臺灣博碩士論文加值系統

丙烯醛 (Acrolein) 是一種具有高度活性與呼吸道刺激性的 α, β 不飽和醛類，於燃燒 (菸草燃燒、汽機車引擎燃燒) 與高溫烹煮 (油煙) 過程中所產生，在體內亦會經由氧化性傷害與多胺類代謝所產生。3-Hydroxypropyl mercapturic acid (3-HPMA) 是丙烯醛經穀胱甘肽 (GSH) 鍵結後產生一連串代謝過程後所產生的一種代謝產物，最後由尿液排出。
在臺灣，嚼食檳榔及抽菸是口腔鱗狀上皮細胞癌 (Oral squamous cell carcinoma, OSCC) 的兩大危險因子，而嚼食檳榔的族群約有九成皆伴隨抽菸；流行病學研究顯示檳榔可協力增強香菸誘發 OSCC，但其機轉不明。含有石灰之檳榔，嚼食後會使口腔環境變成鹼性 (pH 8-11)，而香菸中含有大量之丙烯醛 (pKa=9.6)，可能會因嚼食檳榔而造成的鹼性環境使香菸中丙烯醛的吸收增加，進而導致尿液中 3-HPMA 的濃度升高。
本研究的目的在於探討抽菸伴隨嚼食檳榔的口腔癌患者尿液中的 3-HPMA 濃度是否高於只抽菸而不嚼食檳榔的患者。口腔癌患者尿液樣本來自林口長庚紀念醫院以及淡水馬偕紀念醫院，一般正常人尿液樣本來自高雄地區一般民眾，並利用液相層析串聯質譜儀 (LC-MS/MS) 分析尿液樣本中的 3-HPMA，同時也利用高效能液相層析搭配紫外光可見光檢測器 (HPLC-UV) 分析尿液中的肌酸酐 (Creatinine)。
本研究結果發現，抽菸且嚼食檳榔的口腔癌患者尿液中3-HPMA (1.91 ng/µg Creatinine) 顯著高於只抽菸患者尿液中的3-HPMA (1.45 ng/µg Creatinine) (p＜0.01)；抽菸伴隨嚼食檳榔的一般正常人尿液中3-HPMA (1.97 ng/µg Creatinine) 亦顯著高於只抽菸正常人尿液中的3-HPMA (1.38 ng/µg Creatinine) (p＜0.05)。以上結果顯示可能是因嚼食檳榔所產生的鹼性環境提升丙烯醛的吸收，因而增加尿液中丙烯醛的濃度。此外，在正常人抽菸者尿液中3-HPMA濃度高於抽菸的口腔癌患者，可能是因為在口腔癌病患體內GSH濃度較低，能與丙烯醛鍵結之GSH量較少所導致。

Acrolein, a highly reactive α, β unsaturated aldehyde and respiratory irritant, is formed during combustion (e.g., cigarette smoke), high-temperature cooking of foods, and in vivo as a by product of oxidative stress and polyamine metabolism. 3-Hydroxypropyl mercapturic acid (3-HPMA), the main metabolite of acrolein in urine, is formed by glutathione (GSH) conjugation following the reduction and normal metabolic processing to a mercapturic acid.
In Taiwan, betel quid chewing and cigarette smoking are two major risk factors for the development of oral squamous cell carcinoma (OSCC), and 90% of betel quid chewers are also cigarette smokers. Moreover, epidemiological studies demonstrated that betel quid can enhance cigarette induced OSCC, but the underlying mechanism remains elusive.
The study was designed to test the hypothesis that the alkaline oral environment (pH 8-11) included by chewing slake lime containing betel quid will facilitate the absorption of acrolein (pKa 9.6) exposed from smoking. The aim of the study was to compare the level of urinary 3-HPMA in cigarette-smoking OSCC patients with and without betel quid chewing history. Urine samples of OSCC patients were obtained from Linkou Chang Gung Memorial Hospital and Mackay Memorial Hospital Tamshui Branch. Urinary 3-HPMA and creatinine were analyzed by HPLC-MS/MS and HPLC-UV, respectively.
The results show that the level of 3-HPMA in OSCC patients with cigarette-smoking and betel quid chewing history (1.90 ng/μg creatinine) was significantly higher (p＜0.01) than in patients with smoking history only (1.42 ng/μg creatinine). In smoking and betel quid chewing normal healthy volunteers had significantly higher (p＜0.05) urinary 3-HPMA (1.97 ng/μg creatinine) than that in smoking only healthy volunteers (1.38 ng/μg creatinine). The study concludes that the higher level of urinary 3-HPMA in cigarette-smoking patients with chewing betel quid history was associated with the increase of acrolein absorption at alkaline environment. Chewing betel quid could enhance the alkalinity of oral cavity, causing the additional absorption of acrolein.

目錄
中文摘要 I
Abstract III
目錄 V
圖目錄 VIII
表目錄 IX
壹、緒論 1
第一節、檳榔 1
簡介 1
檳榔之組成物 2
檳榔嚼塊中致癌因子 4
第二節、香菸 6
簡介 6
香菸中之致癌物質與致癌機轉 7
第三節、丙烯醛 9
簡介 9
暴露途徑 9
法規 11
第四節、口腔癌 12
簡介 12
口腔癌之流行病學特徵 12
口腔癌之危險因子 13
第五節、穀胱甘肽 14
第六節、3-Hydroxypropylmercapturic acid (3-HPMA) 15
貳、文獻回顧 16
第一節、3-HPMA與抽菸之間的關係 16
第二節、正常人與口腔癌患者體內GSH濃度的關係 16
叁、研究動機及目的 17
第一節、研究動機 17
第二節、研究目的 17
肆、材料與方法 18
第一節、化學藥品與試劑 18
第二節、實驗耗料 19
第三節、儀器設備 19
第四節、樣本來源及處理 21
樣本來源 21
樣本前處理 21
第五節、檢量線配置 22
3-HPMA 22
Creatinine 23
第六節、尿液中3-HPMA量測 24
液相層析串聯質譜儀 24
液相層析系統 (Liquid Chromatography, LC) 26
電噴灑離子霧化串聯質譜儀 (Electrospray Ionization Tandem Mass Spectrometry, ESI-MS/MS) 27
第七節、尿液中Creatinine量測 27
第八節、統計分析 29
伍、研究結果 30
第一節、口腔癌患者與一般正常人樣本統計 30
第二節、液相層析串聯質譜儀分析3-HPMA 30
標準品3-HPMA及同位素內標準品d3-3-HPMA之質譜鑑定： 30
第三節、口腔癌患者尿液中各代謝物質的濃度差異 31
Creatinine 31
3-HPMA 32
第四節、口腔癌患者之基本變項在3-HPMA高低濃度間比較 33
第五節、一般正常人之基本變項在3-HPMA高低濃度間比較 33
第六節、口腔癌患者與一般正常人尿液中3-HPMA濃度之差異比較 34
第七節、以多變項線性迴歸分析3-HPMA與兩組別間之關係 34
第八節、以多變項線性迴歸分析3-HPMA與兩族群間之關係 34
第九節、喝酒與否對3-HPMA之影響 35
陸、討論 36
第一節、尿液中3-HPMA之分析方法 36
第二節、尿液中3-HPMA之濃度比較 36
第三節、嚼食檳榔對尿液中Creatinine之影響 37
第四節、GSH與尿液中3-HPMA之關係 38
第五節、喝酒對尿液中3-HPMA之影響 38
第六節、研究限制 40
柒、結論 41
捌、參考文獻 42

圖目錄
圖一、待測物3-HPMA標準品之子離子掃描圖 52
圖二、Creatinine檢量線 53
圖三、以UPLC-UV分析Creatinine之圖譜 54
圖四、3-HPMA檢量線 55
圖五、以LC-MS/MS分析空白樣本 (Blank sample) 之層析圖譜 56
圖六、以LC-MS/MS分析3-HPMA表準品之層析圖譜 57
圖七、以LC-MS/MS分析尿液樣本中3-HPMA之層析圖譜 58

表目錄
表一、3-HPMA分析方法梯度 59
表二、3-HPMA分析方法儀器條件 60
表三、Creatinine分析方法梯度 61
表四、口腔癌病患人口學資料 62
表五、一般正常人人口學資料 63
表六、口腔癌患者Creatinine濃度分布 64
表七、一般正常人Creatinine濃度分布 64
表八、口腔癌患者3-HPMA濃度分布 65
表九、一般正常人3-HPMA濃度分布 66
表十、口腔癌病患基本變項在3-HPMA高低濃度間之比較 (未經Creatinne校正) 67
表十一、口腔癌病患基本變項在3-HPMA高低濃度間之比較 (經Creatinine校正) 68
表十二、一般正常人基本變項在3-HPMA高低濃度間之比較 (未經Creatinine校正) 69
表十三、一般正常人基本變項在3-HPMA高低濃度間之比較 (經Creatinine校正) 70
表十四、口腔癌病患與一般正常人3-HPMA之比較 71
表十五、口腔癌族群以多變項線性迴歸分析基本變項對3-HPMA濃度之影響 72
表十六、口腔癌與一般人以多變項線性迴歸分析基本變項對3-HPMA濃度之影響 73
表十七、各研究尿液中3-HPMA之濃度比較 74

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