臺灣博碩士論文加值系統

1H NMR核磁共振是研究生物體液很有用之工具，尤其對含氫之物質。此種技術能迅速提供代謝物狀況，又不需前處理。尿液檢體因為比血液之蛋白含量少而更容易及時處理分析，NMR技術提供之數據除了使吾人了解代謝狀況，也能提供對腎功能之了解。
本研究針對高雄醫學大學附設醫院門診第二型糖尿病患及正常葷食、素食者、大學生利用1H-NMR核磁共振，探討葷食、素食、年紀、性別及第二型糖尿病患者，其對尿中代謝物(包括alanine、lactate、glycine、citrate、acetone、acetate、dimetylamine (DMA)、trimethlamine –N-oxide (TMAO)、hippuric acid)值之影響。
結果顯示素食者其glycine、 DMA較葷食者高，而hippurate值在素食者較葷食者低。葷食、素食混合組與第二型糖尿病者其尿液中代謝物比較，顯示出第二型糖尿病人其尿液中lactate、glycine、TMAO及hippurate值均較葷、素食混合組者高。年紀較輕大學生與成年人之葷、素食者之比較，大學生尿中acetate及hippurate值有意義地增高，而glycine值較低。成年葷、素食者中男性僅hippurate值較有意義高於女性者。第二型糖尿病患尿中無檢出糖分或輕微糖分者與有中、重度糖分者其尿液代謝物NMR值均無有意義之差異。第二型糖尿病患尿液中無檢出蛋白或輕微蛋白者其尿中acetate值，DMA值均較中、重度蛋白者高(P值分別為0.023及0.043)。
1H-核磁共振光譜儀能探討葷食、素食、年紀、性別及第二型糖尿病尿液中許多代謝物之間之關係，也能使吾人得到有關此疾病其病患狀況之許多資訊。本研究應用尿液中各種代謝物值以NMR技術測定，便吾人能夠進一步了解人體內不同飲食(葷食、素食) 、不同年紀、不同性別以及第二型糖尿病對尿液中lactate、alanine、acetate、glycine、acetone、citric acid、TMAO、DMA、Hippurate值均有不同程度之影響。在以NMR判讀尿液中各種代謝物值時，不同飲食、年紀、性別均應為考量因素。

1H nuclear magnetic resonance (NMR) is a powerful technique that allows study of biological fluids, providing an overview of important H-containing substances in samples. The main advantage of this technique is that it can furnish a rapid metabolic picture with little or no pretreatment of samples. NMR spectroscopy of urine samples might represent an eligible analytical method and a powerful diagnostic tool. Urine samples are more readily analyzed than blood because of their low protein content, and the registered signals may give important information about renal function in addition to the metabolic status of the subject. For those out-patients of Kaohsiung Medical University Hospital and normal non-vegetarian, vegetarian and college students, we investigate the effect of type II diabetes, non-vegetarian, vegetarian, age and gender on the NMR values of urinary metabolites including lactate, alanine, acetate, glycine, acetone, citrate, TMAO, DMA and hippurate.
Our results revealed that non-vegetarian group had significantly lower urinary glycine, DMA and higher hippurate values than that of vegetarian. Type II diabetes patients had higher urinary lactate, glycine, TMAO and hippurate values than that of combined non-vegetarian and vegetarian group. College students had significantly higher urinary acetate and hippurate, lower glycine values than that of combined non-vegetarian and vegetarian group. For adult male and female groups, except urinary hippurate, other urinary metabolites revealed no significant differences. In type II diabetes patients, those no or mild glucosuria group revealed no significant difference than that of moderate to severe glucosuria group. For diabetes patients, no or mild proteinuria group had higher urinary acetate and DMA values than that of moderate to severe proteinuria group.
NMR spectroscopy permitted us to explore the relationships among the metabolites present in the urine samples of different diet, age, gender and to obtain information about the disease status in type II diabetic patients. Type II diabetes, non-vegetarian, vegetarian, different age and gender had different effect on NMR urinary metabolites. It is also necessary to consider diet, age and gender factors in interpreting urinary NMR values.

目錄


中文摘要…………………………………………………………………5
英文摘要…………………………………………………………………7
壹、緒論…………………………………………………………………9
<I> 核磁共振光譜簡介 ……………………………………………10
<II> 核磁共振原理 …………………………………………………12
<III> 第二型糖尿病…………………………………………………20
<IV> 不同文化對尿中代謝物NMR值之影響………………………23
<V> 飲食對尿液NMR值之影響……………………………………23
<VI> 年紀對NMR值之影響…………………………………………27
<VII> 不同性別對NMR值之影響 ……………………………… …27
貳、目的 ………………………………………………………………28
参、材料與方法…………………………………………………………29
肆、結果…………………………………………………………………35
陸、討論…………………………………………………………………39
柒、參考文獻……………………………………………………………44
捌、附圖與附表…………………………………………………………53

1.Betty H . Kevles著，楊玉齡譯，露骨－醫學造影檔案，天下遠見出版股份有限公司，1999 p.49-56
2.王文竹，透視生物巨分子結構，科學月刊，2002，396：p.1033~1036
3.科學月刊 1981年10月142期
4.McMurry著，王健行等譯，有機化學 (全)，高立圖書有限公司，p. 457-474
5.Gavaghan CL, Nicholson JK, Connor SC, Wilson ID, Wright B, Holmes E. Directly coupled high-performance liquid chromatography and nuclear magnetic resonance spectroscopic with chemometric studies on metabolic variation in Sprague--Dawley rats. Anal Biochem. 2001 15;291: 245-52.
6.Bollard ME, Holmes E, Lindon JC, Mitchell SC, Branstetter D, Zhang W, Nicholson JK. Investigations into biochemical changes due to diurnal variation and estrus cycle in female rats using high-resolution (1)H NMR spectroscopy of urine and pattern recognition. Anal Biochem. 2001;295: 194-202.
7.Gavaghan CL, Holmes E, Lenz E, Wilson ID, Nicholson JK. An NMR-based metabonomic approach to investigate the biochemical consequences of genetic strain differences: application to the C57BL10J and Alpk: ApfCD mouse. FEBS. 2000;484:169-74.
8.Gavaghan CL, Wilson ID, Nicholson JK. Physiological variation in metabolic phenotyping and functional genomic studies: use of orthogonal signal correction and PLS-DA.FEBS. 2002;530:191-6.
9.Brindle JT, Antti H, Holmes E, Tranter G, Nicholson JK, Bethell HW, Clarke S, Schofield PM, McKilligin E, Mosedale DE, Grainger DJ. Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat Med. 2002;8:1439-44.
10.Wevers RA, Engelke UF, Moolenaar SH, Brautigam C, de Jong JG, Duran R, de Abreu RA, van Gennip AH. 1H-NMR spectroscopy of body fluids: inborn errors of purine and pyrimidine metabolism. Clin Chem. 1999;45:539-48.
11.Messana I, Forni F, Ferrari F, Rossi C, Giardina B, Zuppi C. Proton nuclear magnetic resonance spectral profiles of urine in type II diabetic patients. Clin Chem. 1998;44:1529-34.
12.Hart BA, Vogels JT, Spijksma G, Brok HP, Polman C, van der Greef J. 1H-NMR spectroscopy combined with pattern recognition analysis reveals characteristic chemical patterns in urines of MS patients and non-human primates with MS-like disease. J Neurol Sci. 2003; 212:21-30.
13.Lenz EM, Bright J, Wilson ID, Morgan SR, Nash AF. 1H NMR-based metabonomic study of urine and plasma samples obtained from healthy human subjects. J Pharm Biomed Anal. 2003; 33:1103-15.
14.Zuppi C, Messana I, Forni F, Ferrari F, Rossi C, Giardina B, Zuppi C. Proton nuclear magnetic resonance spectral profiles of urine in type II diabetic patients. Clin Chem. 1998;44:1529-34
15.Nicholson JK, Lindon JC, Holmes E. ''Metabonomics'': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica. 1999;29:1181-9
16.Svensson BG, Akesson B, Nilsson A, Paulsson K. Urinary excretion of methylamines in men with varying intake of fish from the Baltic Sea. J Toxicol Environ Health. 1994;41:411-20..
17.Zuppi C, Messana I, Forni F, Ferrari F, Rossi C, Giardina B. Influence of feeding on metabolite excretion evidenced by urine 1H NMR spectral profiles: a comparison between subjects living in Rome and subjects living at arctic latitudes (Svaldbard).Clin Chim Acta. 1998;278:75-79.
18.Clifford MN, Copeland EL, Bloxsidge JP, Mitchell LA. Hippuric acid as a major excretion product associated with black tea consumption. Xenobiotica. 2000;30:317-26.
19.Clayton TA, Lindon JC, Everett JR, Charuel C, Hanton G, Le Net JL, Provost JP, Nicholson JK. A hypothesis for a mechanism underlying hepatotoxin-induced hypercreatinuria. Arch Toxicol. 2003;77:208-17.
20.Lenz EM, Bright J, Wilson ID, Hughes A, Morrisson J, Lindberg H, Lockton A. Metabonomics, dietary influences and cultural differences: a 1H NMR-based study of urine samples obtained from healthy British and Swedish subjects. J Pharm Biomed Anal. 2004;36:841-9
21.Zuppi C, Messana I, Forni F, Rossi C, Pennacchietti L, Ferrari F, Giardina B. 1H NMR spectra of normal urines: reference ranges of the major metabolites. Clin Chim Acta. 1997;265:85-97..
22.Zuppi C, Messana I, Tapanainen P, Knip M, Vincenzoni F, Giardina B, Nuutinen M. Proton Nuclear Magnetic Resonance Spectral Profiles of Urine from Children and Adolescents with Type 1 Diabetes. Clin Chem. 2002;48:660-2.
23.Ko¨rner A, Eklo¨f AC, Celsi G, Aperia A. Increased renal metabolism in diabetes. Mechanism and functional implications. Diabetes 1994;43:629–33.
24.Christiansen SJ, Gammelgaard J, Frandsen M, Parving HH. Increased kidney size, glomerular filtration rate and renal plasma flow in short-term insulin-dependent diabetes. Diabetologia 1981;20:451–6.
25.Hodgkinson A. Citric acid excretion in normal adults and in patients with renal calculus. Clin Sci 1962;23:203-5.
26.Nicholson JK, Timbrell JA, Sadler PJ. Proton NMR spectra as indicators of renal damage. Mercury induced nephrotoxicity in rats. Mol Pharmacol 1985;27:644–51.
27.Foxall PJD, Mellotte GJ, Bending MR, Lindon JC, Nicholson JK.NMR spectroscopy as a novel approach to the monitoring of renal transplant function. Kidney Int 1993;43:234-45.
28.Tessari P. Effects of insulin on whole-body and regional amino acid metabolism. Diabetes Metab Rev 1994;10:253–85.
29.Lundina TA, Knubovets TL, Sedov KR, Markova SA, Sibeldin LA .Variability of kidney tubular interstitial distorsions in glomerulonephritis as measured by 1H NMR urinalysis. Clin Chim Acta 1993;214:165–73.
30.Gartland KPR, Bonner FW, Nicholson JK. Investigation into the biochemical effects of region specific nephrotoxins. Mol Pharmacol 1989;35:242-250.1
31.Yaqoob M, Patrick AW, McClelland P, Stevenson A, Mason H, Whites MC, et al. Relationship between markers of endothelial dysfunction, oxidant injury and tubular damage in patients with insulin-dependent diabetes mellitus. Clin Sci 1993;85:557-562.
32.Yaqoob M, McClelland P, Patrick AW, Stevenson A, Mason H, Bell GM. Tubular damage in microalbuminuric patients with primary glomerulonephritis and diabetic nephropathy. Am J Nephrol 1990; 10:475-481.
33.Mogensen CE, Christensen CK, Vittinghus E. The stages in diabetic renal disease with emphasis on the stage of incipient diabetic nephropathy. Diabetes 1983;32(Suppl 2):64-78.
34.Bell JD, Lee JA, Sadler PJ, Wolkle DR, Woodhain RH. Nuclear magnetic resonance studies of blood plasma and urine from subjects with chronic renal failure: identification of trimethylamine-N-oxide. Biochem Biophys Acta 1991;1096:101-7.
35.Le Moyec L, Pruna A, Eugene M, Bedrossian J, Idatte JM, Huneau JF, Tome D. Proton nuclear magnetic resonance spectroscopy of urine and plasma in renal transplantation follow-up. Nephron 1993;65:433-9.
36.Anthony ML, Gartland KPR, Beddell CR, Lindon JC, Nicholson JK. Cephaloridine-induced nephrotoxicity in the Fischer 344 rat: proton NMR spectroscope studies of urine and plasma in relation to conventional clinical chemical and histopathological assessments of nephronal damage. Arch Toxicol 1992;66:52.
37.Walsh MC, Brennan L, Malthouse JP, Roche HM, Gibney MJ. Effect of acute dietary standardization on the urinary, plasma, and salivary metabolomic profiles of healthy humans. Am J Clin Nutr. 2006; 84:531-9.
38.Nicholson JK, Connelly J, Lindon JC, Holmes E. Metabonomics: a platform for studying drug toxicity and gene function. Nat Rev Drug Discov. 2002;1:153-61.
39.Keun HC. Metabonomic modeling of drug toxicity. Pharmacol Ther. 2006;109:92-106.
40.Robertson DG. Metabonomics in toxicology: a review. Toxicol Sci. 2005;85:809-22.
41.Brindle JT, Antti H, Holmes E, Tranter G, Nicholson JK, Bethell HW, Clarke S, Schofield PM, McKilligin E, Mosedale DE, Grainger DJ. Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat Med. 2002;8:1439-44.
42.Brindle JT, Nicholson JK, Schofield PM, Grainger DJ, Holmes E. Application of chemometrics to 1H NMR spectroscopic data to investigate a relationship between human serum metabolic profiles and hypertension. Analyst. 2003;128:32-6.
43.''t Hart BA, Vogels JT, Spijksma G, Brok HP, Polman C, van der Greef J.1H-NMR spectroscopy combined with pattern recognition analysis reveals characteristic chemical patterns in urines of MS patients and non-human primates with MS-like disease. J Neurol Sci. 2003 15;212:21-30.
44.Odunsi K, Wollman RM, Ambrosone CB, Detection of epithelial ovarian cancer using 1H-NMR-based metabonomics. Int J Cancer 2005;113:782-8.