U S A. 2004;101:7094-7099.
66.Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res. 1999;85:753-766.
67.Siemianowicz K, Gminski J, Francuz T, Wojcik A, Posielezna B. Activity of antioxidant enzymes in children from families at high risk of premature coronary heart disease. Scand J Clin Lab Invest. 2003;63:151-158.
68.Stempak D, Dallas S, Klein J, Bendayan R, Koren G, Baruchel S. Glutathione stability in whole blood: effects of various deproteinizing acids. Ther Drug Monit. 2001;23:542-549.
69.Jones DP, Carlson JL, Samiec PS, et al. Glutathione measurement in human plasma. Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin Chim Acta. 1998;275:175-184.
70.Serru V, Baudin B, Ziegler F, et al. Quantification of reduced and oxidized glutathione in whole blood samples by capillary electrophoresis. Clin Chem. 2001;47:1321-1324.
71.Owens CW, Belcher RV. A Colorimetric Micro-Method For The Determination Of Glutathione. Biochem J. 1965;94:705-711.
72.Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969;27:502-522.
73.Rose RC, Bode AM. Analysis of water-soluble antioxidants by high-pressure liquid chromatography. Biochem J. 1995;306 (Pt 1):101-105.
74.Senft AP, Dalton TP, Shertzer HG. Determining glutathione and glutathione disulfide using the fluorescence probe o-phthalaldehyde. Anal Biochem. 2000;280:80-86.
75.Floreani M, Petrone M, Debetto P, Palatini P. A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues. Free Radic Res. 1997;26:449-455.
76.Beutler E, West C. Comment concerning a fluorometric assay for glutathione. Anal Biochem. 1977;81:458-460.
77.Wendell PL. Measurement of oxidized glutathione and total glutathione in the perfused rat heart. Biochem J. 1970;117:661-665.
78.Guntherberg H, Rapoport S. [A method for the determination of oxidized glutathione in tissues]. Acta Biol Med Ger. 1968;20:559-564.
79.Lenton KJ, Therriault H, Wagner JR. Analysis of glutathione and glutathione disulfide in whole cells and mitochondria by postcolumn derivatization high-performance liquid chromatography with ortho-phthalaldehyde. Anal Biochem. 1999;274:125-130.
80.Anderson ME. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 1985;113:548-555.
81.Rodriguez-Ariza A, Toribio F, Lopez-Barea J. Rapid determination of glutathione status in fish liver using high-performance liquid chromatography and electrochemical detection. J Chromatogr B Biomed Appl. 1994;656:311-318.
82.Krien PM, Margou V, Kermici M. Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione. Application to human hair follicles. J Chromatogr. 1992;576:255-261.
83.Pelletier S, Lucy CA. HPLC simultaneous analysis of thiols and disulfides: on-line reduction and indirect fluorescence detection without derivatization. Analyst. 2004;129:710-713.
84.Rossi R, Milzani A, Dalle-Donne I, et al. Blood glutathione disulfide: in vivo factor or in vitro artifact? Clin Chem. 2002;48:742-753.
85.Ohsawa M, Tsuru R, Hojo Y, et al. [Relationship between redox state of whole arterial blood glutathione and left ventricular function after acute myocardial infarction]. J Cardiol. 2004;44:141-146.
86.Shimizu H, Kiyohara Y, Kato I, et al. Relationship between plasma glutathione levels and cardiovascular disease in a defined population: the Hisayama study. Stroke. 2004;35:2072-2077.
87.Kharb S. Low blood glutathione levels in acute myocardial infarction. Indian J Med Sci. 2003;57:335-337.
88.Konukoglu D, Akcay T, Erdem T. Susceptibility of erythrocyte lipids to oxidation and erythrocyte antioxidant status in myocardial infarction. Clin Biochem. 1998;31:667-671.
89.Bor MV, Cevik C, Uslu I, Guneral F, Duzgun E. Selenium levels and glutathione peroxidase activities in patients with acute myocardial infarction. Acta Cardiol. 1999;54:271-276.
90.Zachara BA, Ukleja-Adamowicz M, Nartowicz E, Lecka J. Increased plasma glutathione peroxidase activity in patients with acute myocardial infarction. Med Sci Monit. 2001;7:415-420.
91.Altekin E, Coker C, Sisman AR, Onvural B, Kuralay F, Kirimli O. The relationship between trace elements and cardiac markers in acute coronary syndromes. J Trace Elem Med Biol. 2005;18:235-242.
92.Lafont A, Marwick TH, Chisolm GM, Van Lente F, Vaska KJ, Whitlow PL. Decreased free radical scavengers with reperfusion after coronary angioplasty in patients with acute myocardial infarction. Am Heart J. 1996;131:219-223.
93.Simic D, Mimic-Oka J, Pljesa M, et al. Time course of erythrocyte antioxidant activity in patients treated by thrombolysis for acute myocardial infarction. Jpn Heart J. 2003;44:823-832.
94.Beard T, Carrie D, Boyer MJ, et al. [Production of oxygen free radicals in myocardial infarction treated by thrombolysis. Analysis of glutathione peroxidase, superoxide dismutase and malondialdehyde]. Arch Mal Coeur Vaiss. 1994;87:1289-1296.
95.Wang YX, Bocker K, Reuter H, et al. Selenium and myocardial infarction: glutathione peroxidase in platelets. Klin Wochenschr. 1981;59:817-818.
96. 李瑞明, 人類紅血球中麩胱甘肽過氧化酶1之活性及血漿中維生素E濃度對心血管疾病之症狀表現呈負相關性.長庚大學醫學生物技術研究所碩士論文. 2005.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊