臺灣博碩士論文加值系統

摘要
本研究成功建立一液相層析串聯質譜儀搭配連線固相萃取方法，可快速準確及同時分析尿液中 8-oxo-7,8-dihydroguanine (8-oxoGua) 及 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxodGuo)。研究中成功合成 8-oxoGua 之同位素內標準品 15N5-8-oxoGua，尿液樣本經稀釋後，加入同位素內標準品，不需再經前處理即可直接上機分析，層析時間為15分鐘。接著，利用新開發之分析方法確認 8-oxoGua 在不同儲存環境下之穩定性。研究發現，以0.01 N NaOH 溶液溶解 8-oxoGua 標準品後，高濃度標準品 (10 ug/ml) 在4℃、pH ≤ 11條件下有最佳穩定性，其半衰期達105天；在－20℃、pH ≤ 11條件下穩定性最差，半衰期只有4天。低濃度標準品 (50 ng/ml) 在－20℃、pH < 8條件下穩定性最佳，其半衰期達578天；在－20℃、pH = 12狀況下穩定性最差，半衰期為4天。另外再運用此方法分析20個人體尿液樣本，結果發現尿液中 8-oxoGua 和 8-oxodGuo 背景值分別為5.95 ± 5.20和1.74 ± 0.84 (nmol/mmol creatinine)，比值為3.41。本研究建立之分析方法未來可運用於臨床高通量的樣本檢測，以評估人體內之氧化壓力。

A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to simultaneously determine 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxodGuo) in urine. 15N5-labeled 8-oxoGua was also successfully synthesized to serve as an internal standard in this method. After spiking internal standard, the urine sample can be directly analyzed within 15 min without further sample purification. With this newly developed method, we further investigated the stability of 8-oxoGua under several common storage conditions. The results showed that the stability of 8-oxoGua was affected by pH, temperature and its initial concentration. It was found that the high concentration of 8-oxoGua (10 μg/mL) was the most stable at 4°C with pH value ≤ 11 (the half-life was estimated to be around 105 days), while it was the less stable at -20°C with pH value of 12 (the half-life was estimated to be 4 days). On the other hand, the low concentration of 8-oxoGua (50 ng/mL) had the best stability at -20°C with pH value &lt; 8 (half-life: 578 days), and the worst stability at -20°C with pH value of 12 (half-life: 4 days). Meanwhile, this “on-line SPE LC-MS/MS” method was also applied to determine the background levels of 8-oxoGua/8-oxodGuo in urine of healthy adults. The urinary levels of 8-oxoGua and 8-oxodGuo were 5.95 ± 5.20 and 1.74 ± 0.84 (nmol/mmol creatinine), respectively. The ratio of 8-oxoGua and 8-oxodGuo levels in urine was around 3.41. This “on-line SPE LC-MS/MS” method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.

目 錄
中文摘要-I
英文摘要-II
目 錄-III
表目錄-VII
圖目錄-VIII
一、緒論
1-1 前言-1
1-2 研究動機-1
1-3 研究目標-3
二、文獻回顧
2-1 氧化壓力與基因氧化傷害-4
2-1-1 氧化壓力-4
2-1-2 基因氧化性傷害及其主要產物-6
2-2 基因氧化傷害產物 8-oxodGuo 與疾病之相關性-9
2-3 尿液中 8-oxodGuo 及 8-oxoGua-3
2-4 分析方法-17
2-4-1 液相層析儀配合電化學偵測器 (LC-ECD)-18
2-4-2 液相層析儀結合氣相層析質譜儀 (LC-GC-MS)-19
2-4-3 液相層析儀搭配串聯質譜儀 (LC-MS/MS)-20
2-4-4 酵素連結免疫吸附分析 (ELISA)-21
2-5 8-oxoGua 和 8-oxodGuo 之穩定性 (stability)-22
第三章 材料與方法
3-1 化學藥品及試劑-23
3-2 合成同位素內標準品 [15N5]-8-oxoGua-23
3-3 人體尿液樣本-23
3-3-1 樣本來源-23
3-3-2 尿液前處理-24
3-4 連線固相萃取-液相層析-串聯質譜儀-24
3-4-1 自動連線式固相萃取裝置 (automated on-line SPE)-25
3-4-2 液相層析系統 (LC)-28
3-4-3 電灑離子化串聯質譜儀 (electrospray ionization MS/MS)-28
3-5 分析方法確認-29
3-5-1 偵測極限 (LOD) 和定量極限 (LOQ)-29
3-5-2 方法回收率 (recovery)、組內變異 (intra-day variation) 和組間變異 (inter-day variation)-30
3-6 8-oxoGua 穩定性 (stability) 測試-30
3-6-1 實驗架構-30
3-6-2 分析方法-31
第四章、結果與討論
4-1 同位素內標準品 [15N5]-8-oxoGua 合成-33
4-2 以 on-line SPE-LC-MS/MS 同時分析尿液中 8-oxoGua 及 8-oxodGuo-40
4-3 方法確認-43
4-3-1 偵測極限 (LOD) 和定量極限 (LOQ)-43
4-3-2 精準度 (precision) 和回收率 (recovery)-43
4-3-3 離子抑制 (ion suppression)-44
4-4 8-oxoGua 穩定性測試-48
4-5 在離子化過程人為形成的 8-oxoGua-51
4-6 人體尿液樣本分析-55
第五章、結論-59
參考文獻-61
附錄一、環境檢驗品質管制指引通則-67

表目錄
表2-1 8-oxodGuo 與疾病之相關性-11
表3-1 on-line SPE-LC 運作流程表-27
表4-1 8-oxoGua及 [15N5]-8-oxoGua 的離子對 (MRM pairs)-38
表4-2 方法精準度與回收率-44
表4-3 分析方法比較-46
表4-4 8-oxoGua 在各條件下之一次反應常數及半衰期-50
表4-5 8-oxodGuo 在不同溫度下所產生之 8-oxoGua 量-54
表4-6 常溫下 8-oxodGuo 在不同 DP 下所產生之 8-oxoGua 量-55
表4-7 尿液中 8-oxoGua 及 8-oxodGuo 含量-56
表4-8 本研究及文獻中健康成年人尿液 8-oxoGua/8-oxodGuo 值-58

圖目錄
圖2-1 ROS 與抗氧化系統之氧化壓力平衡圖-6
圖2-2 鹼基氧化主要產物結構圖-7
圖2-3 8-OHdG 的形成及結構式-9
圖2-4 8-OHdG 的 6,8-diketo 異構型式 8-oxodGuo-9
圖2-5 體液 8-oxoGua 與 8-oxodGuo 之可能來源路徑-15
圖2-6 LC-ECD 分析尿液之層析結果-19
圖2-7 文獻中使用 LC-MS/MS 分析尿液結果-21
圖3-1 on-line SPE 組成構造與六向閥轉換情形-26
圖3-2 8-oxoGua 穩定性測試實驗設計-31
圖4-1 8-oxoGua 標準品全離子掃描 (Q1 scan)-34
圖4-2 8-oxoGua 標準品子離子掃描 (product ion scan)-35
圖4-3 [15N5]-8-oxoGua 合成品全離子掃描 (Q1 scan)-36
圖4-4 [15N5]-8-oxoGua 合成品子離子掃描 (product ion scan)-37
圖4-5 8-oxoGua 斷片模式-38
圖4-6 [15N5]-8-oxoGua 內標合成品與 8-oxoGua 標準品之 on-line SPE-LC-MS/MS 層析圖譜-39
圖4-7 文獻中8-oxoGua 標準品斷片型式-40
圖4-8 同時分析尿液中 8-oxoGua 和 8-oxodGuo 層析圖譜-42
圖4-9 8-oxoGua 和8-oxodGuo 方法回收率-43
圖4-10 8-oxoGua 穩定性測試結果-49
圖4-11 串聯質譜儀結構簡圖-51
圖4-12 8-oxodG 標準品 Q1 scan-52
圖4-13 8-oxodGuo 單一標準品在分析時人為 (artifactual) 形成的 8-oxoGua (m/z: 168 → 140)-53

參考文獻
Aikens J, Dix TA. Perhydroxyl radical (HOO.) initiated lipid peroxidation. The role of fatty acid hydroperoxides. J Biol Chem. 1991; 266:15091-8.
Basu S, Eriksson M. Oxidative injury and survival duringendotoxemia. FEBS Lett. 1998;438:159-60.
Benamira M, Johnson, K, Chaudhary A, Bruner, K, Tibbetts C, Marnett LJ. Induction of mutations by replication of malondialdehyde-modified M13 DNA in Escherichia coli: determination of the extent of DNA modification, genetic requirements for mutagenesis, and types of mutations induced. Carcinogenesis. 1995;16:93-9.
Bonn D. How DNA-repair pathways may affect cancer risk. Lancet. 1998;351:42.
Breen AP, Murphy JA. Reactions of oxyl radicals with DNA. Free Radic Biol Med. 1995;18:1033-77.
Cabiscol E, Levine RL. Carbonic anhydrase III. Oxidative modification in vivo and loss of phosphatase activity during aging. J Biol Chem. 1995;270:14742-7
Chao MR, Wang CJ, Wu MT, Pan CH, Kuo CY, Yang HJ, Chang LW, Hu CW. Repeated measurements of urinary methylated/oxidative DNA lesions, acute toxicity, and mutagenicity in coke oven workers. Cancer Epidemiol Biomarkers Prev. 2008;17:3381-9.
Chiou CC, Chang PY, Chan EC, Wu TL, Tsao KC, Wu JT. Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers. Clin Chim Acta. 2003;334:87-94.
Cooke MS, Lunec J, Evans MD. Progress in the analysis of urinary oxidative DNA damage. Free Radic Biol Med. 2002;33:1601-14.
Cooke MS, Evans MD, Dove R, Rozalski R, Gackowski D, Siomek A, Lunec J, Olinski R. DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine. Mutat Res. 2005;574:58-66.
Cooke MS, Olinski R, Loft S; European Standards Committee on Urinary (DNA) Lesion Analysis. Measurement and meaning of oxidatively modified DNA lesions in urine. Cancer Epidemiol Biomarkers Prev. 2008;17:3-14.
Dianov G, Bischoff C, Piotrowski J, Bohr VA. Repair pathways for processing of 8-oxoguanine in DNA by mammalian cell extracts. J Biol Chem. 1998;273:33811-6.
Evans MD, Dizdaroglu M, Cooke MS. Oxidative DNA damage and disease: induction, repair and significance. Mutat Res. 2004;567:1-61.
Floyd RA, Watson JJ, Harris J, West M, Wong PK. Formation of 8-hydroxydeoxyguanosine, hydroxyl free radical adduct of DNA in granulocytes exposed to the tumor promoter, tetradecanoylphorbol acetate. Biochem Biophys Res Commun. 1986;137:841-6.
Foksinski M, Gackowski D, Rozalski R, Siomek A, Guz J, Szpila A, Dziaman T, Olinski R. Effects of basal level of antioxidants on oxidative DNA damage in humans. Eur J Nutr. 2007;46:174-80.
Fraga CG, Shigenaga MK, Park JW, Degan P, Ames BN. Oxidative damage to DNA during aging: 8-hydroxy-2''-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci U S A. 1990;87:4533-7.
Gackowski D, Rozalski R, Roszkowski K, Jawien A, Foksinski M, Olinski R. 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2''-deoxyguanosine levels in human urine do not depend on diet. Free Radic Res. 2001;35:825-32.
Gackowski D, Speina E, Zielinska M, Kowalewski J, Rozalski R, Siomek A, et al. Products of oxidative DNA damage and repair as possible biomarkers of susceptibility to lung cancer. Cancer Res. 2003; 63:4899-902.
Garrido N, Meseguer M, Simon C, Pellicer A, Remohi J. Pro-oxidative and anti-oxidative imbalance in human semen and its relation with male fertility. Asian J Androl. 2004;6:59-65.
Hamberg M, Zhang LY. Quantitative determination of 8-hydroxyguanine and guanine by isotope dilution mass spectrometry. Anal Biochem. 1995;229:336-44.
Helbock HJ, Beckman KB, Shigenaga MK, Walter PB, Woodall AA, Yeo HC, Ames BN. DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc Natl Acad Sci U S A. 1998;95:288-93.
Honda M, Yamada Y, Tomonaga M, Ichinose H, Kamihira S. Correlation of urinary 8-hydroxy-2''-deoxyguanosine (8-oxodGuo), a biomarker of oxidative DNA damage, and clinical features of hematological disorders: a pilot study. Leuk Res. 2000;24:461-8.
Hu CW, Wang CJ, Chang LW, Chao MR. Clinical-scale high-throughput analysis of urinary 8-oxo-7,8-dihydro-2''-deoxyguanosine by isotope-dilution liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction. Clin Chem. 2006;52:1381-8.
Johnson RM, Ravindranath Y, el-Alfy M, Goyette G Jr. Oxidant damage to erythrocyte membrane in glucose-6-phosphate dehydrogenase deficiency: correlation with in vivo reduced glutathione concentration and membrane protein oxidation. Blood. 1994;83:1117-23.
Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2''-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res. 1997;387:147-63.
Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol. 2004;44:239-67.
Lee BR, Um HD. Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration. Exp Cell Res. 1999;248:430-8.
Loft S, Svoboda P, Kasai H, Tjønneland A, Vogel U, Møller P, Overvad K, Raaschou-Nielsen O. Prospective study of 8-oxo-7,8-dihydro-2''-deoxyguanosine excretion and the risk of lung cancer. Carcinogenesis. 2006;27:1245-50.
Malayappan B, Garrett TJ, Segal M, Leeuwenburgh C. Urinary analysis of 8-oxoguanine, 8-oxoguanosine, fapy-guanine and 8-oxo-2''-deoxyguanosine by high-performance liquid chromatography-electrospray tandem mass spectrometry as a measure of oxidative stress. J Chromatogr A. 2007;1167:54-62.
Matsui A, Ikeda T, Enomoto K, Hosoda K, Nakashima H, Omae K, Watanabe M, Hibi T, Kitajima M. Increased formation of oxidative DNA damage, 8-hydroxy-2''-deoxyguanosine, in human breast cancer tissue and its relationship to GSTP1 and COMT genotypes. Cancer Lett. 2000;151:87-95.
Musarrat J, Arezina-Wilson J, Wani AA. Prognostic and aetiological relevance of 8-hydroxyguanosine in human breast carcinogenesis. Eur J Cancer. 1996;32A:1209-14.
Paradies G, Ruggiero FM, Petrosillo G, Quagliariello E. Alterations in carnitine-acylcarnitine translocase activity and in phospholipid composition in heart mitochondria from hypothyroid rats. Biochim Biophys Acta. 1997;1362:193-200.
Park EM, Shigenaga MK, Degan P, Korn TS, Kitzler JW, Wehr CM, et al. Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column. Proc Natl Acad Sci U S A. 1992;89:3375-9.
Phillips DH. Smoking-related DNA and protein adducts in human tissues. Carcinogenesis. 2002;23:1979-2004.
Pilger A, Ivancsits S, Germadnik D, Rudiger HW. Urinary excretion of 8-hydroxy-2''-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2002;778:393-401.
Ravanat JL, Guicherd P, Tuce Z, Cadet J. Simultaneous determination of five oxidative DNA lesions in human urine. Chem Res Toxicol. 1999;12:802-8.
Reardon JT, Bessho T, Kung HC, Bolton PH, Sancar A. In vitro repair of oxidative DNA damage by human nucleotide excision repair system: possible explanation for neurodegeneration in xeroderma pigmentosum patients. Proc Natl Acad Sci U S A. 1997;94:9463-8.
Rebelo IA, Piedade JA, Oliveira-Brett AM. Development of an HPLC method with electrochemical detection of femtomoles of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2''-deoxyguanosine in the presence of uric acid. Talanta. 2004;63:323-31.
Roszkowski K, Gackowski D, Rozalski R, Dziaman T, Siomek A, Guz J, Szpila A, Foksinski M, Olinski R. Small field radiotherapy of head and neck cancer patients is responsible for oxidatively damaged DNA/oxidative stress on the level of a whole organism. Int J Cancer. 2008;123:1964-7.
Rozalski R, Siomek A, Gackowski D, Foksinski M, Gran C, Klungland A, et al. Substantial decrease of urinary 8-oxo-7,8-dihydroguanine, a product of the base excision repair pathway, in DNA glycosylase defective mice. Int J Biochem Cell Biol. 2005;37:1331-6.
Shimoi K, Kasai H, Yokota N, Toyokuni S, Kinae N. Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2''-deoxyguanosine in human urine. Cancer Epidemiol Biomarkers Prev. 2002;11:767-70.
Siomek A, Rytarowska A, Szaflarska-Poplawska A, Gackowski D, Rozalski R, Dziaman T, Czerwionka-Szaflarska M, Olinski R. Helicobacter pylori infection is associated with oxidatively damaged DNA in human leukocytes and decreased level of urinary 8-oxo-7,8-dihydroguanine. Carcinogenesis. 2006;27:405-8.
Verhaegh GW, Richard MJ, Hainaut P. Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper. Mol Cell Biol. 1997;17:5699-706.
Weimann A, Riis B, Poulsen HE. Oligonucleotides in human urine do not contain 8-oxo-7,8-dihydrodeoxyguanosine. Free Radic Biol Med. 2004;36:1378-82.
Wu LL, Chiou CC, Chang PY, Wu JT. Urinary 8-oxodGuo: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta. 2004;339:1-9.